Ispp2009 Final Program
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August 9 to 14, 2009 Hotel Delta Centre-Ville Montréal, QC, Canada
FINAL PROGRAM and ABSTRACTS
www.ispp2009.com
August 9 to 14, 2009
WEEK-AT-A-GLANCE SUNDAY August 9
TUESDAY August 11
WEDNESDAY August 12
THURSDAY August 13
FRIDAY August 14
09:30
Plenary 1 R. Blankenship A. Mulkidjanian V. Gorlenko
Plenary 3 E. FLores M. Hagemann K. Forchhammer
Plenary 5 F. Daldal L. Sherman F. Partensky
Plenary 6 C. Bauer D. Kirilovsky B. Masepohl
Plenary 8 C. Harwood P. Tamagnini K. Sivonen
10:00
Coffee Break
Coffee Break
Coffee Break
Coffee Break
Coffee Break
Oral Communications 1&2
Oral Communications 5&6
Oral Communications 9 & 10
Oral Communications 11 & 12
Oral Communications 15 & 16
Lunch
Lunch
Lunch Régence C & Foyer
Régence C or Tour de Ville / Check your Ticket in your badge holder
Régence C or Tour de Ville / Check your Ticket in your badge holder
Régence C & Foyer
Oral Communications 3&4
Oral Communications 7&8
Oral Communications 13 & 14
Oral Communications 17 & 18
Plenary 7 I. Suzuki J.T. Beatty
Closing Keynote F.R. Tabita
07:30
MONDAY August 10
Hotel Delta Centre-Ville Montréal, QC, Canada
Poster Mounting 07:30 to 10:00
08:00 08:30 09:00
10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00 14:30 15:00 15:30 16:00
Registration
Poster Session
16:30 17:00 Welcome & Opening Keynote 18:00 A.E. Walsby 17:30
18:30 19:00 19:30
Opening Reception
Plenary 2 B. Bergman A. Grossman
Free Afternoon
Poster Session
Plenary 4 S. Golden P.C. Wolk
Poster Session
ISPP 2009 Party Sucrerie de la Montagne
20:00 20:30 21:00 21:30 22:00
Buses leave at 17:30 from Hotel Delta Centre-ville
Lunch
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Table of Contents General Information Floor Plans
2
Welcome / Previous ISPP Symposia
3
Committees
4
Symposium Information
5
About Montréal & Useful Information
6
Social Program
7
Scientific Program
9
How This Program Is Organized
10
Instructions to Oral and Poster Presenters
10
Invited Faculty
11
Detailed Program Sunday, August 9
13
Monday, August 10
15
Tuesday, August 11
19
Wednesday, August 12
23
Thursday, August 13
25
Friday, August 14
29
Exhibitors
147
Presenter Index
149
Abstracts Keynotes and Plenaries
33
Oral Communications
45
Posters
85
Symposium Secretariat
Printed in Canada, August 2009 Recycled paper
August 9 to 14, 2009 • Montréal, QC, Canada
c/o IS Event Solutions 1334 Notre-Dame Street West, 2nd Floor Montréal, Québec, Canada H3C 1K7 Tel.: (514) 392-7703 Fax: (514) 227-5083 E-mail: [email protected]
1
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Floor Plans
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Site Plan Floor C
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ROYER
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FOYER
RÉGENCE C
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Welcome to Montréal Welcome to Montréal to ISPP 2009, the 13th International Symposium on Phototrophic Prokaryotes. This is only the second time that the ISPP series finds itself in North America, and we hope that you find Montréal, a major cultural and scientific hub of North America, a pleasant and exciting blend of Old World charm and New World vitality. This is one of my favourite conference series to attend because not only does it inform my specific research passions, it broadly educates me in ways that improve my teaching and increase my breadth of understanding in a variety of outside areas. Indeed, the ISPP series brings together a broad spectrum of interests, techniques, and disciplines. The planned plenary and parallel symposia sessions will permit oral communications by both established researchers and newcomers to this field, as well as promoting active audience participation and lively discussions. Poster sessions will complement the oral program. In addition to the planned social events, we hope that the venue, and the surrounding city filled with small cafés and bistros, will be conducive to productive and insightful informal discussions. One of the highlights of past conferences in this series is the high level of active participation by attendees. We count on you to make ISPP 2009 a great success.
Enjoy!
Patrick Hallenbeck ISPP 2009 Conference Chair Université de Montréal Montréal, Quebec, Canada
Previous ISPP
August 9 to 14, 2009 • Montréal, QC, Canada
1st
1973
Freiburg, Germany
2nd
1976
Dundee, United Kingdom
3rd
1979
Oxford, United Kingdom
4th
1982
Bombannes, France
5th
1985
Grindelwald, Switzerland
6th
1988
Noordwijkerhout, The Netherlands
7th
1991
Amherst, USA
8th
1994
Urbino, Italy
9th
1997
Vienna, Austria
10th
2000
Barcelona, Spain
11th
2003
Tokyo, Japan
12th
2006
Pau, France
3
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Committees
Local Organizing Committee Patrick Hallenbeck, Université de Montréal; Chair Tom Beatty, University of British Columbia David Bird, Université de Québec à Montréal Doug Campbell, Mount Allison University George Espie, University of Toronto George Owttrim, University of Alberta Charles Trick, University of Western Ontario Vladimir Yurkov, University of Manitoba
International Scientific Committee Chair: Jörg Overmann, Germany
4
Group I. Reaction Centres, Antennae, Bioenergetics
Group III. Cell Structure and Biology, Development
Robert Blankenship, USA
Dave G. Adams, United Kingdom
Richard J. Cogdell, United Kingdom
Judith Armitage, United Kingdom
David B. Knaff, USA
Cheryl Kerfeld, USA
Mette Miller, Denmark
Katsumi Matsuura, Japan
Conrad Mullineaux, United Kingdom
Frederic Partensky, France
Wim Vermaas, USA
Satoshi Tabata, Japan
Group II. Metabolism, Biosynthesis, Physiology
Group IV. Taxonomy, Ecology and Evolution
Christiane Dahl, Germany
Patrizia Albertano, Italy
Antonia Herrero, Spain
Ferran Garcia Pichel, USA
Neil Hunter, United Kingdom
Akira Hiraishi, Japan
Masahiko Ikeuchi, Japan
Jörg Overmann, Germany
Oded Beja, Israel
Annick Wilmotte, Belgium
Paula M. Tamagnini, Portugal
Vladimir Yurkov, Canada
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Symposium Information Book of Abstracts
Lost and Found
The abstracts have been included in this publication, immediately following the detailed program section. Abstracts are also available online.
Any lost and found items will be held at the information desk in room Royer for the duration of the event. For any unclaimed items after the conference, please contact IS Event Solutions.
Business Centre
Luggage
The business centre can take care of photocopies, sending faxes etc. It is located one level below the lobby in the administrative offices of the hotel.
During the ISPP conference, whether you are staying at the Hotel Delta Centre-Ville or at a different hotel, you can leave your suitcases with the concierge.
Eco-friendly Event
Meals and Refreshments
We incorporated the following considerations into the planning of this conference: • Online abstract review requiring no printing • Onine registration system requiring no printing • E-mail marketing requiring no printing • Pitchers instead of bottled drinks • Milk and sugar containers instead of individual packaging • Recyclable and environmentally-friendly delegate bags
Coffee breaks will be served daily in the morning and afternoon in the foyer area. For times of service, please refer to the detailed program section.
The hotel is a 4 green key’s property which means that it has shown national industry leadership and commitment to protecting the environment through wide ranging policies and practices. The hotel has mature programs in place that involve management, employees, guests, and the public, and which have shown substantial and measurable results.
Lunch will be served in in different locations: Monday and Friday – Buffet lunch in Régence C and foyer Tuesday and Thursday – Régence C and Tour de Ville (check your lunch ticket!)
Poster Area Posters are installed in room Cartier AB. While delegates can visit posters at their convenience, we have scheduled specific poster sessions on Monday, Tuesday and Thursday.
Emergency/First Aid
Public Notice
For any emergency or first aid services inside the Hotel Delta Centre-Ville, please dial “0” for the operator from any available house phone.
The Hotel Delta Centre-Ville specifically and all Quebec public spaces including terraces in general are smoke-free environments. Smoking is permitted only outside of buildings.
Information
Registration
Should you require any assistance during the Conference, please call 514-991-3851. The Conference Services Desk will be located in the foyer on Sunday and Monday. As of Tuesday, the information desk is located in room Royer (across from Plenary room).
Registration is located in the foyer area of Régence. As of Tuesday, the information desk will be available in the room Royer.
Internet Access Wireless Internet is available for delegates carrying a laptop and can be accessed in the lobby area. Laptops must be wi-fi enabled. Some Internet stations are available inside room Royer. This room will be open during conference hours.
Registration Hours Sunday, August 9
15:00-20:00
foyer
Monday, August 10
07:30-17:00
foyer
Tuesday, August 11
07:30-17:00
Royer
Wednesday, August 12
08:00-12:00
Royer
Thursday, August 13
08:00-17:00
Royer
Friday, August 14
08:00-15:00
Royer
Language Although French is the official language of Quebec, visitors will have no problem communicating in English throughout Montréal. English is the official language of the congress.
August 9 to 14, 2009 • Montréal, QC, Canada
Name Badges For identification and security purposes, delegates must wear their name badges when onsite at the Hotel Delta Centre-Ville. 5
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
About Montreal – Activities You will love Montréal in August. This European-style, multicultural city invites you to enjoy the warm balmy days of summer when Canadians love to be outside enjoying parks and the lively cityscapes. We invite you to enjoy a contagious joie de vivre with a vibrant social life in cafés and on outdoor terraces! The Hotel Delta Centre-Ville is located downtown, close to many attractions, restaurants and shopping.
Rogers Cup
Les Tam-tams du Mont-Royal
August 8-16 • Jarry Park Stadium The best tennis players in the world meet in one of the most prestigious tournaments on the men's professional tour.
Ongoing • Mont-Royal Every Sunday, this free, unofficial festival takes place in the vast green spaces of Mount Royal Park on Avenue du Parc, exactly half-way between Avenue des Pins and rue Mont-Royal.
International Vestibules of Montréal August 12-16 • Downtown 10th edition of Relève en Blues contest
Aqua Ongoing • Montréal Science Centre The global vision that reflects the interdependence between nature and human beings will unfold as the story of water is shared. AQUA is a voyage into the world of water; a non-stop adventure through three distinct spaces where the spectator is completely immersed in the magic of water without a single word being spoken. A signature experience by Guy Laliberté’s ONE DROP foundation.
Piknic Électronik Ongoing • Park Jean-Drapeau Every Sunday, discover the best of the electronic music scene in an environment created to stimulate the senses.
Pirates, Privateers and Freebooters Ongoing • Pointe-à-Callière Discover the amazing stories of these terrors of the high seas as you delve into the history of piracy.
Aires Libres Ongoing • Downtown Sainte-Catherine Street turns into a pedestrian zone for artists and visitors between Berri and Papineau Streets.
About Montreal – Useful Information Credit Cards
Important Numbers
In order to report lost or stolen credit cards, these numbers will provide assistance.
Information Desk Delta Centre-Ville Taxi Service Tourism Montréal Montréal Trudeau Airport
American Express Mastercard Visa
1-800-268-9805 1-800-263-2263 1-800-847-2911
514-991-3851 514-879-1370 514-725-9885 514-873-2015 514-394-7377
Shopping Getting Around Montreal Clean, safe and comfortable, the underground subway system, called the METRO, is an ideal and economical way to reach many of Montreal’s attractions. You can also connect to the city bus system with the same fare. A 1-day pass costs $9 CAD and a 3-day pass costs $17 CAD.
Dress Code The dress code during the conference is casual.
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Montreal’s reputation as an international fashion and shopping centre is well established. With major department stores and countless boutiques lining the city’s downtown streets and the unique underground city, Montreal offers creations by the bestknown names on the local, national and international fashion scene. Find upscale boutiques, art galleries and jewellers a few blocks north, near the Museum of Fine Arts on Sherbrooke Street. A 29-kilometre network of underground passageways provides access to 1,700 boutiques, department stores, restaurants, movie houses, theatres and exhibition halls as well as 7 major hotels, 1,615 housing units and thousands of offices.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Social Program and Excursions
Sunday, August 9, 2009
Wednesday, August 12, 2009
Welcome to Montréal Opening Reception
Conference Party at Sucrerie de la Montagne, Rigaud, Québec
18:30-20:00
19:00 to 23:00
Hotel Delta Centre-Ville, Foyer of Régence, 777 University Street (Included in full conference registration fee for delegates and registered accompanying persons)
Buses leave at 17:30 from Hotel Delta Centre-ville – approx. 45 minute bus ride
Dress Code: Casual
Buses return around 22:00 (Reservation fee of $25 CDN applies.) Dress Code: Comfortable wear!
An exciting evening awaits as ISPP 2009 officially opens with a keynote address followed by a reception with light hors d’oeuvres and beverages. Come and network with your colleagues to set the tone for the meeting.
REMEMBER TO BRING YOUR TICKET!
What’s a Sucrerie? It is a traditional Sugar Shack. In Quebec, sugaring-off is a hearty, typically French Canadian celebration. A traditional event that dates back to pioneer days, it celebrates the end of a hard winter. This activity is held in a sugar bush. The hosts have recreated the rural setting of the last century, with buildings made of authentic barn timbers and fieldstone ovens. This establishment was singled out by the New-York Times Food Editors for its typical decor and the quality of its sugartime feast. Quebecois folklore at its best! All Quebec food specialties are yours to eat as much as you like. A traditional bakery is in operation and a horse drawn caleche ride is part of the fun. Musicians will set the mood.
The traditional Sugar Shack meal includes: • Green Salad • Farm-Style Crusty Bread • Canadian Salted Back Bacon • Québécois Maple-Smoked Ham • Wood-Fired Baked Beans • Farm-Style Omelet • Traditional Meatball Stew • Country-Style Sausages • Old-style Mashed Potatoes • Meat Pie from Quebec's Beauce Region • Homemade Fruit Ketchup & Pickles • Dessert: Pancakes with Maple Syrup, Sugar pie, tea and coffee Vegetarian options are available.
August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Social Program and Excursions Departure from hotel at 1:30 pm. Buses leave from St. Jacques Street.
TOUR 1:
TOUR 3:
OLD MONTRÉAL WALK AND VISIT TO THE MUSEUM OF ARCHEOLOGY
BOTANICAL GARDEN and BIODÔME
Duration: 3 hours Departing from Hotel Delta Centre-Ville, you will first walk through the historic quarter into the life of the first settlers of Montréal. You will stroll through a maze of narrow cobblestone lanes and old buildings, providing a perfect opportunity to discover the history and charm of Old Montréal. En route you will see Place d'Armes, Jacques Cartier Square and Montréal City Hall. One of the highlight of this tour is the museum of Archeology, where you will discover the history of Montréal, starting with the multimedia show, and watch thousands of years of history spin by in 18 fascinating minutes. You will then explore the remains in the Museum at your own pace, through a vibrant and colourful exhibition. You will walk back to the hotel.
Duration: 4 hours Travel along the St. Lawrence River to the second largest Botanical Garden in the world displaying 20,000 species of flowers and plants in 30 outdoor gardens and 10 greenhouses. Of special interest: a remarkable collection of bonsai and orchids. You can choose to visit the Exhibition Greenhouses, the Japanese Pavilion, the Chinese Garden, the Arboretum or the Insectarium. The next visit will bring you to the the Biodôme, a unique concept of a "living" museum. Wander through four full scale natural ecosystems of the Americas: the Tropical Forest, the St. Lawrence Marine, the Laurentian Forest and the Polar World, complete with landscapes, waterfalls, rivers, flora and birds, animals and fish.
TOUR 2: CITY TOUR: MONTRÉAL C'EST MAGNIFIQUE! Duration: 3 hours This exciting tour will introduce you to various parts of the city that make Montréal what it is today. You will have the chance to see how culturally diverse Montréal is, as well as visit landmarks and new developments that are so very important in making Montréal such a unique and wonderful city. See Montréal's elegant upper class communities, our universities and part of our Underground City. Also, the Plateau Mont-Royal, one of the top four "hippest" neighbourhoods in North America, known for its distinctive Montréal architecture, with its spiral staircase and finely wrought cornices. Along the way, the tour includes a stop at the summit of Mt. Royal Park for a panoramic view of the city and a guided visit of Notre-Dame Basilica, an outstanding example of neo-gothic architecture.
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
SCIENTIFIC PROGRAM August 9 to 14, 2009 • Montréal, QC, Canada
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
How This Programme Is Organized ALL ORAL PRESENTATIONS
LEGEND OF POSTER SESSIONS
(INVITED SPEAKERS AND ABSTRACT PRESENTERS) All sessions have been coded to facilitate indexing and reference to presentations in the book of abstracts. The codes consist of two letters (representing the session type) followed by a number from 1 to 18 (2 Keynote, 9 Plenary and 18 Oral Communication sessions in total), followed by a number designating the sequence of presentations.
P = Poster Session
Poster sessions are coded as follows: P.
18
Poster
Poster Number
LEGEND OF ORAL SESSIONS Posters have been organized by Topic, followed by Alphabetic Order.
Keynote Speakers= KN Plenary Speakers = PL OC = Oral Communications OC-
1.
Oral Communications
Session Number
1
First Presentation in Session
Instructions for Oral and Poster Presenters Invited Speakers and Oral Abstract Presenters who Uploaded Their Presentation Pre-Symposium
Invited Speakers and Oral Abstract Presenters who Did NOT Upload Their Presentation Pre-Conference
Even if you have successfully uploaded your presentation, we want to make sure that your presentation functions smoothly onsite. Therefore, we have assigned a review time period for you where you should present yourself at the technician’s table in the room Le Royer. The technician will open your presentation for you to review the functionality of all slides. There won’t be time for you to make major changes. As backup, we request that you bring your presentation on a USB key or CD-rom.
Speakers MUST check in and submit their PowerPoint presentation at least three hours prior to their presentation, we have assigned two computers for you where you could briefly upload and preview your slides. Please see the registration staff for instructions.
Please proceed to the session room 15 minutes before the start of your session in case that the program needs to be adjusted due to last-minute cancellations.
You will be required to provide your presentation on USB key or CD-Rom. PLEASE NOTE THAT ALL PRESENTATIONS WILL BE DELETED FROM THE SERVER AT THE END OF THE DAY. INDIVIDUAL LAPTOP COMPUTERS ARE NOT PERMITTED FOR PRESENTATIONS!
Poster Presenters Poster presenters should mount their posters in the designated poster area (room Cartier) between 07:30-10:00 on Monday, August 10 and remove their posters no later than 18:00 on Thursday, August 13. Conference staff is available to assist. Velcro will already be attached to your board when you arrive.
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Any posters that are not taken down by 18:00 on Thursday will be taken down by conference staff and stored at the registration desk. Posters unclaimed by 18:00 on Friday, August 14 will be discarded.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Invited Faculty Carl Bauer Professor of Biology Indiana University Bloomington, IN, USA
Caroline Harwood Professor of Microbiology University of Washington Seattle, WA, USA
J. Thomas Beatty Professor, Microbiology & Immunology University of British Columbia Vancouver, BC, Canada
Diana Kirilovsky Institut de Biologie et Technologies de Saclay CEA and CNRS Gif sur Yvette, France
Brigitta Bergman Professor, Department of Botany Stockholm University Stockholm, Sweden
Bernd Masepohl Chair, Microbiology Ruhr-Universität Bochum Bochum, Germany
Robert Blankenship Professor of Biology and Chemistry Washington University St. Louis, MO, USA
Armen Y. Mulkidjanian University of Osnabrueck Osnabrueck, Germany
Fevzi Daldal Professor of Biology University of Pennsylvania Philadelphia, PA, USA Enrique Flores Professor of Research Instituto de Bioquímica Vegetal y Fotosíntesis CSIC-Universidad de Sevilla Sevilla, Spain Karl Forchhammer Professor and Head Department of Microbiology / Organismic Interactions University Tübingen Tübingen, Germany Susan Golden Professor Division of Biological Sciences Department of Biology University of California-San Diego La Jolla, CA, USA Vladimir Gorlenko Professor and Head of Laboratory Ecology and Geochemical Activity of Microorganisms Winogradsky Institute of Microbiology Russian Academy of Sciences Moscow, Russia Arthur Grossman Professor, Carnegie Institution of Washington and Department of Biological Sciences Stanford University Stanford, CA, USA Martin Hagemann Bioscience Institute, Plant Physiology Department Universität Rostock Rostock, Germany
August 9 to 14, 2009 • Montréal, QC, Canada
Frederic Partensky Marine Photosynthetic Prokaryotes Team Station Biologique, CNRS and Université Paris06 (UMR 7144) Roscoff, France Louis Sherman Professor, Biological Sciences Purdue University West Lafayette, IN, USA Kaarina Sivonen Professor, Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland Iwane Suzuki Associate Professor University of Tsukuba Tsukuba, Ibaraki, Japan F. Robert Tabita Professor; Ohio Eminent Scholar of Microbiology Ohio State University Columbus, OH, USA Paula M. Tamagnini Departamento de Botânica, Fac. Ciências IBMC – Instituto de Biologia Molecular e Celular Universidade do Porto Porto, Portugal Anthony E. Walsby Emeritus Professor of Microbiology School of Biological Sciences University of Bristol Bristol, United Kingdom Peter C. Wolk Professor Michigan State University E. Lansing, MI, USA
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Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Sunday, August 9, 2009
17:30–18:30
OPENING KEYNOTE
RÉGENCE AB
WELCOME TO ISPP 2009 AND INTRODUCTION OF KEYNOTE SPEAKER. Patrick Hallenbeck, Symposium Chair and Université de Montréal, Montréal, QC. WELCOME FROM THE CHAIR OF THE INTERNATIONAL SCIENTIFIC COMMITTEE. Jörg Overmann, Planegg-Martinsried, Germany. KN-1.1 A SCIENTIST OF THE FLOATING WORLD. Anthony E. Walsby, University of Bristol, United Kingdom. Biography I trained as a botanist at the University of Birmingham and moved to Westfield College London for research with G. E. Fogg on cyanobacteria, which at that time masqueraded as blue-green algae. My first paper, with Peter Fay in 1966, described the serendipitous isolation of heterocysts. Bill Stewart saw the opportunity to test N2-fixation by isolated heterocysts; my contribution was to measure 15N labelling by mass spectrometry. This technique led to my analysing the gas in gas vesicles of cyanobacteria and a life-time of research on these fascinating structures. In the early 70s I spent two years on sabbatical leave in Berkeley, where I met and married Fausta Segrè. Returning to the UK, I spent another seven years with Fogg in marine science at the University College of North Wales and then, despite never having worked on a real plant, I moved to the Chair of Botany at the University of Bristol. Throughout my career chasing gas vesicles I sought collaborations with experts in other fields: on crystallography with Allen Blaurock in London; microscopy with Daniel Branton at Berkeley; protein sequencing with John Walker at Cambridge; and mathematical physics with John Simpson at Menai Bridge. My machines for measuring gas vesicles suggest inspiration by Heath Robinson (or Rube Goldberg). The results were the mechanics of gas vesicles, measurements of cell turgor pressure and estimates of gas diffusion rates into heterocysts. At Bristol I enjoyed a long collaboration with Paul Hayes and some excellent students. We took the physiology and molecular biology of cyanobacteria from the laboratory to analyse populations in lakes, seas and salterns. We had many nice collaborations with local ecologists, including those with Ferdinand Schanz on Lake Zürich, Lucas Stal on the Baltic Sea, and Colin Reynolds on the English Lakes. Sometimes I returned with unexpected trophies: Dactylococcopsis from Solar Lake, the square archeon Haloquadratum from the Sinai, and hundreds of Planktothrix strains from Lake Zürich.
18:30–20:00
WELCOME RECEPTION
FOYER – RÉGENCE
See page 7 for details.
August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
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Notes
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Monday, August 10, 2009
08:30–10:00
PLENARY 1: PHYLOGENY, TAXONOMY AND DIVERSITY
RÉGENCE AB
Co-Chairs: Johannes Imhoff, Kiel, Germany and Min Chen, Sydney, Australia 08:30
PL-1.1
EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHIC BACTERIA DEDUCTED FROM WHOLE GENOME COMPARISONS. Robert Blankenship, Washington University, St. Louis, MO, USA.
09:00
PL-1.2
ORIGIN AND EVOLUTION OF PHOTOSYNTHETIC SYSTEMS. Armen Y. Mulkidjanian, University of Osnabrueck, Osnabrueck, Germany.
09:30
PL-1.3
ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA: DIVERSITY, NEW TAXONS. Vladimir Gorlenko, Russian Academy of Sciences, Moscow, Russia.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 1: PHYLOGENY TAXONOMY AND DIVERSITY
FOYER
RÉGENCE C
Chair: Robert Blankenship, St. Louis, MO, USA OC-1.1 SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETIC MARKERS FOR PURPLE SULFUR BACTERIA. Marcus Tank, Vera Thiel, Johannes F. Imhoff. IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University), Kiel, Germany. OC-1.2 BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTING GENES IN MARINE PHYTOPLANKTON. Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2. 1 School of Ocean and Earth Sciences, National Oceanography Centre, Southampton, United Kingdom; 2School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia. OC-1.3 PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THE CYANOBACTERIAL GENERA ANABAENOPSIS AND CYANOSPIRA. Stefano Ventura, Cristina Mascalchi, Claudio Sili. Institute of Ecosystem Study, National Research Council, Sesto Fiorentino, Italy. OC-1.4 CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATS OF A TROPICAL LAGOON, TIKEHAU ATOLL, TUAMOTU ARCHIPELAGO. Katarzyna A. Palinska1, Raeid M. M. Abed2†, Katja Wendt1, Maria Łotocka3, Loic Charpy4 & Stejpko Golubic5. 1 Institute of Chemistry and Biology of the Marine Environment, Geomicrobiology Dep. and CvO University of Oldenburg, Oldenburg, Germany; 2College of Science-Biology Department, Sultan Qaboos University, Al-Khod, Muscat, Sultanate of Oman; 3Institute of Oceanology, Polish Academy of Sciences, Powsta ców Warszawy, Sopot, Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167 (CYROCO), Traverse de la Batterie des Lions, Marseille, France; 5Biological Science Center, Boston University, Boston, MA, USA. OC-1.5 METAGENOMIC AND PHYLOGENETIC ANALYSES OF CYANOBACTERIAL MATS IN EXTREME HIGH ARCTIC ENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICAL FUNCTION. Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3, Warwick F. Vincent1. 1 Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté de Médecine, Université Laval; 3Québec-Océan, Dép. de Biologie, and Institut de biologie intégrative et des systèmes (IBIS); Université Laval; Québec, QC, Canada. OC-1.6 GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENON SPP. AND ANABAENA SPP. IN NORTHEAST GERMAN LAKES. Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1. 1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes, Neuglobsow; 2Federal Environmental Agency, Berlin; 3Brandenburg Technical University, Department of Freshwater Conservation, Bad Saarow; Germany. August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Monday, August 10, 2009
10:30–12:30
ORAL COMMUNICATIONS 2: ECOLOGY I
RÉGENCE AB
Chair: Mary Allen, Wellesley, MA, USA OC-2.1 MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS IN PHOTOTROPHIC CONSORTIA. Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller. Section Microbiology, Department Biology I, University of Munich, Planegg-Martinsried, Germany. OC-2.2 LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBIC ANOXYGENIC PHOTOTROPHS IN A SEMIARID REALM. J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. OC-2.3 ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MAT- NITROGEN FIXATION. Ina Severin, Lucas J. Stal. Department of Marine Microbiology, NIOO-Center for Marine and Estuarine Ecology, Yerseke, The Netherlands. OC-2.4 BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCED PHOTOSYNTHETIC MICROBES. Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer. SRI International, Menlo Park, CA, USA. OC-2.5 DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACE WATERS OF MARINE AND FRESHWATER ENVIRONMENTS WITH AN EMPHASIS ON CYANOBACTERIA. Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, Torsten Staller2, Rüdiger Schulz2, Jens Appel1. 1 School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Botanisches Institut, Universität Kiel, Kiel, Germany. OC-2.6 HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRING MICROBIAL MATS DOMINATED BY THE FILAMENTOUS ANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUS AGGREGANS. H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1. 1 Department of Biology, Tokyo Metropolitan University, Tokyo; 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki; Japan.
12:30–14:00
16
LUNCH
RÉGENCE C AND FOYER
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Monday, August 10, 2009
14:00–15:00
ORAL COMMUNICATIONS 3: ECOLOGY II
RÉGENCE AB
Chair: Vladimir Gorlenko, Moscow, Russia OC-3.1 CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROM THE HALOCLINE OF THE SALINE MEROMICTIC LAKE, LAKE SUIGETSU, JAPAN. Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya. Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan. OC-3.2 THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIA PROCHLOROCOCCUS. Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA. OC-3.3 THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTAL CYANOBACTERIA COMMUNITY AND ITS CONTRIBUTION TO TOXICITY IN A TROPICAL BRAZILIAN RESERVOIR. Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos. Department of Botany, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
14:00–15:00
ORAL COMMUNICATIONS 4: PHYSIOLOGY I
RÉGENCE C
Chair: David Adams, Leeds, United Kingdom OC-4.1 ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUM TEPIDUM: GENES, METABOLITES AND LINKAGES TO LIGHT HARVESTING. Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan, Rachael Morgan-Kiss. College of Marine and Earth Studies and DBI, Newark, DE, USA. OC-4.2 CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS. Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E. Blankenship1. 1 Departments of Biology and Chemistry; 2Department of Energy, Environment and Chemical Engineering; Washington University, St. Louis, MO, USA. OC-4.3 STRUCTURAL AND FUNCTIONAL STUDIES OF A PHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGH COMPARATIVE METATRANSCRIPTOME ANALYSIS. Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt, Lynn Tomsho, Stephan C. Schuster, David M. Ward, Donald A. Bryant. The Pennsylvania State University, University Park, PA, USA.
15:00–17:00
POSTER SESSION
17:00–18:00
PLENARY 2: ECOLOGY
CARTIER AB
RÉGENCE AB
Co-Chairs: Jörg Overmann, Planegg-Martinsried, Germany and Vladimir Yurkov, Winnipeg, MB, Canada 17:00
PL-2.1
MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMIC AND PROTEOMIC ANALYSES Brigitta Bergman, Department of Botany, Stockholm University, Stockholm, Sweden.
17:30
PL-2.2
PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATION AND POTENTIAL INTERACTIONS Arthur Grossman, Department of Plant Biology, The Carnegie Institution, Stanford, CA, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
17
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
18
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
08:30–10:00
PLENARY 3: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES
RÉGENCE AB
Chairs: Jack Meeks, Davis, CA, USA and James Golden, La Jolla, CA, USA 08:30
PL-3.1
MULTICELLULARITY IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA. Enrique Flores, Professor of Research, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain.
09:00
PL-3.2
EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY GLYCOLATE METABOLISM IN CYANOBACTERIA. Martin Hagemann, Plant Physiology, University Rostock, Inst. Biosciences, Rostock, Germany.
09:30
PL-3.3
THE NETWORK OF INTERACTIONS OF PII SIGNALING IN CYANOBACTERIA. Karl Forchhammer, Department of Microbiology, University Tübingen, Tübingen, Germany.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 5: PHYSIOLOGY II
FOYER
RÉGENCE AB
Chairs: David Knaff, Lubbock, TX, USA and Ann Magnuson, Uppsala, Sweden OC-5.1 HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOC PUNCTIFORME DESTINED TO BECOME HORMOGONIA. H. Christman, E.L. Campbell, J.C. Meeks. Section of Microbiology, University of California, Davis, CA, USA. OC-5.2 PLANT CELL WALL EPITOPES ARE EXPRESSED BY CYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS. Owen Jackson, J. Paul Knox, David G. Adams. University of Leeds, West Yorkshire, United Kingdom. OC-5.3 REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE IN HETEROCYST-FORMING CYANOBACTERIA. Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, Enrique Flores2, David G. Adams3. 1 School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty of Biological Sciences, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom. OC-5.4 SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS IN ANABAENA SP. STRAIN PCC 7120. Jinjie Liu, C. Peter Wolk. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA. OC-5.5 THE INVOLVEMENT OF TWO SIGMA FACTORS IN CYANOBACTERIAL AKINETE FORMATION. Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers. Department of Biology, California State University, Northridge, CA, USA. OC-5.6 SEQUENCES REGULATING NITROGENASE GENE EXPRESSION IN THE CYANOBACTERIUM ANABAENA VARIABILIS. Teresa Thiel, Justin L. Ungerer. Department of Biology, University of Missouri St. Louis, St. Louis, MO, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
19
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
10:30–12:30
ORAL COMMUNICATIONS 6: BIOENERGETICS I
RÉGENCE C
Chairs: Michael Summers, Northridge, CA, USA OC-6.1 THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES AT THE MONOMER-MONOMER INTERFACE OF PHOTOSYSTEM II IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley. Department of Biochemistry, University of Otago, Dunedin, New Zealand. OC-6.2 PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO IN SYNECHOCYSTIS. Danny Yao, Dan Brune, Wim Vermaas. School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. OC-6.3 IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FOR THE OXYGEN DEPENDANCE OF THE PHOTOSYNTHETIC ACTIVITY OF ROSEOBACTER DENITRIFICANS. Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and Andreas Labahn1. 1 Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany; 2Department of Cell Biology, Harvard Medical School, Boston MA, USA. OC-6.4 VARIABLE FLUORESCENCE IN HELIOBACTERIUM MODESTICALDUM CELLS: OBSERVATION AND EXPLANATION. Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, Stefano Santabarbara1,2, Robert Blankenship3. 1 Arizona State University, Dept. of Chemistry and Biochemistry, Tempe, AZ, USA; 2Institut de Biologie Physico-Chimique, Paris, France; 3Washington University, Departments of Biology and Chemistry, St Louis, MO, USA. OC-6.5 NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM II PROTECTS THE CYANOBACTERIUM MICROCOLEUS SP. AGAINST EXCESS LIGHT STRESS. Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2. Departments of 1Biological Chemistry, 2Plant and Environmental Sciences and 3The Interuniversity Marine Institute, Eilat, The Hebrew University of Jerusalem, Israel. OC-6.6 THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-Mari Aro. Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland.
12:30–14:00
20
LUNCH
RÉGENCE C AND TOUR DE VILLE Please check for a ticket inside your badge holder. Only those with a ticket in their badge holder are dining in the Tour de Ville.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
14:00–15:00
ORAL COMMUNICATIONS 7: PHYSIOLOGY III
RÉGENCE AB
Chairs: Enrique Flores, Sevilla, Spain OC-7.1 PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA (PRRA), TRANSCRIPTIONAL REGULATORS OF THE CBB OPERONS (CO2 FIXATION) IN RHODOBACTER SPHAEROIDES. Andrew W. Dangel, F. Robert Tabita . Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, OH, USA. OC-7.2 PpsR AS A NEW TYPE OF HEMIN SENSOR. Liang Yin, Vladimira Dragnea, Carl Bauer. Indiana University, Bloomington, IN, USA. OC-7.3 ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN: AN EXPERIMENTAL SCENARIO FOR A STRICT ANAEROBIC PHOTOTROPH. Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg, Françoise Reiss-Husson, Chantal Astier, Soufian Ouchane. CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; Université Paris-Sud, Orsay; Université Pierre et Marie Curie, Paris; France.
14:00–15:00
ORAL COMMUNICATIONS 8: BIOENERGETICS II, PROTEINS, GENOMICS II
RÉGENCE C
Chair: Susan Golden, La Jolla, CA, USA and Willem Vermaas, Tempe, AZ, USA OC-8.1 CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BY FERREDOXIN:THIOREDOXIN REDUCTASE, FERREDOXIN AND THIOREDOXIN M. David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, Masakazu Hirasawa, Marcellus Ubbink. Texas Tech University, Lubbock, TX, USA. OC-8.2 CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA. Shinichi Takaichi1, Mari Mochimaru2. 1 Department of Biology, Nippon Medical School, Kawasaki; 2Department of Natural Sciences, Komazawa University, Setagaya, Tokyo; Japan. OC-8.3 EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROM PURPLE BACTERIA AND CYANOBACTERIA. Gerhard Sandmann, Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Frankfurt, Germany.
15:00–16:30
POSTER SESSION
16:30–17:30
PLENARY 4: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES
CARTIER AB
RÉGENCE AB
Chair: Fevzi Daldal, Philadelphia, PA, USA 16:30
PL-4.1
A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME. Susan Golden, Department of Biology, University of California-San Diego, La Jolla, CA, USA.
17:00
PL-4.2
EFFECTS OF INSERTION SEQUENCES ON THE GENOME OF ANABAENA SP. STRAIN PCC 7120. Peter C. Wolk, Michigan State University, E. Lansing, MI, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
21
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
22
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Wednesday, August 12, 2009
08:30–10:00
PLENARY 5: BIOENERGETICS, PROTEINS AND GENOMICS
RÉGENCE AB
Chairs: F. Robert Tabita, Columbus, OH, USA 10:00
PL-5.1
CYTOCHROMES STRUCTURE FUNCTION AND BIOGENESIS. Fevzi Daldal, University of Pennsylvania, Philadelphia, PA, USA.
10:30
PL-5.2
BETTER LIVING THROUGH CYANOTHECE-UNICELLULAR DIAZOTROPHIC CYANOBACTERIA WITH HIGHLY VERSATILE METABOLIC SYSTEMS. Louis Sherman, Professor, Biological Sciences, Purdue University, West Lafayette, IN, USA.
11:00
PL-5.3
PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENT EVOLUTION SCHEMES FROM A COMMON ANCESTRAL GENOME. Frederic Partensky, Marine Photosynthetic Prokaryotes Team, Station Biologique, CNRS and Université Paris, Roscoff, France.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES
FOYER
RÉGENCE AB
Chairs: Bernd Masepohl, Bochum, Germany OC-9.1 THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM – A STRUCTURAL MODEL BASED ON SOLID-STATE NMR DATA COMBINED WITH MOLECULAR SIMULATION STUDIES OF CD AND ABSORPTION SPECTRA. Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1, Peter Højrup2, Anders Giessing2, Juha Linnanto3, Niels-Ulrik Frigaard4, Mette Miller2 and Niels Chr. Nielsen1. 1 Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus, Århus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 3Department of Chemistry, University of Jyväskylä, Finland; 4Department of Biology, University of Copenhagen, Copenhagen, Denmark. OC-9.2 THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLS IN CHLOROSOMES OF CHLOROBACULUM TEPIDUM. Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, Michael Reus, Aline Gomez Gomez Maqueo Chew, Alfred R. Holzwarth, Egbert J. Boekema, Huub J. M. de Groot. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. OC-9.3 MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANES IN CYANOBACTERIA. Sawsan Hamad, Wim Vermaas. School of Life Sciences and the Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. OC-9.4 PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OF FUNCTION IN THE CARBOXYSOME. Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C. Bagby3, Sabine Heinhorst4, Fei Cai4, Gordon Cannon4, Sallie W. Chisholm3. 1 US Department of Energy, Joint Genome Institute, Walnut Creek, CA; 2Department of Plant and Microbial Biology, University of California, Berkeley, CA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; 4Department of Biochemistry, University of Southern Mississippi, Hattiesburg, MS; USA.
August 9 to 14, 2009 • Montréal, QC, Canada
23
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Wednesday, August 12, 2009
10:30–12:30
ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES
RÉGENCE AB
Chairs: Bernd Masepohl, Bochum, Germany OC-9.5 PROTEOMIC ANALYSIS OF THE DEVELOPING INTRACYTOPLASMIC MEMBRANE DURING CHROMATIC ADAPTATION IN RHODOBACTER SPHAEROIDES. Kamil Woronowicz, Robert A. Niederman. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA. OC-9.6 BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODING THE MEMBRANE-ASSOCIATED THIOREDOXIN PROTEIN PLAYS AN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OF PS I. Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1. 1 Department of Biochemistry and Molecular Biology; 2Department of Chemistry; The Pennsylvania State University, University Park, PA, USA.
10:30–12:30
ORAL COMMUNICATIONS 10: PHYSIOLOGY IV
RÉGENCE C
Chair: J. Thomas Beatty, Vancouver, BC, Canada OC-10.1
A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THE APPARENT PHOTOSYNTHETIC AFFINITY FOR CO2 BY MODULATING LOW-CO2-INDUCED GENE EXPRESSION. Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2, Yukako Hihara2, Ariel Gaathon3, Mario Lebendiker4, Aaron Kaplan1. 1 Dept. of Plant and Environmental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; 2 Dept. of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan; 3Bletterman Laboratory, Interdepartmental Equipment Unit, Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre, Hebrew University of Jerusalem, Jerusalem, Israel.
OC-10.2
THE EFFECTS OF TEMPERATURE AND OXYGEN ON NITROGENASE ACTIVITY IN THE THERMOPHILIC CYANOBACTERIUM FISCHERELLA SP. Lucas J. Stal, Department of Marine Microbiology, Netherlands Institute of Ecology, Yerseke, The Netherlands.
OC-10.3
PHOSPHATE SCAVENGING IN AN UNPREDICTABLE ENVIRONMENT, HOW DO CYANOBACTERIA MEET THE CHALLENGE? Frances D. Pitt, David J. Scanlan. Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom.
OC-10.4
REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY IN CYANOBACTERIA. IDENTIFICATION OF THE REGIONS INVOLVED IN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS. Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J. Florencio. Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla-CSIC, Sevilla, Spain.
OC-10.5
BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS. Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A. Bryant1. 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2 Department of Biological Sciences, University of New Orleans, New Orleans, LA; USA.
OC-10.6
ACCUMULATION OF TREHALOSE IN RESPONSE TO DESICCATION AND CONTROL OF TREHALASE IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE. Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima. Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan.
FREE AFTERNOON AND EXCURSIONS
17:30 24
ISPP 2009 PARTY (SEE PAGE 7) August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Thursday, August 13, 2009
08:30–10:00
PLENARY 6: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION
RÉGENCE AB
Chairs: Donald A. Bryant, University Park, PA, USA and Kaori Inoue-Sakamoto, Ishikawa, Japan 08:30
PL-6.1
OXYGEN AND LIGHT CONTROL OF TETRAPYRROLE BIOSYNTHESIS IN RHODOBACTER SPECIES. Carl Bauer, Indiana University, Bloomington, IN, USA.
09:00
PL-6.2
A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHT INTENSITY SENSOR IN CYANOBACTERIA PHOTOPROTECTION. Diana Kirilovsky, Institut de Biologie et Technologies de Saclay, CEA and CNRS, Gif sur Yvette, France.
09:30
PL-6.3
NITROGEN AND MOLYBDENUM CONTROL OF NITROGEN FIXATION. Bernd Masepohl, Chair, Microbiology, Ruhr-Universität Bochum, Bochum, Germany.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 11: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I
FOYER
RÉGENCE AB
Chairs: Iwane Suzuki, Tsukuba, Ibaraki, Japan OC-11.1
FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAIN IN RHODOSPIRILLUM CENTENUM PPR. J.A. Kyndt, T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
OC-11.2
SHORT-TERM LIGHT ADAPTATION STRATEGIES OF PHYCOBILISOME-CONTAINING PHOTOSYNTHETICS, CYANOBACTERIA AND RED ALGAE. Igor Stadnichuk, PA.N.Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
OC-11.3
REGULATORY RNAS IN CYANOBACTERIA AND BEYOND. W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1, J. Vogel2, A. Wilde3. 1 Freiburg Initiative in Systems Biology and Faculty of Biology, University Freiburg; 2Max-Planck-Institute for Infection Biology Berlin; 3Justus-Liebig University Giessen, Institute of Microbiology and Molecular Biology; Germany.
OC-11.4
FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUM PROCHLOROCOCCUS: WHAT CAN WE LEARN? C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, Joerg Vogel3, Wolfgang Hess1. 1 University of Freiburg, Freiburg, Germany; 2University of Algarve, Faro, Portugal; 3Max Planck Institute for Infection Biology, Berlin, Germany.
OC-11.5
REGULATION BETWEEN PHOTOAUTOTROPHIC AND PHOTOMIXOTROPHIC GROWTH AND ITS DEPENDENCE ON THE CO2 LEVEL IN SYNECHOCYSTIS PCC 6803. Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy Lieman-Hurwitz1, Aaron Kaplan1. 1 Plant and Environmental Sciences, The Hebrew University of Jerusalem; Israel; 2Department of Biochemistry and Molecular Biology, Saitama University, Japan.
OC-11.6
CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY IN OSCILLATORIA. Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson, Dave Adams. University of Leeds, Leeds, West Yorkshire, United Kingdom.
August 9 to 14, 2009 • Montréal, QC, Canada
25
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Thursday, August 13, 2009
10:30–12:30
ORAL COMMUNICATIONS 12: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I
RÉGENCE C
Chairs: Karl Forchhammer, Tübingen, Germany and Soufian Ouchane, Gif-sur-Yvette, France OC-12.1
GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGEN CONDITIONS AND THE ROLE OF THE HISTIDINE KINASE, HIK31, IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Tina C. Summerfield1, Louis A. Sherman2. 1 Botany Department, University of Otago, PO Box 56, Dunedin, New Zealand; 2Purdue University, Department of Biological Sciences, West Lafayette, IN, USA.
OC-12.2
INCREASING HYDROGEN PRODUCTION BY PERTURBING FERMENTATIVE METABOLISM IN THE MARINE, UNICELLULAR CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002. Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G. Charles Dismukes. Princeton University, Princeton, NJ, USA.
OC-12.3
PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATION TRANSFER TO PHOTOSYSTEM I IN THE FILAMENTOUS CYANOBACTERIUM NOSTOC PUNCTIFORME. Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring, Ann Magnuson. Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden.
OC-12.4
GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC 7942. Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, Yasunori Nakamura. Akita Prefectural University, Akita, Japan.
OC-12.5
IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTIS AERUGINOSA UNDER IRON LIMITATION. Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1. 1 School of Biotechnology and Biomolecular Sciences; 2School of Civil and Environmental Engineering; University of New South Wales, Sydney, Australia.
OC-12.6
THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITE WITH ECOPHYSIOLOGICAL ROLES. S.B. Watson1, F. Juttner2. 1 Environment Canada, Canada Centre for Inland Waters, Burlington, ON, Canada; 2Limnological Station, University of Zurich, Switzerland.
12:30–14:00
26
LUNCH
RÉGENCE C AND TOUR DE VILLE Please check for a ticket inside your badge holder. Only those with a ticket in their badge holder are dining in the Tour de Ville.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Thursday, August 13, 2009
14:00–15:00
ORAL COMMUNICATIONS 13: PHYSIOLOGY VI
RÉGENCE C
Chair: Peter Lindblad, Uppsala, Sweden OC-13.1
COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND PURPLE SULFUR BACTERIA. N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3. 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Plant Biology Department, University of Geneva, Geneva, Switzerland; 3Department of Biochemistry and Molecular Biology, Pennsylvania State University, State College, Pennsylvania, USA.
OC-13.2
THE GENOME OF THE TOXIC BLOOM-FORMING CYANOBACTERIUM ANABAENA SP. 90. David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2, Kaarina Sivonen1. 1 Department of Microbiology and Applied Chemistry, University of Helsinki, FIN-00014, Helsinki, Finland; 2 Beijing Institute of Genomics of the Chinese Academy of Sciences, Beijing Genomics Institute, Beijing, China.
OC-13.3
CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPMENT AND BEHAVIOR OF HORMOGONIA OF NOSTOC PUNCTIFORME. Jack Meeks, Elsie Campbell, Rui Chen. Department of Microbiology, University of California, Davis, CA, USA.
14:00–15:00
ORAL COMMUNICATIONS 14: BIOENERGETICS IV
RÉGENCE AB
Chair: Peter C. Wolk, East Lansing, MI, USA and Teresa Thiel, St. Louis, MO, USA OC-14.1
COMPARISON OF NONCODING FEATURES OF CYANOBACTERIAL GENOMES. Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, José Costa5. 1 Virginia Commonwealth University, VA, USA; 2University of California at Davis, CA, USA; 3University of Pennsylvania, PA, USA; 4Uppsala University, Uppsala, Sweden; 5University of Porto, Porto, Portugal.
OC-14.2
HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) AS ELECTRON DONOR? SEARCHING FOR THE ANSWER IN THE MEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM 263. Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, Ulrich Fischer1. 1 Zentrum für Umweltforschung und nachhaltige Technologien (UFT) and Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie, Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie; 3Fachbereich Biologie/Chemie, Laboratorium für Allgemeine Mikrobiologie, Universität Bremen; Bremen, Germany.
OC-14.3
PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS: INSIGHTS INTO THE SULFUR METABOLISM OF A PHOTOTROPHIC GREEN BACTERIUM. Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, Anders Mellerup2, Kirsten S. Habicht3, Raymond P. Cox1, Jens S. Andersen1 & Niels-Ulrik Frigaard2. 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK5230 Odense; 2 Department of Biology, University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center for Earth Evolution and Institute of Biology, University of Southern Denmark, Odense; Denmark.
August 9 to 14, 2009 • Montréal, QC, Canada
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Thursday, August 13, 2009
15:00–16:00
PLENARY 7: ENVIRONMENT AND BIOENERGETICS
RÉGENCE AB
Chairs: Davide Zannoni, Bologna, Italy and Igor Stadnichuk, Moscow, Russia
28
15:00
PL-7.1
HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVES THE ENVIRONMENTAL STIMULI? Iwane Suzuki, University of Tsukuba, Tsukuba, Ibaraki, Japan.
15:30
PL-7.2
GENOMICS AND METAGENOMICS APPROACHES TO THE EVOLUTION AND REGULATION OF GENE TRANSFER AGENTS (GTAS). J. Thomas Beatty, University of British Columbia, Vancouver, BC, Canada.
16:00–18:00
POSTER SESSION
CARTIER AB
18:00
POSTER REMOVAL
CARTIER AB
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Friday, August 14, 2009
08:30–10:00
PLENARY 8: BIOREMEDIATION, SECONDARY METABOLITES, AND APPLIED ASPECTS (BIOFUELS AND CLIMATE CHANGE)
RÉGENCE AB
Chairs: Jens Appel, Tempe, AZ, USA and Anatoly Tsygankov, Pushchino, Russia 08:30
PL-8.1
PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGEN PRODUCTION. Caroline Harwood, University of Washington, Seattle, WA, USA.
09:00
PL-8.2
THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICAL APPLICATIONS. Paula M. Tamagnini, Departamento de Botânica, Fac. Ciências , IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
09:30
PL-8.3
CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES, ACTIVITIES AND BIOSYNTHESIS. Kaarina Sivonen, University of Helsinki, Helsinki, Finland.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 15: APPLIED ASPECTS I
FOYER
RÉGENCE AB
Chairs: Caroline Harwood, Seattle, WA, USA and Hidehiro Sakurai, Hiratsuka, Japan OC-15.1
A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALE PHOTOBIOLOGICAL HYDROGEN PRODUCTION UTILIZING CYANOBACTERIA. Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue. Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka, Kanagawa, Japan.
OC-15.2
SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTION AMONG DIVERSE HETEROCYSTOUS CYANOBACTERIA. Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples, Polly Berseth, Tommy Sessions. Savannah River National Laboratory, Aiken, SC, USA.
OC-15.3
THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLE BACTERIA WITH DARK FERMENTATIVE HYDROGEN PRODUCTION AND HYDROGEN ELECTRODE. A. Tsygankov, Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
OC-15.4
HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUM RUBRUM UNDER RESPIRATORY DARK CONDITIONS. Lisa Zeiger, Christiane Rudolf, Hartmut Grammel. Max-Planck-Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
OC-15.5
DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA IN MISSISQUOI BAY, QUEBEC, USING Q-PCR. Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, Frances Pick3, David Bird4, Charles W. Greer1. 1 National Research Council, Biotechnology Research Institute, QC; 2Environmental Health Center, Health Canada, ON; 3Biology Department, University of Ottawa, ON; 4Biological Sciences, University of Quebec at Montreal, QC, Canada.
OC-15.6
EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENES AND THE GENETIC POPULATION STRUCTURE OF TOXIC CYANOBACTERIA. Rainer Kurmayer, Guntram Christiansen. Austrian Academy of Sciences, Institute for Limnology, Mondsee, Austria.
August 9 to 14, 2009 • Montréal, QC, Canada
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Friday, August 14, 2009
10:30–12:30
ORAL COMMUNICATIONS 16: PHYSIOLOGY VII
RÉGENCE C
Chairs: Louis Sherman, West Lafayette, IN, USA and Kaarina Sivonen, Helsinki, Finland OC-16.1
THE RESPIRATORY TERMINAL OXIDASES OF THE CYANOBACTERIUM SYNECHOCOCCUS SP. STRAIN PCC7942. Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder, Dominik Aschenbrenner. Institute of Physical Chemistry, Vienna, Austria.
OC-16.2
PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA: STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN POST-TRANSLATIONAL MODIFICATION. Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1, Gaozhong Shen2, Donald A. Bryant2, Wendy M. Schluchter1. 1 Department of Biological Sciences, University of New Orleans, New Orleans, LA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; USA.
OC-16.3
SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVEL CYANOBACTERIOCHROME-TYPE PHOTORECEPTOR AnPixJ. Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose, Tomoo Shiba, Shigeru Itoh, Genji Kurisu, Masahiko Ikeuchi. University of Tokyo, Meguro, Tokyo, Japan.
OC-16.4
CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OF SYNECHOCOCCUS ELONGATUS REVEALS A NEW ANTIDOTE MOLECULE. Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz. Bar-Ilan University, Ramat-Gan, Israel.
OC-16.5
HOW CYANOBACTERIA BORE (AND WHY LATERAL HETEROCYSTS EXIST). F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao. School of Life Sciences, Arizona State University, Tempe, AZ, USA.
OC-16.6
FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEEN IRON HOMEOSTASIS AND OXIDATIVE STRESS. Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2, Nir Keren1. 1 The Alexander Silberman Institute of Life Sciences, Department of Plant and Environmental Sciences, Hebrew University, Givat Ram, Jerusalem, Israel; 2Department of Biological Sciences Purdue University, West Lafayette, IN, USA; 3 Department of Botany, University of Otago, Dunedin, New Zealand.
12:30–14:00
30
LUNCH
RÉGENCE C AND FOYER
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Friday, August 14, 2009
14:00–15:00
ORAL COMMUNICATIONS 17: APPLIED ASPECTS II
RÉGENCE C
Chair: Paula Tamagnini, Porto, Portugal OC-17.1
RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS, HARBORING THE CRTI REPORTER GENE TO MONITOR ENVIRONMENTAL TOXIC METALS. Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.
OC-17.2
THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTER CAPSULATUS CELLS VIA ACETATE PERMEASE. Roberto Borghese, Davide Zannoni. Department of Biology, University of Bologna, Bologna, Italy.
OC-17.3
CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CR-PLATING INDUSTRY WITH EXOPOLYSACCHARIDE-PRODUCING CYANOBACTERIA. Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis. Deparment of Agricultural Biotechnology, University of Florence, Firenze, Italy.
14:00–15:00
ORAL COMMUNICATIONS 18: BIOENERGETICS V AND APPLIED ASPECTS III
RÉGENCE AB
Chair: Peter Lindblad, Uppsala, Sweden OC-18.1
TRANSCRIPTIONAL REGULATION AND MATURATION OF CYANOBACTERIAL HYDROGENASES. Peter Lindblad, Department of Photochemistry & Molecular Science, Uppsala University, Uppsala, Sweden.
OC-18.2
PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803. Carrie Eckert, Jianping Yu, and Pin-Ching Maness. National Renewable Energy Laboratory, Golden, CO, USA.
OC-18.3
ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATION OF THE BIDIRECTIONAL [NIFE]HYDROGENASE FROM SYNECHOCYSTIS SP. PCC 6803. Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2, Rüdiger Schulz3 and Frauke Germer3. 1 School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Max-Volmer-Laboratorium, Technische Universität, Berlin, Germany; 3Botanisches Institut, Universität Kiel, Kiel, Germany.
15:00–16:00
CLOSING KEYNOTE
RÉGENCE AB
Chair: Patrick Hallenbeck, Montréal, QC, Canada KN-2.1
INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN, AND SULFUR METABOLISM: THE CENTRAL ROLE OF THE CALVIN-BENSON-BASSHAM CYCLE. F. Robert Tabita, Ohio State University, Columbus, OH, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
KN-1.1
KN-2.1
A SCIENTIST OF THE FLOATING WORLD.
INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN, AND SULFUR METABOLISM: THE CENTRAL ROLE OF THE CALVINBENSON-BASSHAM CYCLE.
Anthony E. Walsby, School of Biological Sciences, University of Bristol, United Kingdom. My invitation to give the Keynote Address proposed a lecture with reflections and a history of research on phototrophs. Before accepting, I ought to have reflected on Kazuo Ishiguro’s novel An Artist of the Floating World, in which Mr Ono, an aging painter, looks back on his life noticing how his reputation has faltered and attitudes towards him and his work have changed. The Floating World in which I wandered for over forty years is that of planktonic prokaryotes that derive buoyancy from gas vesicles. I was launched into this by the discovery of Bowen & Jensen (1965) that gas vacuoles of cyanobacteria were made up of cylindrical gas vesicles, packed like the cells of a honeycomb. Their report ended a hiatus of over twenty years since the scholarly review on gas vacuoles by G.E. Fogg, then a 21 year-old graduate student of F. E. Fritsch. Hans Klebahn had been the dominant figure but his fêted ‘hammer, cork and bottle’ experiment, demonstrating the loss of gas vauoles under pressure, belonged to Ahlborn (1894). Years later, the report of Houwink (1956) of gas vesicles in halobacteria was also eclipsed, by the later investigations on cyanobacteria. Discoveries made in isolation can become consigned to obscurity. I was using a mass spectrometer for 15N2-fixation studies in 1965 and I thought I would use the technique to identify the gas-vacuole gas. Two years later, using a modified “blood-gas manometer” of Barcroft and Haldane (1902), I showed that gas vesicles were freely permeable to gases. This led to investigations of the gas vesicles’ collapse under pressure, which affected many aspects of these structures. For example, cell turgor presure which presses on gas-vesicles, could be determined from the modified collapse pressure. The rigid, protein wall of the gas vesicle, little more than one molecule thick, obeyed the principles of engineering used in making pipes and submarines, principles also relevant to the provision of buoyancy of cyanobacteria in lakes, where gas vesicles must survive the pressure increasing with depth. In deeper lakes are cyanobacteria that evolved stronger gas vesicles, which are necessarily narrower and more expensive to make; in shallow salty pools, where cells have no turgor pressure, the weakest, widest and cheapest gas vesicles suffice.
F. Robert Tabita, Ohio State University, Columbus, OH, USA For many years our laboratory has studied the biochemistry and regulation of CO2 assimilation in phototrophic prokaryotes. In this talk, the central role that RubisCO and the Calvin-Benson-Bassham (CBB) pathway play in controlling many different aspects of metabolism will be stressed. Some 15 years ago, we discovered that the molecular control of key processes such as CO2 fixation, nitrogen fixation, hydrogen metabolism, and sulfur oxidation/reduction are linked in photosynthetic bacteria. Indeed, when the primary means by which these organisms balance their intracellular redox state is blocked (via inactivating the CBB reductive pentose pathway such that the preferred metabolic electron acceptor, CO2 , is no longer used) interesting adaptive mutant strains may be selected. One adaptive “trick” that we discovered in four separate organisms is that the nitrogenase complex may be derepressed so that this enzyme system acts exclusively as a hydrogenase and reduces protons to balance excess redox equivalents that are produced via the oxidation of organic carbon during photosynthetic growth. Such strains eructate massive quantities of molecular hydrogen, even in the presence of fixed nitrogen; some strains more than others. However, it is clear that the carbon assimilatory process influences this capability. It will also be shown that RubisCO and the control of the CBB cycle plays a central role in mediating other aspects of normal metabolism of these organisms.
In my researches on gas vesicles I depended on collaborations with specialists in diverse disciplines – the mathematical physicist John Simpson, the freeze-etching pioneer Daniel Branton, the crystallographer Allen Blaurock, and the molecular biologist John Walker; Paul Hayes, who started as my apprentice in things molecular, soon became my mentor, the co-superviser of some clever students and collaborator with visitors to our lab. Other valued contributers were the biologists drawn into my obsession with gas vesicles and buoyancy when I visited their institutes – Colin Reynolds in the English Lake District; Lucas Stal, who organised our Baltic cruises; and Ferdinand Schanz who sampled Planktothrix for a decade from Lake Zürich. I suspect that the fruitless attempts of Fogg (1941) to induce gasvacuole formation in Anabaena, led to his discovery that fixed nitrogen inhibits heterocyst formation. My career began as his graduate student with papers on the first isolation not of gas vesicles, but of heterocysts (with Peter Fay), and then with Bill Stewart, Fay and Fogg, the theory of nitrogen fixation by heterocysts, now one of microbiology’s forgotten discoveries. As Mr Ono discovered, attitudes changed. August 9 to 14, 2009 • Montréal, QC, Canada
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-1.1
PL-1.2
EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHIC BACTERIA DEDUCTED FROM WHOLE GENOME COMPARISONS.
ORIGIN AND EVOLUTION OF THE PHOTOSYNTHETIC FUNCTION.
Robert Blankenship, Departments of Biology and Chemistry, Washington University, St. Louis, USA. Photosynthesis is a central biological process that has a long and complex evolutionary history. The photosynthetic machineries found in the existing groups of phototrophs have both common and divergent characters, suggesting an evolutionary process that combines de novo gene appearance, gene duplication, gene and pathway recruitment and loss, with both vertical and horizontal genetic transfer. The earliest phototrophs were almost certainly anoxygenic and were in existence on Earth by at least 3.4 billion years ago and possibly somewhat earlier. Oxygenic phototrophs were undoubtedly in existence by 2.4 billion years ago when free molecular oxygen, the waste product of oxygenic photosynthesis, began to accumulate in the atmosphere. These organisms may have been present up to several hundred million years before that time. Several lines of evidence, including molecular evolution analysis, structural comparisons, and biochemical and biophysical data, suggest that all modern photosynthetic reaction centers are derived from a single ancient common ancestor and that the anoxygenic phototrophs preceded oxygenic ones. The transition from anoxygenic to oxygenic photosynthesis was accompanied by a number of evolutionary innovations, including multiple gene duplication and divergence events, modification of the pigment biosynthesis pathways from anaerobic to oxygen-requiring, invention of the oxygen evolution center and a dramatic increase in the reaction center protein subunit complexity. The precise evolutionary pathway that led to the current diversity of different types of phototrophs was not linear and involved significant amounts of horizontal gene transfer.
Armen Y. Mulkidjanian1,2, Michael Y. Galperin3, Eugene V. Koonin3. 1
School of Physics, University of Osnabrueck, Osnabrueck, Germany; A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; 3NCBI, NLM, National Institutes of Health, Bethesda, MD, USA. 2
Introduction: Based on a comparative analysis of 15 cyanobacterial genome sequences as well as complete genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus and Heliobacillus mobilis, we have suggested that the (bacterio)chlorophyll-based photosynthesis had originated in the anoxygenic ancestors of cyanobacteria (‘procyanobacteria’) and then spread to other phyla via lateral gene transfer [1]. Results: This scheme will be considered in a broader context of the evolution of complex cellular systems and, in particular, of the energyconverting machinery [2-4]. It will be argued that whether or not an increased complexity of a cellular system would be preserved in the course of evolution is determined by a trade-off between the gains that are brought by the system and its maintenance costs. Conclusions: The evolution of the primeval pro-cyanobacterial photosynthetic machinery can be considered as going in two directions, namely (1) towards the sophisticated photosynthetic apparata of the modern cyanobacteria that, besides being able to capture the energy of light, can exploit water as an unlimited source of electrons and 2) towards the “spare” photosynthetic appliances of other bacteria that have tighter energy budgets. References: 1. Mulkidjanian, A.Y., E. V. Koonin, K. S. Makarova, S. L. Mekhedov, A. Sorokin, Y. I. Wolf, A. Dufresne, F. Partensky, H. Burd, D. Kaznadzey, R. Haselkorn, M. Y. Galperin (2006) The cyanobacterial genome core and the origin of photosynthesis, Proc. Natl. Acad. Sci. U.S.A., 103: 13126-13131. 2. Mulkidjanian, A.Y., M.Y. Galperin, K.S. Makarova, Y.I. Wolf, E.V. Koonin (2008) Evolutionary primacy of sodium bioenergetics, Biol Direct 3 13. 3. Mulkidjanian, A.Y., P. Dibrov, M.Y. Galperin (2008) The past and present of the sodium energetics: May the sodium-motive force be with you, Biochim. Biophys. Acta, 582, 238-242. 4. Mulkidjanian, A.Y., M.Y. Galperin, E.V. Koonin (2009) Co-evolution of membranes and membrane bioenergetics, Trends Biochem. Sci., in press, DOI: 10.1016/j.tibs.2009.01.005.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-1.3
PL-2.1
ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA: DIVERSITY AND NEW TAXONS.
MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMIC AND PROTEOMIC ANALYSES. Birgitta Bergman, Department of Botany, Stockholm University, Stockholm, Sweden.
Vladimir Gorlenko, Irina Bryantseva, Ekaterina Boldareva. Vinogradsky Institute of Microbiology RAS, Moscow, Russia. The first isolates of alkaliphilic purple sulfur bacteria (PSB) obtained from the Vadi Natrun soda lakes included moderately halophilic members of the genus Ectothiorhodospira (Ect. haloalkalophila, Ect. vacuolata, and Ect. shaposhnicovii) and extremely halophilic Halorhodospira species (Hlr. halophila, Hlr. abdelmalekii, and Hlr. halochloris). We have described two new alkaliphilic genera of the family Ectothiorhodospiraceae, Thiorhodospira and Ectothiorhodosinus. The members of these new genera PSB differed from the known species in the topography of their photosynthetic membrane structures (PS). Recent research revealed big purple spirilla, “Ect. magna”, sp. nov., closely related to Ect. shaposhnicovii. Simialr to Thiorhodospira sibirica, their PS contained irregular intracellular lamellae and precipitated sulfur in the periplasm, like to members of the family Chromatiaceae. Microorganisms with gas vacuoles, described as Ect. variabilis, are widespread in the soda lakes where the mineralization is higher than ion seawater; this species is phylogenetically different from Ect. vacuolata. Thus, our findings confirm the low taxonomic value of the presence of intracellular gas vacuoles. Bacteria morphologically and phylogenetically related to Ect. shaposhnicovii were recently isolated from the Mono Lake (California); they are capable of a new type of photosynthesis with arsenite as an electron donor. The first alkaliphilic member of Chromatiaceae, Thioalkalicoccus limnaeus (containing BChl b), was isolated from Transbaikalian soda lakes. We investigated new strains of this species of alkaliphilic phototrophic bacteria. The second member of Chromatiaceae, Thiocapsa imhoffii (containing BChl a), was recently described. In the lakes with low salinity, alkalitolerant members of the genera Allochromatium and Thiocapsa are also present; the systematic position of these species has not yet been determined. Among nonsulfur purple bacteria (NPB), two Rhodobaca species are alkaliphilic. The new NPB Rubribacter polymorphus, was isolated from a soda lake in the Barguzin Valley. Some species of the genus Rhodovulum were also shown to be alkaliphilic. Close phylogenetic similarity was revealed between NPB and anaerobic, bacteriochlorophyll a-containing bacteria (ABC) of the genus Roseinatronobacter; this finding indicates their evolutionary relatedness. Similarity exists between NPB and ABC in their reaction centers and the electron transfer system. The new members of the genus Roseococcus, Rsc. suduntuensis and Rsc. vulcaneus were demonstrated to be alkaliphilic (natronophilic) but not halophilic. The alkaliphilic genus Heliorestis contains three natronophilic species. It may be concluded that obligate alkaliphily and natrononophily developed as the result of evolutionary selection caused by prolonged existence of the microorganisms under the alkaline conditions of soda environments. The taxonomic rank of obligate alkaliphiles is usually that of a genus. Such secondary adaptations as active motility by means of flagella, formation of gas vesicles and of cyst-like microcolonies are important from the ecological point of view. The intracellular membranes are diverse and correlate with the cell size and bacteriochlorophyll type.
August 9 to 14, 2009 • Montréal, QC, Canada
It has become increasingly clear that diazotrophic cyanobacteria play a crucial role in maintaining life in oceans, major players being members of the genus Trichodesmium. We are interested in understanding the structural and molecular background related to the maintenance of diazotrophy in Trichodesmium IMS101, a non-heterocystous cyanobacterium fixing nitrogen in light/day. We have approached this by examining events at the structural, transcriptomic and proteomic level, with focus on cellular dynamics during the development of the nitrogen-fixing cells, the diazocytes. The data obtained demonstrated a diurnal separation of basic physiological processes, with nitrogenfixation and photosynthesis confined to the light and cell division and diazocyte development to the dark period. When non-diazotrophic cultures were subject to nitrogen depletion a subset of cells differentiated into nitrogenase containing diazocytes, while addition of combined nitrogen abolished development. Comparative proteomic analysis revealed that ninty-four proteins were differentially expressed in diazotrophic (with diazocytes), as opposed to in the nondiazotrophic cultures, such as the nitrogenase enzyme and proteins related to supporting reducing equivalents and lowering oxygen tensions. Some recent metagenomic analyses of size fractionated microbes from a transect spanning the Indian Ocean will also presented.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-2.2
PL-3.1
PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATION AND POTENTIAL INTERACTIONS.
MULTICELLULARITY IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA.
Arthur R. Grossman1, Anne-Soisig Steunou2, Rosario Gomez-Garcia1, Melissa Adams1, Michael Kühl3, John Heidelberg4, Devaki Bhaya1.
Enrique Flores, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain.
1 The Carnegie Institute for Science, Department of Plant Biology, Stanford, CA, USA; 2CNRS, Centre de Génétique Moléculaire, Gif-surYvette, France; 3University of Copenhagen, Marine Biology Laboratory, Department of Biology, Helsingør, Denmark; 4University of Southern California, Department of Biological Sciences, Avalon, CA, USA.
Filamentous, heterocyst-forming cyanobacteria have been described to reproduce by random trichome breakage implying that their unit of growth is the filament. Under combined nitrogen deprivation, growth of filaments depends on the activity of two different types of cells, nitrogen-fixing heterocysts and photosynthetic vegetative cells, which exchange nutrients and regulatory compounds making the filaments true multicellular organisms. They carry a Gram-negative type of cell envelope in which, as shown by electron microscopy, the outer membrane is continuous along the filament without entering the septa between cells. The periplasmic space, which lies between the cytoplasmic and outer membranes, is therefore continuous along the filament, and this is so not only between vegetative cells but also between vegetative cells and heterocysts. In the model organism Anabaena sp. PCC 7120, a periplasmic form of the green fluorescent protein produced in (pro)heterocysts can be detected at a distance in the periplasm of vegetative cells showing that the periplasm is also functionally continuous and suggesting that it could constitute a communication conduit between the cells in the filament. Recent studies have shown that the Anabaena outer membrane is a permeability barrier for some sugars and amino acids that are important in the diazotrophic physiology of these organisms. On the other hand, electron microscopy also shows the presence in the intercellular septa of thin structures perpendicular to the cytoplasmic membranes of adjacent cells. In Anabaena sp. PCC 7120, a protein located at the cell poles in the intercellular septa, which consists of a predicted N-terminal extracytoplasmic domain and a C-terminal permease domain, has been identified and named SepJ, and two proteins, FraC and FraD, required for focused localization of SepJ have also been identified. Mutants of the genes encoding these proteins show a filament fragmentation phenotype indicating that they contribute to keep cells together in the filament. A method for testing intercellular molecular exchange in filamentous cyanobacteria has been developed that consists in loading of a fluorescent dye, calcein, in the cytoplasm of the cells and measuring calcein fluorescence in Fluorescence Recovery After Photobleaching (FRAP) experiments. Calcein exchange takes place rapidly between the cells in Anabaena filaments but is impaired in sepJ, fraC and fraD mutants suggesting that these proteins are involved in intercellular molecular exchange. Our current view of the Anabaena filament is that of a string of cells which are encapsulated by a continuous outer membrane and share the periplasm, and that are connected by proteinaceous structures that could also be involved in intercellular communication. Identifying which compounds move in the filament through the periplasm or the cell-to-cell joining structures is a major topic for future research.
Introduction: Hot spring microbial mats are natural biofilms composed of oxygenic photosynthetic cyanobacteria as well as heterotrophic microbes. These organisms are distributed along horizontal thermal and vertical light and oxygen gradients. The mats that we have been studying are from Octopus and Mushroom Springs of Yellowstone National Park. They are generally 1-2 cm thick, with densely packed cyanobacteria of the genus Synechococcus in the upper mat layer. The Synechococcus of the hot spring mats can harvest light energy for photosynthetic CO2 fixation at temperatures exceeding 70oC. Methods: Our work involves sequencing genomes from specific Synechococcus ecotypes that reside at different temperatures in the mat and also generating metagenome sequence information. Moreover, we have analyzed the abundances of specific transcripts using reverse transcriptase quantitative PCR and measured levels (immunologically) and activities of specific proteins over the diel cycle in situ. Results and Conclusions: The genomes of two Synechococcus variants from the hot springs of Yellowstone National Park were sequenced, and while they have similar gene content, they are remarkably different with respect to gene arrangement. Furthermore, the genomes of both of the cyanobacterial isolates harbor a full complement of nearly identical nitrogen-fixation (nif) genes. The nif genes are expressed in the mat toward evening, when the mat becomes anoxic, although the highest in situ N2 fixation rates are measured in early morning, when the mat is still anoxic but light starts to drive photosynthesis and the production of ATP. The mat community also switches from oxidative metabolism in the day, when cells are photosynthesizing, to fermentation metabolism in the evening. This metabolic switching is reflected by rapid changes in gene expression in response to anaerobic/aerobic conditions. We have also cultured Synechococcus variants from the hot spring mats and show that they can use various forms of nutrients, including phosphonates as a sole source of phosphate. I will discuss the physiology, metabolism and energetic constraints within the mat environment over the diel cycle.
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-3.2
PL-3.3
EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY 2-PHOSPHOGLYCOLATE METABOLISM IN CYANOBACTERIA.
THE NETWORK OF PII SIGNALING PROTEIN INTERACTIONS IN CYANOBACTERIA .
Martin Hagemann, University of Rostock, Institute of Biological Sciences, Plant Physiology, Rostock, Germany.
Karl Forchhammer, Institut für Mikrobiologie der Eberhard-KarlsUniversität Tübingen, Tübingen, Germany.
Introduction: Cyanobacteria fix most of the carbon using the CalvinBenson cycle with RubisCO as the carboxylating enzyme. However, Rubisco has a rather low affinity to CO2 and exhibits in the presence of oxygen also an oxygenase activity resulting in the toxic byproduct 2phosphoglycolate (2PG). This intermediate is detoxified by the photorespiratory 2PG cycle in plants. Cyanobacteria evolved an inorganic carbon concentrating mechanism (CCM) allowing them to cope with low carbon concentrations in the presence of oxygen. Active uptake systems for inorganic C accumulate high internal bicarbonate amounts. Moreover, RubisCO is concentrated in carboxysomes, where bicarbonate is transferred to CO2 leading to high CO2 concentration in the vicinity of RubisCO making oxygenase activity in cyanobacteria unlikely. Accordingly, it was widely accepted that cyanobacteria do not perform photorespiratory 2PG metabolism. During the last years we analyzed the mode of 2PG metabolism and its possible diverse function with the cyanobacterial model strain Synechocystis sp. PCC 6803.
Introduction: The family of PII signal transduction proteins is represented in all domains of life, and is especially abundant in organisms capable of inorganic nitrogen assimilation. The elementary mode of PII action is sensing the state of central metabolism/nitrogen assimilation and in response, orchestrating cellular processes related to nitrogen assimilation by binding to key factors involved in nitrogen control. Sensing the state of central metabolism is achieved by interdependent binding of effector molecules to PII, in particular, 2oxoglutarate and adenyl-nucleotides (ATP and ADP), and eventually by additional covalent modification of PII by modifying enzymes.
Methods:Defined mutants of Synechocystis defective in candidate genes for glycolate metabolism were generated by interposon mutagenesis (1, 3). Moreover, microarray analyses were used to search for genes coding for alternative routes in the glycolate metabolism (2). Results:Single-mutants in candidate genes for 2PG metabolism of Synechocystis showed only small changes, while the generation and characterization of double- and triple-mutants showed that an active 2PG metabolism employs three different routes: A) Glycolate is degraded by a plant-like C2 cycle. B) A route exists corresponding to the bacterial glycerate pathway. C) Additionally, the complete decarboxylation of glycolate via oxalate and formate occurs. A complete block in all three postulated routes resulted in a high-CO2requiring (HCR) phenotype as it is known from photorespiratory mutants in higher plants. Bioinformatic analyses were used to search for corresponding genes in the completed genomes of other cyanobacterial strains, which indicated that generally all cyanobacteria are able to metabolize 2PG despite the existing strain-specific differences in the occurrence of single pathways. The photorespiratory metabolism contributes also to high light acclimation in Synechocystis, since a combined mutation of 2PG metabolism and the so-called Mehler reaction resulted in higher light sensitivity. Conclusions: We could show that cyanobacteria perform an active photorespiratory metabolism despite the activity of the CCM. The 2PG metabolism uses three different routes in Synechocystis. A complete block of 2PG metabolism resulted in a HCR phenotype, which shows that this metabolism is essential for organisms performing oxygenic photosynthesis in the present day atmosphere. These findings gave rise to the hypothesis that an early photorespiratory has evolved already in ancient cyanobacteria and was conveyed endosymbiontically into plants. The main function of the glycolate metabolism is directed to the detoxification of critical intermediates. However, it is also involved in the acclimation to high light conditions. 1 – Eisenhut et al. (2006) Plant Physiol 142:333-342 2 – Eisenhut et al. (2007) Plant Physiol 144:1946-1959 3 – Eisenhut et al. (2008) PNAS 105:17199-17204
August 9 to 14, 2009 • Montréal, QC, Canada
In most cyanobacteria, one highly conserved gene, termed glnB, encoding a PII homologue has been identified in the genomic sequences. Some strains, such as Gloeobacter violaceus, Synechococcus sp. WH5701 or Acharyochloris marina harbor one or two further potential glnB paralogues. Functions and properties of cyanobacterial PII signalling were mostly worked out with the proteins from strains Synechococcus elongatus and Synechocystis sp. PCC 6803. There, PII is covalently modified by S49 phosphorylation, but this apparently does not apply to all cyanobacteria. Here, I will present interaction studies of PII with different target molecules, PII-phosphatase PphA, N-acetylL-glutamate kinase (NAGK) and PipX. PII - phosphatase interaction: The structure of the phospho-PII phosphatase PphA from Thermosynechococcus elongatus was resoved recently (1). Based on this structure, mutants were generated to elucidate the basis of substrate recognition of PphA. Further, an assay was established to stabilize PII-PphA interaction intermediates. Metal 1 (M1) of the trinuclear metal centre of PphA, a PPM family phosphatase, is absolutely required for binding PII-P. Furthermore, several residues at the periphery of the enzyme could be identified to be required for productive PII-P binding, but without affecting the activity of the catalytic centre towards artificial substrates. PphA binding of PII responds specifically to the presence of PII effector molecules ATP/ADP and 2-oxoglutarate, indicating that the entry of the phosphorylated T-loop of PII into the catalytic cavity is controlled by the effector molecules. PII-NAGK interaction: NAGK, the controlling enzyme of arginine biosythesis, by binding PII, is catalytically activated and relieved from arginine inhibition. The structure of the PII-NAGK complex from Synechococcus elongatus was resolved (2) and the catalytic activation of NAGK by PII was investigated in detail. 2-oxoglutarate antagonizes the PII mediated activation of NAGK. Compared to PII/NAGK from Arabidopsis, the principal mode of PII activation - in particular its response to 2-oxoglutarate - is conserved in these oxygenic phototrophs (3). By random mutagenesis combined with bacterial-two hybrid screening, we could identify several PII mutants with novel phenotypes regarding NAGK and effector metabolite binding, leading to novel insights in PII function. This involves in particular residues at the B-loop of PII, which appears to play a key role in the intra-molecular transmission of metabolite signals. PII-PipX interaction: A further PII target, PipX, appears to be a transcriptional co-activator of NtcA. PII competes with NtcA for binding to PipX, thereby antagonizing the activation of NtcA (Espinosa et al., 2007). Whereas binding of PipX to NtcA requires 2-oxoglutarate, 2-OG in concert with ATP im37
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts pairs PipX binding to PII. When PII and NtcA compete for PipX in presence of constant 2-oxoglutarate levels, PipX swaps form PII to NtcA when ATP is added. The NtcA-PipX complex binds to DNA, with PipX not affecting the DNA binding properties of NtcA. When PipX and NAGK compete for PII binding in absence of 2-OG, PII preferentially binds to NAGK.
PL-4.2 EFFECTS OF INSERTION SEQUENCES ON THE GENOME OF ANABAENA SP. STRAIN PCC 7120. C. Peter Wolk, Sigal Lechno-Yossef.
Other PII interactions:
Michigan State University, East Lansing, MI, USA.
Further to the above-mentioned targets, PII was shown to bind to a MscS-like protein from Synechocystis PCC 6803, PamA (5). In contrast to PipX or NAGK, PamA is not highly conserved in cyanobacteria, and it remains to be elucidated what the physiological function of this protein is. Mutants in the PII signalling system show impaired ability to regulate nitrate utilization (6) implying PII interactions with components of the nitrate utilization pathway.
Introduction: Many cyanobacteria closely related to Anabaena sp. strain PCC 7120 make akinetes and gas vacuoles, but PCC 7120 lacks those properties despite having homologs of the akinete-marker gene avaK (1) and gas vacuole-related orfs all2247 through asl2254. It bears ca. 145 orfs that are annotated as encoding transposases (http://bacteria.kazusa.or.jp/cyanobase/), which are diagnostic of insertion sequences (ISs). Some of the ISs surely transpose (2-4, and compare 5 and 6), and can thereby mutagenize. Our laboratory earlier identified three evidently IS891-interrupted regulatory genes in Anabaena sp. (7). Using the newly available sequence data for various cyanobacteria, we have returned to an in-silico attempt to examine features of the genome of PCC 7120 as it may have appeared prior to entry of those ISs.
The overall organization of the PII signal transduction network will be discussed. 1. Schlicker et al., 2008 J. Mol. Biol. 376: 570-581 2. Llacer et al, 2007 Proc. Natl. Acad. Sci. U.S.A. 104: 17644-17649 3. Beez et al., 2009 J Mol Biol. 389: 748-758 4. Espinosa et al., 2007 Microbiology 153: 711-718 5. Osanai et al., J. Biol. Chem. 280: 34684–34690 6. Kloft & Forchhammer, 2005 J. Bacteriol. 187: 6683-6690
PL-4.1 A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME. Susan Golden, Department of Biology, Professor Division of Biological Sciences University of California-San Diego, La Jolla,CA, USA. Cyanobacteria, like diverse eukaryotes, possess circadian clocks that allow cells to coordinate physiological processes with the predictable daily patterns of environmental fluctuations on Earth. An autonomous oscillator comprised of three distinctive proteins, KaiA, KaiB, and KaiC, underpins the circadian clock of the cyanobacterial model organism Synechococcus elongatus PCC 7942. Our work applies genetic, genomic, biochemical, and structural approaches, integrated with cell biology, to achieve a comprehensive understanding of how the S. elongatus cell harnesses this oscillator to control cellular activities. The emerging picture is one of a clock that is set daily by sampling the cellular redox environment as a proxy for light and which uses protein-protein interactions and subcellular localization to coordinate gene expression, cell division, and chromosome dynamics. A functional genomics project has produced inactivation alleles of nearly all S. elongatus loci. Gene expression assays of the 700 mutants tested so far suggest that about 10% of loci affect circadian period or phasing.
Methods: To “remove” an IS, one must know how far it extends outwards from its transposase orf(s) and how many base pairs it replicates upon insertion. Such information is provided by: transposition of ISs to known genetic backgrounds; alignments of sequences containing related ISs to see where their commonality ceases, with the ISs often terminating in inverted repeats and frequently bracketed by direct repeats; and the finding that in other organisms, sequences that are nearly identical to those that flank ISs are joined. Results: Depending on whether neighboring transposase orfs are part of the same IS, PCC 7120 may have ca. 100-150 ISs. We have excised most, in silico, with convincing accuracy; and have identified further elements that, although lacking an annotated transposase, appear to be mobile. Once ISs are removed, we find: nearby orfs extended by replacement of their initiation or termination codons; flanking orfs fused in-frame; orfs previously unrecognized, including those of underlying ISs, found; orfs lost that consisted principally of ends of ISs; and extended intergenic regions that appear empty of orfs. Anabaena variabilis orthologs of two of the kinase genes intercepted in PCC 7120 may also have been intercepted by ISs. Conclusions: We have not identified a phenotype that is surely attributable to the presence of an IS. However, our “archeological genomics“ permits a view of at least part of the genome of PCC 7120 in a more ancestral form. We have identified presumptive orfs, operons, and regulatory genes whose restoration might enlarge PCC 7120‘s phenotype, including its ability to protect itself against entering DNA. Finally, we have added to knowledge of the ISs of PCC 7120 (and other bacteria): have they inverted terminal repeats, do they generate direct repeats upon insertion, and how widespread is their taxonomic dispersion? The excavation continues. References: 1. R Zhou and CP Wolk, J Bacteriol 184: 2529 (2002). 2. Y Cai and CP Wolk, J Bacteriol 172: 3138 (1990). 3. J Alam et al., J Bacteriol 173: 5778 (1991). 4. Y Cai, J Bacteriol 173: 5771 (1991). 5. I Bancroft and CP Wolk, J Bacteriol 171: 5949 (1989). 6. T Kaneko et al., DNA Res 8: 205 (2001). 7. M Ohmori et al., DNA Res 8: 271 (2001).
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-5.1
PL-5.2
CYTOCHROME C MATURATION, DISULFIDE BOND FORMATION AND PERIPLASMIC PROTEIN DEGRADATION IN RHODOBACTER CAPSULATUS.
BETTER LIVING THROUGH CYANOTHECE--UNICELLULAR DIAZOTROPHIC CYANOBACTERIA WITH HIGHLY VERSATILE METABOLIC SYSTEMS.
Ozlem Onder, Serdar Turkarslan, Carsten Sanders, Fevzi Daldal.
Louis A. Sherman1, Hongtao Min1, Jorg Toepel1, Michelle Liberton2, Hanayo Sato2, Jana Stöckel2, Himadri B. Pakrasi2.
University of Pennsylvania, Department of Biology, Philadelphia, PA, USA. Current knowledge on cytochrome c maturation process in the facultative phototrophic bacterium Rhodobacter capsulatus will be reviewed, and the crucial role of periplasmic thio-redox homeostasis (DsbA-DsbB versus CcdA-CcmG) involved in forming and breaking of apocytochrome c disulfide bonds during this process will be described. In addition, we will report the unusual observation that DsbA-null mutants are proficient in photosynthesis but are defective in respiration, especially in enriched growth medium at 35 ºC. Using combined proteomic and molecular genetic approaches we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originate from the overproduction of the periplasmic protease DegP, which render them temperature sensitive for growth. The DsbAnull mutants revert frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. We have shown that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for the proteasechaperone activity. Remarkably, although R. capsulatus DsbA-null or DegP-null mutants are viable, DegP-null DsbA-null double mutants are lethal at all growth temperatures. We found that this is unlike E. coli, indicating that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations amongst the DegP homologues, including a critical disulfide bond-bearing region, correlate well with the differences seen between various species like R. capsulatus and E. coli, and illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.
1 2
Dept. of Biological Sciences, Purdue University, West Lafayette, IN; Dept. of Biology, Washington University, St. Louis, MO; USA.
Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium with a versatile metabolism and very pronounced diurnal rhythms. Since nitrogen fixation is exquisitely sensitive to oxygen, Cyanothece utilizes temporal regulation to accommodate these incompatible processes in a single cell. When grown under 12h light-dark (LD) periods, it performs photosynthesis during the day and N2 fixation and respiration at night. During this process, carbohydrates and amino acids are compartmentalized in granules in the light and dark, respectively. In essence, Cyanothece creates an O2limited intracellular environment to perform oxygen-sensitive processes such as N2-fixation and H2 production during the night. This strain also grows exceedingly well heterotrophically (on glycerol). The excellent synchrony of a culture under LD diazotrophic conditions permits analysis of cellular morphology, mRNA levels, proteomics and metabolomics as a function of time. The genome sequence of Cyanothece sp. ATCC 51142 (Welsh et al (2008) PNAS 105: 15094-99) and that of 5 other Cyanothece sp. are complete and that of a seventh is in progress. Cyanothece sp. ATCC 51142 has 2 chromosomes—a 4.9 Mb circular chromosome and a 0.43 Mb chromosome with a total of 5,300 genes. This strain has the capability of producing high-energy compounds, such as ethanol and hydrogen and Cyanothece sp. PCC 7822 can produce these compounds as well as rather large quantities of parahydroxyalkanotes (PHAs). We are particularly interested in the regulation of these metabolic processes and the way in which these organisms respond to environmental cues such as light, the lack of combined nitrogen and changing O2 levels. Analysis at both the transcriptomics (with microarrays, Toepel et al (2008) J Bact 190:3904 and Stockel et al (2008) PNAS 105:6156) and proteomics levels in Cyanothece sp. ATCC 51142 has demonstrated the relationship of the metabolic synchrony with gene expression and has given us insights into diurnal and circadian regulation throughout a daily cycle. The Cyanothece strains (51142 and 7822 have been the most studied) produce copious amounts of H2 under different types of physiological conditions. Both the bidirectional hydrogenase (encoded by the hox genes) and the nitrogenase (nif genes) are capable of producing measurable hydrogen under appropriate conditions. However, nitrogenase produces far more H2 than the hydrogenase, in part because the nitrogenase levels are extremely high under N2-fixing conditions. With Cyanothece 51142 cultures grown in NO3-free media, either photoautotrophically or mixotrophically with glycerol, we have obtained rates of H2 produced of 150 mmoles/mg Chl/hr and 300 mmoles/mg Chl/hr, respectively. We anticipate even higher values once we are able to construct specific mutants and develop optimal physiological conditions. This work was sponsored by grants from the DOE and from the EMSL at the Pacific Northwest National Labs.
August 9 to 14, 2009 • Montréal, QC, Canada
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-5.3
PL-6.1
PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENT EVOLUTION SCHEMES FROM A COMMON ANCESTRAL GENOME.
OXYGEN AND LIGHT CONTROL OF TETRAPYRROLE BIOSYNTHESIS IN RHODOBACTER SPECIES.
Frédéric Partensky1, Alexis Dufresne1, 2, Martin Ostrowski3, David J. Scanlan3 and Laurence Garczarek1 . 1
UMR 7144 CNRS & Université Paris 6, Station Biologique, Roscoff, France; 2UMR 6553 EcoBio, Université Rennes, Rennes, France; 3 Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. Prochlorococcus and Synechococcus are the two most abundant oxyphototrophs in the ocean. These two marine picocyanobacteria are phylogenetically closely related and together make a branch well separated from all other cyanobacteria, including freshwater Synechococcus but also marine cyanobacteria of larger cell size, including Trichodesmium thiebautii and Crocosphaera watsonii. The large number of genomes of Prochlorococcus (13) and marine Synechococcus (11) that have recently been sequenced, covers most of the diversity known within these groups. This makes it possible to draw insights about the mechanisms that have presided genome evolution in these ecologically important groups. It is now clear that Prochlorococcus is a fairly recent group, which is highly specialized to life in warm, oligotrophic waters. It appeared quasi concomitantly with the highly diversified Synechococcus subcluster 5.1, whose members are seemingly the best represented in the ocean nowadays. Light has been a key environmental factor in the evolution of both genera. Prochlorococcus has first colonized the bottom of the euphotic zone then, more recently, the upper layer of oligotrophic zones. As a result, Prochlorococcus has differentiated itself into low- and high light ecotypes, a phenomenon that has allowed this genus to colonize the whole sunlit layer. For Synechococcus, however, light quality has seemingly been the key factor, since the diversification of its pigment content has allowed this genus to colonize the whole range of environments over the coastal to offshore gradient. But other environmental factors, including salinity, temperature or nutrient availability are also important in shaping the genomes. In this talk, current scenarios for genome evolution in these two key organisms that originate from a common ancestor will be presented. In particular, the impact of environmental factors (mainly light) on the genome content and structure of the different ecotypes within each genus will be discussed.
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Carl Bauer, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. The purple photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides synthesizes three tetrapyrrole endproducts, bacteriochlorophyll, heme and cobalamine (vitamin B12). All three tetrapyrroles are the product of a complex pathway that is responsible for controlling synthesis of these endproducts in response to light intensity and oxygen tension. Over the past decade our laboratory has identified numerous transcription factors that are responsible for the control of tetrapyrrole gene expression. Recent biochemical analysis of several of these transcription factors has demonstrated that many also utilize heme or cobalamine as cofactors. Our current understanding of the control of tetrapyrrole biosynthesis in these species will be presented as well as what is known about the biochemical properties of key regulators.
PL-6.2 A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHT INTENSITY SENSOR IN CYANOBACTERIA PHOTOPROTECTION. Adjélé Wilson, Clémence Boulay, Claire Punginelli, Diana Kirilovsky. Commissariat à l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS) and Centre National de la Recherche Scientifique (CNRS), 91191 Gif sur Yvette, France. Because too much light can be lethal for photosynthetic organisms, photoprotection against excess absorbed light energy is an essential and universal attribute of oxygenic photosynthetic organisms. In plants and algae, the membrane embedded chlorophyll antenna, LHCII, reversibly switchs from a very efficient energy collection state into a photoprotective state. This state by converting the excess energy into heat decreases the energy arriving at the reaction centers under high light conditions. The existence of an equivalent process in cyanobacteria, which use the extramembranal phycobilisomes to harvest energy instead of LHCII, was only recently discovered. The study of the photoprotective thermal energy dissipation mechanism in phycobilisome- and Photosystem II-mutants of the cyanobacterium Synechocystis PCC6803 by fluorescence measurements allowed to demonstrate that this mechanism involves a specific decrease of the fluorescence emission of the phycobilisomes and a decrease of the energy transfer from the phycobilisomes to the reaction centers. The process is induced by the absorbance of blue-green light by a soluble orange carotenoid protein, the Orange-Carotenoid-Protein (OCP). In Synechocystis , the OCP is encoded by the slr1963 gene which is constitutively expressed. OCP genes appear to be highly conserved among phycobilisome-containing cyanobacteria with few exceptions, indicating that the OCP related photoprotective mechanism is widespread. The strains containing a whole OCP gene can perform the blue-light induced photoprotective mechanism. In contrast, strains containing only N-terminal and/or C-terminal OCP like genes or no OCP like genes at all lack this mechanism and they were more sensitive to high light illumination. These strains to longer survive under stress conditions degrade the phycobiliproteins very fast to avoid the appearance of a population of dangerous, functionally disconnected phycobilisomes. The OCP containing strains increase the expression of the OCP and as a consequence increase the conversion to heat of the excess energy absorbed by the phycobilisomes. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes The OCP is a photoactive protein. The absorbance of blue-green light by the OCP induces structural changes in the carotenoid and the protein, converting its dark stable orange form into a relatively unstable active red form. The presence of the red OCP form is essential for the induction of the photoprotective mechanism. In the absence of the formation of the red form due to point mutations in the OCP, no fluorescence quenching is induced by strong white or blue-green light. OCP is fully active when binds hydroxyechinenone or echinenone. When these carotenoids are absent, OCP binds zeaxanthin but light is unable to photoconvert the dark form into a light active form. In the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. These results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity. Moreover, Trp288 and Tyr 201 forming hydrogen bonds to the carbonyl are essential for the photoconvertion and cannot be replaced by other amino-acid without loosing activity. The OCP is the first photoactive protein containing a carotenoid as the photoactive chromophore and its photocycle is completely different from those of other photoactive proteins.
Final Program and Abstracts The Mo-responsive regulators MopA and MopB are modular in structure consisting of an N-terminal DNA-binding and a C-terminal Mo-binding domain. They bind to conserved palindromic promoter sequences, so-called Mo-boxes. Affinity of the regulators to their target promoters is clearly enhanced by molybdenum. MopA and MopB can substitute for each other in repressing transcription of several target genes including the mopA-modABC operon and the anfA gene. In addition to its role as a repressor, MopA acts as an activator of the mop gene coding for the molbindin-like Mop protein. The genes coding for the Mo-responsive regulators, MopA and MopB, belong to two divergently transcribed operons, mopA-modABC and mopB. While transcription of the mopA operon is repressed by molybdenum, mopB is constitutively expressed, suggesting that the MopA-MopB ratio changes in response to the cellular Mo status. MopA and MopB form homodimers and heteromers. Formation of heteromers is thought to deplete the pool of homodimers, and thus might represent a fine-tuning mechanism controlling expression of Mo-regulated genes.
PL-7.1
PL-6.3
HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVES ENVIRONMENTAL STIMULI?
NITROGEN AND MOLYBDENUM CONTROL OF NITROGEN FIXATION.
Iwane Suzuki.
Bernd Masepohl, Alexandra Müller, Jessica Wiethaus. Ruhr University Bochum, Bochum, Germany. The phototrophic purple bacterium Rhodobacter capsulatus has the capacity to grow with atmospheric dinitrogen (N2) as sole source of nitrogen. Reduction of N2 to ammonia is catalyzed by two nitrogenases, molybdenum-nitrogenase (Mo-nitrogenase) and an alternative iron-only nitrogenase (Fe-nitrogenase). Fe-nitrogenase exhibits lower specific activity than Mo-nitrogenase, and thus, Monitrogenase is the preferred enzyme as long as molybdenum is available. Upon Mo-depletion, R. capsulatus synthesizes the highaffinity Mo-uptake transporter ModABC. Since nitrogen fixation is a highly energy-demanding process, synthesis and activity of both nitrogenases is tighly controlled. Only in the absence of ammonium the sensor kinase NtrB autophosphorylates, and subsequently, phosphorylates its cognate response regulator NtrC. In turn, NtrC activates transcription of nifA and anfA coding for the transcriptional activators of all the other nitrogen fixation genes. Transcription of anfA is repressed by the Mo-responsive regulators MopA and MopB preventing expression of Fe-nitrogenase in the presence of molybdenum. Finally, activity of both nitrogenases is controlled by a switch-off/switch-on mechanism involving reversible covalent modification, thus enabling R. capsulatus to respond very fast to subtle changes in ammonium availability. Like many other bacteria, R. capsulatus contains two genes, glnB and glnK, coding for PII-like signal transduction proteins, which play central roles in regulation of nitrogen fixation and assimilation. GlnB and GlnK control activity of many target proteins by transient interaction in response to changes in the cellular nitrogen status. Among these target proteins are the sensor kinase NtrB, the transcription activator NifA, and the switch-off protein DraT. Affinity of GlnB and GlnK to their target proteins is modulated by binding of oxoglutarate and ATP, and uridylylation at a conserved tyrosine residue. The uridylylation state of GlnB and GlnK reflects the cellular glutamine concentration, with both proteins being uridylylated when glutamine concentrations are low. Upon ammonium addition both PII proteins are sequestered to the membrane by binding to the ammonium transporter AmtB. August 9 to 14, 2009 • Montréal, QC, Canada
University of Tsukuba, Tsukuba, Ibaraki, Japan. Introduction: Two-component systems, which generally consist of a sensory histidine kinase and a response regulator, are major signaling pathways in most of bacteria. In general, histidine kinases possess a conserved transmitter domain associating kinase activity at the Cterminus and a unique signal input domain (SID) at the N-terminus. The SIDs in the histidine kinases are thought to play important roles in perception of the specific stimuli and regulation of the kinase activity of the transmitter domain, however, the actual molecular mechanisms of signal perception are not clarified yet. I developed a system to express chimeric histidine kinases containing a transmitter domain of SphS, a phosphate-deficient sensor, and SID from other histidine kinase in the cyanobacterium Synechocystis sp. PCC 6803. It might be a useful strategy to investigate the functions of the histidine kinases. Methods: I constructed genes for chimeric sensors containing transmitter domain of SphS, which include either the N-terminal region of a Ni2+-sensor, NrsS, or SID of Hik33. The chimeric histidine kinases were driven by the native promoter of the sphS gene. Effects of the chimera proteins on the activity of SphS transmitter domain were examined by measuring expression levels of the phoA gene for an alkaline phosphatase as an internal reporter. Results: The N-terminal region of SphS contains both a hydrophobic region and a PAS domain. In order to evaluate the function of the Nterminal region of SphS, deletion mutants under the control of the native promoter were analyzed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from NrsS resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi-availability. These results indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and the PAS domain is involved in perception of Pi-availability. This strategy was also applied to characterize the SID in Hik33, which regulate gene expression under cold, high light, oxidative, high salt, and hyperosmotic stress conditions. The SID includes two transmembrane helices, a periplasmic loop, a HAMP and a PAS 41
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Final Program and Abstracts domain. When the SID was fused with the transmitter domain of SphS, it activated the phoA expression under the standard growth condition and repressed under the stress conditions. The repression of expression of phoA was dependent on the PAS domain in SID. The detail results will be discussed in the presentation. Conclusions: The chimeric sensor system may be a useful approach to investigate the function of SID of the histidine kinases.
PL-7.2 EVOLUTION AND REGULATION OF THE RHODOBACTER CAPSULATUS GENE TRANSFER AGENT. J. Thomas Beatty1, Molly Leung1, Sarah Florizone1, Jeanette A. Johnson1, Ryan Mercer2, and Andrew S. Lang2. Department of Microbiology & Immunology, the University of British Columbia1, BC; Department of Biology, Memorial University of Newfoundland2, NF; Canada. Introduction: The gene transfer agent (GTA) produced by the purple non-sulfur bacterium Rhodobacter capsulatus is a model for several virus-like elements that seem to function solely for mediating gene exchange. Some GTA structural genes in R. capsulatus are related to bacteriophage genes, but the cellular regulatory mechanisms that control GTA production indicate that GTA is more than just a defective prophage. Genome sequencing projects have shown that most prokaryotes contain prophage-like gene clusters, but possible relationships to the GTA gene cluster were unclear. The R. capsulatus GTA is produced maximally in stationary phase cultures, but it was not known what aspect of stationary phase induces the expression of GTA structural genes. Our research was done to investigate the presence of GTA genes in a variety of prokaryotes, and to study factors that affect the stationary phase production of GTA in R. capsulatus. Methods: R. capsulatus GTA protein sequences were used as queries in BLAST searches of genome sequences, and the genome context of high-scoring hits was subsequently evaluated by eye. Sequences were aligned with Clustal X v1.81 and the alignment was used to construct neighbor-joining trees with PAUP* v4.0. Cultures of R. capsulatus were grown photoheterotrophically, and cells separated from GTA by centrifugation and filtration through a 0.2 µm filter. The production of GTA was evaluated in gene transduction experiments, or by probing western blots with antiserum raised against the GTA major capsid protein over-expressed in E. coli. Results: Complete genome sequences were searched for GTA gene homologues to identify candidate GTA-producing species. Although homologous GTA gene clusters are widespread, they are found exclusively within the α-proteobacteria. A phylogenetic tree comparing 16S rRNA and GTA gene homologues shows that these two types of gene have evolved in concert. Work on R. capsulatus GTA regulatory gene mutants indicates three independent pathways that affect the production of extracellular GTA particles. One pathway involves the response-regulator CtrA, which is essential for transcription of GTA structural genes. A second pathway transmits a quorum-sensing signal, produced by a homoserine lactone synthase, which is needed for maximal induction of transcription of GTA genes. A third pathway requires the sensor-kinase homologue CckA for release of GTA particles from cells. An evaluation of the effects of carbon, nitrogen and phosphorus deprivation on the synthesis and release of GTA particles indicates that different environmental signals affect the production of GTA, similarly to the effects of regulatory mutations. 42
Conclusions: It appears that GTA arose in the α-proteobacteria after divergence of this line from other prokaryotes. The similarity of 16S rRNA and GTA gene descent to present-day species indicates that the transmission of GTA was largely vertical, with little or no horizontal gene transfer. We propose a speculative model in which three environmental signals affect GTA production: 1) the availability of phosphate and ammonium affect the balance between phosphorylated and non-phosphorylated CtrA, which in turn determines whether GTA genes are transcribed; 2) cell population density-dependent accumulation of a homoserine lactone molecule stimulates transcription of GTA genes; 3) a decrease in the availability of carbon stimulates a pathway involving CckA, which is needed for release of GTA particles from cells.
PL-8.1 PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGEN PRODUCTION. Caroline S. Harwood, University of Washington, Seattle, WA USA. In the world today, large quantities of hydrogen gas are used in petroleum refining and ammonia production for fertilizer and in the future there is the potential for hydrogen to be used in huge amounts as a transportation fuel. Although still under development, there are several microbial routes for hydrogen production that depend on the environmentally benign use of biomass, solar energy and other readily available resources. The development of an efficient process for hydrogen production by purple nonsulfur bacteria will require that the biocatalyst for hydrogen production, the nitrogenase, be synthesized and active under all growth conditions and it will be important to develop strains that cannot recapture and dissipate the hydrogen that they produce. Beyond this, the metabolic modules of photophosphorylation, reductant generation and nitrogenase activity that feed the process of hydrogen production, must each operate with maximum efficiency for hydrogen generation as opposed to for ammonia production. Obviously, the integrated functioning of these modules must also be maximally efficient. With this framework in mind there are numerous strategies for strain development that can be expected to lead to improvements and stabilization of the hydrogen production process. In addition to their practical usefulness, the application of such strategies will lead to an improved understanding of the hydrogen production process as it operates in the context of whole cells. In this talk I will discuss three strategies to improve hydrogen production. These are 1) Removal of regulatory constraints on nitrogenase, 2) Optimization of electron flow to nitrogenase, and 3) Design of efficient bioreactors.
PL-8.2 THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICAL APPLICATIONS Paula Tamagnini1,2, 1IBMC - Instituto de Biologia Molecular e Celular; 2 Faculdade de Ciências, Departamento de Botânica; Universidade do Porto, Porto, Portugal. Introduction: Cyanobacteria combine the ability to perform oxygenic photosynthesis with typical prokaryotic features like the ability to fix nitrogen. Many strains produce exopolymers, mainly of polysaccharidic nature, named exopolysaccharides (EPS). Due to their characteristics, cyanobacteria have high potential for biotechnological applications, such as the (i) production of biohydrogen and (ii) removal of heavy metals from contaminated waters. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes (i) BioModularH2 uses a synthetic biology approach to design reusable, standardized molecular building blocks that integrated into a chassis will produce a photosynthetic bacterium containing engineered chemical pathways for competitive, clean and sustainable hydrogen production [1]. (ii) The EPS project aims at optimizing the production of cyanobacterial EPS to establish an effective system to remove metallic ions from polluted waters. Methods: (i) BioModularH2 - Generation of mutants without redundant parts (e.g. genes) and identification of neutral sites using standardized vectors compatible with the BioBrick™; design and construction of synthetic parts and its assembly into modules/devices; characterization of the devices in the cyanobacterial chassis. (ii) EPS project - Metal removal assays, determination of EPS monosaccharidic composition by ion-exchange chromatography, identification of the functional groups responsible for metal-binding by potentiometric titration and Diffuse Reflectance Infrared Fourier Transform spectrometry, and sequence comparisons using bioinformatics tools. Results: (i) Preparation of the chassis starting with the (i) deletion of genes encoding the bidirectional hydrogenase in Synechocystis sp. PCC 6803 (redundant part) and (ii) construction of mutants on putative neutral sites (to integrate synthetic parts/devices such as an efficient heterologous hydrogenase). Concomitantly, several parts (e.g. promoters), and oxygen consuming devices (to provide a microaerobic environment required for an optimal heterologous hydrogenase activity) are being synthesized, characterized and tested. (ii) Copper removal assays performed with Gloeothece sp. PCC 6909 wild type and its sheathless mutant revealed that the mutant is more efficient in the process. Subsequent studies showed that although the mutant does not possess a sheath, it releases large amounts of polysaccharidic material (RPS) into the medium, and that its RPS possess higher amount and/or more accessible functional groups (e.g. carboxyl and amide groups) [2]. An in silico analysis of cyanobacterial genomes revealed the presence of genes encoding proteins that, in other organisms, are involved in the last steps of EPS production, although with a different physical organization. Based on these findings a putative mechanism for the biosynthesis and export of cyanobacterial EPS was proposed [3]. Conclusions: (i) BiomodularH2 - The standardized parts, modules and devices generated will be introduced to the chassis for photobiological H2 production, as well as will be available for other biotechnological applications. (ii) The future implementation of heavy metal removing systems based on cyanobacterial EPS depends on the knowledge about the biosynthetic pathways leading to their production. References: [1] http://www.biomodularh2.org [2] Micheletti E et al (2008) Appl Environ Microbiol 74: 2797-2804. 19: 139-185 [2] Pereira et al (2009) FEMS Microbiol Rev (accepted)
Final Program and Abstracts
PL-8.3 CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES, ACTIVITIES AND BIOSYNTHESIS. Kaarina Sivonen, Department of Applied Chemistry and Microbiology, Viikki Biocenter, University of Helsinki, Finland. Introduction: Cyanobacteria are a prolific source of bioactive compounds. These include potent toxins as well as biomedically interesting molecules, drug leads or useful probes in cell biology studies. Mass occurrences of neurotoxic and hepatotoxic cyanobacteria have caused number of animal poisonings and are a risk for human health. The cyclic hepatotoxic heptapeptides microcystins are most studied due to their worldwide occurrence. Methods: The structures of cyanobacterial compounds were determined by LC-MS and NMR. Bioactivities were investigated by enzyme inhibition tests and cell assays. Molecular methods (PCR, qPCR, cloning, sequencing, heterologous expression, microarrays) were used to identify the biosynthetic gene clusters and to detect hepatotoxic cyanobacteria. Shotgun sequencing and bioinformatic analyses were applied to genome of Anabaena strain 90. Results: Microcystins and nodularins (pentapeptide hepatotoxin occurring in brackish waters) are specific inhibitors of eukaryotic protein phosphatases and act as tumour promoters. Cyanobacterial bioactive cyclic and linear peptides may also display various bioactivities including serine protease inhibition and cytotoxicity. Novel bioactivities found recently include compounds acting as antidotes for microcystins and lipopeptides distroying the eukaryotic cell membrane. Microcystins and nodularin are products of a mixed non-ribosomal peptide and polyketide synthetase. The microcystins and nodularin gene clusters encode peptide synthetases, polyketide synthases and tailoring enzymes. This knowledge has been used to develop number of molecular techniques to detect hepatotoxin producing cyanobacteria. Studies on the evolution of microcystin/nodularin synthetase genes suggest that these genes are ancient and that present non-toxic strains have lost the genes and toxin production. The cyclic anabenopeptilides, and anabaenopeptins as well as linear spumigins are synthesised on non-ribosomal peptide synthetases. Interestingly, in anabaenopeptin biosynthesis a new way to create non-ribosomal peptide structural diversity was detected. The complete genome of Anabaena sp. strain 90 showed that 5% of the genome was dedicated to biosynthesis of bioactive compounds and that all these gene clusters are carried on the chromosome. It also revealed ribosomal peptide synthesis of a novel family of cyanobactins, anacycloamines. We demonstrated the widespread (48 out of 132 strains) but sporadic occurrence of the cyanobactin biosynthetic pathway among planktonic cyanobacteria. Conclusions: Cyanobacteria are a rich source of bioactive compounds for drug leads and the number of new compounds identified from cyanobacteria is increasing continuously. Cyanobacterial bioactive compounds may prove useful in combating various diseases in the future. In addition, their biosynthetic machineries (ribosomal and nonribosomal) provide enzymes to be used in combinatorial biosynthesis or chemoenzymatic synthesis to produce novel compounds. References: Sivonen, K. and T. Börner. 2008. Bioactive compounds produced by cyanobacteria. In: “The cyanobacteria: Molecular Biology, Genomics and Evolution”, Herraro, A. & E. Flores (Eds.). p. 159-197. Caister Academic Press, Norfolk, U. K. Sivonen, K. 2009. Cyanobacterial toxins. In: “Encyclopedia of Microbiology“, 3rd Edition, edited by M. Schaechter, pp. 290-307. Oxford:Elsevier.
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OC-1.1 SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETIC MARKERS FOR PURPLE SULFUR BACTERIA. Marcus Tank, Vera Thiel, Johannes F. Imhoff IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University), Kiel, Germany. Introduction: Purple Sulfur Bacteria (PSB) are photoautotrophic bacteria phylogenetically grouped to the order Chromatiales within the Gammaproteobacteria. They perform anoxygenic photosynthesis under anaerobic conditions generally using reduced sulfur compounds (e.g. H2S, S2-, thiosulfate) as e--donator. PSB are ubiquitously distributed but mainly restricted to aquatic environments containing adequate light conditions, low/no oxygen tension and moderate sulfide concentrations. pufL and pufM are essential in photosynthesis of PSB and encode for polypeptides of the photosynthetic reaction centres which are located in intracytoplasmic membranes (ICM). The phylogenetic relationship of purple sulfur bacteria (PSB) of the Chromatiales (Gammaproteobacteria) was analysed based on photosynthetic gene sequences of the pufL and pufM genes and the results compared to phylogenetic trees and grouping of the 16S rRNA gene. Methods: Primers for pufL and pufM genes were constructed and used to successfully amplify the pufLM genes of members of 16 genera of Chromatiales. pufLM and 16S rRNA gene sequences of 66 PSB strains, including 29 type strains and 28 new isolates, were determined and phylogenetically analyzed. The phylogenetic relationships based on the functional and ribosomal genes were compared to each other as well as to current taxonomic classification. Results: The inferred phylogenetic trees of pufLM and 16S rRNA genes reflect a largely similar phylogenetic development suggesting coevolution of these essential genes within the PSB. The two functional genes displayed similar phylogenetic relationship of the PSB regardless of the used gene (pufL, pufM or pufLM) and of weather nucleotides or deduced amino acid sequences were used. pufLM phylogeny is in good agreement to current taxonomic classification of PSB. Conclusion: It is concluded that horizontal gene transfer of pufLM genes within the PSB is highly unlikely, which contrasts the situation in other groups of anoxygenic phototrophic bacteria belonging to Alphaproteobacteria and Betaproteobacteria. A phylogenetic classification of PSB to the genus level is possible based on their pufL or pufM sequences, in many cases even to the species level. In addition, our data support a correlation between Puf protein structure and the type of internal photosynthetic membranes (vesicular, lamellar or tubular).
OC-1.2 BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTING GENES IN MARINE PHYTOPLANKTON. Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2. 1
School of Ocean and Earth Sciences, National Oceanography Centre, Southampton, United Kingdom; 2School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia. Introduction: Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial genera Prochlorococcus and Synechococcus that are defined by two distinct August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts light-harvesting systems, chlorophyll-bound protein complexes or phycobilin-bound protein complexes, respectively. Here, we use the Global Ocean Sampling (GOS) Project as a unique and powerful tool to analyze the environmental diversity of photosynthetic lightharvesting genes in relation to available metadata including geographical location and physical and chemical environmental parameters. Methods: All light-harvesting gene fragments and their metadata were obtained from the GOS database, aligned using ClustalX and classified phylogenetically. Each sequence has a name indicative of its geographic location; subsequent biogeographical analysis was performed by correlating light-harvesting gene budgets for each GOS station with surface chlorophyll concentration. Results: Using the GOS data, we have mapped the biogeography of light-harvesting genes in marine cyanobacteria on ocean-basin scales and show that an environmental gradient exists in which chlorophyll concentration is correlated to diversity of light-harvesting systems. Three functionally distinct types of light-harvesting genes are defined: (1) the phycobilisome (PBS) genes of Synechococcus; (2) the pcb genes of Prochlorococcus; and (3) the iron-stress-induced (isiA) genes present in some marine Synechococcus. At low chlorophyll concentrations, where nutrients are limited, the Pcb-type lightharvesting system shows greater genetic diversity; whereas at high chlorophyll concentrations, where nutrients are abundant, the PBStype light-harvesting system shows higher genetic diversity Conclusions: We interpret this as an environmental selection of specific photosynthetic strategy. Importantly, the unique lightharvesting system isiA is found in the iron-limited, high-nutrient low-chlorophyll region of the equatorial Pacific. This observation demonstrates the ecological importance of isiA genes in enabling marine Synechococcus to acclimate to iron limitation and suggests that the presence of this gene can be a natural biomarker for iron limitation in oceanic environments.
OC-1.3 PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THE CYANOBACTERIAL GENERA ANABAENOPSIS AND CYANOSPIRA. Stefano Ventura, Cristina Mascalchi, Claudio Sili Institute of Ecosystem Study, National Research Council, Sesto, Fiorentino, Italy. Introduction: Planktic heterocytous cyanobacteria from tropical alkaline environments have been studied in our group since long ago. We described the genus Cyanospira, with its two species C. rippkae and C. capsulata, that are among the few cyanobacterial taxa which hold a formally valid taxonomic description under the Bacteriological Code. The existence of the genus Cyanospira and of its two species and its separation from the genus Anabaenopsis has been repeatedly questioned. To contribute to clarify the relationships between the two genera, we present a phylogenetic analysis of Anabaenopsis and Cyanospira and a detailed study of the morphology of several undescribed strains of Cyanospira. Methods: Strains have been isolated from natron coming from Chad that contained large numbers of dormant akinetes. Phylogenetic analysis has been performed using the ARB and SILVA phylogenetic tools and databases, which supply a constantly updated and aligned database with quality evaluation of the sequences. Morphology has been studied in laboratory cultures inoculated with dormant desiccated akinetes. The entire life cycle has been studied and documented, from the akinete germination to the full development of vegetative forms. 45
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Final Program and Abstracts Results: Cultures of Cyanospira, obtained from germination of desiccated akinetes, were characterized by variable filament dimensions and coiling degrees. Akinete development in vegetative filaments was typically apoheterocytic; akinetes developed in chains that continuously expanded along the filament; in old cultures, all cells in the filament can be transformed into akinetes. Filaments of Cyanospira were often surrounded with a thick mucilaginous capsule. The development pattern of akinetes in Cyanospira was definitely different from that of Anabaenopsis. After a selection of sequences based on length and quality, that is inherent to the SILVA database, all publicly available sequences identified as belonging to the genera Anabaenopsis and Cyanospira, plus new sequences of our strains have been compared with selected entries of Nostoc, Thricormus, Nodularia, and Azolla cyanobionts. The phylogenetic analysis produced several well defined clusters. The assemblage of the two genera Anabaenopsis and Cyanospira formed a phylogenetically coherent and well defined unit, mostly related to the genus Nodularia. Inside this unit, sequences of Anabaenopsis were subdivided into three clusters, two housing freshwater strains, and one, more apart, housing representatives of the species A. abijatae which came from mineralized (alkaline) lakes in Ethiopia. Sequences of Cyanospira formed three distinct phylogenetic clusters that were more related to the A. abijatae cluster. Conclusions: As already demonstrated for hypersaline environments, alkaliphilic cyanobacteria stayed apart from their freshwater counterparts. Among the alkaliphiles, the genus Cyanospira was subdivided into three phylogenetic subunits. The relationships of two of these units with A. abijatae should be investigated in more details by means of a precise morphological study. Our data support the taxonomic robustness of the genus Cyanospira as the alkaliphilic counterpart of Anabaenopsis, and the existence of more than one species inside it.
OC-1.4 CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATS OF A TROPICAL LAGOON, TIKEHAU ATOLL, TUAMOTU ARCHIPELAGO. Katarzyna A. Pali ska1, Raeid M. M. Abed2†, Katja Wendt1, Maria Łotocka3, Loic Charpy4 & Stejpko Golubic5. 1
Institute of Chemistry and Biology of the Marine Environment, Geomicrobiology Dep.; CvO University of Oldenburg, Oldenburg, Germany; 2College of Science-Biology Department, Sultan Qaboos University, Al-Khod, Muscat, Sultanate of Oman; 3Institute of Oceanology, Polish Academy of Sciences, Powsta ców Warszawy, Sopot, Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167 (CYROCO), Traverse de la Batterie des Lions, Marseille, France; 5 Biological Science Center, Boston University, Boston, MA, USA. Introduction: Cultivation of microorganisms is known to underestimate the real diversity of bacterial communities thriving under natural conditions (Amann et al., 1995; Abed et al., 2008) and the isolates are often selected by the supplied culture conditions. However, these minor bacterial populations in the field may become dominant in response to environmental changes or in the case of certain perturbation events that favor their growth. While recent studies in microbial ecology focuses on identifying dominant field bacterial populations using culture-independent molecular tools, with the assumption that they are responsible for most bacterial activities, minor populations are often ignored. In this study, we investigated the diversity of opportunistic cyanobacteria that respond to the high load of nutrients provided by the cultivation medium. We postulate that 46
these autochthonous assemblages of taxa are of ecological importance. Methods: Twelve strains of filamentous and unicellular cyanobacteria were isolated from microbial mats of the lagoon of Tikahau atoll, Tuamotu Archipelago, the most oligotrophic Pacific waters; identified morphologically and using 16S rRNA-based phylogenetic analysis. Taxonomic identity of the studied strains was further supported by obtaining CpcBA-IGS sequences. Cultures were isolated from natural populations growing on the bottom of the lagoon formed of calcareous sand from the surface to a depth of ca. 20m. We investigated strains with respect to their pigmentation, as well as their potential to perform chromatic adaptation, and nitrogen fixation. Results: Using direct microscopy, 12 different cyanobacterial morphotypes were identified. They were classified in six morphogenera sensu Geitler (1932) and Anagnostidis & Komarek (1988): Cyanocystis, Leptolyngbya, Aphanothece, Phormidium, Chlorogloea and Pseudanabaena. The genera Cyanocystis and Chlorogloea were genotypically characterized for the first time Most of the strains posses’ phycoerythrin and have the ability to fix nitrogen.. Non-heterocystous cyanobacteria with narrow trichomes were classified under Leptolyngbya and Phormidium following botanical recommendation, but homogeneity of their grouping was not genotypically confirmed. Conclusions: The strains represent autochthonous cryptic diversity, which dominated cultures but not the natural populations. Taxa studied here clearly responded to higher nutrient supply provided by the standard artificial medium, which may simulate conditions in nature following the impact of tropical storms, thus recruiting opportunistic taxa, which are normally present at low frequencies. They may be considered important members of microbial successions during restoration of the ecosystem following catastrophic impacts. With increasing man-made disturbances, such as eutrophication, pollution or dredging, it is expected that these taxa will increase in abundance. While recent studies in microbial ecology focuses on identifying dominant field bacterial populations using culture-independent molecular tools, with the assumption that they are responsible for most bacterial activities, minor populations are often ignored. We presume that these less-dominant populations are equally important and can only be identified by enrichment cultivation simulating possible environmental perturbations and changes.
OC-1.5 METAGENOMIC AND PHYLOGENETIC ANALYSES OF CYANOBACTERIAL MATS IN EXTREME HIGH ARCTIC ENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICAL FUNCTION. Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3, Warwick F. Vincent1. 1
Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté de Médecine, Université Laval; 3Québec-Océan, Département de Biologie, and Institut de biologie intégrative et des systèmes (IBIS); Université Laval, Québec, QC, Canada. Introduction: Mat-forming cyanobacteria are among the oldest known prokaryotes and are widely distributed in more extreme environments, and in environments where grazing pressure is reduced or absent. The physical form of the cyanobacteria living within the mats and the three dimensional structure of mats themselves is much conserved with fossils dating from more than 2 billion years. However given the wide
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13th International Symposium on Phototrophic Prokaryotes variety of habitats where mats tend to be abundant, a major question is whether such morphologically similar structures harbour similar species and what particular genetic adaptations or metabolic pathways may be present in different environments. Polar aquatic ecosystems are dominated by microbes and abundant mat-forming cyanobacteria are widely distributed in shallow lakes, streams and meltwater ponds on ice shelves of the High Arctic. During summer, oxygenic photosynthesis by cyanobacteria is the primary energy source supporting a complex community within the mats. In contrast to physically similar mats from other habitats, such as hot springs, there is relatively little data on the taxonomic and functional diversity of these polar cyanobacteria-dominated mat communities. Without such information, questions on the evolutionary persistence of cyanobacteria mats during both warming and glacial epochs, and on their global biogeography cannot be addressed. Therefore, we characterised High Arctic microbial mats using metagenomics to uncover adaptive functional genes and targeted 16S rRNA gene libraries and phylogenetics to determine cyanobacterial diversity. Methods: Microbial mats were investigated from freshwater ecosystems of different nutrient and physical regimes in the Canadian High Arctic. Microbial community analysis was performed using pyrosequencing and bioinformatic analysis. The phototrophic diversity was determined using signature pigment analysis. Cyanobacterial diversity was targeted using strain isolation, and culture independent methods including microscopic examination, and phylogenetic analyses based on 16S rRNA gene clone-libraries. Results: High Arctic microbial mats had chlorophyll a values ranging from 3.9 to 46.2 µg cm-2 with high concentrations of cyanobacterial pigments such as scytonemin. Metagenomic analyses of two microbial mat communities from ice shelves identified Cyanobacteria and Proteobacteria as dominant bacterial groups. Other bacteria, Archaea, Eukaryota and viruses were also identified. A high proportion of genes were implicated in metabolic pathways of major biogeochemical cycles with evidence of specific molecular adaptations to the cryosphere. Cyanobacterial diversity consisted of phylotypes within Chroococcales, Oscillatoriales, Nostocales and Stigonematales based on morphological and 16S rRNA gene analyses. Phylotypes within the orders Chroococales and Nostocales had highest similarity to temperate environments, whereas the majority of oscillatorians had closest similarity (>99) to Antarctic and alpine sequences, including to taxa previously considered to be endemic to the Antarctica. Conclusions: High Arctic microbial mats contain diverse assemblages of microbes that perform an ensemble of biogeochemical cycling processes, similar to cyanobacteria-dominated mats from other climate zones. Our results also imply that low-temperature cyanobacterial ecotypes have a cosmopolitan distribution throughout the cold terrestrial biosphere.
OC-1.6 GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENON SPP. AND ANABAENA SPP. IN NORTHEAST GERMAN LAKES. Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1. 1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes, Neuglobsow; 2Federal Environmental Agency, Berlin; 3Brandenburg Technical University, Department of Freshwater Conservation, Bad Saarow; Germany.
Introduction: In the last decades hepatotoxic cylindrospermopsin (CYN) and neurotoxic saxitoxins (STX) were detected in several lakes in August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts northeast Germany. CYN and STX are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, or Aphanizomenon. So far only Aphanizomenon sp. was confirmed as producer of CYN in two German lakes, while the source of STX is not yet identified in German water bodies. In this study we attempted to identify STX- and CYN producers in German water bodies and characterized them morphologically, genetically, and biochemically in comparison to non toxin producing strains. Methods: In 2007 and 2008, 200 strains of Aphanizomenon spp., Anabaena spp., and Anabaenopsis spp. from 8 selected lakes in Brandenburg, Germany were isolated and cultivated. The isolated strains were identified morphologically using light microscopy and genetically using different genetic markers (cpcBA-IGS and RBCL). Primers were designed for a fast detection of the sxtA part of the STX gene cluster. For the detection of the CYN gene cluster primers to amplify the peptide synthetase (PS) gene were used. To confirm potential STX and CYN producers ELISA as a biochemical method and liquid chromatographic tandem (LC-MS/MS) methods were applied. Results: The isolated strains were assigned morphologically to four different species of Aphanizomenon and four different species of Anabaena and one Anabaenopsis species. Our sequence data and phylogenetic analyses confirmed the formation of intermixed clusters by planktic Anabaena and Aphanizomenon strains. Aphanizomenon gracile and Aphanizomenon flos-aquae could not be divided as separate species. Using polymerase chain reaction (PCR), 14 strains of Aphanizomenon cf. gracile, three strains of Aphanizomenon issatschenkoi, and one strain of Anabaenopsis sp. were identified as potential STX producers due to the possession of fragments of the sxtA gene. The ability to produce STX was confirmed with ELISA and LC-MS for the 14 strains of Aphanizomenon cf. gracile. Each strain produced 6 STX variants in variable amounts The STX concentrations found were between 400 and 1300 µg/g cyanobacterial dry weight. No CYN producer was detected using genetical methods and ELISA. Conclusions: This is the first study to confirm Aphanizomenon cf. gracile as saxitoxin producer in German water bodies. As strains of Aphanizomenon issatschenkoi and Anabaenopsis sp. also possessed fragments of the STX gene cluster other nostocalean producers of STX are very likely in German water bodies.
OC-2.1 MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS IN PHOTOTROPHIC CONSORTIA. Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller. Section Microbiology, Department Biology I, University of Munich, Planegg-Martinsried, Germany. Introduction: Phototrophic consortia represent the most highly developed bacterial symbiosis and consist of green sulfur bacterial epibionts surrounding a central chemotrophic Betaproteobacterium. A rapid and specific exchange of multiple signals occurs between both bacterial partners and allows tactic behaviour towards light and chemical stimuli. Laboratory cultures of the consortium “Chlorochromatium aggregatum“ as well as of the isolated epibiont have recently been established, providing the opportunity to elucidate the molecular mechanisms underlying the symbiotic interaction. Methods: Subcellular structures were studied by high resolution analytical scanning electron and transmission electron microscopy. Putative symbiosis genes of the epibiont were identified by suppression subtractive hybridization of genomic DNA against that of 16 relatives and bioinformatics. Transcription was studied by reverse
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Final Program and Abstracts transcriptase-PCR. Cross-linking experiments were conducted with bis(sulfosuccinimidyl)suberate or 3,3´dithiobis(sulfosuccinimidylpropionate). Results: In “C. aggregatum”, numerous periplasmic tubules extend from the outer membrane of the central bacterium and are in direct contact to the epibiont. Our search for specific recognition and signal transduction proteins identified a total of 189 genes of the epibiont of “C. aggregatum“ to be absent in all other green sulfur bacteria. Most noticable was the presence of four genes which were related to virulence factors of human or plant pathogenic bacteria. These genes were transcribed constitutively in the epibiont. The hemagglutinin-like putative gene products of ORFs Cag0614 and Cag0616 probably arose by gene duplication and represent the largest bacterial open reading frames so far documented for bacteria. Cag1920 codes for a putative hemolysin whereas the gene product of Cag1919 is a putative repeat in toxin (RTX)-like protein containing a Ca2+- binding beta roll motif. In fact, intact consortia disaggregated upon the addition of EGTA. The RTX-type C-terminus coded by Cag1919 exhibits a significant similarity to RTX modules of various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium. This conclusion is further supported by the presence of three genes encoding transposases which are located immediately upstream of ORF 1919. Most recently, cross-linking experiments of intact consortia revealed the presence of a corresponding filamentous hemagglutinin/adhesin in the central bacterium which appears to be involved in maintaining the multicellular structure of the phototrophic consortium. Conclusions: While the adaptation of green sulfur bacteria to a symbiotic lifestyle in phototrophic consortia requires only a comparatively low number of niche-specific genes, it involves several unique genes which most likely were acquired by lateral gene transfer from a proteobacterium. Our study of the molecular mechanisms of symbiosis indicates that typical bacterial virulence factors have been exploited by green sulfur bacteria to establish a mutualistic interaction and enabled these bacteria to occupy a novel ecological niche.
OC-2.2 LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBIC ANOXYGENIC PHOTOTROPHS IN A SEMIARID REALM. J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. The discovery of aerobic anoxygenic phototrophs from biological soil crusts (BSC) is reported. Living on an ecological knife-edge of habitability, BSC are drought-tolerant communities composed primarily of microorganisms that form a cohesive, erosion-resistant veneer on the surface of soil. These communities inhabit semiarid to arid soils and are composed of cyanobacteria, bacteria, fungi and mosses. They are receiving increased attention from a land management perspective because they reduce soil erosion and enhance moisture and nutrient status. From sandy soils (including sand dunes) in Manitoba and Alberta, Canada, 21 strains were isolated that possessed the diagnostic light harvesting pigment, bacteriochlorophyll. Anoxygenic phototrophs constituted 0.1 to 5.9% of the cultivable biological soil crust bacterial community. Sequencing the 16S rRNA gene of 16 isolates revealed that nine strains were closely related to the genus Methylobacterium, while seven fell within the alpha-1- and alpha-4-proteobacteria, related to typical aerobic anoxygenic phototrophs such as Paracraurococcus, and to soil crust 48
heterotrophs such as Belnapia. This study is the first demonstration of proteobacterial anoxygenic phototrophs from semiarid environments. The presence of anoxygenic phototrophs in BSC implies a higher efficiency of light harvesting by soil curst organisms than previously realized. Utility of near-IR radiation not used by oxygenic phototrophs facilitates light-accelerated turnover of soil organic carbon content.
OC-2.3 ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MATNITROGEN FIXATION. Ina Severin, Lucas J. Stal. Department of Marine Microbiology, NIOO-Center for Marine and Estuarine Ecology, Yerseke, The Netherlands. Microbial mats on intertidal beaches and sand flats are often pioneer systems. Following their establishment, colonization by higher plants occurs and rigorously changes the morphodynamics of the ecosystem. One perquisite for this succession is the enrichment of the sediment with combined nitrogen as the result of N2 fixation. Cyanobacteria are usually the most conspicuous structural part of microbial mats. These organisms have long been the focus of research on N2 fixation in microbial mats. However, there is growing evidence that Cyanobacteria are sometimes minor players in terms of N2 fixation. We investigated a coastal microbial mat with regard to N2 fixation using the acetylene reduction assay as well as with regard to the diversity of the 16S rRNA and nifH genes and transcripts. Quantitative RT-PCR was used to access daily changes of nifH-gene expression of the dominant phylotypes and groups as well as the whole mat community. The daily patterns of nitrogenase activity differed with respect to the point of time when the maximum was reached. The mat higher up in the littoral zone (station I) showed several nitrogenase activity-maxima between sunset and sunrise and harboured a variety of heterocystous and non-heterocystous Cyanobacteria. The mat situated close to the low water mark (station II) was mainly composed of non-heterocystous Cyanobacteria with Lyngbya aestuarii being the most dominant diazotroph. Accordingly, a night time-maximum of nitrogenase activity was recorded. A clear light dependency of nitrogenase activity implied phototrophs as main diazotrophs. 16S rRNA gene clone libraries showed that both microbial mats were dominated by Cyanobacteria and Proteobacteria. Filamentous non-heterocystous Cyanobacteria and Gammaproteobacteria were predominant at both stations but the relative contribution of taxa differed. Analysis of the nifH gene clone libraries confirmed that Cyanobacteria were the dominant diazotrophs in both communities. In accordance with 16S rRNA gene clone libraries, Gammaproteobacteria-related nifH sequences were most frequently retrieved at station I whereas the delta-subdivision made up for almost all Proteobacteria-related nifH sequences at station II. The analysis of nifH transcripts of station I revealed a dynamic community with changing relative abundances of nifH transcripts of members of the community over time. While Cyanobacteria-related sequences were predominant from midday to midnight, Proteobacteria-related nifH transcripts were found to become more important from midnight on. A high proportion of these proteobacterial sequences were most closely related to known anoxygenic phototrophs. Relative contribution of sequences to cDNA clone libraries differed considerably from those observed for DNA clone libraries, highlighting active diazotrophs. Gene expression levels yielded further insight into diel nifH gene expression patterns of key diazotrophs in comparison to whole mat community nitrogenase activity. The results suggest that species composition affects the maximum attainable August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes nitrogenase activity and its diel pattern. Oxygenic as well as anoxygenic phototrophs were found to be the key players in N2 fixation in the investigated microbial mats.
Final Program and Abstracts
OC-2.5
OC-2.4
DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACE WATERS OF MARINE AND FRESHWATER ENVIRONMENTS WITH AN EMPHASIS ON CYANOBACTERIA.
BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCED PHOTOSYNTHETIC MICROBES.
Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, Torsten Staller2, Rüdiger Schulz2, Jens Appel1.
Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer.
1
SRI International, Menlo Park, CA, USA. The BioCyc [1] collection of 409 Pathway/Genome Databases (PGDBs) is available at URL BioCyc.org. BioCyc includes the genomes, and predicted metabolic networks and operons of eleven photosynthetic microbes. BioCyc also includes the EcoCyc [2] database (DB), which describes the metabolic and genetic regulatory network of Escherichia coli; and the MetaCyc DB, which describes more than 1200 metabolic pathways that were experimentally elucidated in more than 1,600 organisms. BioCyc contains PGDBs for most organisms with completely sequenced genomes. Each BioCyc PGDB combines the annotated genome sequenced with predicted metabolic pathways, predictions of pathway hole fillers, and operon predictions. BioCyc is generated on a regular ongoing basis using the MetaCyc reference database of experimentally elucidated pathways. BioCyc offers many capabilities to researchers for analysis and investigation of genomes, metabolic networks, and regulatory networks, and for analysis of high-throughput datasets. A genome browser allows exploration of a microbial genomes, and comparison of conserved genome regions. The predicted metabolic network of each organism can be queried and visualized using a variety of bioinformatics tools that display information about metabolites, enzymes, reactions, and individual pathways. Computational tools for analysis of metabolic networks include tools for finding dead-end metabolites, and for performing reachability analysis of metabolic networks. In addition, each PGDB provides a diagram of the complete metabolic map of the organism, and of the complete regulatory network when it has been curated for that organism. These tools support analysis of large-scale omics datasets [3], such as gene-expression and metabolomics data, by painting those data onto the full metabolic map diagram and regulatory network diagram to place omics data within a biological context. A toolkit of comparative analysis tools permits comparisons of the genome and biochemical networks of multiple Pathway/Genome Databases. BioCyc can be accessed at BioCyc.org, as a set of downloadable data files, and as an installable software distribution. The BioCyc.org Web site and data files are freely available to all; the downloadable software is freely available to research institutions. [1] R. Caspi et al, “The MetaCyc Database of Metabolic Pathways and Enzymes and the BioCyc Collection of Pathway/Genome Databases,” Nucleic Acids Research 36:D623-31 2008. [2] Karp, P.D. et al, “Multidimensional annotation of the Escherichia coli K-12 genome,” Nucleic Acids Research 35:7577:90 2007. [3] Paley, S.M. et al, “The Pathway Tools Cellular Overview Diagram and Omics Viewer,” Nucleic Acids Research 34:3771-8, 2006.
August 9 to 14, 2009 • Montréal, QC, Canada
2
School of Life Sciences, Arizona State University, Tempe, AZ, USA; Botanisches Institut, Universität Kiel, Kiel, Germany.
Introduction: After methane hydrogen is the second most abundant trace gas in the atmosphere, making up around 0.5 ppm to 0.6 ppm. Surface waters of different aquatic environments have been shown to evolve hydrogen. The ocean is estimated to be the major natural source of H2. Still its origin and the proportion of biological and nonbiological production and consumption processes are unknown. Hydrogen concentrations of surface waters above the expected equilibrium prompted us to investigate the presence and distribution of all known hydrogenases in marine and fresh water environments with a special emphasis on cyanobacteria. Methods: DNA was isolated from samples from the North Sea, the Baltic Sea, the North Atlantic, the Mediterranean Sea and two German lakes (Westensee and Selenter See). Degenerated primers were used to amplify part of the gene of the large subunit of the bidirectional NAD(P)-linked hydrogenase (hoxH) from these samples. In addition the available genomes of cyanobacteria, marine bacteria and the GOS database were searched for the presence of all the known hydrogenase genes. Results: The search of the cyanobacterial genomes confirms that their uptake hydrogenase (hupL) is strictly linked to the nitrogenase genes. This is not surprising since it is used to recycle the hydrogen inevitably produced by the nitrogenase in each catalytic cycle. But nevertheless there are some strains that do have the nitrogenase genes but no uptake hydrogenase indicating that the hydrogenase is not essential to compete and might not be necessary under all environmental conditions. The distribution of the hoxH sequences in the complete genomes as well as in the different samples analyzed indicates a strong bias to freshwater environments and coastal waters. It seems to be absent in the open ocean, which is confirmed by the database searches also in other marine bacterial genomes. Therefore, the presence of this hydrogenase is especially linked to the probability to encounter anaerobiosis. In approximately 20 % of all marine genomes hydrogenases were found. The presence of H2-sensing hydrogenases in photosynthetic bacteria such as Roseovarius strains and other marine bacteria suggests that H2 triggers the expression of the structural genes of their uptake hydrogenases. Conclusions: Our results show that in marine waters all the genetic complement is present for biological hydrogen production as well as uptake. Therefore, biological processes alone might be responsible for the measured H2 turnover in these habitats. The sequences found suggest that hydrogen might be used as an additional energy source by marine bacteria in nutrient-limited environments. In addition, our data supports the conclusion that the bidirectional NAD(P) linked hydrogenase is especially needed for adaptation to rapidly changing redox conditions and unnecessary in the open ocean.
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Final Program and Abstracts
OC-2.6
OC-3.1
HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRING MICROBIAL MATS DOMINATED BY THE FILAMENTOUS ANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUS AGGREGANS.
CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROM THE HALOCLINE OF THE SALINE MEROMICTIC LAKE, LAKE SUIGETSU, JAPAN.
H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1.
Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan.
1
Department of Biology, Tokyo Metropolitan University, Tokyo; 2 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki; Japan. Introduction: The filamentous anoxygenic phototrophic bacteria Chloroflexus aggregans and C. aurantiacus are often present in hot springs dominated by cyanobacteria. In some hot springs however, there are distinctive microbial mats that lack cyanobacteria and are instead dominated by Chloroflexus spp. At Nakabusa hot spring (Nagano, Japan) at 65°C, C. aggregans develops mats by growing autotrophically using hydrogen sulfide as an electron donor for photosynthesis. These mats also contain a thermophilic sulfatereducing bacterium, Thermodesulfobacterium sp., which produces hydrogen sulfide. It is known that this sulfate-reducing bacterium can utilize hydrogen, but it is still unknown whether this bacterium consumes hydrogen in the mats or whether biological hydrogen production even occurs in this community. In this study, we investigated net hydrogen production in the 65°C microbial mats at Nakabusa. Methods: Samples of these mats, mainly composed of C. aggregans, were collected and hydrogen production was measured in artificial hot-spring water at 65°C under various treatments in anaerobic bottles. Results: In the absence of any treatment, hydrogen was not detected, but significant production was observed in the presence of molybdate, an inhibitor of sulfate reduction or with a homogenization treatment of the mat. This net hydrogen production largely decreased under illumination. These results suggest that the metabolism resulting in hydrogen production was not photosynthetic; we hypothesize that hydrogen was produced by fermentative bacteria breaking down organic compounds supplied by anoxygenic photosynthetic bacteria. This hydrogen was then consumed by sulfate-reducing bacteria as they reduced sulfate to sulfide. The decrease of net hydrogen production under illumination can be explained by the anoxygenic photosynthetic bacteria using hydrogen as an electron donor or competing with fermentative bacteria for organic compounds. Conclusions: Hydrogen was shown to be produced and consumed within the mats, and net hydrogen production could not be detected without the inhibition of sulfate reduction or physical blocking of hydrogen consumption. In the 65°C mats at Nakabusa, anoxygenic photosynthetic bacteria produce organic compounds using hydrogen sulfide as an electron donor. Fermentative bacteria use this carbon as an energy source and release hydrogen as a byproduct. This hydrogen is then used by sulfate-reducing bacteria that produce hydrogen sulfide. Thus, electrons cycle via organic compounds, hydrogen and hydrogen sulfide in the community.
Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya.
Introduction: The surface layer of Lake Suigetsu (35 35’N, 135 52’E, coast of the Japan Sea in Fukui Prefecture, Japan) has O2-containing fresh water, while the deeper layer is anoxic salt water where a permanent halocline (salinity is ca.0.4 to 1.4%) is created between these two water layers. The concentration of O2 decreases to zero at about the middle of the halocline; and H2S, a potent inhibitor of oxygenic photosynthesis, is detected deeper below the oxic-anoxic boundary layer (OABL). Only weak green light (less than 1% of surface intensity) penetrates to the OABL. We found small size cyanobacteria (<2 μm, PCy) were distributed through the year within the halocline as the dominant phytoplankton (0.5 to 5×105 cells/ml) during four years 2005 - 2008. The attempt to determine the adaptation mechanism(s) of PCy to their habitat, phylogenetic and physiologic properties of PCy were studied using isolated strains from the halocline of Lake Suigetsu. Methods: Water samples were collected from the different depths within the halocline of Lake Suigetsu during the summer of 2005-6. Unicyanobacterial isolates were obtained by isolating colonies on agar plates using various salinities in artificial media (0.4 to 1.6 %). Morphology observations were performed using epifluorescence and electron microscope. Phycobiliprotein (PB) compositions were determined spectrophotometrically using intact cells and/or isolated phycobilisomes. The most part of the 16S rRNA and the internal transcribed spacer (ITS) between 16S and 23S rRNA genes were sequenced from the genomic DNA of the isolated PCy; and molecular phylogenetic trees were constructed using the neighbor-joining method. Results: C-phycoerythrin (R-type) or C-phycocyanin (G-type) was the major PBs. The isolated clones (about 100) were grouped into six subgroups using the PB composition and sequence homology of the ITS between the 16S and 23S rRNA genes. Further analyses were performed using six clones from the different sub-groups (three Rtypes and three G-types). The six clones were included within the freshwater Synechococcus-Cyanobium clade, but they were not monophyletic in the 16S rRNA-based tree. Cells were spherical or ellipsoidal at 0.5 to 1.5 μm in diameter or width and reproduced using transverse binary fission in a single plane. The thylakoids were peripheral and were oriented parallel to the cytoplasmic membrane. They were able to grow in a wide range of salinities (0.2 to 2 % or more). Significant growth was observed under weak green light (2 μmol·m-2·sec-1, ca. 460 to 600 nm). Whereas, the cells were bleached irreversibly under the white light at relatively high intensities (≥15 μmol·m-2·sec-1 for the R-type; ≥25 μmol·m-2·sec-1 for the G-type). At least four clones survived in medium containing sulfide (0.3 to 1.2 mM as S2-, pH7.6) for more than a week. Conclusion: The physiological properties found in this study show PCy survive under unique physicochemical environments in the halocline of the saline meromictic lake, Lake Suigetsu. The ability to use weak green light for photosynthesis and sulfide tolerance may provide an advantage over other phytoplankton under sulfide-rich environments. The present phylogenetic analyses suggested this ability was acquired by PCy in Lake Suigetsu more than once.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts analyses of sequences obtained from part of the 16S rRNA gene.
OC-3.2 THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIA PROCHLOROCOCCUS. Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA. The marine cyanobacteria Prochlorococcus is thought to be the most numerically abundant phototropic prokaryote in the global oceans and as such it plays a critical role in the ecology and biogeochemistry of marine ecosystems. Recently we have completed several global ocean surveys covering vast oceanic regions where we measured the abundance and photosynthesis (primary production) of this important phototrophic organism. We find that over these regions, the integrated water column abundance of Prochlorococcus is remarkably conserved, in spite of significant variability in the vertical distributions. Further, although these surveys covered many oceanographic biogeochemical provinces, each with unique nutrient, temperature and other environmental variables, the contribution of Prochlorococcus to primary production remains highly conserved and ranges between 25 – 50% of the total production rates. Quantitative phylogenetic analyses using clade specific qPCR shows that while the taxonomic composition of Prochlorococcus can change dramatically in response to these environmental variables (and in particular temperature), the integrated abundance and production of the genus remains relatively consistent. Together, these global abundance and primary production estimates suggest that the Prochlorococcus niche is more consistent and larger than previously thought.
OC-3.3 THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTAL CYANOBACTERIA COMMUNITY AND ITS CONTRIBUTION TO TOXICITY IN A TROPICAL BRAZILIAN RESERVOIR. Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos. Department of Botany, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. Introduction: Autotrophic picoplankton (APP) represents an important part of the planktonic communities in aquatic systems and may be responsible for a large amount of carbon fixation. Even though these organisms are widespread and abundant, in the past their populations were often not recognized because of their diminutive size (0.2-2 µm). A large part of the picoplanktonic community is represented by cyanobacteria. The importance of picocyanobacteria for global primary production and biomass has received less attention in freshwater than in marine systems. The development of molecular techniques opened new possibilities for the study of the diversity of these organisms and even their quantification. In the present work we present results obtained from a large mostly oligotrophic reservoir located in Southeastern Brazil, Furnas Reservoir. Methods: Samples were taken regularly over a two years period, concentrated on glass fibre filters and frozen until extraction. Samples for phytoplankton counting were preserved unfiltered with a Lugol’s iodine solution. Extraction for molecular analyses were performed by the phenol-chloroform technique. Extracts were analyzed by conventional PCR and real-time quantitative PCR (Step One Applied Biosystem). Primers were designed aimed at the detection of the cpcB region (total cyanobacteria) and mcyD (toxic cyanobacteria) genes in natural populations. New primers were designed for the q-PCR. Some samples were selected for the investigation of cyanobacterial diversity by the technique of “Amplified Ribosomal DNA Restriction Analysis” (ARDRA) using primers for amplification of part of the 16S rRNA gene and the entire internal transcribed spacer (ITS 16S-23S), and the August 9 to 14, 2009 • Montréal, QC, Canada
Results: Results from q-PCR revelead the importance and the numerical dominance of picocyanobacteria in the overall cyanobacteria community in the reservoir but not a sgnificant contribution in its toxicity. The percent of mcyD genes copies relative to cpc-B varied between 0.1 to 1.6 % but increased up to 55% when picoplanktonic cells were eliminated. The analyses of ARDRA patterns showed that although eighteen patterns were found, fifteen of them were related with the sequences of just two picoplanktonic genera (Cyanobium and Synechococcus). Conclusions: Our results suggest that the cyanobacterial APP community can be extremely important in tropical freshwater systems and may represent a significant contribution in the plankton diversity. Further development of the genomic approach to photosynthetic picoplankton will lead to new understanding of their roles in warm oligotrophic systems.
OC-4.1 ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUM TEPIDUM: GENES, METABOLITES AND LINKAGES TO LIGHT HARVESTING. Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan, Rachael Morgan-Kiss. College of Marine and Earth Studies and DBI, Newark, DE, USA. Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is a model green sulfur bacterium for studying light harvesting in phototrophs that utilize chlorosomes as the major antenna complex and pathways of anaerobic sulfur oxidation. Current studies are focused on delineating pathways of anaerobic sulfur oxidation, including required gene products and metabolites, and how these pathways interact with global physiology in this organism. Methods: C. tepidum is capable of phototrophic growth with sulfide or thiosulfate as the sole electron donor or with both substrates in combination. This flexibility enabled the genetic dissection of sulfur oxidation pathways by targeted transposon mutagenesis, random transposon mutagenesis, and the addition of His-tags to specific genes in the C. tepidum chromosome. The resulting mutant strains and wild type grown under various sulfur, light and temperature regimes were analyzed by techniques of photobiology and a method for detecting and characterizing thiol metabolites. Results: C. tepidum mutant strains were recovered that implicated previously unsuspected gene products in thiosulfate oxidation and clearly showed that two of three sulfide:quinone oxidoreductase (SQR) homologs display in vitro SQR activity and are required for C. tepidum to grow on high concentrations of sulfide. Furthermore, mutant strains and the wild type grown under different conditions displayed altered chlorosome properties and altered energy transfer between the chlorosome and reaction center. Analysis of thiol metabolites revealed that C. tepidum contains structurally novel thiols and that thiol pool sizes are significantly altered in mutant strains defective for sulfur oxidation and in the wild type grown under a variety of conditions. Conclusions: Taken together, the results suggest that C. tepidum is capable of a range of responses to altered environmental and physiological states. These responses presumably allow C. tepidum to coordinately regulate and balance fluxes of light energy and reducing power in order to grow under a wider variety of conditions than was appreciated when it was first isolated. Furthermore, it appears that even a small genome can encode metabolic surprises as evidenced by novel thiol metabolites that we hypothesize are integral to both regulatory and anaerobic sulfur oxidation pathways. 51
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
OC-4.2
OC-4.3
CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS.
STRUCTURAL AND FUNCTIONAL STUDIES OF A PHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGH COMPARATIVE METATRANSCRIPTOME ANALYSIS.
Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E. Blankenship1. Departments of Biology and Chemistry; 2Department of Energy, Environment and Chemical Engineering; Washington University, St. Louis, MO, USA.
Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt, Lynn Tomsho, Stephan C. Schuster, David M. Ward, Donald A. Bryant.
Introduction: Aerobic anoxygenic phototrophs (AAPs) are the only known organisms performing photosynthesis requiring oxygen but not producing oxygen. The AAPs are potentially major contributors to global carbon metabolism as they make up at least 10% of the microbial community in some euphotic upper ocean. Previous genetic analysis indicated the lack of the key genes required for the Calvin cycle, and physiological characterization suggested the requirement of organic carbon for Roseobacter denitrificans (R. denitrificans) growth. It is not clear how the R. denitirificans (and other AAPs) can assimilate carbon for the formation of the cellular components without autotrophic growth.
Introduction: The microbial mats in alkaline siliceous hot springs in Yellowstone National Park are model systems for microbial community studies, especially communities constructed by photosynthetic prokaryotes. Recently developed metatranscriptome analyses using high-throughput sequencing technology have revealed valuable information about the composition, in situ gene expression activities and gene regulation behavior of members of this microbial community.
1
Methods: We optimized the growth conditions of R. denitrificans with different defined carbon sources, and used metabolomic approaches, biochemical studies, and gene expression profiles to investigate the organic carbon metabolism, organic carbon and CO2 incorporation rate, and the carbon assimilation pathway(s). Results: We have investigated the R. denitrificans growth with various defined carbon sources in different growth conditions. The optimized growth conditions without non-specific carbon sources (such as yeast extract) are critical to minimize the background for the following metabolomic studies. The spectra of the LHII antenna complex and LHI-RC complex for the cultures with either pyruvate or glucose as the sole carbon source are very similar to those of the cultures grown in the rich growth medium. Metabolomic studies to probe the proposed carbon assimilation pathway in R. denitrificans with [1-13C]-labeled pyruvate, [1-13C]-labeled glucose, or 13C-labeled sodium bicarbonate were carried out. The N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide was used to derivatize amino acids for gas chromatography/mass spectrometry analysis. We have detected very low 13C-incorporation level of leucine and isoleucine and identified the CO2 assimilation to glutamate and proline. About 70% of alanine and valine are 13C-labeled. The incorporation rate of pyruvate, glucose, or CO2 was estimated with various biochemical approaches. Moreover, we used gene expression studies to identify the transcriptional level of genes encoding four anaplerotic carbon fixation enzymes in R. denitrificans under various growth conditions. In all of the conditions we tested, the transcript level is higher for malic enzyme (RD1_0421) and PEP carboxykinase (RD1_1376) than for pyruvate carboxylase (RD1_3376) and PEP carboxylase (RD1_4248), and different gene expression profiles for the anaplerotic carbon fixation enzymes are observed. Conclusions: We have successfully grown the R. denitrificans with either pyruvate or glucose as the sole carbon source, without the addition of yeast extract as previous literature reported. Our metabolomic studies confirm the biosynthesis of leucine and isoleucine, in which the citramalate pathway is favorable compare to derive from theronine for isoleucine biosynthesis, and incorporation of CO2 to oxaloacetate is suggested. The incorporation of pyruvate is more efficient than that of CO2. With the studies of the metabolomic (relatively low 13C-incorporation on several amino acids), biochemical and gene expression profiling, a working hypothesis on the carbon assimilation pathway in R. denitrificans is presented herein. 52
The Pennsylvania State University, University Park, PA, USA.
Methods: Samples of the photosynthetic microbial mat at Mushroom Spring in Yellowstone National Park at 61-64 °C were collected on 1011 July 2008 at four different time points in one 24-h period: at sunset (2100 h), prior to sunrise (0515 h), in dim light after sunrise (0640 h) and shortly after full illumination of the mat (0840 h). Total RNAs were extracted from these samples and were reverse transcribed into cDNAs. Reads generated from 454-pyrosequencing of the cDNAs were searched by blastN against reference genomes and databases. Taxonomic composition information was obtained by analysis of rRNA reads while analyses of mRNA reads revealed functional characteristics and regulatory patterns. Results: Pyrosequencing generated 409,567 reads, totaling 94,692,205 basepairs for the four samples. For each sample, from 90 to 95 percent of the reads were derived from rRNA, most of which could be assigned to different taxa. Ribosomal RNAs related to six chlorophototrophic microbial sources, Roseiflexus sp. RS-1, Synechococcus sp. JA-2-3B’a, Synechococcus sp. JA-3-3Ab, one or more Chlorobi species, Chloroacidobacterium thermophilum and a Chloroflexus species, accounted for nearly 90% of the total rRNA reads. The fact that the composition of this microbial community is so well defined, and that reference genomes of the major community members are available, allowed an in-depth survey of the 3.5 to 8 percent of reads corresponding to mRNA reads. Comparative analyses uncovered not only which genes of the major species are most actively transcribed, but also how they are regulated at light transition periods. This information sheds new light on the metabolism of the chlorophototrophic microbes in the community and their interactions. Insights on other components of the mat will also be discussed. Conclusions: This study vastly improves our understanding of a model photosynthetic microbial community because it provides a comprehensive view of the physiology of major mat phototrophic community members. It should also serve as a model for future studies of this and other microbial communities.
OC-5.1 HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOC PUNCTIFORME DESTINED TO BECOME HORMOGONIA. H. Christman, E.L. Campbell, J.C. Meeks. Section of Microbiology, University of California, Davis, CA, USA. Introduction: Nostoc punctiforme is a developmentally diverse filamentous cyanobacterium capable of differentiating three distinct cellular forms- heterocysts, hormogonia and akinetes. Different signals give rise to these different states, however there is some August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes overlap. Cultures growing with ammonium can be induced to make hormogonia or heterocysts upon removal of combined nitrogen. Methods: Our DNA microarray of 6,893 genes predicted from the 9.05 Mb annotated genome sequence has been used to generate a global transcription data set consisting of seven time points over a 24 hour period of nitrogen deprivation that results in heterocyst formation and a similar profile that results in hormogonia formation. Results: 1 hour after nitrogen deprivation leading to heterocyst differentiation, there is enhanced expression of nitrate utilization and urea transport genes, and the nitrogen response regulator nrrA, which is required for timely development of heterocysts. Within 3 hours, the positive acting heterocyst master regulator hetR shows enhanced expression. From 6 to 12 h, enhanced levels of heterocyst structural genes, such as polysaccharide and glycolipid synthesis genes, and functional genes, such as those encoding two additional sets of cytochrome oxidase specific to heterocysts were observed. At 18 to 24 h the nif genes encoding nitrogenase are transcribed. In the nitrogen starvation induced (NSI) hormogonia time course, the nitrate utilization genes are enhanced at 1 h and nrrA is up regulated from 3 to 18 hours. hetR is maintained at a repressed level throughout the time course. The urea transport and cytochrome oxidase genes are enhanced transiently at the 12 hour time point and there is no change in expression of the heterocyst structural genes. At 24 h, the nif genes show up to 8-fold enhanced expression. Conclusion: The heterocyst time course is consistent with previous data, demonstrating a sequence of events required to form a fully functional heterocyst. NSI hormogonia show an early nitrogen response, but lack enhanced expression of heterocyst regulatory genes that would be expected to elicit transcription of nif genes. This is in contrast to previous work that has shown nif gene transcription to be under heterocyst developmental control. These data indicate a link in the regulatory decision to become either hormogonia or heterocysts. In order to sort out this regulation, examination of expression profiles on heterocyst deficient mutants is underway.
OC-5.2 PLANT CELL WALL EPITOPES ARE EXPRESSED BY CYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS. Owen Jackson, J. Paul Knox, David G. Adams. University of Leeds, West Yorkshire, United Kingdom. Introduction: Gunnera is the only angiosperm to host a cyanobacterial symbiont. Uniquely for plant-cyanobacteria symbioses the cyanobiont, Nostoc punctiforme, is intracellular. The plant benefits from the nitrogen-fixing properties of the bacterium, and in return Nostoc receives physical protection, an uncompetitive ecological niche and nutrients. The Gunnera-Nostoc system has potential uses in developing new, beneficial symbioses between nitrogen-fixing cyanobacteria and crop plants. However, when compared to the highly characterised relationship between leguminous plants and bacteria of the genus Rhizobium, the NostocGunnera relationship is not well understood. We present results showing the presence of arabinogalactan-protein (AGP) and pectin epitopes to be associated with both partners in the symbiosis. AGPs and pectic polysaccharides are important plant cell wall components implicated in cell growth, cell development and cell interactions. Methods: We have used a variety of immunochemical techniques (including ELISAs and epifluorescence microscopy) with anti-AGP and anti-pectin monoclonal antibodies, along with β-glycosyl Yariv reagent (known to specifically interact with AGPs), to investigate the presence and localisation of these polymers. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Results: Use of anti-AGP antibodies indicated the presence of AGPs associated with cyanobacterial strains grown in liquid media. The same set of antibodies also indicated the presence of AGPs in the gland mucilage of uninfected Gunnera glands, and associated with the walls of plant cells surrounding symbiotic Nostoc colonies in infected Gunnera glands. Anti-pectin antibodies revealed the presence of pectins on the surfaces of symbiotic cyanobacterial colonies. Conclusions: Cyanobacterial strains are capable of producing and expressing AGPs on their surfaces independent of their plant symbiotic partner, and are associated with pectins when in a symbiotic relationship with Gunnera. Both AGPs and pectin are present at the symbiotic surface at different times during the symbiotic relationship, and hence we discuss their potential roles in the initiation and continuation of a stable symbiosis
OC-5.3 REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE IN HETEROCYST-FORMING CYANOBACTERIA. Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, Enrique Flores2, David G. Adams3. 1 School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty of Biological Sciences, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.
Introduction: When heterocyst-forming filamentous cyanobacteria such as Anabaena sp PCC7120 are growing diazotrophically, they must exchange metabolites between cells in the filament. Amino acids synthesised in the heterocysts must be transferred to the vegetative cells, and sugars synthesised in the vegetative cells must be transferred to the heterocysts. Recently we showed that intercellular molecular diffusion in Anabaena could be visualised by loading the cytoplasm with calcein, a fluorescent tracer dye. Fluorescence Recovery after Photobleaching (FRAP) was used to quantify rates of exchange of the dye among vegetative cells and heterocysts. We further showed that FraG, a protein with a DME permease domain located at the cell-cell interface, is essential for the diffusion of molecules from cell to cell (Mullineaux et al, 2008, EMBO J. 27, 12991308). FraG is probably a central component of pore structures (“microplasmodesmata”) that link the cells in the filament. Here we test the possibility that the activity of the microplasmodesmata is regulated according to the requirement for exchange of metabolites between cells. Methods: We used confocal FRAP to quantify rates of exchange of calcein at vegetative-vegetative and heterocyst-vegetative cell junctions, and GFP-tagging and confocal microscopy to quantify levels of FraG at cell junctions. Changes in both parameters were measured following nitrate step-down and heterocyst formation, and then after adding nitrate back to the cultures. Results: FraG is constitutively present at cell junctions, even when filaments are grown on nitrate. This is consistent with EM studies showing that the microplasmodesmata are always present at cell junctions. However, intercellular molecular exchange is slow in nitrategrown filaments. The rate of exchange between vegetative cells increases by more than a factor of 10 following nitrate step-down. When nitrate is added back to diazotrophically-growing cultures, molecular exchange becomes slower again. Conclusions: 1. Rates of intercellular diffusion are controlled according to the requirement for metabolite exchange. When nitrate is added 53
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Final Program and Abstracts back to differentiated, diazotrophic filaments, exchange becomes much slower. We suggest that the reduced supply of sugars to the heterocysts is a major factor leading to their subsequent death and disappearance. Under these conditions the vegetative cells may benefit by minimising the loss of sugars to the heterocysts. 2. The intercellular channel structures are constitutively present, even when molecular exchange is slow. Therefore there must be posttranslational mechanisms that regulate the opening and closing of the channels. We argue that the channels are constitutively present because they can only be formed during cytokinesis. Channels are synthesised in undifferentiated filaments so they are ready and waiting to be activated when the supply of combined nitrogen runs out.
but not of alr3442 and all0192, form heterocysts closer together than does the wild type strain, and that over-expression of patA resulted in closer heterocysts than in the wild-type strain. Conclusions: Yeast two-hybrid analysis provided evidence of proteins that interact with PatA. Six genes that consistently showed interaction with PatA were tested by mutation in Anabaena sp., looking for a possible alteration in pattern. Because the phenotype of an all3305 mutant has a phenotype like that of patA, there is a substantial likelihood that they interact in vivo. We are trying to test whether they interact in vitro. Over-expression of patA by use of PpetE elicited closer spacing of heterocysts.
OC-5.4
1. Liang J et al. PNAS 89: 5655 (1992); 2. Risser DD, Callahan SM. J Bacteriol 190: 7645 (2008); 3. Makarova KS et al. Bioinformatics 22: 1297 (2006); 4. Prentki P et al. Gene 103: 17 (1991).
SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS IN ANABAENA SP. STRAIN PCC 7120.
OC-5.5
Jinjie Liu, C. Peter Wolk.
THE INVOLVEMENT OF TWO SIGMA FACTORS IN CYANOBACTERIAL AKINETE FORMATION.
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA. Introduction: In wild type Anabaena sp., heterocysts form with an average interval of ca. 10 intervening vegetative cells. patA (all0521) affects the regulation of the pattern of heterocyst differentiation: heterocysts of a patA mutant form nearly exclusively at the ends of filaments (1). PatA has also been reported to control the level of HetR (2). We have sought to identify the protein(s) with which PatA interacts and what their roles may be in the process of pattern formation. Methods: We used PatA as bait to identify interacting proteins in a yeast two-hybrid (Y2H) library. The library was constructed by partially digesting Anabaena sp. DNA separately with seven 4-bp restriction endonucleases whose products match 3 different cloning sites in the prey vectors. Different size ranges of the digested genomic DNA were recovered separately and ligated into appropriately digested prey vectors. Products of ligation were electro-transformed into Stratagene competent cells of E. coli XL10 Gold, isolated, and re-transferred to Saccharomyces cerevisiae strain AH109. Bait vector was transferred to S. cerevisiae strain Y187. The two yeast strains were mated on plates of SD/-Ade-His-Leu-Trp with X-alpha-galactosidase. Blue colonies were selected, purified, their inserts amplified by colony PCR, and the PCR products of several hundred clones were sequenced and analyzed. Because PatA appears to be a two-component response regulator that lacks an intact DNA-binding domain, we focused our attention on 11 prey genes that presumptively encode kinases and on genes for which multiple, independent clones showed interaction with PatA. Prey genes that survived those tests were mutated in Anabaena sp. by double reciprocal recombination with insertion of the Omega interposon (4). Mutations in six of those genes have been fully segregated. Results: The 11 candidate prey clones, all of which when re-tested by Y2H showed interaction with PatA, were tested for interaction with the REC, the PATAN, or both domains of PatA. According to Y2H analysis, All3305 interacted with the PATAN, but not with the REC, domain. Double-recombinant mutants of the prey genes alr3304, all3305, all5210, all2699, alr3442 and all0192, respectively, have been fully segregated. The all3305 mutant showed a phenotype very similar to that of a patA mutant. Preliminary observations and statistical analyses with the Wilcoxon rank-sum test suggest that mutants of alr3304, all5210, and all2699, 54
Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers. Department of Biology, California State University, Northridge, CA, USA. Introduction: Akinetes are desiccation and cold-resistant spore-like resting cells formed from vegetative cells in certain heterocyst forming cyanobacteria. In Nostoc punctiforme, akinetes first appear midway between heterocysts, or randomly along a filament lacking heterocysts, following phosphate or potassium limitation and low light. A previous DNA microarray study comparing vegetative cells to akinetes differentiated in a zwf metabolic mutant indicated that two putative sigma factors (NpR4091 and NpF4153) were up-regulated during this process. Almost nothing is known about the genetic regulation involved in the morphogenesis of vegetative cells into akinetes. To elucidate if these sigma factors were involved in akinete differentiation, each was over-expressed to test the hypothesis that over-expression of the sigma factor would lead to increased akinete formation. Methods: Random amplification of cDNA ends (RACE) was used to map the transcriptional start sites for each gene. DNA fragments bearing promoter regions were cloned into the green fluorescent protein (GFP) vector pSUN119, electroporated into N. punctiforme and reporter strains visualzed by epifluorescence microscopy following induction of akinetes by phosphate starvation. PCR fragments containing the entire reading frame of both sigma factors were amplified from the N. punctiforme genome and cloned into the BamHI site of pRL490 in the correct orientation to drive constitutive expression from the strong tac promoter. The expression plasmids were electroporated into the wild-type strain and neomycin selection used to obtain over-expression strains. DNA microarray analysis of the over-expression strain expression relative to wild-type is in progress to determine the regulon of each sigma factor. Results: NpR4091 encodes for an alternate group 2 sigma factor similar to SigB2 in Anabaena sp. strain 7120. Two promoters were identified, and a GFP reporter containing both was more highly expressed in akinetes relative to neighboring vegetative cells. Electroporants overexpressing NpR4091 exhibited large numbers of akinetes in filaments growing on non-inducing plates containing A&A medium containing Mops and ammonium. Over-expression strains in A&A/4 Mops and ammonium liquid culture grew slowly relative to wild-type cultures. Microscopic examination revealed large numbers of abnormally large cells that had the characteristics of akinetes when growing in non-inducing conditions. NpF4153 encodes for an Extracytoplasmic Function (ECF) sigma factor similar to SigG in August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes Anabaena sp. strain 7120. Two promoters were identified; the upstream promoter conferred akinete-specific expression of a GFP reporter, and the downstream promoter did not, although it was required for the higher-level gene expression in akinetes when both were present. The over-expression strain exhibited large numbers of akinetes under non-inducing conditions, and slow growth in liquid culture, similar to that of NpR4091. Conclusions: Cell-type specific gene expression or transcriptional reporters and altered cell morphology of over-expression strains support the hypothesis that the sigma factors encoded by NpR4091 and NpF4153 are involved in akinete formation.
Final Program and Abstracts similar stem-loop structure exists in the nifU-nifH intergenic region. We hypothesize that the required sequences upstream the transcription start site of nifH act to prevent transcriptional termination of nifBSU or function in transcript processing. NtcA activates expression of various genes under conditions of nitrogen deprivation. We show that NtcA does not directly regulate nifHDK, however it is likely that NtcA regulates nifBSU because this promoter contains an NtcA-binding site. This supports the hypothesis that nifHDK expression results from nifBSU promoter activity. 1
Wilson, J., Pierrard, J., Hubner, P (1993) Gene, 133: 39-46
OC-6.1 OC-5.6 SEQUENCES REGULATING NITROGENASE GENE EXPRESSION IN THE CYANOBACTERIUM ANABAENA VARIABILIS.
THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES AT THE MONOMER-MONOMER INTERFACE OF PHOTOSYSTEM II IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Teresa Thiel, Justin L. Ungerer.
Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley.
Department of Biology, University of Missouri St. Louis, St. Louis, MO, USA.
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Introduction: The goal of this research is to identify and describe the function of key genetic elements that govern expression of the nitrogenase structural genes in heterocysts of cyanobacteria. Once we understand how specific sequences affect high-level, heterocystspecific expression of nitrogenase, we can use these regulatory elements to provide heterocyst specific expression of other oxygensensitive genes such as hydrogenases or alternative nitrogenases. The nifHDK regulatory elements would also be useful for expressing genes for bio-diesel production in heterocysts; an environment rich in lipids that may serve as the precursors to bio-diesel.
Introduction: X-ray diffraction data of Photosystem II (PS II) crystals from Thermosynechococcus elongatus have revealed a dimeric structure of PS II at 2.9 – 3.5 Å resolution [Ferreira et al. (2004) Science 303: 1831-1838; Guskov et al. (2009) Nat. Struct. Mol. Biol. 16: 334342]. Three low-molecule-weight polypeptides, PsbL, PsbM and PsbT have been identified at the monomer-monomer interface. We have used the model organism Synechocystis sp. PCC 6803 to investigate the role of these polypeptides in PS II dimer formation, assembly, turnover and electron transport.
Methods: Transcriptional lacZ fusions of various sized nifH1 promoter fragments, either wild type or containing specific mutations, were used to identify important nifH1 promoter sequences. Activity of the promoter fragments was determined using a standard β-galactosidase assay. A fluorescent substrate for β-galactosidase was used to visualize localization of expression of the reporter. The results from the lacZ expression study were confirmed by reconstructing the most interesting mutations or deletions directly in the upstream nifHDK region on the chromosome. Results: We have determined that the first 100 bp upstream of the nifH transcription start site are necessary but insufficient to provide normal regulated expression of nifH. Deletions in this region severely limit expression of nifHDK, whereas upstream sequences up to the start codon of nifB can be deleted from the chromosome without affecting expression. Conversely, the same required region and/or upstream sequences are not capable of driving high-level expression of nifH or the lacZ reporter in trans, though the limited expression that is detected is localized to heterocysts. This nifH1 upstream region contains a non-canonical NtcA binding site, however mutations of the NtcA binding site do not affect expression of nifHDK, therefore NtcA does not directly regulate nifHDK. Conclusions: The 100 bp upstream of the nifH transcription start site are sufficient to determine heterocyst specific expression of the nifHDK transcript; however, it is insufficient to provide high-level expression. Our data suggest that transcriptional activation of nifH may be under control of the nifB promoter. No region between the nifH start codon and the nifB promoter can drive high-level expression of lacZ; therefore, expression must result from the nifB promoter. It is possible that the known nifH1 transcription start site is a result of processing of a larger nifBSUHDK transcript, as there is evidence of nif transcript processing at stem-loop structures in other species 1. A August 9 to 14, 2009 • Montréal, QC, Canada
Methods: Mutagenesis, biochemical characterization and physiological measurements were carried out as described in [Hao et al. (2008) Photosynth. Res. 98: 337-347]. Chlorophyll a fluorescence induction and decay kinetics were measured with a fluorometer supplied by PSI Instruments, Brno, Czech Republic. Polysomes from thylakoid and cytosolic fractions were isolated according to [Tyystjärvi et al. (2001) Mol. Microbiol. 40: 476-484]. Results: Mutations in the cytosolic N-terminal extension of PsbL destabilized PS II dimers, although the monomeric complexes retained their ability to split water. Mutations in this region of PsbL also activated diuron-insensitive cyclic electron transfer around PS II. Additionally, truncation of 4 residues at the C-terminus was sufficient to prevent association of PsbL with a subcomplex of the photosystem thereby blocking assembly of active PS II complexes and creating an obligate photoheterotrophic strain. The absence of PsbM decreased the number of assembled PS II centers by one third and the remaining centers exhibited an increased susceptibility to high-light-induced photodamage suggesting turnover and assembly were slowed when compared with PS II centers in wild type. Similarly, absence of PsbT resulted in a strain that was highly susceptible to high-light-induced stress. ∆PsbT cells were able to recover from photodamage when transferred from high- to low-light conditions. Recovery was observed to be dependent on protein synthesis and light, and was prevented in the absence of Psb27. Moreover, evidence for a specific role for PsbT in PS II turnover following photodamage was found since accumulation of psbA transcripts with thylakoid-associated polysomes was observed in wild type but not seen in ∆PsbT cells. Conversely, following exposure to high light, elevated levels of psbA transcripts with polysomes isolated from the cytosolic fraction were only observed in ∆PsbT cells. Electron transfer between the PS II primary and secondary plastoquinone acceptors, QA and QB, was also impaired in the ∆PsbT strain. Unexpectedly, loss of oxygen-evolving activity under high light was reversed by addition of bicarbonate in 55
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Final Program and Abstracts ∆PsbT cells and oxygen evolution was abolished by addition of formate under ambient CO2 levels in the presence of the artificial electron acceptors 2,5-dimethyl-p-benzoquinone (DMBQ) and K3Fe(CN)6. Our results suggest that DMBQ may bind and inhibit at the QA binding site in ∆PsbT cells and in some PsbL mutants. Conclusions: C-terminal mutations of PsbL prevent association with PS II blocking assembly at the CP43-less inactive monomer. N-terminal mutations within PsbL alter the acceptor side and stability of PS II, as does the absence of PsbM and PsbT. The absence of PsbT may also destabilize the association of polysomes with thylakoid membranes following high light stress and therefore PsbT could play a key role in efficient repair of PS II following photodamage.
OC-6.2 PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO IN SYNECHOCYSTIS. Danny Yao, Dan Brune, Wim Vermaas. School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. Introduction: The lifetime of at least one of the photosystem II protein components, PsbA (D1), is short (less than an hour at high light intensity) but little information is available on the lifetimes of most other photosystem II subunits. As the subunits all form a complex in the membrane, different lifetimes of components raise interesting questions regarding mechanisms of repair. The lifetime of chlorophylls (determined by in vivo labeling with 13C or 15N) in Synechocystis is more than a week, suggesting that chlorophylls are recycled efficiently if photosystem II breaks down. Small Cab-like proteins (SCPs) have been shown to play a role in the stability of the chlorophyll, with the chlorophyll lifetime decreasing in the absence of SCPs. The questions addressed in this research are what the range of lifetimes of photosystem II proteins is, and whether SCPs also affect the stability of proteins in the photosystems. Methods: Stable isotope labeling and mass spectrometry have proven to be a powerful combination to determine lifetimes. Using 15 N-ammonium nitrate labeling that is added to growing cultures at a specific time, both chlorophyll and protein are labeled, and the disappearance of unlabeled (old) compounds can be followed over time after isolation of photosystem II that carries a His-tagged CP47. For these experiments we used photosystem I-less strains that were grown at a light intensity of 4 mol photons m-2 s-1. Results: The lifetimes of specific photosystem II proteins (PsbA (D1), PsbB (CP47), PsbC (CP43), PsbD (D2), PsbE and PsbF (cytochrome b559), PsbH, PsbO and Psb27) in assembled photosystem II complexes were followed in living Synechocystis cells under standard growth conditions by monitoring the amount of unlabeled protein that could be extracted from membranes using the His tag on CP47. The halflifetime of D1 was short as expected (about 2 hours at low light intensity), but particularly PsbE and PsbO first showed a ~25% increase in unlabeled protein integrated into photosystem II complexes in the first 9 hours after the start of labeling before unlabeled protein amounts decreased and labeled protein became more prevalent. This suggests that a significant amount of PsbE (and PsbO) is present in cells that is not incorporated into photosystem II complexes, and that may serve as an anchor for photosystem II assembly. The role of SCPs in photosystem protein stability will be presented as well.
and –in contrast to radioactive labeling- can detect both “old” (unlabeled) and newly synthesized protein. The lifetime of polypeptides in photosystem II complexes varies greatly, implying that the polypeptides are replaced independent of each other. Also, the presence of a pool of photosystem II proteins that are not yet incorporated into a mature photosystem II complex but that may form the nucleus of a new photosystem II complex is demonstrated.
OC-6.3 IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FOR THE OXYGEN DEPENDANCE OF THE PHOTOSYNTHETIC ACTIVITY OF ROSEOBACTER DENITRIFICANS. Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and Andreas Labahn1. 1 Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany; 2Department of Cell Biology, Harvard Medical School, Boston MA, USA.
Introduction: Roseobacter denitrificans belongs to the group of obligate aerobic photosynthetic bacteria. The typical bacterial photosynthesis is an anaerobic process where the formation of the photosynthetic apparatus is down-regulated in the presence of oxygen. Roseobacter produces bacteriochlorophyll a as well but is incapable of performing photosynthetic energy transduction without oxygen. Only under aerobic conditions, the protein components of the photosynthetic apparatus are synthesized and photoinduced electron transport is operative. Methods: Core complexes, consisting of reaction center and light harvesting complex 1 (RC-LH1) were solubilized from the photosynthetic membranes of Roseobacter denitrificans with decylmaltoside and purified by anion exchange chromatography and sucrose density gradient centrifugation. Single particle electron microscopy was used to perform a first structural characterization of the core complex . 30,000 particles were selected from micrographs of negatively stained samples, aligned to each other and classified to produce class averages. Results: The class averages showed different views of the core complex which could thus be combined to calculate a threedimensional reconstruction. The density map at 30 Å resolution shows that the LH1 complex surrounding the RC forms a closed circle. Although the resolution of the reconstruction is not yet sufficient to resolve possible gaps in the LH1 ring, this tight ring around the reaction center may prevent the exchange of dihydroquinone and thus light-induced cyclic electron transfer. Conclusion: With the RC-LH1 complex from R. denitrificans we have isolated and structurally characterized a core complex from an obligate aerobic photosynthetic bacterium for the first time. A tight LH1 ring surrounding the reaction center was found which may inhibit the exchange of dihydroubiquinone with the quinone pool. Thus, we conclude that light-driven energy production is insufficient for bacterial growth and that oxygen is not only required for enabling photosynthetic electron transport but mainly for alternative metabolic pathways. We will now 2D-crystallize the core complex for cryo-electron microscopy to obtain improved resolution and a detailed reconstruction of its 3D structure.
Conclusion: Stable-isotope labeling in combination with mass spectrometry is a powerful method for determining protein lifetimes,
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Final Program and Abstracts
OC-6.4
OC-6.5
VARIABLE FLUORESCENCE IN HELIOBACTERIUM MODESTICALDUM CELLS: OBSERVATION AND EXPLANATION.
NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM II PROTECTS THE CYANOBACTERIUM MICROCOLEUS SP. AGAINST EXCESS LIGHT STRESS.
Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, Stefano Santabarbara1,2, Robert Blankenship3. 1
Arizona State University, Dept. of Chemistry and Biochemistry, Tempe, AZ, USA; 2Institut de Biologie Physico-Chimique, Paris, France; 3 Washington University, Departments of Biology and Chemistry, St Louis, MO, USA. Introduction: Heliobacteria have the simplest photosynthetic apparatus of all known phototrophs. They possess a single type I homodimeric reaction centre (RC), which harvests light using a pool of 22-34 bacteriochloropyll g (Bch g) as part of each RC’s intrinsic antenna. There is no extrinsic antennae. The immediate electron donor is a membrane-attached cytochrome c, which is re-reduced by a diheme-containing cytochrome bc complex, and the immediate electron acceptor is a di-cluster ferredoxin. At this point, it is not clear how the cycle is closed to allow light-driven proton pumping. Also unclear is the role of the pool quinone (menaquinone-9) – each RC contains 2 of these molecules, but there is conflicting evidence as to their role in electron transfer. Although heliobacteria are photoheterotrophs and do not fix CO2, all species examined so far fix N2, and nitrogenase is expected to be a large consumer of lowpotential electrons under N2-fixing conditions. Methods: We have examined in vivo kinetics of electron transfer within living cells of Heliobacterium modesticaldum using a combination of transient absorption and fluorescence spectroscopy. Results: We found that H. modesticaldum cells exhibit the phenomenon of variable fluorescence, although the characteristics are quite different from those seen in oxygenic phototrophs. It usually took ~100 ms of illumination with actinic light before fluorescence began to rise. Variable fluorescence could be abolished by blocking re-reduction of P800 + (oxidized primary electron donor), either by addition of ferricyanide (oxidant) or stigmatellin (Qo inhibitor). Contrariwise, reduction of intracellular electron acceptors by addition of dithionite and PMS (as mediator) increased variable fluorescence, as did inhibition of nitrogenase by addition of ammonia. Under conditions that led to high variable fluorescence, it was observed that the yield of photo-oxidizable P800 (in the µs timescale) was lower. We can explain all of these data by a model in which saturation of the electron acceptor pool leads to reduction of the FX Fe4S4 cluster in the RC, provoking a back-reaction from the P800 + A0 state, which produces delayed fluorescence. We tested this model by using pumpprobe spectroscopy in the ns timescale and found that a ~20-ns back-reaction from the P800 + A0 state was indeed observed under conditions that produce high variable fluorescence. Moreover, we can quantitatively account for almost all of the variable fluorescence as being due to back-reaction. The effects of QB-type inhibitors upon variable fluorescence provide evidence in favor of the participation of menaquinone in forward electron transfer within the RC. Conclusions: These results demonstrate a new phenomenon in heliobacterial physiology – variable fluorescence – and explain it as a consequence of delayed fluorescence stemming from a fast backreaction within the heliobacterial RC. This demonstrates that the intracellular electron acceptor pool can easily become saturated under strong illumination conditions, and indicates the importance of electron donor/acceptor dynamics. These facts likely explain why this organism does not require a large antenna (as it is not required) August 9 to 14, 2009 • Montréal, QC, Canada
Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2. Departments of 1Biological Chemistry, 2Plant and Environmental Sciences and 3The Interuniversity Marine Institute, Eilat, The Hebrew University of Jerusalem, Israel. Photosynthetic organisms are exposed to light-induced oxidative stress due to photoinactivation of the oxygen evolving photosystem II (PSII). We investigated the persistence of PSII activity of the desiccation-tolerant Microcoleus vaginatus, a cyanobacterium inhabiting biological desert crusts where it is exposed to high light intensities. Surprisingly, CO2-dependent oxygen evolution persisted at light intensities 2-3 times higher than saturation. In contrast, light intensities close to or higher than required to saturate oxygen evolution triggered extensive loss (85-90%) of radiative PSII charge recombination measured as variable fluorescence or thermoluminescence (TL) emission. Radiative charge recombination, which involves the generation of singlet oxygen, recovered slowly following exposure to low light intensity but did not recover in darkness. Light induced loss of fluorescence and its recovery are not inhibited by herbicides that bind to the PSII-QB site, indicating that reduction of plastoquinone or O2 is not involved. The temperature required for maximum TL emissions resulting from QA-/S2 charge recombination (Q band, 22oC) indicated a significant upshift of the PSII-QA redox potential as compared to “model” photosynthetic organisms (Q band, 10-15 oC). This property of Microcoleus may reduce harmful radiative charge recombination as a function of light intensity, thereby lowering the generation of 1O2 and related oxidative stress. On the basis of our findings we present a novel model whereby a non-radiative charge recombination within PSII lowers photoinactivation damage under excess illumination and a possible reason why this effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacterium to the higher plant chloroplast.
OC-6.6 THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-Mari Aro. Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland. Introduction: The prokaryotic photoautotrophic cyanobacteria (bluegreen algae) are favored model organisms to investigate photosynthetic mechanisms. Stress conditions like high light or limitation in inorganic carbon often lead to restriction in the availability of the terminal electron acceptors of the photosynthetic electron transfer chain. To ensure the delicate balance of energy input and consumption, thus preventing the generation of dangerous reactive oxygen species, photosynthetic organisms developed different strategies. It is suggested that flavodiiron proteins (Flv) are involved in those adaptation strategies. Though quiet well studied in (facultative) anaerobic Bacteria and Archaea, knowledge on Flvs in cyanobacteria is very poor. However, the modular character (a β-lactamase-like domain containing a diiron center, a flavodoxin domain with FMN binding site and an additional C-terminal flavin reductase domain in 57
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Final Program and Abstracts cyanobacterial Flvs) of those proteins strongly suggests their participation in electron transfer processes [1]. Methods: Detailed material and methods are described in [2]. Results: The genome of the cyanobacterial model strain Synechocystis sp. PCC6803 comprises four genes, encoding the hypothetical flavodiiron proteins Flv1, Flv2, Flv3 and Flv4. While Flv1 and Flv3 were shown to function in the Mehler reaction as NAD(P)H:oxygen oxidoreductases [3], the physiological function of Flv2 and Flv4 remains open. RT-PCR and Western Blot analyses show that the expression of flv2 and flv4 is high under LC (air level of CO2) and negligible at HC (3% CO2). Moreover, the rate of accumulation of flv2 and flv4 transcripts upon shift of cells from HC to LC is strongly dependent on light intensity. Characterization of Flv-mutants in Synechocystis revealed a specific decline in PSII centers and impaired translation of the D1 protein in the mutants ∆flv2 and ∆flv4 when grown at LC, whereas at HC the Flvs were dispensable. ∆flv2 and ∆flv4 were also more susceptible to high light induced inhibition of PSII than WT or ∆flv1 and ∆flv3. In fact, ∆flv1 and ∆flv3 seemed to have more functional PSII centers, and in both mutants the photoinhibition of PSII was slightly less severe than in the WT. Analysis of published amino acid sequences revealed that cyanobacterial genomes always contain two up to six genes encoding distinct Flvs within the same organism. Furthermore we could find cyanobacteria-like Flvs also in some oxygenic photosynthetic eukaryotes like green algae, mosses and lycophytes. The occurrence of Flvs in pairs seems to be a special feature for oxygenic photosynthetic organisms. Conclusions: We demonstrate a novel and crucial function of flavodiiron proteins, particularly Flv2 and Flv4, for oxygenic photosynthesis in Synechocystis cells at LC. There seems to be an evolutionary trend in the diversity and function of the flavodiiron proteins: In anaerobic microbes the activity of Flvs is directed against O2/NO toxicity. In oxygenic photosynthetic organisms Flv1 and Flv3 reduce oxygen to water while Flv2 and Flv4 protect the oxygen evolving PSII complex against photoinhibition. Higher plants lack Flvs and distinctly different mechanisms have evolved for photoprotection of PSII. [1] Vicente JB, Justino MC, Gonçalves VL, Saraiva LM, Teixeira M (2008) Methods Enzymol 437: 21-45 [2] Zhang P, Allahverdiyeva Y, Eisenhut M, Aro EM (2009) PLoS ONE [3] Helman Y, Tchernov D, Reinhold L, Shibata M, Ogawa T et al. (2003) Curr Biol 13: 230-235
OC-7.1 PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA (PRRA), TRANSCRIPTIONAL REGULATORS OF THE CBB OPERONS (CO2 FIXATION) IN RHODOBACTER SPHAEROIDES.
and RegA are DNA binding proteins with helix-turn-helix motifs for DNA interaction. DNA binding sites for both proteins are found in the regulatory regions of the cbbI and cbbII operons in Rhodobacter sphaeroides. RegA has four DNA binding sites in the cbbI promoter. The CbbR binding site and RegA binding site 1 overlap each other upstream of the cbbI operon in R. sphaeroides, as demonstrated by footprint analysis. Results: CbbR and RegA interact and CbbR must be bound to DNA for this protein-protein interaction to occur. Conversely, cbbI promoter binding by RegA is not required for interaction with CbbR. The presence of both CbbR and RegA in cbbI promoter/protein complexes was confirmed. In addition, the presence of RegA enhances the ability of CbbR to bind the cbbI promoter. Gel mobility shift analysis demonstrates that RegA binds to cbbI promoter DNA and forms incrementally larger multimeric complexes with DNA as the concentration of RegA increases, referred to as oligomerization. When RegA binding sites 1, 2 and 3 are present in the cbbI promoter, RegA forms a complex with the DNA at a significantly lower concentration and a greater mobility compared to complex formation with RegA binding sites 1/2, 3 or 3/4, separately. Specific site-directed mutants of RegA have been generated that cannot bind CbbR and also have reduced oligomerization function. These mutations of the RegA protein are located in the N-terminus receiver domain as well as in the DNA binding domain. Methods: Methods include gel mobility shift experiments using 32Plabeled DNA probes and chemical cross-linking analysis using dimethylpimelimidate. Isolation of the protein/DNA complexes from native acrylamide gels and immunoblot analysis using antibodies specific to CbbR and RegA was employed to determine the composition of promoter/protein complexes. Conclusions: There is strong interaction between RegA and CbbR, and the presence of RegA increases the quantity of CbbR that binds the cbbI promoter. Possibly, RegA lowers the activation energy required for CbbR to bind DNA, thereby increasing the DNA binding affinity of CbbR. CbbR must be bound to DNA to interact with RegA, possibly ensuring that RegA only binds transcriptional regulators such as CbbR at the appropriate promoter site. RegA sites 1, 2 and 3 are necessary for optimal binding of RegA to the cbbI promoter. There is communication or cooperation between RegA binding site 1/2 and site 3 that allows RegA/DNA complex formation at a lower concentration of RegA. An intermediate loop structure could be formed to facilitate the communication between RegA site 1/2 and site 3. In addition, several specific regions of the RegA protein are important for interaction with CbbR.
OC-7.2 PpsR AS A NEW TYPE OF HEMIN SENSOR. Liang Yin, Vladimira Dragnea, Carl Bauer.
Andrew W. Dangel, F. Robert Tabita.
Indiana University, Bloomington, IN, USA.
Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, OH, USA.
My research focuses on PpsR-AppA system. To date, this regulatory system is the only cascade identified that coordinates light- and redox- signaling in transcription control. PpsR was identified as a DNA-binding transcription factor in R. sphaeroides. Under aerobic conditions, PpsR blocks the transcription of the tetrapyrrole biosynthetic and the photosynthetic genes, which are responsible for biosynthesis of the pigments and the light harvesting II complex (1). AppA, an antirepressor of PpsR, is able to inactivate PpsR by forming a PpsR2-AppA complex under anaerobic and dark conditions (1). In the past year, our results indicate that hemin provides another level of control in this system, showing the direct link between free hemin pool
Introduction: CbbR and RegA (PrrA) are transcriptional regulators of the cbbI and cbbII (Calvin-Benson-Bassham CO2 fixation pathway) operons in Rhodobacter sphaeroides. CbbR is a LysR type transcriptional regulator (LTTR) which is the most commonly utilized protein family for gene regulation in prokaryotes. RegA is part of a two-component signal transduction system also involving the membrane-bound histidine kinase, RegB (PrrB). RegA is phosphorylated by RegB to activate its regulatory function. Both CbbR 58
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13th International Symposium on Phototrophic Prokaryotes and photosynthesis for the first time. We demonstrated that PpsR is a hemin-binding protein with stoichoimetry of 1:1 ([PpsR]/[hemin]). The presence of hemin inhibited the DNA-binding ability of PpsR significantly, while other tetrapyrrole products did not show any inhibition. Experiments with truncated PpsR suggested that it bound to hemin via its C-terminal region, possibly through the Cys located in its DNA-binding domain. In summary, our results suggest that PpsR is likely to be a hemin sensor. Most of well characterized hemin-binding proteins have tightlycoordinated hemin to perform many different tasks, such as electron transfer, oxygen storage and gas molecule sensing (2). Only in recent years people started to appreciate the hemin sensors, which are able to sense the free hemin in vivo, from bacteria (3) to mammal (4). Yet all the identified hemin sensors to date contain the conserved -Cys-Promotif, which PpsR does not have. Our research will provide us new insights about hemin sensors, which seem to be more diverse than we used to think. 1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623 2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L., Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073 3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A 96, 13056-13061 4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc Natl Acad Sci U S A 105, 76-81
OC-7.3 ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN: AN EXPERIMENTAL SCENARIO FOR A STRICT ANAEROBIC PHOTOTROPH. Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg, Françoise Reiss-Husson, Chantal Astier, Soufian Ouchane. CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; Université Paris-Sud, Orsay; Université Pierre et Marie Curie, Paris; France. Introduction: The appearance of oxygen in Earth’s atmosphere as a result of oxygenic photosynthesis required strict anaerobes and obligate-phototrophs to cope with the presence of this potentially toxic molecule. Nitrogen-fixing organisms, for example, have evolved a number of strategies to protect nitrogenase from environmental oxygen. Among these strategies, oxygen depletion by specific oxidases was shown to play an important role. We have previously detailed the AcsF/BchE enzyme replacement and suggested a similar HemF to HemN replacement during the shift from aerobic to anaerobic environment to emphasize the O2 dependent and independent metabolic networks. Here, we further investigate the problem of O2 and photosynthesis (PS) in the anoxygenic phototroph bacterium Rubrivivax (R) gelatinosus and show how the terminal oxidases (cbb3, bd and caa3) expand the physiological range of O2 environments under which this bacterium and probably many other “aerobic” phototrophs can carry out photosynthesis. Methods: PCR-based cloning allowed the identification of four oxidase encoding operons in R. gelatinosus. Mutations were generated to disrupt each operon to study the respiratory chain of this bacterium. Mutants were further analyzed to check how photosynthesis and respiration or O2 detoxification are functionally related in this microorganism. Results: We have shown that: August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts 1. Only two terminal oxidases are functional in wild type R. gelatinosus: the cbb3 cytochrome c oxidase, and the bd type quinol oxidase. 2. Unlike the wild type and the single mutants, the cbb3--bd- deficient double mutant can carry out photosynthesis only under strict anaerobicity. 3. In oxygenated environments, this mutant survives nonphotosynthetically until the O2 tension drops. Co-inoculation with aerobes (E. coli) and PS deficient strains restores PS growth of this mutant even in oxygenated medium. 4. Spontaneous suppressor mutations especially in caa3 cytochrome c oxidase that restore respiration simultaneously restore photosynthesis in oxygenated environment. Conclusions: The cbb3, bd and caa3 oxidases are therefore not only required for respiratory growth but also play an essential role in the reduction of the environmental O2 pressure during anaerobic photosynthesis. These results demonstrate that photosynthetic and other anaerobic metabolisms in environments which are (at least transitorily) exposed to oxygen are critically dependent on the O2detoxifying action of terminal oxidases.
OC-8.1 CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BY FERREDOXIN:THIOREDOXIN REDUCTASE, FERREDOXIN AND THIOREDOXIN M. David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, Masakazu Hirasawa, Marcellus Ubbink. Texas Tech University, Lubbock, TX, USA. Introduction: Ferredoxin:thioredoxin reductase (FTR), catalyzes the two-electron reduction of thioredoxins in chloroplasts and in cyanobacteria (thioredoxins f and m and perhaps other thioredoxins are substrates), using reduced ferredoxin as the electron donor. Reduced thioredoxins then play important roles in redox regulation. FTR, a heterodimer with a unique [4Fe-4S] cluster as its sole prosthetic group, has a single binding site for ferredoxin and a separate single binding site for thioredoxin. The binding sites for FTR from the cyanobacterium Synechocystis sp. PCC 6803 on ferredoxin and on thioredoxin in the 1:1 complexes of the enzyme with its two protein substrates have been mapped and the ternary complex between all three proteins has been characterized. Methods: Perturbations of the two-dimensional NMR spectra of both ferredoxin and thioredoxin arising from complex formation with FTR have been used to identify specific amino acids on the two proteins involved in complex formation with FTR. Results: A mono-gallium analog of the [2Fe-2S] Synechocystis sp. PCC 6803 ferredoxin was obtained by reconstituting apo-ferredoxin in a gallium-containing refolding buffer. The use of this diamagnetic Ga structural analog eliminates the paramagnetic broadening of NMR resonances of amino acids in the vicinity of the [2Fe-2S] cluster in native ferredoxin. This has allowed the first complete mapping of the interaction interface of a [2Fe-2S] ferredoxin for a target enzyme. NMR has also been used to contain a three-dimensional structure of the Gaferredoxin in solution, confirming that its structure is almost identical to that of the native, iron-containing protein. NMR spectroscopy was also used to map the interaction domain for FTR on thioredoxin m in a 1:1 complex of the two proteins. Both similarities and differences are seen in the thioredoxin m interaction domain for FTR in the noncovalent complex examined by NMR and in a disulfide-linked covalent complex of FTR and thioredoxin m for which an X-ray crystal structure 59
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Final Program and Abstracts has been obtained. NMR has also been used to characterize a ternary complex between ferredoxin, FTR and thioredoxin m in solution. Conclusions: The interaction domains on both ferredoxin and thioredoxin for FTR appear to consist largely of hydrophobic amino acids. The fact that FTR can form a ternary complex with ferredoxin and thioredoxin simultaneously confirms the presence of separate binding sites on FTR for its two substrates. Furthermore, Gasubstituted ferredoxin has been established as a valuable, non-paramagnetic analog for ferredoxin for NMR investigations of the interaction of ferredoxin with its target enzymes.
OC-8.2 CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA. Shinichi Takaichi1, Mari Mochimaru2. 1
Department of Biology, Nippon Medical School, Kawasaki; Department of Natural Sciences, Komazawa University, Setagaya, Tokyo; Japan. 2
Introduction: Cyanobacteria grow by photosynthesis, and essentially contain chlorophyll and various carotenoids, whose main functions are light-harvesting and photoprotection. We have summarized carotenoids, characteristics of carotenogenesis enzymes and genes, and carotenogenesis pathways in some cyanobacteria, whose both carotenoids and genome DNA sequences have been determined (Takaichi & Mochimaru (2007) Cell. Mol. Life Sci. 64: 2607-2619). Methods: Carotenoids were extracted from cyanobacterial cells and purified. They were identified based on absorption spectra, retention times on C18-HPLC, MS, 1H-NMR and CD analyses (Iwai et al. (2008) Plant Cell Physiol. 49: 1678-1687). Knockout mutants of carotenogenesis genes were constructed for characterization of enzymes (Mochimaru et al. (2008) J. Bacteriol. 190: 6726-6733).
Results & Discussion: Cyanobacteria contain various carotenoids; βcarotene, its hydroxyl derivatives, such as zeaxanthin and nostoxanthin, or keto derivatives, such as echinenone and canthaxanthin, and carotenoid glycosides, such as myxol glycosides and oscillol diglycoside. Both ketocarotenoids, such as echinenone and 4-ketomyxol, and the carotenoid glycosides are the unique carotenoids among phototrophic organisms. Some cyanobacteria, e.g. Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, contain both unique carotenoids, while others, e.g. Synechococcus elongatus PCC 7942 and Prochlorococcus marinua, do not contain such carotenoids. From these findings and characteristics of carotenogenesis enzymes, certain carotenogenesis pathways can be proposed. The different compositions of carotenoids might be due to the presence or absence of certain gene(s), or to different enzyme characteristics. For instance, two distinct β-carotene hydroxylases, CrtR and CrtG, are bifunctional enzymes; CrtR catalyzes β-carotene to zeaxanthin and deoxymyxol to myxol, CrtG catalyzes zeaxanthin to nostoxanthin and myxol to 2-hydroxymyxol, and substrate specificities of CrtR vary across species. Two distinct b-carotene ketolases, CrtO and CrtW, are found only in the first group, and properly used in two pathways, β-carotene to echinenone and myxol to 4-ketomyxol, depending on the species. Lycopene cyclases of CrtL from Synechococcus elongatus PCC 7942, CruA and CruP from Synechococcus sp. PCC 7002, and CrtL-b and CrtL-e from Prochlorococcus marinus have been functionally confirmed, while those in other species are not found yet. In Synechococcus sp. PCC 7002, 1’,2’-hydratase (CruF) for myxol synthesis is functionally confirmed, and novel unique carotenoid of synechoxanthin is found. At present, the number of functionally confirmed genes is limited, and
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only a few species are examined. Therefore, further studies of carotenoids, characteristics of carotenogenesis enzymes and genes, and carotenogenesis pathways are needed.
OC-8.3 EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROM PURPLE BACTERIA AND CYANOBACTERIA. Gerhard Sandmann. Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Frankfurt, Germany. Introduction: Carotenoids are essential pigments for photosynthetic organisms. Their predominant function is protection of photosynthesis from photooxidation. This antioxidative property depends on the conjugatd double bond system of the carotenoid molecule which is formed in a series of desaturation steps. During evolution of carotenogenesis, two basically different desaturation pathways were acquired. The “ancient” pathway exists in archeae, bacteria and fungi involving a single CrtI type enzyme which can catalyze up to six desaturations and isomerizes cis to trans double bonds. Among the purple bacteria, CrtI desaturases exist either as 4-step desaturases starting the spirilloxanthin pathway as in Rhodospirillum rubrum or as 3-step desaturases as in Rhodobacter species synthesizing spheroidene derivatives. In contrast, double bonds are formed by structurally and mechanistically unrelated CrtP type desaturases in cyanobacteria, Chlorobaculum species and photosynthetic eukaryotes. The most likely evolutionary root of CrtP is a CrtU desaturase which is responsible for aromatization of b-ionone rings in several groups of bacteria including Chlorobiaceae. Methods: Carotenogenic genes were cloned from different species. They were heterologously expressed and the resulting enzymes used to carry out enzyme kinetic studies including determinations of substrate and product specificities Results: Within cyanobacteria, one finds among the existing species a relic of the CrtI desaturation pathway, Gloeobacter violaceous, which is the only known cyanobacterium with a CrtI-type desaturase. In contrast to the CrtI-catalyzed desaturations, the desaturation products of CrtP-related reactions are poly-cis carotenes. Consequently, the resulting tetra-cis lycopene has to be isomerized to all-trans by an additional isomerase CrtH which developed from CrtI by loosing its entire desaturation function but retaining its isomerization function. There is an enzyme resembling an evolutionary link between CrtI and CrtH in the cyanobacterium Nostoc PCC7120. It resembles an additional CrtI-related enzyme named CrtQa which is a z-carotene desaturase producing all-trans products. Its participation in the polycis desaturation pathway of Nostoc PCC7120 could be specified. The evolutionary modification of the product specificity of the CrtI desaturases was studied in Rubrivivax gelatinosous in which CrtI resembles a transitional state yielding 3-fold and 4-fold desaturated products, simultaneously. The in vitro and in vivo results were compared to a 3-step desaturase from Rhodobacter. We could find that the kinetic property of this phytoene desaturase is one factor to determine the type of reaction product. However, structural properties can prevent that the last desaturation step is catalyzed. The reaction stops at the level of neurosporene which is no longer a substrate for the desaturase. Conclusion: Among the prokaryotes, two basically different carotenoid desaturation pathways were acquired. Our enzyme kinetic investigations were focussed on two desaturases, a missing link between both pathways and one desaturase comprising all
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13th International Symposium on Phototrophic Prokaryotes characteristics of a 3- and of a 4-step desaturase. The results will help to understand the evolutionary origin and the divergence of carotene desaturation.
OC-9.1 THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM – A STRUCTURAL MODEL BASED ON SOLID-STATE NMR DATA COMBINED WITH MOLECULAR SIMULATION STUDIES OF CD AND ABSORPTION SPECTRA. Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1, Peter Højrup2, Anders Giessing2, Juha Linnanto3, Niels-Ulrik Frigaard4, Mette Miller2 and Niels Chr. Nielsen1. 1
Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus, Århus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 3Department of Chemistry, University of Jyväskylä, Finland; 4 Department of Biology, University of Copenhagen, Copenhagen, Denmark. Introduction: Green sulfur bacteria possess two external lightharvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. A small 6.1 kDa chlorosome protein, CsmA, plays a major role by connecting these two antennae physically and functionally. CsmA is located in the chlorosome baseplate - a paracrystalline structure observed by freeze fracture electron microscopy. Each CsmA coordinates one molecule of bacteriochlorophyll (BChl) a, and reconstitution experiments have shown that the CsmA-BChl a complex forms dimers and higher aggregates (1). The CsmA baseplate is one of the simplest known antenna systems. Thus, a structural characterization might provide new insights into how photosynthesis evolved, as well as serve as inspiration to the development of new photosynthetic devices. Methods: We have deduced a solution NMR structure of monomeric CsmA in a mixed solvent system, which shows CsmA to be an αhelical protein (2). To find the native organization of the CsmA-BChl a complex in the baseplate, we have studied isolated BChl c-less chlorosomes (so-called carotenosomes (3)) by solid-state NMR. In this sample, CsmA is the dominating protein so it can be investigated directly without further purification. The native state of the pigmentprotein complex was investigated by absorption and CD spectroscopy and used for molecular simulation studies. Results and Conclusions. Based on NMR and CD data, we present a model of the chlorosome baseplate in which CsmA is arranged symmetrically in long rows of dimers with BChl a molecules in a hydrophobic pocket between the dimers. Literature: (1) Pedersen, M.Ø.; Pham, L.; Steensgaard, D.B.; Miller, M. (2008). “A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a.” Biochemistry 47(5): 1435-1441. (2) Pedersen, M. Ø.; Underhaug, J.; Dittmer, J.; Miller, M.; Nielsen, N.C. (2008). “The three-dimensional structure of CsmA: A small antenna protein from the green sulfur bacterium Chlorobium tepidum.” FEBS Letters 582(19): 2869-2874. (3) Frigaard, N. U., Li, H.; Martinsson; P., Das, S.K.; Frank, H.A.; Aartsma, T.J.; Bryant, D.A. (2005). “Isolation and characterization of carotenosomes from a bacteriochlorophyll c-less mutant of Chlorobium tepidum.” Photosynthesis Research 86(1-2): 101-111. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts
OC-9.2 THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLS IN CHLOROSOMES OF CHLOROBACULUM TEPIDUM. Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, Michael Reus, Aline Gomez Gomez Maqueo Chew, Alfred R. Holzwarth, Egbert J. Boekema, Huub J. M. de Groot. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. Introduction: Chlorosomes are remarkably efficient light-harvesting structures that allow green sulfur bacteria to live at light intensities at which no other phototrophs can survive. Each chlorosome can contain up to 250,000 bacteriochlorophyll (BChl) c/d/e molecules, and cells can contain up to 50 million or more BChl c/d/e molecules. Because these BChls are not bound to proteins and are self-organized, the structural basis for BChl organization has been of intense interest as models for biomimetic systems for solar energy applications. Methods: Chlorosomes were isolated from wild type or triple-mutant strain bchQ bchR bchU of Chlorobaculum tepidum (1) by standard procedures using 2M sodium isothiocyanate. Cryo-electron microscopy and negative stain microscopy were performed as previously described (2). Magic-angle spinning NMR studies were performed with uniformly 13C labeled chlorosomes as described (3). Results: The NMR assignments of the 13C and 1H signals of BChl d molecules in chlorosomes from the bchQRU mutant were obtained from two-dimensional 13C-13C and 1H-13C magic-angle spinning (MAS), solid-state NMR dipolar correlation spectra collected from 13C enriched chlorosome preparations. The observed interstack correlations provided convincing evidence that syn-anti monomer stacking is the basic building block in the bchQRU chlorosome structure. Additional structural constraints were obtained from Fourier transforms of cryoEM images of chlorosomes. Supramolecular models were constructed for different orientations of the stacks relative to the tube axis. With stacks running perpendicular to the tube axis along the circumference of a cylinder in rings, the simulated image and its Fourier transform reproduced the strong periodicity of 0.83 nm and the distinct striped appearance with a spacing of 2.1 nm that is observed in the cryoEM images of the chlorosomes of the bchQRU mutant. Wild-type chlorosomes also had BChl c nanotubes with the same 2.1-nm lamellar spacing. However, indicating that the stacks are differently oriented relative to the long-axis of the chlorosome, they also exhibited a weak layer line at 1.25 nm, which corresponds to the distance between repeating syn-anti pair units in the direction of the stacks. Conclusions: Computational integration of two different bio-imaging techniques, solid-state NMR and cryoEM, revealed a previously undescribed syn-anti stacking mode and showed how ligated BChl c and d self-assemble into coaxial cylinders to form concentric tubularshaped suprastructures. The close packing of BChls via π-π-stacking and the helical H-bonding networks present in both the mutant and wild-type chlorosomes forms the basis for ultrafast, long-distance transmission of excitation energy. The structural framework is robust and can accommodate extensive chemical heterogeneity in the BChl side chains for adaptive optimization of the light-harvesting functionality in low-light environments. In addition, syn-anti BChl stacks form sheets that allow for strong exciton overlap in two dimensions, which enables triplet exciton formation for efficient photoprotection.
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Final Program and Abstracts References Gomez Maqueo Chew, A, Frigaard, NU & Bryant, DA (2007) J Bacteriol 189: 6176-6184. Oostergetel, GT, et al. (2007) FEBS Lett 581: 5435-5439. Ganapathy, S, et al. (2009) Proc. Natl. Acad. Sci. USA, in review.
OC-9.3 MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANES IN CYANOBACTERIA. Sawsan Hamad, Wim Vermaas. School of Life Sciences and the Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. Introduction: The process of biosynthesis of photosynthetic membranes in oxygenic phototrophs remains elusive. The first protein that was linked directly to thylakoid membrane biosynthesis was Vipp1, a membrane-associated polypeptide. The vipp1 gene (sll0617) could not be fully deleted from the cyanobacterial genome. In order to determine the mechanism of Vipp1 function we have now overexpressed this protein in Synechocystis. Methods: A second vipp1 gene, identical in sequence to the native one, was placed under the control of the strong psbA3 promoter. In addition, a YFP or His tag sequence was fused to the second copy of the vipp1 gene as appropriate for localization or for identification of co-isolating proteins. Results: Overexpression of vipp1 resulted in a significantly enhanced content of thylakoid membranes and of vesicles inside cyanobacterial cells, and in an increased amount of photosystem II. Moreover, large inclusions with membranous content were frequently observed in Vipp1 overexpresser cells, often near the constriction site for the next cell division; these inclusions may be used to provide the building blocks for membrane biosynthesis. The Vipp1-YFP fusion protein in Vipp1-overexpressing Synechocystis was localized at specific locations near the periphery of the cell, again often near the constriction site for the next cell division, suggesting that Vipp1 is associated with the large membranous inclusions in the cell that may be used for thylakoid biogenesis. Slr1641, which is one of the ClpB chaperones in Synechocystis, was found to be the only protein that co-isolated with His-tagged, overexpressed Vipp1 from a cyanobacterial protein extract. Conclusion: Our results suggest Vipp1 to be critical for and to regulate thylakoid membrane biogenesis, and Vipp1 appears to be associated with inclusions at specific membrane biogenesis sites in the cell. This work also demonstrates the value of overexpression of native proteins if corresponding segregated deletion mutants cannot be obtained.
OC-9.4 PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OF FUNCTION IN THE CARBOXYSOME. Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C. Bagby3, Sabine Heinhorst4, Fei Cai4, Gordon Cannon4, Sallie W. Chisholm3. 1
US Department of Energy, Joint Genome Institute, Walnut Creek, CA; Department of Plant and Microbial Biology, University of California, Berkeley, CA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; 4Department of Biochemistry, University of Southern Mississippi, Hattiesburg, MS; USA. 2
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Introduction: Cyanobacteria and many chemoautotrophic microbes have evolved a special mechanism for dramatically enhancing CO2 fixation by encapsulating RuBisCO and carbonic anhydrase into subcellular microcompartments called carboxysomes. Methods: Bioinformatic and Structural Studies Results: Our recent structural studies revealed the basic architectural principles underlying construction of the carboxysome shell. Expansion of these studies into the Prochlorococcus model system has led to the identification and elucidation of the structures of a new type of building block for the carboxysome shell, one with the potential for gated transport across the shell. The carboxysome is the best studied bacterial microcompartment; bioinformatic surveys of genomic sequence reveal that the tendency to form bacterial microcompartments is widespread. For example, structurally related micrcompartments are found in Rhodopseudomas palustris and Rhodospirillum rubrum that appear to be involved in functions other than CO2 fixation. Conclusions: The carboxysome provides general insights into a surprisingly widespread strategy for biological compartmentalization; recent data provide an increasingly sophisticated view of its function and regulation.
OC-9.5 PROTEOMIC ANALYSIS OF THE DEVELOPING INTRACYTOPLASMIC MEMBRANE DURING CHROMATIC ADAPTATION IN RHODOBACTER SPHAEROIDES. Kamil Woronowicz, Robert A. Niederman. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA. Introduction: Although the primary photochemical events and energy transduction processes in Rba. sphaeroides are well understood, much less is known about mechanisms involved in the formation of the participating light-harvesting (LH) and photochemical reaction center (RC) complexes, or the interactions of their apoproteins with numerous complex-specific and general assembly factors in the site-specific assembly of functional photosynthetic units in the growing intracytoplasmic membrane (ICM). Here, we report a detailed structural and functional proteomic analysis of the ICM assembly process during a downshift in a light intensity. Methods: Proteomic approaches have focused upon identifying proteins temporally expressed during ICM development and spatially localized in both membrane growth initiation sites, isolated as an upper pigmented band (UPB) after rate-zone sedimentation in sucrose density gradients, and in the mature ICM, isolated as the main band (chromatophores). The isolated membrane fractions have been further purified by a two-phase partitioning procedure and subjected to nondenaturing clear gel electrophoresis (CNE). Bands are excised from the gels and subjected to LC-MS/MS analysis. Results: CNE gives rise to four distinct bands: a top band containing the LH1 (B875)-RC core complex; a bottom band containing the LH2 (B800-850) peripheral antenna complex; and two bands of intermediate migration in which the LH2 and core complexes remain associated. Proteomic analysis yielded a large array of proteins associated with the gel bands and the resulting profiles can be correlated with the local organization of ICM as revealed by atomic force microscopy (AFM). The PuhA RC-H subunit was the most abundant protein, while surprisingly, the Puc2A LH2-α polypeptide, encoded by puc2BA operon, was the second most abundant protein with PucB LH2-β and Puc2B LH2-β comprising the other detectable August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes LH2 subunits. The spectral counts of several of the F1FO– ATP synthase subunits were unexpectedly high, given the low abundance of the ATP synthase in chromatophores and the inability to detect this coupling factor, as well as the more abundant cytochrome bc1 complex in AFM. Moreover, the high levels of ATP synthase and subunit IV of the bc1 complex found associated with the LH2-enriched fractions is consistent with their localization at ICM vesicle edges, thought to contain LH2-only domains, which are outside the flat ICM vesicle regions imaged by AFM. Significant levels of a preprotein translocase YidC homolog were found, along with lesser amounts of other general membrane assembly factors such as TatA, the twin-arg translocation system subunit, the SecY preprotein translocase subunit and the bacterial type 1 signal peptidase. In contrast, no complex-specific assembly factors were found in any of the bands. Forty-five proteins of unknown function were associated with the gel bands. One hypothetical protein, RSP6124, was observed in high abundance and is associated with the LH2 band, with its appearance preceding the increase of LH2 levels during chromatic adaptation. Conclusions: Proteomic analysis of bands containing the LH1-RC and LH2 complexes obtained by CNE of chromatophore from low light adapting cells, yielded a variety of proteins that could be correlated with their localization determined in AFM topographs and the possible roles of general membrane assembly factors that may be involved in the insertion of their nascent polypeptides into the ICM. (Supported by DOE Grant No. DE-FG02-08ER15957).
Final Program and Abstracts organisms. Inactivation of the Syn7002_A0564 gene in Synechococcus sp. PCC 7002 leads to about 40% reduction of chlorophyll content, which is due to impairment of PS I accumulation as demonstrated by measurements of the low temperature fluorescence emission spectroscopy and PS I analysis. However, no obvious difference could be observed in the composition of PS I, as shown in the results of SDS-PAGE analysis of isolated PS I complexes. Under photoautotrophic growth conditions the mutant exhibited a slower growth rate and higher sensitivity to high light. The stress response of the mutant is possibly related to an increase of ROS in cells. As measured by using a cell-permeant ROS probe and compared to the wild type, the ROS content is two-times higher in the mutant cells even when grown in reduced light. A 30-min treatment in high light led to a 6-fold increase in ROS content. As shown by RT-PCR analysis, expression of the sodB gene is significantly up-regulated in the mutant cells grown under the reduced light conditions. An enhanced photoautotrophic growth rate was observed for the mutant when cells were grown under semi-anaerobic growth conditions by bubbling with 99% N2 and 1% CO2. Conclusion: Our results demonstrate that the PDI-like protein Syn7002_A0564 is involved in biogenesis of PS I reaction center. It may function as a membrane-associated dithiol/disulfide oxidoreductase or may play an important role in maintaining the balance of oxidizing/reducing equivalents of thiol redox in PS I assembly and regulation, especially in facilitating cofactor insertion and maturation.
OC-9.6 BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODING THE MEMBRANE-ASSOCIATED THIOREDOXIN PROTEIN PLAYS AN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OF PS I.
OC-10.1
Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1.
Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2, Yukako Hihara2, Ariel Gaathon3, Mario Lebendiker4, Aaron Kaplan1.
1
2
Department of Biochemistry and Molecular Biology; Department of Chemistry; The Pennsylvania State University, University Park, PA, USA. Introduction: The biogenesis of PS is a complex, multi-step process in which many factors are involved, mostly in assembly of the protein component and insertion of cofactors. In recent years, advances in genome sequencing of several cyanobacteria (including Synechococccus sp. PCC 7002) and functional genomics of photosynthetic genes have provided much new information for exploring assembly and regulation of the PS I reaction center. In this study, we describe a novel gene (Syn7002_A0564) encoding a protein disulfide isomerase (PDI)-like protein that is found be involved in assembly and/or regulation of PS I Methods: DNA and protein sequence analyses were performed with MacVector software. Homologous sequences of the DPI-like Syn7002_A0564 were retrieved from the IMG and Cyanobase databases. A Syn7002_A0564 mutant was generated through insertion of a kanamycin-resistance cartridge at the unique EcoRV site within Syn7002_A0564 coding sequence. Growth rates of the wild type and mutant were measured at different conditions. The low-temperature fluorescence emission spectroscopy was applied to compare changes of PS I and PS II complexes in cells. PS I complexes were isolated for analysis of subunit composition. The cell-permeant indicator CMH2DCFDA was used for probing intracellular reactive oxygen species (ROS). Results: Sequence analyses indicate that Syn7002_A0564 encodes a PDI-like protein that is composed of two thioredoxin-like domains and that it is predicted to be membrane-associated. Homologs of Syn7002_A0564 can be found in all oxygenic photosynthetic
August 9 to 14, 2009 • Montréal, QC, Canada
A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THE APPARENT PHOTOSYNTHETIC AFFINITY FOR CO2 BY MODULATING LOW-CO2-INDUCED GENE EXPRESSION.
1 Dept. of Plant and Environmental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; 2 Dept. of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan; 3Bletterman Laboratory, Interdepartmental Equipment Unit, Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre, Hebrew University of Jerusalem, Jerusalem, Israel.
In Synechocystis sp. strain PCC 6803, over 450 genes are up-regulated following transfer of the cells from a high (1-5% CO2 in air, HC) to a low level of CO2 (as in air or lower, LC). This includes sbtA, ndhF3 and cmpA involved in inorganic carbon (Ci) uptake. Earlier studies implicated NdhR in the regulation of LC-induced genes but there are indications that additional components are involved. Following extraction of proteins from cells grown under HC and (NH4)2SO4 fractionation, we have identified LexA and two AbrB-like proteins, Sll0359 and Sll0822 that bind to a fragment of the sbtA promoter. Using extracts prepared from LC-grown cells, Sll0822 did not bind to the sbtA promoter despite its presence in the cells, suggesting that it may serve as a repressor of LC-induced genes. This is supported by the fact that sbtA, ndhF3, and cmpA normally expressed only under LC in the wild type are transcribed under both HC and LC in a ∆sll0822 mutant. When grown under HC this mutant exhibits an elevated apparent photosynthetic affinity to Ci, typically observed in the wild type only under LC. Clearly, expression of genes essential for Ci uptake was sufficient to raise the apparent photosynthetic affinity for external Ci.
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13th International Symposium on Phototrophic Prokaryotes
OC-10.2
OC-10.3
THE EFFECTS OF TEMPERATURE AND OXYGEN ON NITROGENASE ACTIVITY IN THE THERMOPHILIC CYANOBACTERIUM FISCHERELLA SP.
PHOSPHATE SCAVENGING IN AN UNPREDICTABLE ENVIRONMENT, HOW DO CYANOBACTERIA MEET THE CHALLENGE?
Lucas J. Stal, Department of Marine Microbiology, Netherlands Institute of Ecology, Yerseke, The Netherlands.
Frances D. Pitt, David J. Scanlan.
Introduction: Nitrogenase – the enzyme complex responsible for the fixation of dinitrogen in diazotrophic prokaryotes - is irreversibly inactivated by oxygen. Cyanobacteria evolved strategies that allowed the coexistence of the two incompatible processes of dinitrogen fixation and oxygen-evolving photosynthesis. Heterocysts are differentiated cells that contain nitrogenase but have lost the oxygenic photosystem. The heterocyst possesses a thick glycolipid cell envelope that serves as a gas diffusion barrier. Any oxygen that enters the heterocyst is respired thereby guaranteeing an anoxic interior of the heterocyst. However, a too efficient glycolipid cell envelope would also limit dinitrogen from entering the heterocyst. Hence, the gas diffusion barrier must be a trade-off between the entrée of dinitrogen and a limitation of the entrée of oxygen. Ideally, the amount of gas that diffuses into the heterocyst should be as high as possible so that the respiratory system can still keep it to sufficient low intracellular concentrations. This would require a dynamic gas diffusion system of the heterocyst in order to adequately respond to changes in the environmental conditions. Methods: I have selected the thermophilic heterocystous cyanobacterium Fischerella sp. to investigate the question of the dynamic response of gas diffusion in the heterocyst by measuring nitrogenase activity by an on-line, near real-time acetylene reduction assay (ARA) using a laser photoacoustic ethylene detector. Fischerella sp. was grown under continuous light at 27oC. Results: I measured ARA at temperatures from 18-39oC at 3oC intervals. At each temperature light response curves of ARA were recorded. The light response curves were fitted to the rectangular hyperbola function yielding the following parameters. Nd and Nm were the dark- and maximum light-dependent acetylene reduction rates, respectively and Ntot was the sum of these and represented the actual nitrogenase activity measured at light saturation. The light saturation constant Ik equals Km in Michaelis-Menten kinetics and is the light intensity at which 0.5Nm. Ik equals Nm/α. The light affinity constant α is the initial slope of the light response curve at zero light. I found that nitrogenase activity increased with temperature with a Q10 of 6, three times higher than what one would expect from enzyme kinetics. The dimensionless factor Ntot/Nd equaled ~2 and was constant over the temperature range. Nd increased with temperature and this is only possible when the amount of oxygen that enters the heterocyst increased. This would indicate a possible dynamic regulation of the oxygen (gas) diffusion into the heterocyst. In order to investigate this further, I measured ARA at a range of oxygen concentrations from 090%. I found that Ntot remained constant in the range of 5-30% oxygen. This was the result of an increasing Nd and simultaneously decreasing Nm. Above 30% oxygen acetylene reduction decreased, obviously because an increasing part of the reducing equivalents were necessary for respiration and nitrogenase activity became electron limited. Conclusions: I conclude that the heterocyst did not dynamically regulate the entrée of oxygen at a constant temperature but that it happened as a function of temperature. The changes of the photosynthetic parameters Ik or α were fully attributed to changes in Nm and therefore photosynthesis itself did not affect dinitrogen fixation. 64
Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom. Introduction: Cyanobacteria are ubiquitous in aquatic and marine environments where they play a key role as primary producers. Their growth is limited by the availability of nutrients, particularly phosphorus (P), and many species persist and flourish in environments with an unpredictable and constantly fluctuating supply of P. Whilst genome-wide analyses have revealed many details of the P uptake and regulatory machinery there are still very few reports that demonstrate the functional significance of individual, and apparently redundant, components of the cyanobacterial Pho regulon. For example, Synechocystis sp. PCC6803 encodes duplicate ABC transporters (i.e. two PstCAB operons) with at least three associated periplasmic binding proteins (two of the PstS type and one of the SphX type). This contrasts with virtually all other known bacteria which encode a single ABC transport system. Current research into the specific functional attributes of the multiple phosphate transporter arrangement in Synechocystis sp. PCC6803 aims to elucidate their interplay with other proteins involved in phosphate acquisition and regulation. By answering the question of evolutionary adaptation by biochemical or regulatory means, we aim to reveal new strategies for the acquisition of phosphate during conditions of environmental stress. Methods: In order to assess the role of multiple ABC transporter elements in Synechocystis sp. PCC6803 we created gene deletions of the two pstCAB operons as well as single knock-outs of the associated pstS / sphX genes. Gene expression profiles were generated using real time quantitative PCR to compare mutants with WT under phosphate replete and deplete conditions. Phosphate uptake assays using radiolabelled 32P were then performed to compare the kinetics of phosphate uptake in the various mutants. Results: Disruption of one of the PBPs in Synechocystis sp. PCC6803, pstS1 (sll0680) led to an impairment in the expression of specific genes of the pho regulon during P-deplete growth. In addition, the mutant was unable to alter the expression of various genes normally induced in WT in response to changes in external P concentration. This phenotype was not observed when the remaining phosphate binding proteins were disrupted suggesting that only one of the two transporters is involved in sensing and regulation of the pho regulon. Preliminary P uptake assays suggest potential differences in the binding affinities of the two transporters. Further uptake work using specific gene knock out mutants should clarify their contribution to phosphate uptake under different growth conditions. Conclusion: Differential expression of P binding protein genes in WT Synechocystis sp. PCC6803 during P replete and deplete conditions suggests that each ABC transporter performs a distinct functional role. This conclusion is supported by the phenotype of the pstS1 deletion mutant which displays impaired ability to respond to changes in external P. Preliminary results from P uptake assays go further and suggest that there may be differences in the P binding protein’s affinity for P, thus leading to the development of a novel strategy for P uptake in a freshwater cyanobacterium.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
OC-10.4 REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY IN CYANOBACTERIA. IDENTIFICATION OF THE REGIONS INVOLVED IN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS. Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J. Florencio. Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de SevillaCSIC, Sevilla, Spain. Glutamine synthetase plays a central role in the regulation of nitrogen assimilation in cyanobacteria via the control of the carbon/nitrogen metabolic flow. Thus, this enzyme is tuned up depending on the nitrogen source and the carbon disponibility. The intracellular concentration of 2-oxoglutarate defines the carbon/nitrogen status of the cell and it is the key metabolite in the control of the GS activity, being high level of GS activity when 2-oxoglutarate concentration is high and low GS activity at low 2-oxoglutarate level. The regulation of cyanobacterial GS activity occurs by a original system consisting in the protein-protein interaction between the GS protein and two inactivating factors (IFs) named IF7 and IF17. This interaction provokes the inactivation of the enzyme when the carbon/nitrogen ratio is low, for example using ammonium as nitrogen source, but if it is nitrate, the enzyme is active and the IFs are absents. The expression of the IFs are controlled at the level of transcription by the global nitrogen regulator NtcA. Here, we have investigated the molecular interaction between GS and the IFs, we try to know which are the regions in both GS and IFs implicated in the protein-protein interaction by using two different approaches. The first one was to construct different GS chimeras using Synechocystis and Anabaena GSs, it is worth to note that both cyanobacteria possess the same system to inactivate GS, but the IF are different in the way that IF7 from Anabaena is able to inactivate both GS in vitro, but IF7 or IF17 are unable to inactivate the Anabaena GS. Since IFs from Synechocystis are unable to inactivate Anabaena GS, we expect to find a region responsible for the interaction in Synechocystis GS. The second method was to construct different site-directed mutants of IFs from Synechocystis in order to study their capacity to inactivate or not GS. We will present results indicating that the region required for the interaction resides in the carboxy-terminal part of the GS. We also found that a change of single residue is this region is sufficient to disrupt the interaction between GS and IFs in Synechocystis. With respect to the IFs interaction studies, we have found that at least 3 basic amino acid residues are essential to maintain the interaction with the GS and that these residues are localized in the central part of these proteins. The relevance of these results will be discussed in relation to a possible model that explain how these GS-IFs interaction promotes the GS inactivation.
OC-10.5 BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS. Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A. Bryant1. 1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2 Department of Biological Sciences, University of New Orleans, New Orleans, LA; USA. Introduction: Cyanobacteria employ a number of different tetrapyrrole/biliprotein combinations both as accessory pigments to augment their ability to utilize wavelengths of light which are not efficiently captured by chlorophyll and as sensors to perceive and August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts respond to light conditions in their environment. Up to four different linear tetrapyrroles may be incorporated into various phycobiliproteins: phycocyanobilin, phycoerythrobilin, phycoviolobilin and phycourobilin. When bound to its cognate protein, each of these bilins confers unique spectral properties, which enhance the ability of the cyanobacteria to exploit the light available in its environment. On the other hand, plants are only known to utilize biliproteins known as the phytochromes as sensors. Plant phytochromes uniquely incorporate the bilin, phytochromobilin. In the last several years the components involved in the assembly of biliproteins have been characterized. This study was conducted to determine the factors involved in maintaining the specificity between phycobiliproteins and their cognate chromophores and to determine the effects on a cyanobacterium when chromophores are misincorporated into a noncognate apoprotein. Methods: A dual-plasmid E. coli expression system was used to study the attachment of chromophores to phycobiliproteins in isolation. Use of this heterologous system allowed the co-expression of the minimal components necessary to assemble a holophycobiliprotein and made it possible to assay the effects of substitutions of various components, specifically, bilin lyases and bilin biosynthetic enzymes, on the final holophycobiliprotein made in E. coli. In addition, we studied the effects of potential mis-pairings of chromophores and apoproteins inside Synechococcus sp. PCC 7002 using site-specific integrations into the genome. Results: With various lyase and chromophore synthesis combinations, CpcA from Synechococcus sp. PCC 6803 can bind six different linear tetrapyrrole chromophores, including the plant-specific chromophore phytochromobilin and an A-ring isomerized version of phytochromobilin not known to occur naturally. Additionally, the heterologous over-expression of the phycoerythrobilin or phytochromobilin biosynthetic enzymes in Synechococcus sp. PCC 7002, a cyanobacterium that utilizes only phycocyanobilin as a chromophore, results in strains with readily apparent altered absorbance profiles. Conclusions: Different chromophore/apoprotein/lyase combinations can be used to yield several different colored versions of a single chromophore binding protein. The specificity of attachment of a linear tetrapyrrole chromophore to its cognate apoprotein is more general than once believed. Although in nature the misincorporation of chromophores is believed to occur only rarely, if at all, it can be made to occur quite readily in genetically modified E. coli and cyanobacteria. These studies shed new light on the assembly and evolution of phycobiliproteins, and the results have implications for the production of recombinant proteins with properties not found in natural proteins.
OC-10.6 ACCUMULATION OF TREHALOSE IN RESPONSE TO DESICCATION AND CONTROL OF TREHALASE IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE. Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima. Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan. Introduction: Desiccated organisms have little to no metabolic activity and rapidly resume metabolism upon rehydration. This phenomenon is termed “anhydrobiosis”. The terrestrial cyanobacterium Nostoc commune sustains the capacity for cell growth for over 100 years in a
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Final Program and Abstracts desiccated state; thus, N. commune is considered an anhydrobiotic microorganism with oxygenic photosynthetic capabilities. Since N. commune does not differentiate into akinetes (spores), the mechanisms of its extreme desiccation tolerance most likely involve multiple processes. Anhydrobiotic organisms accumulate trehalose, which protects biological membranes and proteins against the destructive effects of water removal by replacing the primary waters of hydration, as well as through the formation of amorphous glasses (vitrification). In this study, we investigated changes in photosynthetic activity and trehalose levels in the terrestrial cyanobacterium Nostoc commune responding to desiccation and the biochemical properties of the enzymes involved in trehalose metabolism to elucidate the mechanisms of trehalose accumulation. Methods: Trehalose was determined by gas-liquid chromatography (GLC) and by high performance liquid chromatography (HPLC). Photosynthetic O2 evolution during desiccation was measured using a gas-phase Clark-type oxygen electrode. The three genes for trehalose metabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY (maltooligosyltrehalose synthase, Mts), and treH (trehalase), were found as a gene cluster, and the mRNAs for these genes were detected by RT-PCR. The activity of trehalose synthesis, consisting of Mts and Mth, was measured by the production of trehalose from soluble starch. The treH gene was heterologously expressed in E. coli cells in an active form with a molecular mass of 52 kDa and the activity of trehalase was measured by the production of glucose from trehalose.
13th International Symposium on Phototrophic Prokaryotes chromophore that typically absorbs 446 nm light, although there is some variation amongst species. The Ppr from the purple photosynthetic bacterium, Rhodospirillum centenum is a chimera of PYP, bacteriophytochrome (Bph), and histidine kinase. The function of Ppr is to regulate a polyketide synthase. When both photoreceptors are activated, the PYP domain appears to hasten the recovery of Bph to the dark state. However, the cause of this phenomenon is unclear. We have now analyzed the Ppr recovery kinetics and kinase activity in greater detail under three different light conditions. Methods: Ppr was produced in E. coli using a two plasmid based system. Purification was performed on a Q-sepharose column followed by a TALON His-tag column. Absorption spectra and kinetics were obtained using a Cary 300 spectrophotometer. Recovery kinetics were measured for up to 300 minutes and the data were fit using Sigmaplot 8.0 (Systat Software). Autophosphorylation was measured by adding 1mM ATP (containing ~ 2.5 μCi P32 ATP) and keeping the sample illuminated for 30 min. Six time points were taken and analyzed on SDS-page gels. The resulting autoradiogram was digitized using a Typhoon Imager (GE Healthcare) and intensities were quantified by volume using ImageQuant TL.
Results: As the water content decreased in N. commune colonies during desiccation, photosynthetic O2-evolving activity decreased and no activity was detected in desiccated colonies. No detectable trehalose was found in fully hydrated colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air-dried colonies. Moreover, NaCl treatment also induced trehalose accumulation and the levels of trehalose induced were equivalent to that in the desiccated colonies. The transcripts for treZ, treY and treH genes were detectable at similar levels during desiccation. Trehalase activity was strongly inhibited in the presence of 10 mM NaCl while trehalose synthesis activity remained active in the presence of salt.
Results: Bph normally absorbs 700 nm light in the Pr state and when illuminated with red light, it is red-shifted in most species to the photoreversable Pfr state absorbing near 750 nm. However, the Bph domain within Ppr bleaches upon red-light illumination to a form which weakly absorbs near 650 nm and cannot be photoreversed by near red light. Dark reversion normally takes from 20 to 60 minutes. However, recovery of the Bph 701 nm absorbance is accelerated in part to about 1 minute following white light illumination. White and blue light bleaches PYP, which initially recovers with kinetics similar to that of the PYP construct, however, only about half the absorbance at 434 nm is recovered in ~ 4 minutes, the remainder takes several days to recover due to formation of an intramolecular complex with Bph whose absorption is red-shifted. The Bph within the metastable complex can no longer be bleached by red light. The PYP remains locked in its bleached state which absorbs near 355 nm, but it can be photoreversed by UV light, in which case the Bph also returns to its initial 701 nm dark state. Autokinase activity is turned on by both red and white light, but is inactivated when photoreversed by UV light.
Conclusions: Trehalose accumulation and cessation of photosynthesis occurs during desiccation. Since transcriptional regulation of the gene cluster for trehalose synthesis and trehalose hydrolysis cannot fully explain trehalose accumulation in response to desiccation, posttranscriptional regulation most likely controls the cellular level of trehalose. Under water-stress conditions characterized by increased cellular solute concentrations, the rate of trehalose production exceeds that of trehalose hydrolysis because of the inhibition of trehalase. Control of trehalase plays an important role in trehalose accumulation under conditions of extreme desiccation.
Conclusions: The kinase activity of Bph is normally turned off in the dark and activated in red light. It is switched off again by far-red light with simultaneous return of the 700 nm absorbance. Because the Bph within Ppr does not form the Pfr state but is instead bleached by red light, it cannot be photoreversed in the usual way. Thus, the role of PYP is threefold, to block activation of Bph in blue light through formation of the metastable complex, to accelerate recovery of Bph absorbance in white light, and to photoreverse the effects of blue and white light by subsequent illumination in the UV. We therefore postulate that Ppr functions as a UV-red light sensor.
OC-11.1
OC-11.2
FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAIN IN RHODOSPIRILLUM CENTENUM PPR. J.A. Kyndt, T.E. Meyer, M.A. Cusanovich.
SHORT-TERM LIGHT ADAPTATION STRATEGIES OF PHYCOBILISOME-CONTAINING PHOTOSYNTHETICS, CYANOBACTERIA AND RED ALGAE.
Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
Igor Stadnichuk.
Introduction: Ppr is unique in that it has a blue light photoreceptor (PYP) N-terminally fused to a bacteriophytochrome (Bph) domain. Photoactive Yellow Protein (PYP) is related to the PAS domain superfamily of signalling proteins. It generally occurs as a freestanding cytoplasmic protein and has been found in 18 species of bacteria of diverse lifestyles. It contains a p-hydroxy-cinnamic acid
A.N.Bakh Institute of Biochemistry, Russian academy of Sciences, Moscow, Russia.
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Phycobilisome (PBS)-containing photosynthetics, the cyanobacteria and red algae, have evolved several short-term reversible adaptation mechanisms to survive under different light quality and quantity conditions. Realized on a timescale of minutes, all mechanisms do not August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes involve alterations in gene expression or photoinhibition of the pigment apparatus and are revealed as non-photochemical fluorescence quenching (NPQ) that includes alterations of the fluorescence emission from PBS. The appropriate phenomena changing rapidly the cross-sections of the photosystems I (PSI) and II (PSII) are: state transitions realized in cyanobacteria and red algae when light is normal to growth (≤ 150 microE m-2s-1); orange carotenprotein (OCP)-induced PBS quenching realized in cyanobacteria for relatively high light intensities (≥ 500 microE m-2s-1); induced by weak (~10 microE m-2s-1) far-red light increase of PBS emission, in the red algae. State transitions are well-known phenomenon of the adaptation of pigment apparatus to unbalanced light absorption by PSI and PSII first described 40 years ago ([1] and ref. there). Contrary to elucidation of the effect in green plants, the cause of state transitions in cyanobacteria and red algae is still in debate and different models have been proposed. In the spillover model, the excitation energy is transferred directly from PSII to PSI core chlorophylls. The partial dissociation of PBS from PSII and its reassociation without docking to PSI is the main event of the detachment model. In the mobile model, the PBS could reversibly migrate in the plane of the thylakoid membrane from the surface of PSII to PSI. It could be possible that none of the existed models would stay correct after additional investigations [2]. In cyanobacteria, most probably, the monomerisation of the PSII dimers is the main event of transition to state 1 [3]. In 2004, due to the registered action spectrum of the process, we have described [4] an additional to state transitions short-term mechanism of energy dissipation in PBS of cyanobacteria involving a carotenoid identified as OCP [5]. Light-activated OCP was found to cause reversible NPQ via interception the energy transfer from PBS to chlorophyll a of PSII as well as of PSI cores [2, 5]. Recently [5], we have demonstrated quite new display of short-term light adaptation of the pigment apparatus in the extremophilic microalga, Galdieria sulphuraria. Comparing to the dark-adapted cells, weak far-red light primarily absorbed by photosystem I (PSI) causes an increase in the amplitude of the PBS low-temperature fluorescence emission. Contrary to emission, the PBS peak in fluorescence excitation spectrum of PSI increases simultaneously with PBS quenching while excitation spectrum of PSII stays invariable. Therefore, in G. sulphuraria, the registered changes of 77 K fluorescence emission correspond to the reversible detachment of the PBS from the core complexes of PSI. The data indicates the adaptive decrease of cross-section and the diminishing of the PSI activity when it is not accompanied by PSII function. This kind of NPQ is not known for cyanobacteria where PBS docking to PSI has to be faster than the docking to monomeric PSI in the red algae. References 1. Murata N. (2009) Photosynth. Res. 99, 155-160. 2. Stadnichuk I. et al. (2009) Photosynth. Res. 99, 227-241. 3. Stadnichuk I. et al. (2009) FEBS Lett. in press. 4. Rakhimberdieva M. et al. (2004) FEBS Lett. 574, 85-88. 5. Wilson A. et al. (2006) Plant Cell 18, 992-107.
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Final Program and Abstracts
OC-11.3 REGULATORY RNAS IN CYANOBACTERIA AND BEYOND. W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1, J. Vogel2, A. Wilde3. 1
Freiburg Initiative in Systems Biology and Faculty of Biology, University Freiburg; 2Max-Planck-Institute for Infection Biology Berlin; 3 Justus-Liebig University Giessen, Institute of Microbiology and Molecular Biology; Germany. Regulatory RNA has been discovered in all three domains of life. However, transcriptional units that give rise to non-coding RNAs (ncRNAs) or antisense RNAs (asRNAs) are not identified during normal genome annotation, and in phototrophic bacteria only a small number of ncRNAs has been described. We have used 5 different methods for the identification of novel non-coding and antisense RNAs in various cyanobacteria, with a focus on model cyanobacteria (1-4). The reliability of computational predictions for the detection of ncRNAs and asRNAs in Synechocystis sp. PCC 6803 and for marine Synechococcus species was scrutinized in microarrays, complemented by deep sequencing of the small RNA population and validated by an extended set of Northern hybridizations and 5’ RACE experiments. The total number of asRNAs in Synechocystis sp. PCC 6803 alone is more than 350 and of classical trans-acting ncRNAs about 100. Thus, regulatory RNAs play a much more important role in this model organism and probably also in most other bacteria. Although functions are still unknown for the vast majority of regulatory RNAs, we will provide functional evidence for cyanobacterial ncRNAs acting against invading phages, controlling the expression of key photosynthetic genes, or serving as signals for RNA maturation, processing and degradation. References (1) Axmann et al. (2005), Genome Biol. 6, R73: 1-16. (2) Voss et al. (2007), BMC Genomics 8:375. (3) Steglich et al. (2008), PLoS Genetics 4 (8) e10000173. (4) Voss et al. (2009), BMC Genomics 10, 123.
OC-11.4 FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUM PROCHLOROCOCCUS: WHAT CAN WE LEARN? C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, Joerg Vogel3, Wolfgang Hess1. 1
University of Freiburg, Freiburg, Germany; 2University of Algarve, Faro, Portugal; 3Max Planck Institute for Infection Biology, Berlin, Germany. Small non-coding RNAs (ncRNAs) are functional RNA molecules, mostly without a protein-coding function, that have been found in all domains of life. In bacteria these functional RNA molecules range in size between 50 – 400 nt and frequently play a crucial role in regulatory networks particularly in response to environmental stress. ncRNAs are also known to control plasmid and viral replication, bacterial virulence and quorum sensing, while the function of others has remained unknown. A detailed survey for ncRNA was performed in the ecologically important marine cyanobacterium Prochlorococcus, which it is the most abundant phototroph in the vast nutrient-poor areas of the ocean. Genome analyses of several isolates revealed compact streamlined genomes that contain a core set of about 1,200 genes augmented with a variable number of ‘flexible’ genes. All sequenced Prochlorococcus isolates possess only a small number of 67
Final Program and Abstracts genes coding for regulatory proteins. Applying different methods for the detection of ncRNAs in Prochlorococcus MED4, the total number of ncRNAs, which have been experimentally confirmed by two different methods, rises to 26 (including Yfr1-6, 6S RNA, SRP RNA, tmRNA, RNase P RNA). Thus, unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNA genes are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism. Of the 12 completely sequenced Prochlorococcus strains only 2 possess the RNA chaperone Hfq. A 454 deep sequencing approach was conducted to compare the number of regulatory RNAs between the high light-adapted hfq-lacking strain MED4 and MIT9313 who is adapted to low light and contains an hfq homolog.
OC-11.5 REGULATION BETWEEN PHOTOAUTOTROPHIC AND PHOTOMIXOTROPHIC GROWTH AND ITS DEPENDENCE ON THE CO2 LEVEL IN SYNECHOCYSTIS PCC 6803. Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy LiemanHurwitz1, Aaron Kaplan1. 1 Plant and Environmental Sciences, The Hebrew University of Jerusalem; Israel; 2Department of Biochemistry and Molecular Biology, Saitama University, Japan.
Cyanobacteria represents a unique case were carbohydrates formation in photosynthesis and their breakdown in respiration occurs in the same compartment at the same time, unlike eukaryotes where the catabolic and anabolic activities are spatially separated. The mechanisms whereby these activities are regulated in the context of the environment is poorly understood. Glucose sensitive mutants of the cyanobacterium Synechocystis sp. PCC 6803 were isolated. Many of them are far more resistant to the presence of glucose when exposed to low level of CO2. Analyses of the expression of certain CO2-dependent genes in the wild type and the mutants as affected by the ambient CO2 concentration and presence of glucose revealed an intriguing but complex regulation between these processes. Our results shows for the first time the interconnectivity between CO2 availability and organic carbon supply and provide some insight into the molecular mechanisms involved.
OC-11.6 CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY IN OSCILLATORIA. Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson, Dave Adams. University of Leeds, Leeds, West Yorkshire, United Kingdom. Introduction: When attached to a surface many filamentous cyanobacteria move by a process known as gliding. The mechanism of gliding is unknown, although two main theories, slime extrusion and surface waves, have been proposed to explain the generation of thrust. In the first, filaments are pushed along by the extrusion of slime from rows of junctional pores that are known to encircle each cell septum. The second theory proposes that gliding results from the rhythmical progression of waves along the cell surface and their interaction with the substrate to which the filament is attached. It has been suggested that such waves are created by protein fibrils beneath the outer membrane. We have been using Atomic Force Microscopy 68
13th International Symposium on Phototrophic Prokaryotes (AFM) and Field Emission Gun Scanning Electron Microscopy (FEGSEM) to study the cell walls and cell surfaces of Oscillatoria strains to identify structures that may be associated with motility. Methods: For FEGSEM, samples were fixed in 2.5% (vol/vol) glutaraldehyde and post-fixed with 1.0% (wt/vol) osmium tetroxide. Samples were then dehydrated in a graded ethanol series, criticalpoint dried and mounted on aluminum pin stubs. Finally, samples were coated with either gold or platinum-palladium and examined with a LEO 1530 series FEGSEM instrument operating at 3 kV. For AFM imaging of cells under liquid, cyanobacterial filaments were first immobilized by partial embedding in dental wax which had been softened by warming in an oven. Results: AFM scanning under liquid of filaments of Oscillatoria sp. strain A2 immobilised in wax confirmed the presence of an array of parallel 40 nm wide corrugations on the surface. These surface corrugations are a consequence of the presence of protein fibrils beneath the outer membrane. By increasing pressure on the AFM tip it was possible to remove the fibrillar array from a defined area of the cell surface. By contrast, FEGSEM images of the very large filaments of O. princeps revealed an irregular array of corrugations on the surface. These proved difficult to visualise by AFM under liquid, possibly because of a slime layer obscuring the finer surface details. However, the O. princeps surface is covered by raised structures which correspond in spacing to large pores, approximately 150 nm in diameter, seen in FEGSEM images to penetrate the extremely thick peptidoglycan layer. The function of these pores seems to be to bring the cytoplasmic and outer membranes into closer proximity. However, they may also have a function in slime production. The significance of these cell wall and surface features, in terms of the mechanism of gliding motility, will be discussed. Conclusions: It is possible to immobilise cyanobacterial filaments in dental wax in a way that preserves the filaments in a fully hydrated state and allows AFM scanning under liquid. The AFM tip can be used to remove surface layers, such as the slime layer, outer membrane and fibrillar array, and by repeat scans of the same area, it is possible to visualise surface changes. We hope in the near future to employ these techniques to visualise the putative surface waves that drive gliding motility.
OC-12.1 GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGEN CONDITIONS AND THE ROLE OF THE HISTIDINE KINASE, HIK31, IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Tina C. Summerfield1, Louis A. Sherman2. 1 Botany Department, University of Otago, PO Box 56, Dunedin, New Zealand; 2Purdue University, Department of Biological Sciences, West Lafayette, IN, USA.
Introduction: In habitats such as hot springs, soils (e.g. rice paddies or estuarine mud), and eutrophic lakes, cyanobacteria experience low oxygen conditions. These conditions are associated with the ability of cyanobacteria to produce ethanol and H2 indicating decreased O2 may play an important role in this energy production. Methods: Using DNA microarrays, we examined gene expression in Synechocystis sp. PCC 6803 on transition from aerobic growth to low oxygen conditions. We observed up-regulation of the gene encoding the histidine kinase Hik31 under low oxygen conditions. To examine the role of this histidine kinase we compared gene expression in the wild type and a ∆Hik31 strain that lacked Hik31. Additionally, we measured growth, oxygen evolution and whole cell spectra during August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes incubation under low oxygen conditions. Results: In the wild type strain, expression of >13% of chromosomal genes were altered within 1 hour under low oxygen conditions. Similar numbers of genes showed increased and decreased transcript abundance. This included down-regulation of genes encoding photosynthesis proteins, regulatory proteins and chaperones. Upregulated genes included the hox genes encoding the bidirectional hydrogenase and the LexA-like transcription regulator (sll1626) that binds the hox promoter. Genes encoding proteins involved in transport, regulation and energy metabolism were up-regulated under low oxygen conditions, including an operon encoding Hik31 and response regulator (Rre34). The transition from aerobic to low oxygen conditions resulted in differential regulation of ~8% of chromosomal genes in the ∆Hik31 strain. The majority of these genes (~90%) showed increased transcript abundance under low oxygen conditions. Almost half these genes, including the hox operon, were up-regulated in the wild type under the same conditions. Genes up-regulated in the ∆Hik31 strain but not the wild type under low oxygen conditions encoded proteins involved in photosynthesis, such as phycobilisome components, and ribosomal proteins. Consistent with this, extended incubation under low oxygen conditions resulted in cells with increased phycobilisome content relative to chlorophyll content in the ∆Hik31 strain but not the wild type. Furthermore, unlike the wild type, the ∆Hik31 strain did not down-regulate genes encoding chaperones and ATP synthase. Conclusions. These data suggest that Synechocystis sp. PCC 6803 responds to low oxygen conditions by down-regulating genes involved in key processes such as photosynthesis and translation and that the histidine kinase, Hik31, plays a role in this negative regulation.
OC-12.2 INCREASING HYDROGEN PRODUCTION BY PERTURBING FERMENTATIVE METABOLISM IN THE MARINE, UNICELLULAR CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002. Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G. Charles Dismukes. Princeton University, Princeton, NJ, USA. Introduction: Certain strains of cyanobacteria contain a bidirectional hydrogenase and make hydrogen as a byproduct of sugar catabolism during fermentation via the reaction: NADH + H+ NAD+ + H2. In the cyanobacterium Synechococcus 7002, nitrate reduction and lactate production are two pathways that utilize reductant during fermentation and therefore compete with hydrogenase. These pathways have been eliminated in this study. Lactate dehydrogenase utilizes one equivalent of NADH to reduce pyruvate to lactate during fermentation. Nitrate metabolism is largely unexplored during fermentative metabolism, but is expected to be analogous to photoautotrophic nitrogen metabolism in which nitrate is reduced to nitrate with 2 electrons from ferredoxin. Nitrite is further reduced to ammonium with 6 electrons. Methods: Cultures were grown photoautotrophically to stationary phase in medium A sparged with 2% CO2. Cultures were prepared for fermentation experiments by washing cells in media lacking nitrate and then resuspending cells media lacking nitrate or with noted concentration of nitrate added. Anoxic conditions were induced in 10mL vials with 3mL headspace by wrapping the vials in foil and purging with argon. The hydrogen concentration in the headspace was measured using gas chromatography. The metabolite concentrations in the extracellular media were quantified using proton NMR spectroscopy. Nitrate/nitrite concentrations in the media and August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts sugar concentrations in the cells were assayed chemically. The ldhA gene was insertionally inactivated to create the mutant ldhA. Results: In order to examine the effect of nitrate limitation under fermentative conditions, cells were resuspended in fresh medium A containing 0 µM, 200 µM, or 1 mM sodium nitrate. Eliminating nitrate from the media prior to fermentation gives a 10-fold increase in the rate of hydrogen production as compared to media containing 200 µM or 1 mM nitrate. The cells assimilate 100% of the extracellular nitrate added within one day of fermentation. Nitrite is detected in the extracellular media after four days of fermentation. The cells that assimilate 200 µM nitrate excrete 70 µM nitrite; the cells that assimilate 1 mM nitrate excrete 700 µM nitrite. The presence of nitrate in the fermentation media acts as an outlet for reductant during fermentation. Nitrate competes for reductant with hydrogenase as evidenced by the 10-fold increase in in vivo hydrogen when nitrate is absent. The primary product excreted by Synechococcus 7002 under fermentative conditions when nitrate is not present is lactate, at a constant rate of 21.6 mol*day-1 per 1017 cells over the 4 days experiment. To redistribute reductant flux away from lactate and towards hydrogen, a mutant lacking D-lactate dehydrogenase was examined under fermentative conditions. The ldhA mutant excretes no lactate, while all other metabolites (hydrogen, acetate, alanine, and succinate) are excreted in higher amounts compared to WT. Hydrogen production increases 5-fold in ldhA cultures, to a rate of 14.1 mol*day-1 per 1017 cells. Conclusions: We have gained insight into carbon and nitrogen metabolism of these photoautotrophs during fermentative metabolism, a branch of metabolism largely unexplored in cyanobacteria. We show increases of 10-fold, with removal of nitrate, and 5-fold, with removal of pyruvate reduction to lactate. Engineering photoautotrophs gives promise for obtaining substantial increases for producing biofuels.
OC-12.3 PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATION TRANSFER TO PHOTOSYSTEM I IN THE FILAMENTOUS CYANOBACTERIUM NOSTOC PUNCTIFORME. Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring, Ann Magnuson. Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden. Cyanobacteria as well as eukaryotic algae and plants have the capacity to adapt to varying light conditions by controlling the amount of excitation energy distributed to the photosystems. On the minute time scale this control leads to so-called state transitions, that in cyanobacteria involve the movement of the phycobilisomes (PBS) as well as non-photochemical quenching. On longer time scales, cyanobacteria regulate the size and protein composition of PBS as a response to light conditions (‘chromatic adaptation’) on the genetic level. State transitions have been suggested to be dependent on the redox state of the plastoquinone pool, which in part results from electron transfer between the photosystems, but in spite of decades of research, the molecular mechanisms behind the observed phenomena are still not fully understood. In the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 ca 5-10% of the vegetative cells differentiate into heterocysts. The process brings extensive structural reorganization of the thylakoid membranes, and due to oxygen sensistivity of the nitrogenase,
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Final Program and Abstracts Photosystem II is inactivated in the heterocyst. In the heterocyst thylakoids, cyclic electron transfer around Photosystem I (PSI) is essential for nitrogen fixation, but linear electron transfer between the photosystems does not take place. Nevertheless, isolated heterocysts display changes in the excitation transfer to PSI reminiscent of a state transition.
determined as DP 3 through 6 (maltotriose to maltohexaose).
Here we present a characterization of the composition and functionality of thylakoid membranes of the heterocysts. We have investigated the ability of the heterocyst to regulate excitation transfer to PSI, and present the outline of a mechanistic model for excitation re-distribution that does not depend on linear electron transfer.
OC-12.5
OC-12.4 GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC 7942. Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, Yasunori Nakamura. Akita Prefectural University, Akita, Japan. Introduction Our previous analysis of the mutants of Synechococcus elongatus PCC 7942 defective in glycogen biosynthesis indicated that they were highly susceptible to salt and oxidative stresses. It is therefore suggested that the storage of the polysaccharide and its efficient mobilization are very important for cyanobacteria to cope with various environmental stresses. It is postulated that the degradation of storage β-glucans in bacteria (glycogen or starch) is mediated by phosphorylase (PHO), debranching enzyme (DBE) and 1,4-glucanotransferase (disproportionating enzyme, DPE), encoded by glgP, glgX and malQ, respectively. However, their roles in carbohydrate metabolism of cyanobacteria have been poorly investigated. In the present study, mutants of glgP, glgX and malQ have been constructed and characterized for the catabolic activity of glycogen accumulated in the cell. S. elongatus PCC 7942 is suitable for the functional analysis of the genes involved in the glycogen catobolism, as the organism contains just one each of these genes. Methods The three genes of S. elongatus PCC 7942 were disrupted independently by insertional inactivation using antibiotic resistant markers. Inactivation of the targeted enzyme was confirmed by in vitro assays as well as non-denaturating electrophoresis using amylopectincontaining polyacrylamide gels, followed by iodine staining. Distribution of chain length (as expressed by degree of polymerization, or DP) in the debranched glycogen and maltooligosaccharides was determined by capillary electrophoresis. Results Comparison of the electrophoretic patterns of crude extracts between wild type (WT) and the mutants led us to the identification of the specific bands on the zymograms, corresponding to the activities of all three enzymes. When grown under continuous illumination, the phenotype of the mutants was not clearly distinguished from that of WT, except for the increased accumulation of glycogen and relative increase of short chains in the glycogen molecule (DP 2 through 6) in glgX mutant. The cellular content of glycogen was therefore examined in the continuous darkness, where the turnover of the polysaccharide was stimulated. After 72 hours of dark treatment, the glycogen content in WT cells was decreased to 27% of the initial level. The addition of 0.2 M NaCl to the culture further enhanced the glycogen breakdown (to 6% of the original level after 72 hours) in WT cells. Under the same conditions, the activity of glycogen degradation in the glgP, glgX and malQ mutants was markedly reduced as compared to WT. In addition, accumulation of malto-oligosaccharides was observed specifically in malQ mutant, irrespective of the growth conditions. The molecular species of the malto-oligosaccharides was 70
Conclusions These results demonstrated the pivotal roles of the three enzymes PHO, DBE and DPE in the glycogen catabolism of cyanobacteria.
IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTIS AERUGINOSA UNDER IRON LIMITATION. Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1. 1
School of Biotechnology and Biomolecular Sciences; 2School of Civil and Environmental Engineering; University of New South Wales, Sydney, Australia. Introduction: The production of toxins during cyanobacterial blooms poses a significant public health threat in water bodies globally and requires the urgent development of effective bloom management strategies. Microcystin is a hepatotoxin synthesised non-ribosomally by members of several cyanobacterial genera. Although the microcystin biosynthesis pathway is now characterised, the contribution of toxicity to the adaptation of cyanobacteria to environmental stresses, such as changing light intensity and nutrient limitation, is still unclear. Previously, microcystin synthesis has been proposed to be regulated by iron availability. The aim of this study was to compare the transcription of genes involved in iron uptake and redox control in toxic and non-toxic Microcystis aeruginosa subjected to moderate and severe iron stress.
Methods: Three strains of M. aeruginosa, the toxic PCC7806, the nontoxic PCC7005 and the mutant mcyH-, were grown in Fraquil media containing 10-1000 nM Fe. The transcription of a number of genes involved in iron uptake, oxidative stress response and toxin synthesis was accessed by quantitative real-time PCR (qRT-PCR). The possible involvement of the ferric uptake regulator homologues FurA, FurB and FurC in regulating toxicity and survival under redox stress was also assessed. Results: Both non-toxic strains (PCC7005 and mcyH-) used in this study showed pronounced bleaching by the end of the experiment, with the growth of mcyH- being significantly retarded under iron limitation. Toxin production in PCC7806 was increased in an irondependent manner and appeared to be regulated by FurA. The transcription of the genes studied here showed a high degree of variability between strains and the growth stage at which the cells were harvested. The inability to produce microcystin, either due to natural mutations in the mcy gene cluster or insertional inactivation of mcyH, affected the remodeling of the photosynthetic machinery in iron-stressed cells and the transport of Fe3+ in early exponential growth. Conclusion: This is the first comprehensive study on the effects of iron starvation in toxic and non-toxic M. aeruginosa. Our results show that the process of adaptation to iron stress is strain-specific and highly dynamic. Toxicity appears to protect microcystin-producing cyanobacteria in the early stages of exposure to severe iron stress and may protect the cell from reactive oxygen species-induced damage. We have initiated proteome studies to confirm this hypothesis.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
OC-12.6 THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITE WITH ECOPHYSIOLOGICAL ROLES. S.B. Watson1, F. Juttner2. 1
Environment Canada, Canada Centre for Inland Waters, Burlington, ON, Canada; 2Limnological Station, University of Zurich, Switzerland. Introduction: Microcystis species produce sulphides, a sometimes significant source of malodour. Although well documented in marine systems, little is known about freshwater photoautotrophic production of sulphides, which to date has been generally regarded as insignificant. However, the apparent increasing number of Microcystis blooms may in fact represent a significant source of these compounds in many inland waters. Methods: We investigated the physiology and cellular/ecological function of sulphide production by four axenic and non-axenic Microcystis aeruginosa strains. Using short-term incubations and an adapted Headspace-GC-MS technique and more focussed studies with the axenic strain using different light regimes, metabolic inhibitors (sodium azide, DCMU), antioxidant enzymes (superoxide dismutase, catalase) and isotopically labelled precursors (hydrogencarbonate, acetate and sulphate) Results: We found that isopropyl sulphides were of cyanobacterial in origin. Our work demonstrated that i) isopropyl thiol (ISH) is the initial cell product, with subsequent extracellular conversion to diisopropyl disulphide and trisulphide); iv) ISH is actively produced over the growth cycle, iii) is continuously excreted with no internal storage or post-lysis catalytic generation; iii) production occurs in both light and dark, increases up to an optimal light level and decreases significantly under very high irradiance; iv) labelling patterns indicate ISH is synthesized via the acetate pathway.
Final Program and Abstracts Methods: Genome sequence data of Thiodictyon sp. Cad16 was generated by pyrosequencing and annotated using software packages from Softberry Inc. (www.softberry.com). Results: The core genes of all analyzed GSB strains largely exhibit congruent phylogeny. Photosynthesis genes are largely in agreement with this core phylogeny. However, sulfur metabolism genes are often not congruent and are scattered across the GSB lineages suggesting extensive horizontal gene transfer events. The PSB Thiodictyon sp. Cad16 has an unexpectedly large genome (~8 Mbp), which reflects the large physiological diversity of this strain. CO2 fixation metabolism is supplemented by carboxysomes, previously not known to occur in PSB. Genes encoding sulfur metabolism exhibit unexpected combinations and similarities with both GSB and other PSB. Conclusions: GSB have relatively small genomes (2–3 Mbp) reflecting their limited physiological capabilities. Some PSB have very large genomes (~8 Mbp), whereas other PSB have much smaller genomes (3–4 Mbp), probably reflecting large variations in their environmental adaptation. Finally, whereas the cellular core and key metabolisms such as photosynthesis and carbon assimilation have rather different origins in GSB and PSB, their sulfur metabolism appears to share a common origin.
OC-13.2 THE GENOME OF THE TOXIC BLOOM-FORMING CYANOBACTERIUM ANABAENA SP. 90. David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2, Kaarina Sivonen1. 1
Department of Microbiology and Applied Chemistry, University of Helsinki, FIN-00014, Helsinki, Finland; 2Beijing Institute of Genomics of the Chinese Academy of Sciences, Beijing Genomics Institute, Beijing, China.
Conclusions: These experiments and related work using the two aquatic invertebrates Thamnocephalus and Daphnia indicate that isopropyl thiol plays several important physiological and ecological roles. It acts as an effective antioxidant against high levels of reactive oxygen species, particularly in surface blooms; it elicits avoidancerelated behavioural responses in grazer communities and at high levels, can be toxic to some invertebrates.
Introduction: Toxic cyanobacterial blooms are linked to the deaths of wild and domestic animals and are a health risk for human beings via recreational or drinking water. Strains of the genus Anabaena are important primary producers in freshwater bodies throughout the world and contribute to the formation of noxious cyanobacterial blooms through the production of a range of hepatotoxins and neurotoxins. Here we report the genome sequence of a hepatotoxic bloom-forming cyanobacterium Anabaena sp. 90.
OC-13.1
Methods: We constructed a whole-genome shotgun assembly from 119,316 sequencing reads from 2 kb, 6 kb and 40 kb libraries. These were organized in pairs by virtue of end-sequencing 6-kb and 50-kb inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 12.5 times.
COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND PURPLE SULFUR BACTERIA. N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3. 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Plant Biology Department, University of Geneva, Geneva, Switzerland; 3Department of Biochemistry and Molecular Biology, Pennsylvania State University, State College, Pennsylvania, USA.
Introduction: Green sulfur bacteria (GSB) and purple sulfur bacteria (PSB) are photoautotrophs that oxidize reduced sulfur compounds for growth. Whereas some aspects of their ecology are similar, other aspects of their physiology and evolution are rather different. We are interested in the evolution of these lineages and their photosynthetic, sulfur, and carbon metabolisms. Genome sequence data are available for >12 strains of GSB, but not for any PSB of the Chromatiaceae. We have therefore sequenced the genome of the PSB Thiodictyon sp. Cad16, which grows both photolithoautotrophically and chemolithoautotrophically.
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Results: The length of the genome amounts to 5,305,674 bp and is partitioned into two circular chromosomes and three plasmids. Essential house keeping genes were distributed across both chromosomes while the plasmids consisted largely of hypothetical open reading frames. A total of 5,750 putative ORFs were annotated from the genome with just over 20% of the predicted ORFs being unique to Anabaena sp. 90. This strain is known to produce toxins and enzyme inhibitors and over 5% of the genome is dedicated to the production of bioactive compounds. During the assembly we documented insertions, deletions and single nucleotide polymorphisms which in some cases has lead to the inactivation of biosynthetic pathways. Interestingly, approximately 7% of the genome is composed of repetitive autonomous and non-autonomous mobile DNA which have inactivated a number of genes. Together this suggests a mechanism by which Anabaena can tailor and customize its
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Final Program and Abstracts genomic repertoire through genomic rearrangements. Analysis of the genome revealed an investment in restriction modification systems explaining in part difficulties in genetic manipulation of this organism. Bioinformatic analysis revealed strong similarities to other heterocyst forming cyanobacteria and preliminary evidence that phage infection has shaped the genome of this bloom forming cyanobacterium. Conclusions: Further research will target a system biological investigation of this organism and is likely to reveal in-depth information of this ecologically relevant, bioactive compound producing cyanobacterium.
OC-13.3 CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPMENT AND BEHAVIOR OF HORMOGONIA OF NOSTOC PUNCTIFORME. Jack Meeks, Elsie Campbell, Rui Chen. Department of Microbiology, University of California, Davis, CA, USA. Introduction: The genome of the symbiotic nitrogen-fixing cyanobacterium Nostoc punctiforme contains five loci of genes that encode core complex proteins analogous to the chemotaxis proteins CheY, CheW, MCP and CheA of Escherichia coli. Genes in four of the loci appear to have evolved in the cyanobacterial lineage, while those in the fifth locus have roots in the proteobacteria. We have set out to characterize these genetic loci in terms of organization and physiological role. Methods: A DNA microarray of N. punctiforme was used to examine global transcription profiles during nitrogen starvation induction of heterocysts and hormogonia, and plant produced exudate containing a hormogonium inducing factor. The data were analyzed in the R statistical platform and normalized log2 of reference/experimental values were k-means-clustered using the program Genesis. Insertion mutants were constructed by gene replacement, and cellular differentiation and behavior phenotypes analyzed by standard techniques. Results: Global transcriptional analyses indicate that 13 out of 35 chemotaxis-like genes are expressed at a low level in ammoniumgrown cultures; these include 3 CheB methylesterase and 2 CheR methyltransferase-like adaptive proteins, all of which lack the response regulator receiver domain and their genes are not collocalized in the mentioned loci. Transcription of genes in the four cyanobacterial lineage loci are up-regulated only during hormogonium differentiation, except for three genes in different loci (a MCP in locus 1, and a cheA and cheW in locus 3) that are not transcribed under any condition examined. Insertion mutations in genes encoding MCP proteins in the four loci in the cyanobacterial lineage yielded two distinct phenotypes: a locus 2 MCP mutant fails to differentiate hormogonia and a locus 4 mutant is defective in phototaxis. Hormogonia of a locus 1 mutant have a slight morphological alternation, but are phototactic and infect a bryophyte symbiotic plant partner, while a mutant in the MCP of locus 3 has no detectable phenotypic defect. The chromophore bound to the MCP of locus 3 undergoes green-red light photoconversion. The genes in locus 4 (phototaxis) include duplicates encoding CheW and partial MCP and CheA proteins. Insertion mutations in the duplicate MCP and cheA genes yield phototaxis defective phenotypes. Only one MCP of locus 5 (NpR0247) is significantly up-regulated late in hormogonium differentiation, but the gene is difficult to manipulate in E. coli. Conclusions: The transcription and mutant phenotypes indicate a variable organization of the multiprotein sensory transduction
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complexes encoded in the four cyanobacterial lineage loci. We propose the proteins encoded by locus 4 assemble into a soluble complex as heterodimeric MCP, CheW and CheA proteins, with one or both CheY proteins communicating with the motility motor(s). Conversely, the proteins encoded in the membrane-associated locus 2 complex (development) assemble as homodimers. The homodimeric CheA and CheW of locus 1 may associate with the MCP encoded in a different locus. The MCP of locus 3 may assemble as a homodimer with homodimeric CheW and CheA proteins also encoded in a different locus, perhaps locus 1. The MCP of locus five may be a chemotactic sensor and interact with proteins encoded in other loci.
OC-14.1 COMPARISON OF NONCODING FEATURES OF CYANOBACTERIAL GENOMES. Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, José Costa5. 1
Virginia Commonwealth University, VA, USA; 2University of California at Davis, CA, USA; 3University of Pennsylvania, PA, USA; 4Uppsala University, Uppsala, Sweden; 5University of Porto, Porto, Portugal. Introduction: Insights from genomic sequences have been gained primarily analysis of the genes and protein functions inferred from them, but much can be learned beyond this analysis. Short repeated sequences that are not explained by inclusion within multicopy genes pose interesting biological questions: (1) By what mechanism are the repetitive units formed? (2) What selective forces act to preserve them? and (3) What effect do they have on cellular function? Methods: The sequences of 42 cyanobacterial genomes (36 complete) were analyzed using BioBIKE, an integrated knowledge base and programming interface. DNA methylation was inferred by the presence of orthologs of DNA methyltransferases with known methylation activity. Results: Sequences of at least 24 nucleotides that appear multiple times in the cyanobacterial genomes were dominated by three types: (1) long dispersed repeats often associated with transposes (hence probably insertion sequences), (2) repeated sequences interspersed with non-repeated elements, characteristic of CRISPRs (clustered regularly interspersed short palindromic repeats), and (3) short dispersed repeats (termed SDRs) not previously described. All of these are potentially mobile sequences. We have shown by intergenomic comparisons that SDR sequences, ranging in size from 21 to 28 nucleotides, have moved in recent evolutionary times. They may be the smallest mobile sequences known. Three families of SDR sequences (SDR4, SDR5, and SDR6). can assume the same conserved secondary structure, even though they do not share significant sequence similarity. This suggests that they act through an RNA intermediate. One family, SDR5, specifically targets HIP1 sites (GCGATCGC). For ease in discussion, we define Group F of cyanobacteria to consist of the filamentous cyanobacteria plus those unicellular cyanobacteria within the genera Synechocystis, Cyanothece, Crocosphaera, Microcystis, and Acaryochloris, as well as Synechococcus PCC 7002. Group P consists of all small marine Prochlorococcus and Synechococcus. The remaining cyanobacteria, Synechococcus PCC 7942/6301, Thermosynechococcus, hot spring Synechococcus, and Gloeobacter are ungrouped. All Group F cyanobacteria were found to possess a high frequency of HIP1 (GCGATCGC) sequences, as do Synechococcus PCC 7942/6301 and Thermosynechococcus but not other genomes. Insertion
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13th International Symposium on Phototrophic Prokaryotes sequences were found to be prominant in all Group F cyanobacteria but were seldom found in Group P cyanobacteria. CRISPR sequences were confined to Group F (though absent in many). Orthologs of dmtA (encoding a GATC-specific DNA methyltransferase) are found in all cyanobacterial genomes outside of Group P. GGCC- and CGATCGmethylation (inferred from the presence of orthologs of dmtB and dmtC, respectively is found exclusively within Group F cyanobacteria. SDR sequences were frequent in heterocystous (filamentous) cyanobacteria and very rare in other genomes. Conclusions: DNA sequence characteristics – the frequency of repeated sequences and methylation patterns – can be used to define cyanobacterial groups that make sense by comparison with other phylogenetic measures. The groupings may relate to underlying molecular properties, e.g. recombination (HIP1) or possibly DNA replication (GATC methylation). It is interesting that three conserved features have overlapping specificity: GATC-specific methylation, CGATCG- specific methylation, and HIP1 (GCGATCGC), suggesting overlapping function.
OC-14.2 HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) AS ELECTRON DONOR? SEARCHING FOR THE ANSWER IN THE MEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM 263. Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, Ulrich Fischer1. 1
Zentrum für Umweltforschung und nachhaltige Technologien (UFT) and Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie, Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie; 3 Fachbereich Biologie/Chemie, Laboratorium für Allgemeine Mikrobiologie, Universität Bremen; Bremen, Germany. Intoduction: Green sulfur bacteria are anaerobic anoxygenic photolithoautotrophs provided with a photosystem of type I and with peculiar organelles, the chlorosomes, in which the light-harvesting pigments are organized. These bacteria couple the fixation of CO2 to the oxidation of inorganic sulfur compounds or, in only one known case, ferrous iron. When sulfide or thiosulfate are the electron donors, elemental sulfur (S0) is an intermediate product, which can be further oxidized to sulfate. S0 is thus a key substrate, whose insolubility in water constitutes a special task for the living cells. At present, it is unknown how green sulfur bacteria mobilize S0 and if the protein components involved are subjected to transcriptional control. Methods: The mesophilic strain Chlorobaculum parvum DSM 263, whose genomic sequence is now available, was chosen to investigate the mechanism of S0 mobilization in green sulfur bacteria. To preliminarily assess whether the cells need a direct contact with sulfur for its oxidation, previously prepared biogenic sulfur was embedded into agar and brought in contact with a living culture. The appearance of cleared zones at the border between cell culture and agar was then monitored. For the proteomics experiment, Clb. parvum DSM 263 was grown only on sulfide or on biogenic sulfur previously extracted from a culture of the same species. French press was used to disrupt the cells and ultracentrifugation to separate the soluble fraction from the membranes. 2-dimension gel electrophoresis with non-equilibriumpH-gradient-electrophoresis (NEPHGE) as first dimension and matrix-assisted-laser-desorption/time-of-flight (MALDI/TOF) mass spectrometry were used to identify which proteins are differentially August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts expressed in the membranes of the 2 populations. Results: Neither a clear zone could be observed in the agar with biogenic sulfur, nor a culture could grow if the embedded sulfur was the only electron donor present. These results allowed a focus on the membrane fraction, in which differences in the analysed protein patterns were found, a major one regarding a conserved hypothetical protein. Conclusions: We could show that cells of Clb. parvum DSM 263 need a direct contact with S0 for its mobilization and that at least some of the proteins involved in the utilization of this electron donor appear to be subjected to transcriptional control.
OC-14.3 PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS: INSIGHTS INTO THE SULFUR METABOLISM OF A PHOTOTROPHIC GREEN BACTERIUM. Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, Anders Mellerup2, Kirsten S. Habicht3, Raymond P. Cox1, Jens S. Andersen1 & Niels-Ulrik Frigaard2. 1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK5230 Odense; 2Department of Biology, University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center for Earth Evolution and Institute of Biology, University of Southern Denmark, Odense; Denmark. Introduction: Green sulfur bacteria (GSB) use reduced sulfur compounds as electron donors for their photosynthetic carbon dioxide fixation. They are often found in stratified lakes at, or below, the level in the chemocline where sulfide is exhausted. The sulfur metabolism in GSB is complex; almost all green sulfur bacteria oxidize sulfide (HS-) and elemental sulfur (S0) but in addition some are able to utilize thiosulfate (S2O32-). GSB have a high affinity for sulfide and this substrate is taken up in preference for other sulfur substrates such as thiosulfate. Predictions from the genome sequence of Chlorobaculum (Cba.) tepidum TLS suggest that at least 60 gene products participate in sulfur oxidation pathways (1). Methods: To obtain information about gene expression during various stages of the sulfur metabolism of Cba. tepidum, we have grown the cells in batch culture in a medium containing both sulfide and thiosulfate. Proteome analyses were performed at two stages: once in the early exponential growth phase where the cells are consuming sulfide, and once at the end of the exponential growth phase where the cells have utilized almost all thiosulfate. Proteins were extracted, digested with trypsin and the peptides released were analyzed using an Agilent HPLC coupled to either a LTQ FT Ultra (Thermo) or a LTQ Orbitrap XL (Thermo) mass spectrometer. Proteins where identified using Mascot and quantified using MaxQuant. Samples were run in duplicate on each system and the relative expression of proteins was calculated on basis of the measured intensities. Results: In total 945 proteins of the 2,288 coding sequences on the Cba. tepidum genome were identified on the basis of at least two unique peptides. We detected 34 of the 60 proteins proposed to be involved in the sulfur metabolism of Cba. tepidum and most of these proteins were equally expressed in the two samples. However, in cells in the early exponential growth phase with active sulfide uptake we found a two-fold up-regulation of the dissimilatory sulfite reductase enzymes DsrE, DsrF, DsrH and the Rubisco-like enzyme RLP compared to cells growing on thiosulfate where the sulfur oxidizing enzymes SoxX, SoxY & SoxZ and quinone modifying oxidoreductase QmoA & QmoB and the soluble cytochrome c555, CycA were up-
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Final Program and Abstracts regulated. Results will also be presented on proteome analysis of a mutant of Cba. tepidum that lacks the DSR enzyme system and is deficient in elemental sulfur utilization. (1) Frigaard & Bryant, 2008. p 60-76. In C Dahl and CC Friedrich (eds.), Microbial sulfur metabolism. Springer, New York
OC-15.1 A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALE PHOTOBIOLOGICAL HYDROGEN PRODUCTION UTILIZING CYANOBACTERIA. Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue. Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka, Kanagawa, Japan. Introduction: In order to meet for the demand for large-scale production of renewable energy, we have proposed nitrogenasebased photobiological production of H2 using maricultured cyanobacteria. Although most of the technologies are still in the beginning stages of development, a preliminary trial cost analysis was made in order to assess the future prospects for the economical viability of large-scale renewable energy production. Methods: Assumptions: 1) Total solar radiation on the surface of Earth: 2.7 x 106 EJ per year (or 5.3 x 109 J m-2 year-1, 1,680 kWh m-2 year-1 or 14.5 MJ m-2 day-1) on average. 2) Cyanobacteria photobiologically produce H2 at 1% efficiency vs. total solar radiation (5.3 x 107 J m-2 year-1). 3) Finally, available H2 energy: one and two thirds of biologically produced H2 energy, consuming two and one third needed for harvesting, purification, storage, and transportation to land (1.8 and 3.5 x 107 J m-2 year-1, respectively, in the final product). 4) Area required: 38.8 x 1018 divided by 1.8 and 3.5 x 107 = 2.2 and 1.1 x 1012 (m2) (=2.2 and 1.1 x 106 km2), respectively. Results: 1. A plausible general scheme 1) Cyanobacteria produce H2 with sunlight as the sole source of energy. The substrate is water and products are H2 and O2. The culture medium is simple in composition and contains mineral nutrients, and the gas phase in the bioreactor is Ar plus low concentrations of CO2 and N2. 2) The bioreactor consists of three layers plastic bag (inner: retains culture medium, middle: hydrogen gas barrier, outer: mechanically protects the inner twos) and is floated on a calm sea surface. 3) Repeated harvesting of gas. When H2 gas attains a certain concentration level (e.g. 25 - 30%, v/v), a factory ship harvests the gas mixture (main components: Ar, H2, O2) from bioreactors, and purifies H2 on board. 2. Cost analysis 1a) Nutrients. Potentially growth-limiting nutritional elements are added to the natural water (20 cm in depth) as ‘fertilizers’ akin to agricultural practices. [1.5 - 12 cents m-2 of the bioreactor surface] 1b) Water. Reference price of water for industrial use in Japan at about 7.5 - 60 cents per ton. [1.5 - 12 cents m-2] 1c) Bioreactor. The average plastic price of $2 - $4 per kg and the plastics are recycled at the half prices of the feed stocks [50 - 100 cents per m2 of bioreactor,] 1d) Gas (mostly Ar). FOB, bulk rate of Ar: $100 - 500 per ton. [9 cents 45 cents per m2] 74
13th International Symposium on Phototrophic Prokaryotes 1e) Other costs: the cost of growing cyanobacteria, the labor cost, the cost of ships, the interest on capital goods, and the cost of marine transportation of production materials. [Cost E]. 1f) Cost of gas harvesting, H2 separation, storage, and marine transportation to final destination. [Cost F] 2) Depending on the Cost E and F, the final cost of H2 at end-users: 1.8 - 13.4 (in cents MJ-1). Comparison; wind electricity: 2.0 - 5.2, solar photovoltanics: 10 - 50, crude oil: 0.8 - 2.4 (50 - 150 dollars barrel-1) or 2.4 - 7.2 (conversion efficiency of 1/3 into electricity). Conclusions: Our trial calculation indicates that it will be difficult for bio-solar H2 production to be competitive with fossil fuels at current prices, but as specific policies come into play such as carbon taxes, there will likely be an acceleration of its development. In spite of the technical as well as economical difficulties, we propose that largescale H2 production by mariculture is the most promising strategy to be pursued for “mitigating dangerous anthropogenic interference with the climate system” (the purpose of UNFCCC).
OC-15.2 SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTION AMONG DIVERSE HETEROCYSTOUS CYANOBACTERIA. Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples, Polly Berseth, Tommy Sessions. Savannah River National Laboratory, Aiken, SC, USA. Introduction: H2 generated by cyanobacteria is a highly attractive form of alternative energy. Much progress has been made to this end, yet an important element of the research continuum has been largely overlooked in recent years - the physiology and diversity of naturally occurring, H2-producing cyanobacteria. The overall goal of the research described here was to develop and apply a systematic approach for evaluating the H2 production capacity among diverse heterocystous cyanobacteria. Specifically we wished to: 1) compare several environmental parameters (O2 concentration, N2 concentration, light intensity, and carbon source) that control H2 production among diverse heterocystous cyanobacteria, and 2) establish benchmark activities/phenotypes against which to routinely evaluate newly discovered H2 producing cyanobacteria. Methods: Cyanobacteria (10 diverse heterocystous strains were tested) were cultured in Roux flasks containing BG11- media bubbled rapidly with air under a light (70-100 µmol m-2 s-1)/dark photocycle (16:8 h). To assay biological hydrogen production, cells were concentrated via centrifugation, washed, and placed in sealed vials where an argon atmosphere (≥ 98.5% argon) was generated. Headspace gas compositions of the vials were adjusted with N2, O2, or CO2 and gas samples were analyzed with MTI Model M200D micro gas chromatograph equipped with a TCD detector. Results: All strains exhibited an immediate inhibition of H2 production rates upon the addition of N2. Compared to the activity of cells incubated in vials containing <1% N2 headspace volume, H2 production rates measured for the individual strains decreased 38-61% in the presence of 5% N2, 71-92% in the presence of 20% N2, and 8997% in the presence of 80% N2. It was determined that although nitrogenase is sensitive to O2, low levels of O2 (typically 2.5-5% atm) are required for respiratory metabolism to generate sufficient reductant and/or ATP to drive H2 production. The addition of glucose (10 mM) accelerated H2 production in 7/10 strains examined, with rate increases ranging from 1.1-14.5 fold and yields increasing 1.4-88.1 fold. When cells were incubated in the absence of an exogenous carbon source, all of the strains exhibited dramatic increases in H2 yields (13.2-18.0 fold) when the light intensity was increased from 30 August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes to 200 µmol m-2 s-1. The stimulatory effect of increased light intensity on cyanobacterial H2 yield was generally not as pronounced when strains were incubated in the presence of glucose (1.4-10.5 fold increases). Of particular interest, cells of 4 strains exhibited near maximal H2 yields at light intensities <100 µmol m-2 s-1 when incubated with glucose Conclusions: The immediate, inhibitory effect of N2 on H2 production was quite similar across a phylogenetically diverse collection of heterocystous cyanobacteria, enabling us to establish a baseline from which to evaluate the “N2 sensitivity” of H2 production among these organisms. Our results also highlight that it is critical to optimize reductant availability for H2 production. Addition of sugars and low levels of O2 (2.5-5% atm) may be useful for many strains. The commonly held belief that the economic feasibility of cyanobacterial H2 production is dependent primarily on the light to H2 conversion efficiency may be fundamentally flawed. Mixotrophic conditions should be explored as a means to produce biohydrogen with cyanobacteria.
OC-15.3 THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLE BACTERIA WITH DARK FERMENTATIVE HYDROGEN PRODUCTION AND HYDROGEN ELECTRODE. A. Tsygankov. Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia. Introduction: Green and cost-effective H2 production and H2dependent electricity generation are of the key problems restricting the development H2-based economy. Purple bacteria produce H2 using light energy and simple organic compounds with high rates. The source of organic compounds is one of factors preventing development of biotechnological H2 producing systems based on purple bacteria. Last years in different laboratories the integration of purple bacteria with dark fermentation of organic wastes are under investigation. This presentation describes the integrated system consisting of dark fermentation of wastes with H2 and concomitant organic acids production, of purple bacterial H2 production under the light using organic acids, and of hydrogen enzyme electrode for the electricity generation using H2. Methods: Experiments used bioreactors for dark fermentation, photobioreactors for suspension and immobilized purple bacteria, and hydrogen enzyme electrodes with various hydrogenases separately and in combination. Results: The stability of dark fermentation and light-dependent H2 production by purple bacteria in continuous mode of operation was studied and it was shown that these processes are stable for months. Operational stability of hydrogen enzyme electrode in buffer system, as well as in medium from photobioreactor was studied also. Different combinations of reactors with photobioreactors, hydrogen electrodes with bioreactors were tested and results will be presented in detail. Conclusion: Results indicate that there are no principal factors preventing an integration of bioreactors and hydrogen enzyme electrode exist. However, the system needs in optimization for electricity output. The research was supported by Russian RAS Program for Basic Research “Chemical aspects of energetics” and by Russian Foundation of Basic Research.
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OC-15.4 HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUM RUBRUM UNDER RESPIRATORY DARK CONDITIONS. Lisa Zeiger, Christiane Rudolf, Hartmut Grammel. Max-Planck-Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany. Introduction: The aim of this study was to determine the maximal achievable cell concentration of the facultative photosynthetic bacterium Rhodospirillum rubrum in a bioreactor, thus allowing the estimation of potential volumetric yields in bioprocesses. Mass cultivation of purple non-sulfur bacteria, incl. R. rubrum under photosynthetic conditions is generally inefficient due to the inevitable limitation of light-supply when cell densities become very high. For R. rubrum however, a cultivation process has been described that allows the high-level expression of photosynthetic membranes (PM), equivalent to light-grown cultures, under completely dark conditions when grown semi-aerobically in a bioreactor. On the basis of this cultivation system, we developed a process for obtaining very high cell densities (HCD) in aerobic and semi-aerobic fed-batch cultivations. We also present a biochemical analysis of HCD cells and show that the physiology of R. rubrum is affected significantly by the applied conditions. Methods: For HCD fed-batch cultivations, an exponential feeding algorithm implemented in a computer process control system was applied. An unstructured computational model, based on mixedsubstrate kinetics was developed for optimization of the process in simulation studies. For aerobic fed-batch cultivation, the exponential feed algorithm was applied in combination with a pH-stat element to prevent substrate accumulation. Semi-aerobic fed-batch cultures were conducted with a different control strategy, using the culture redox potential as the manipulated variable. The obtained high density cells were characterized by biochemical analyses, absorption and fluorescence spectroscopy, and HPLC-MS. Results: Key growth parameters (saturation constants, inhibition constants, etc. ) were determined prior to the fed-batch experiment and used to parameterize the computational model and the controller algorithm. Two carbon substrates, fructose and succinate, were present in the feed because of the previously described elevated PM levels in cells grown semi-aerobically in this medium composition. The applied exponential fed-batch strategy resulted in a maximum cell density of 59 g L-1 cell dry weight in aerobically grown cultures. To our knowledge, comparable values have never been reached before with R. rubrum or related species. An interesting finding was that high density cells accumulated and excreted large amounts of tetrapyrrole intermediates which were identified as protoporphyrin IX and Mgprotoporphyrin IX monomethyl ester. Furthermore, attempts to induce PM expression by applying oxygen-limiting conditions surprisingly failed. Conclusion: In this study we demonstrate that it is possible to cultivate R. rubrum under chemoheterotrophic conditions to, so far inaccessible high cell densities. Biochemical analyses revealed regulatory imbalances in HCD cultures at the branchpoint of aerobic and anaerobic tetrapyrrole biosynthetic pathways. An additional finding was that the induction of PM expression is dependent on the cell density and PM formation is severely impaired in HCD cells. The results are important for the developments of bioprocesses using this organism, and provide an interesting experimental system for the investigation of regulatory mechanisms at the hemebacteriochlorophyll metabolic branch. 75
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13th International Symposium on Phototrophic Prokaryotes
OC-15.5
OC-15.6
DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA IN MISSISQUOI BAY, QUEBEC, USING Q-PCR.
EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENES AND THE GENETIC POPULATION STRUCTURE OF TOXIC CYANOBACTERIA.
Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, Frances Pick3, David Bird4, Charles W. Greer1. 1
National Research Council, Biotechnology Research Institute, QC; Environmental Health Center, Health Canada, ON; 3Biology Department, University of Ottawa, ON; 4Biological Sciences, University of Quebec at Montreal, QC, Canada. 2
Toxic cyanobacterial blooms, and their increasing global occurrence, pose a serious threat to human health, domestic animals and livestock. The frequency and severity of bloom events continue to rise, most probably as a direct result of increased nutrient loading of water systems worldwide. The number of lakes in Quebec, Canada, affected by blooms has been increasing steadily from 34 (2004), to 45 (2005), 84 (2006) and 197 (2007). Government agencies are under tremendous pressure to cope with escalating demands for water analysis, specifically for cyanotoxins. In Missisquoi Bay, Lake Champlain, public health advisories were issued from 2000 to 2008, and local microcystin concentrations found in lake water often exceeded the Canadian drinking water guideline of 1.5µg L-1. A quantitative-PCR (Q-PCR) approach was developed for the early detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the polyketide synthase (mcyD(KS)) and the first dehydratase domain (mcyD(DH)) of the mcyD gene, involved in microcystin synthesis. Q-PCR was used to monitor the blooms in the littoral and pelagic zones of Philipsburg, Missisquoi Bay, during the summers of 2006 and 2007. Two toxic bloom events were detected by Q-PCR during the summer of 2006: more than 60,000 copies of the mcyD(KS) gene mL-1 were detected in August and an average of 40,000 copies mL-1 were detected in September, when microcystin concentations were more than 4 µg L-1 and approximately 2 µg L-1, respectively. There was a good correlation between gene copy number and microcystin concentrations. The Microcystis aeruginosa population followed the same trend as the number of mcyD(KS) copies and microcystin concentration in the littoral zone. The HPLC profile revealed a change in the toxin composition of the two bloom events. Four microcystin analogues were observed during the bloom in August (MCYST-LA, LR, RR and YR). The MCYST-LA analogue was predominant in August and dominated the bloom in September. The other analogues identified in the second bloom were MCYST-LR and YR. Favorable conditions for a cyanobacterial bloom occurred only late in the summer of 2007. Approximately 300 copies of the mcyD(KS) gene mL-1 were identified in the pelagic zone at the end of August despite the low concentration of toxin. The Q-PCR method was more sensitive than standard chemical assays and allowed the detection of microcystin-producing cyanobacteria as early as June. This technique could be used for the efficient monitoring of the most at-risk water bodies.
Rainer Kurmayer, Guntram Christiansen. Austrian Academy of Sciences, Institute for Limnology, Mondsee, Austria. Introduction: Green- or red-pigmented cyanobacteria of the genus Planktothrix spp. frequently contain the toxic heptapeptide microcystin. We could previously show that non-toxic strains isolated from 28 water bodies in nine European countries have lost more than ninty percent of the large gene cluster (mcy) responsible for microcystin synthesis (Christiansen, G, Molitor, C, Philmus, B, Kurmayer, R. 2008, Mol. Biol. Evol. 25:1695-1704). This gene loss event probably happened million years ago. Due to its rather long history we hypothesized that the non-toxic strains meanwhile could have adapted to a variety of ecological factors not causally related to microcystin anymore. This would obscure a clear correlation of microcystin production and ecological fitness. Methods: In this study 139 red-/green-pigmented strains isolated from lakes of three continents (Europe: 101 strains, North America:29, Sub-Saharan East Africa:9), were analysed (i) for their pigmentation and the production of microcystin, (ii) for the genetic variation within five housekeeping gene loci (16S rDNA, 16S rDNA-ITS, PC-IGS, PSAIGS, RNase P), and (iii) for remnants of the mcy gene cluster (in non-toxic strains). Using established multi locus sequence typing techniques the strains were phylogenetically assigned to several lineages that may be considered as cryptic ecotypes (Cohan, FM 2002, Ann. Rev. Microbiol. 56:457-487). According to common theory clonal dependence can only be observed if ecological differentiation prevents genetic exchange and therefore favours genetic differentiation. Results: In total 58 non-toxic strains were found that were isolated from all three continents. These non-toxic strains were found to lack the mcy gene cluster but still contained at least one of the former flanking regions. The majority of them (42 strains) shared a common remnant of the mcy gene cluster, mcyT, a type II thioesterase. The sequencing of the five housekeeping gene loci revealed six major phylogenetic lineages. Those lineages consisted either of toxic strains (two lineages) or non-toxic strains (three lineages). Only one lineage contained both toxic and non-toxic strains. Taking all strains together the presence/absence of the mcy gene cluster was not related to pigmentation: All toxic lineages and one non-toxic lineage contained both green- and red-pigmented strains. Conclusions: The fact that the mcy gene cluster seems to be patchily distributed is due to a so far unrecognized clonal dependence. We conclude that the clonal lineages have been adapted to significantly different ecological niches forcing their genetic differentiation. The identification of mcy gene cluster remnants supports the gene loss hypothesis and provides evidence against the role of a recent horizontal gene transfer. Interestingly the reddish type of pigmentation evolved several times independently in one toxic and two non-toxic lineages (after the loss of the mcy gene cluster).
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13th International Symposium on Phototrophic Prokaryotes
OC-16.1 THE RESPIRATORY TERMINAL OXIDASES OF THE CYANOBACTERIUM SYNECHOCOCCUS SP. STRAIN PCC7942. Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder, Dominik Aschenbrenner. Institute of Physical Chemistry, Vienna, Austria. Introduction: All cyanobacteria respire with dioxygen as the terminal electron acceptor. The key enzymes of respiration are the respiratory terminal oxidases (RTOs) that transfer electrons to oxygen with water as the reaction product. A search for genes encoding putative RTOs in those cyanobacteria whose genomic sequence has been determined, has revealed a surprising variability both in the types of RTOs (at least 7 different types) and in the number of different RTOs per cell (1 to 10). Many cyanobacterial RTOs have not yet been characterized beyond their sequence similarity to RTOs from other organisms. At least some cyanobacteria have respiratory chains both in their cytoplasmic membranes (CM) and in their intracellular menbranes (thylakoids or ICM). In the ICM the respiratory chain shares components with the photosynthetic electron transport chain. The best studied cyanobacterium in this respect is Synechocystis sp. strain PCC6803 that has three RTOs: one genuine aa3-type cytochrome c oxidase, one quinol oxidase of the cytochrome bd-type - both localized in the ICM - and one ARTO, (Alternate Respiratory Terminal Oxidase, a homolog of cytochrome c oxidase that apparently does not react directly with soluble cytochrome c) localized in the CM. The aim of this work was to study cyanobacterial genes that show sequence similarity to RTO of the cytochrome cbb3 type that is well known from several organisms (including purple bacteria). In cyanobacteria, the corresponding gene products have so far not been characterized at all to the point that at the beginning of our study it was even unclear whether they actually encode an RTO. Only a few cyanobacteria (Synechococcus sp. PCC7942, Synechococcus sp. PCC6301 and Trichodesmium erythraeum IMS101) seem to contain these genes and among these the strain of choice was Synechococcus sp. strain PCC7942, since it is well amenable to genetic manipulation. In other organisms cbb3-type RTO accepts electrons from soluble cytochrome c and transfers them to dioxygen. In addition to the putative RTO of the cbb3 type , the genomic sequence of strain PCC7942 shows the presence of one set of genes encoding an aa3-type (mitochondrialtype) cytochrome c oxidase and a set of genes encoding a bd-type qunol oxidase. Methods: Mutants in two putative cbb3 genes (ccoN and ccoO) and also a mutant with a deletion of the subunit I of cytochrome c oxidase (coxA) were kindly provided by Dr. Susan Golden. We have developed a method to separate and purify cyanobacterial CM and ICM from a large culture (10 liters) using gradient centrifugation in a zonal rotor. The membranes were tested for in vitro cytochrome c oxidase activity by dual wavelength spectroscopy in a Varian Cary5 spectrophotometer using horse heart cytochrome c reduced by ascorbic acid as the electron donor. Results: Wild type strain PCC7942 displayed in vitro cytochrome c oxidation both in the CM and in the ICM. Compared to other cyanobacteria, the cytochrome c oxidation activity of PCC7942 CM is high on a per mg protein basis. The mutant strain lacking subunit I of aa3 type cytochrome c oxidase also shows in vitro cytochrome c oxidation in both CM and ICM. In contrast, the mutant strain lacking the ccoN gene displayed no cytochrome c oxidase activity in the ICM , but some activity in the CM. Conclusions: We have shown for the first time that the ccoN gene of August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Synechoccoccus sp. strain PCC7942 encodes a protein that is part of an RTO enzyme able to oxidise reduced cytochrome c in vitro. Since no activity could be measured in the ICM of the ccoN deletion mutant, our results imply that the cbb3 enzyme of this strain is the sole cytochrome c oxidase in these membranes. The cytochrome c oxidase activity activity of the CM of the ccoN mutant of strain PCC7942 must be due to the aa3-type cytochrome c oxidase. This is in striking contrast to Synechocystis sp. strain PCC6803, where the aa3-type cytochrome c oxidase is the only such enzyme in the ICM and is not present in the CM.
OC-16.2 PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA: STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN POSTTRANSLATIONAL MODIFICATION. Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1, Gaozhong Shen2, Donald A. Bryant2, Wendy M. Schluchter1. 1
Department of Biological Sciences, University of New Orleans, New Orleans, LA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; USA. Introduction: Cyanobacterial phycobiliproteins can contain one or more of four different types of isomeric bilins: phycourobilin, phycoerythrobilin, phycoviolobilin (also called phycobiliviolin), and phycocyanobilin (PCB). These linear tetrapyrroles are derived from heme and are covalently attached via thioether linkages at cysteine residues within the α- and β-subunits. Phycobiliproteins also contain a unique, post-translational modification of a conserved asparagine (Asn) present at β-72, which occurs only on the β-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases responsible for attaching PCB to phycobiliproteins, and the Asn methyl transferase for β-subunits in Synechococcus sp. PCC 7002. Methods: Genes encoding putative enzymes involved in posttranslational modifications of phycobiliproteins in Synechococcus sp. PCC 7002 were identified and characterized using comparative genomics, reverse genetics, in vitro enzyme assays of the recombinant proteins, and in vivo heterologous expression in E. coli. Results: Two new families of bilin lyases that are involved in phycobiliprotein biosynthesis in Synechococcus sp. PCC 7002, first identified in Fremyella diplosiphon as cpeS and cpeT, were characterized. There are three cpeS-like genes (denoted cpcS, cpcU, cpcV) and one cpeT-like gene (denoted cpcT) within the genome of Synechococcus sp. PCC 7002. The CpcS and CpcU proteins form a heterodimer (1:1) and catalyze the addition of PCB to Cys-82 on CpcB (β-phycocyanin) and most allophycocyanin subunits (ApcA, ApcB, ApcD, and ApcF). CpcT attaches PCB to Cys-153 on CpcB. ApcE was shown to have intrinsic bilin lyase activity and has a region with similarity to CpcS/CpcU. Many cyanobacteria have one CpcS/CpeS protein that catalyzes bilin attachment. We hypothesized that these proteins may form homodimers. Consistent with this, a 3D structure of CpcS-III from Thermosynechococcus elongatus BP1 (tll1699) was recently solved, and it crystallized as a homodimer. This is the first structure solved for any bilin lyase, and we have verified it is the Cys82 bilin lyase for attaching PCB to CpcB, ApcA and ApcB by in vitro enzyme assays. The cpcM gene encodes a protein with sequence similarity to other Sadenosylmethionine-dependent methyltransferases. Recombinant CpcM methylated the β-subunits of phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding α-subunits (CpcA, ApcA, and ApcD). Based upon in vitro studies with various substrates, 77
Final Program and Abstracts we conclude that this methylation probably occurs after chromophorylation but before trimer assembly in vivo. Conclusions: All enzymes required for post-translational modification of all phycobiliproteins in Synechococcus sp. PCC 7002 have now been identified and characterized, and we are beginning to characterize the order in which these post-translational modifications occur during phycobiliprotein biosynthesis. Now that the first bilin lyase structure has been solved, we hope to gain more mechanistic insight into how these enzymes catalyze the specific addition of bilins to their phycobiliprotein substrates.
OC-16.3 SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVEL CYANOBACTERIOCHROME-TYPE PHOTORECEPTOR AnPixJ. Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose, Tomoo Shiba, Shigeru Itoh, Genji Kurisu, Masahiko Ikeuchi. University of Tokyo, Meguro, Tokyo, Japan. Introduction: Phytochromes of plants, cyanobacteria and some bacteria are the photoreceptor superfamily that binds a linear tetrapyrrole and exhibits reversible photoconversion between the redabsorbing (Pr) and the far-red-absorbing forms. They exhibit photochemical isomerization between the C15-Z and C15-E isomers of the linear tetrapyrroles. GAF domain plays a crucial role in the function of the chromophore. Cyanobacteriochromes are the recently emerging photoreceptors in cyanobacteria whose GAF domains are related to but distinct from those of the phytochromes. Mutational studies in cyanobacteria have revealed that they are involved in various photoacclimation responses such as chromatic acclimation and phototaxis. GAF domains in the phototaxis regulator SyPixJ1 (from Synechocystis sp. PCC 6803) and TePixJ (from Thermosynechococcus elongatus BP-1) bind a phycoviolobilin and undergo the reversible photoconversion between blue-absorbing and green-absorbing (Pg) forms. Similar GAF domains identified in cyanobacterial genomes may function in novel photoreceptor proteins. Methods: The chromophore-binding GAF domains of the cyanobacteriochromes expressed in Synechocystis or phycocyanobilin (PCB) -producing Escherichia coli have been purified. The purified proteins were characterized by the spectral and crystallographic analyses Results: Putative phototaxis regulator AnPixJ (from Anabaena sp. PCC 7120) binds PCB and shows reversible photoconversion between Pr and Pg forms. Time-resolved spectral analysis suggest that the Pr form of AnPixJ is almost equivalent to that of the phytochromes and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the phytochromes, but is finally photoconverted to the unique Pg form. On the other hand, chromatic acclimation regulator SyCcaS (from Synechocystis) covalently binds PCB and shows similar reversible photoconversion between Pr and Pg forms. However, relationship of chromophore configuration and spectral properties is opposite between AnPixJ and SyCcaS. To understand the structural basis, we are now trying the X-ray crystallographic analyses. We determined the crystal structure of the GAF domain of AnPixJ in the Pr form. The backbone structure of the protein and the chromophore configuration are independently similar to those of the Pr form of bacterial phytochromes, although relative position of the chromophore bound to the protein is significantly deviated. Correspondingly, critical amino acid residues interacting with the chromophore are also diverged in position or orientation, and 78
13th International Symposium on Phototrophic Prokaryotes resultantly in the function. Together with the role of water molecules, a novel protonation mechanism of the chromophore is proposed. Unique features of a hydrophobic pocket to accommodate photoisomerization of the chromophore are discussed with regard to its unusual formation of the Pg form. We will discuss about structurefunction relationship of the tetrapyrrole-based photoreceptors. Conclusion: We found various spectral properties of the cyanobacteriochromes. Spectral analyses suggest that these properties are yielded by different chromophores, different conjugated systems or different photoconversion mechanisms. Structure of the Pr form of AnPixJ provides direct insights into the photoconversion mechanism. This new structural basis shed light on the universal aspects of the photosensory mechanism of linear tetrapyrrole-based photoreceptors.
OC-16.4 CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OF SYNECHOCOCCUS ELONGATUS REVEALS A NEW ANTIDOTE MOLECULE. Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz. Bar-Ilan University, Ramat-Gan, Israel. Introduction: Toxin-Antitoxin (TA) loci are ubiquitously present on chromosomal DNA of diverse bacteria. These modules were assigned a role in programmed cell death and it has been suggested that the population as a whole benefits from sacrificing individual cells. In other cases it has been shown that the un-neutralized toxin does not cause cell death, rather, it re-sets cell physiology and adjusts it to stress conditions. In the course of our global transcriptome analyses of the cyanobacterium Synechococcus elongatus, we noted that vapC, encoding for a toxin homolog, is highly induced upon nitrogen and sulfur starvation. Chromosomal TA systems of cyanobacteria have not been characterized thus far. Additionally, S. elongatus does not possess a homolog of VapB, the typical cognate antidote of known Vap-systems. Thus, it was highly interesting to examine if VapC acts as part of a TA system or functions as ‘solitary toxin’ and to address the cellular role of VapC in the context of a cyanobacterial cell. Methods: We employed genetic and molecular tools including gene inactivation, over-expression and protein tagging to characterize the nature and role of VapC and the new antidote we identified. Results: To test if VapC has a potential cytotoxic effect on S. elongatus cells, we expressed it using a strong promoter. VapC accumulation upon induction, as verified by Western analysis, was accompanied by rapid cell death. TA modules are typically encoded by operons, in which the gene for the antidote molecule precedes the toxin gene. Upstream of vapC, we noted an ORF and termed it VapX; this ORF is not defined as a potential coding region in the current annotation of S. elongatus genome. We bring evidence for the function of VapX, which is not homologous to proteins of known function, as an antitoxin. Over-expression of VapX together with VapC counteracts the killing effect observed when VapC is over-expressed by itself. Furthermore, Northern analysis and RT-PCR indicate that vapXC form a bicistronic operon, a typical feature of TA modules. Experiments involving expression of VapX and VapC in Escherichia coli further support the function of these components as a TA system. Additionally, these experiments indicate complex formation between VapC and VapX, in agreement with the suggested antidote function of the latter. Conclusions: VapC of S. elongatus has a cytotoxic effect when expressed by a strong promoter. Additionally, we present evidence
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
based on several observations supporting identification of VapX as a new antitoxin of the Vap-system. To the best of our knowledge, our study provides the first characterization of cyanobacterial chromosomal TA system. The role of the Vap-system of S. elongatus will be discussed in light of quantitative analyses of natively expressed VapX and VapC and inactivation of vapXC as well as additional TA systems of this cyanobacterium.
Calcium is then subject to trans-cellular transport along the filament and extruded at the lagging end to the outside aqueous medium. Proton antiport at the apical cell likely maintains charge and neutralizes alkalization by excess carbonate ions. Incidentally, an evolutionary pressure not to interfere with calcium trafficking, may be behind the unusual property of Mastigocoleus (and BC008) to develop lateral heterocysts.
OC-16.5
OC-16.6
HOW CYANOBACTERIA BORE (AND WHY LATERAL HETEROCYSTS EXIST).
FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEEN IRON HOMEOSTASIS AND OXIDATIVE STRESS.
F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao.
Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2, Nir Keren1.
School of Life Sciences, Arizona State University, Tempe, AZ, USA. Introduction: Some cyanobacteria bore into carbonates. They are few, but ancient players in geologic phenomena such as the destruction of coastal limestones, the reworking of carbonate sands and the cementation of microbialites. They constitute important tools for paleoenvironmental reconstruction, and play a significant role as pests in marine aquaculture. In spite of their importance, the mechanism by which cyanobacteria excavate minerals remains unknown. In fact, this ability represents a geochemical paradox, in that autotrophic metabolism will push the carbonate system towards precipitation, not dissolution. We have advanced mechanistic models that may allow cyanobacterial boring to proceed and be still consistent with geochemistry. These are based on either temporal or spatial separation of photosynthesis and boring, or on the active extrusion of calcium through a cellular uptake and transport process. Until now, experimental approaches were hampered by the lack of cyanobacteria that could bore in the laboratory. We report here on investigations using a novel isolate of the filamentous heterocystous cyanobacterium Mastigocoleus/Fischerella strain BC008 that maintains boring in the laboratory. Methods: Growth and boring of axenic cultures of BC008 were investigated on different crystalline minerals, and under a variety of conditions. The microenvironments of cyanobacterial filaments actively boring on calcite chips were monitored quantitatively for free Ca2+ using in vivo fluorophore-enabled confocal fluorescence microscopy. The temporal dynamics of a variety of physical and chemical inhibitors on calcium transport dynamics in the cyanobacterium/mineral system were tracked using this system. PCR-based methods were used to detect and track the differential expression of genes putatively involved in boring. Results: We could easily reject hypotheses involving the action of accompanying heterotrophic bacteria or the temporal separation of photosynthesis and boring. The range of mineral carbonates bored (Ca-bearing, or those containing its biochemical analog Sr, but not those with Mg, Mg/Ca, Mn, Fe, K, or Na as metal) implicated Ca as necessary for boring. Extracellular Ca2+ levels in and around the boreholes of actively boring BC008 were consistent with an active uptake Ca2+ at the apical end cell (severe under-saturation) and extrusion at the surficial end of the filaments (supersaturation). This out-of-equilibrium state could be relaxed by i) ceasing illumination, ii) adding inhibitors of ATP generation, and iii) adding specific inhibitors of P-type calcium ATPases, indicating that cellular energy is required for this process, as are ATP-ase calcium transport systems. P-type ATPase genes could be detected in BC008’s genome.
1
The Alexander Silberman Institute of Life Sciences, Department of Plant and Environmental Sciences, Hebrew University, Givat Ram, Jerusalem, Israel; 2Department of Biological Sciences Purdue University, West Lafayette, IN, USA; 3 Department of Botany, University of Otago, Dunedin, New Zealand. Introduction: Iron is an essential nutrient for the survival of all organisms, including cyanobacteria. However, the same redox properties that makes iron a valuable cofactor also lead to oxidative interactions resulting in the formation of harmful radicals. Therefore, iron accumulation in the cells should be tightly regulated. Ferritin family proteins play an important role in iron homeostasis. Synechocystis sp. PCC 6803 contains two ferritin type storage complexes, bacterioferritin (BFR) and MrgA. Previous studies demonstrated the role of BFR and MrgA in iron storage. In addition, MrgA was found to play a key role in oxidative stress response. Methods: In the study presented here, we examined the dual role of the ferritin family proteins using physiological and transcriptomic approaches. These include measurements of growth of cyanobacterial cultures in media limited for iron by various chelators; analysis of internal transition metal quotas by ICP-MS; measurements of photosynthetic performance and transcriptional profiling by microarray analysis. Results: Microarray analysis of iron limited wild type and ∆mrgA cultures revealed a remarkable up-regulation of oxidative stress related genes in the mutant cells. The PerR regulator was found to play an important role in that process. Furthermore, we were able to demonstrate the connection between internal iron quota, the presence of the two storage proteins and the sensitivity to externally applied oxidative stress. These data suggests a pivotal role for the two ferritin type protein-complexes of Synechocystis 6803 cells in coordinating iron homeostasis and the oxidative stress response. Conclusions: The combined action of the two complexes allows for the safe accumulation and release of iron from storage by minimizing damage caused by the interaction reduced iron and oxygen radicals which are abundant in cyanobacterial cells due to the function of the photosynthetic apparatus.
Conclusions: Our experiments suggest that cyanobacteria bore by actively taking up Ca2+ ions from the leading end of a filament at the cost of energy, locally lowering extracellular calcium levels to the point that mineral equilibrium is displaced towards dissolution there. August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
OC-17.1 RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS, HARBORING THE CRTI REPORTER GENE TO MONITOR ENVIRONMENTAL TOXIC METALS. Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan. Introduction: Genetically engineered bacterial biosensors utilize living cells as sensing elements that harbor a reporter gene linked to a transcriptional switch. The genetic engineering makes it possible for bacterial cells to elicit signal in response to a small amount of environmental chemicals as effectors. Reporter gene systems that do not require instruments and exogenous chromogenic reagents are advantageous in establishing simple monitoring protocols. By taking it into account, the usefulness of several genes involved in carotenoid biosynthesis pathways has been examined, and the crtI gene has been selected as the reporter gene system of the purple bacterium, Rps. palustris. In the crtI reporter gene system, the crtI gene placed downstream of a transcriptional switch is expressed, and complements the crtI deletion of host bacterium, only in the presence of effectors. In this study, Rps. palustris strains responding to arsenic and lead/cadmium were constructed, and their possibilities as biosensors for environmental monitoring were examined. Methods: The green mutant, no. 711, was constructed through deletion of crtI from the chromosome. The arsenic crtI biosensor (AsCrtIBS) was made of the no. 711 strain transformed with the crtI gene under regulation of the operator/promoter region of the ars operon and the arsR gene from Escherichia coli. The hmrR gene of Rps. palustris, encoding the heavy metal resistance transcriptional regulator HmrR, and its upstream region were chosen as the transcriptional switch of the lead/cadmium crtI biosensor (Pb/Cd-CrtIBS).
13th International Symposium on Phototrophic Prokaryotes approximately 1 mM. Further, its susceptibility to tellurite under respiratory conditions is affected by the carbon source. Under these conditions the MIC for tellurite varies from 8 μM, with malate and other dicarboxylic acids, to 300 μM with acetate. R. capsulatus is also characterized by a high tellurite uptake rate as compared to other bacterial species. Uptake activity measurements have shown that tellurite enters the cells at a much lower rate when acetate is present. This effect is not seen when dicarboxylic acids are used instead (1). These early data have led to propose that tellurite exploits an acetate transport system to enter R. capsulatus cells. In this work we show, for the first time in a bacterial system, that the main entry gate for tellurite is acetate permease (actP), and that an insertional mutation in this gene is sufficient to drastically reduce the oxyanion uptake. Methods: Resistant mutants were isolated on aerobic RCV standard medium in the presence of 100 μM tellurite. The actP insertional mutant was constructed using the pJP5603 suicide vector (2). Tellurite uptake was measured as described by Turner et al. (3). MIC determinations, conjugations, subcloning and PCR amplification were performed using standard molecular procedures. Results: The resistant mutant RTe36, used in this work, showed a MIC 50 fold higher than the wild type B100 and an uptake rate that was limited to 25% of the wild type control. Complementation of the mutant with a R. capsulatus cosmid library allowed the isolation of two cosmids which restored high level uptake and reduced the MIC well below that measured in the mutant, but still above the w.t. basal level. From the cosmids it was subcloned the acetate permease gene (actP), which was identified by DNA sequencing, and was shown to complement the mutant by restoring both the sensitivity to tellurite and the uptake level of the w.t.. Finally, to confirm the role of actP, an acetate permease insertion mutant was constructed. Initial experiments indicated that the actP – mutant has indeed a decreased uptake and a MIC for tellurite higher than wild type.
Results: As-CrtIBS and Pb/Cd-CrtIBS changed color in response to 10 μg/L or less As(III) and Pb(II)/Cd(II), respectively. This change could be distinguishable to the sense of sight after 24-hour culture without further manipulation. The hue angle of cultures in the CIE-L*a*b* color space shifted from greenish yellow toward red when concentration of the metals increased (Fig). As(III) supplemented to mineral waters on the market and environmental arsenic in Bangladesh well waters were monitored. Although the basal color in the absence of As(III) shifted to red with an increase in the hardness of mineral water, As-CrtIBS held responsibility to As(III). As-CrtIBS could screen the well waters indicating an arsenic concentration higher than 1 μg/L.
Conclusions: This work demonstrates, for the first time, that acetate permease is the main gate for tellurite entry into R. capsulatus cells. The residual uptake, still measurable in the mutant, indicates that tellurite can take advantage of other less efficient entry facilities. The failure to bring the MIC for tellurite in the complemented mutant, back to level of the wild type, as opposed to the complete restoration of the uptake rate, indicates that other mutations are present, determining the high level of resistance measured in the mutant. Work is already planned to address this question. Finally, it remains to be determined if acetate permease is involved in tellurite uptake in other bacterial species and, if so, how spread is this mechanism.
Conclusion: The crtI biosensors indicate practical detection limits and are applicable to monitoring of toxic metals in groundwater and freshwater by the naked eye.
Turner R.J., Weiner J.H. and Taylor D.E. (1992) Anal. Biochem., 204:292-295
OC-17.2
Borghese R., Marchetti D. and Zannoni D. (2008) Arch. Microbiol. 189:93-100 Penfold R.J. and Pemberton J.M. (1992) Gene, 118:145-146
OC-17.3
THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTER CAPSULATUS CELLS VIA ACETATE PERMEASE.
CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CRPLATING INDUSTRY WITH EXOPOLYSACCHARIDE-PRODUCING CYANOBACTERIA.
Roberto Borghese, Davide Zannoni.
Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis
Department of Biology, University of Bologna, Bologna, Italy.
Deparment of Agricultural Biotechnology, University of Florence, Firenze, Italy.
Introduction: Rhodobacter capsulatus is a purple non-sulfur phototrophic bacterium that shows a highly variable response to the toxic oxyanion tellurite. It is highly susceptible to the oxyanion while growing by respiration with a MIC (minimal inhibitory concentration) of 8 μM, and is very resistant when grown phototrophycally with a MIC of 80
Introduction: Chromium (VI) compounds are contained in the waste waters deriving from a large number of industrial processes. In the last twenty years, the possibility to use specific microorganisms for the removal of heavy metals from industrial waste waters has been widely August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes studied, but the possibility to use exopolysaccharide (EPS)-producing cyanobacteria for the removal of heavy metals in the anionic form was never tested. The aim of this study was the assessment of the capability of some EPS-producing cyanobacteria to remove chromate from waste waters of a plating industry in lab and in semi-pilot systems and to define the role of the different cell fractions (i.e. biomass and polysaccharidic layers) in the metal removal process. Methods: Exopolysaccharide-producing Cyanothece ET5, TI4, PE14, VI22, CE4, Cyanospira capsulata and Nostoc PCC7936, were tested for their ability to remove Cr (VI) from waste waters of a plating industry containing Cr (VI) 89.71% (w/w), Cr (III) 8.97%, other metals 1.32%. Lab experiments were carried out for 48h with 50 ml of acidpre-treated cyanobacterial cultures confined in dialysis tubing and dipped in 450 ml of the diluted industrial wastewater (pH 3; Cr (VI) concentrations in the range 10-200 mg L-1). Field experiments were carried out with acid-pre-treated cultures confined in three different semi-pilot systems (a filter press, a filter column and a dialysis membrane system) operating with 30 L of the same diluted waste water. Results: In lab experiments, Cr (VI) was removed by Cyanothece CE4 at a specific metal uptake of 18.0 ± 0.8 mg of Cr(VI) per g of cell dry weight, while three other strains, Cyanothece ET5 and PE14 and Nostoc PCC7936 showed a metal uptake in the range 10 to 12 mg (Cr) (g dry wt)-1. All the other strains showed a lower capability to remove the metal. The three semi-pilot systems operating with Nostoc PCC7936 biomass showed a very different behavior in the metal removal process: in the dialysis membrane system, a decrease of the concentration of Cr (VI) and of total Cr was observed, both concentrations reaching, after 72 hours, values corresponding to about 60% of the initial ones. In the filter press system, the metal removal was done by flushing 30L of the diluted waste water containing 112 mg L-1 of Cr (VI). In the experiment, a decrease in the concentration of Cr (VI) down to a value of 0.40 mg L-1 was observed after 48 h; at the same time, the total Cr concentration only decreased from 123 to 53 mg L-1, owing to a very significant increase in the amount of Cr (III) present in solution. In the column system, after 24 h almost all the Cr (VI) was reduced to Cr (III), without any significant decrease of the total Cr concentration. Thus, dried biomass of Cyanothece TI4, which is known to have a very high Cr3+ uptake, was added on the top of the column. In the following 120 h, the concentration of total Cr and of Cr (III) decreased to a value of 3.6 mg L-1, while the concentration of Cr (VI) reached the value of zero. Conclusions: The results obtained pointed out the complex role played by the cyanobacterial biomass in the removal of Cr (VI): the cells, when subjected to treatments removing the external polysaccharidic structures, carried out the reduction of Cr (VI) to Cr (III), while the polysaccharidic fraction, previously released by the cells and solubilized in the culture medium, was capable to remove the Cr (III) cations formed in the reduction process. The results obtained in the semi-pilot systems showed that EPS-producing cyanobacteria can be profitably used for the removal Cr (VI) from industrial waste waters.
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OC-18.1 TRANSCRIPTIONAL REGULATION AND MATURATION OF CYANOBACTERIAL HYDROGENASES. Peter Lindblad. Department of Photochemistry & Molecular Science, Uppsala University, Uppsala, Sweden. Introduction: In cyanobacteria three enzymes are directly involved in the hydrogen metabolism: a nitrogenase that produces H2 as a byproduct of nitrogen fixation, an uptake hydrogenase (HupSL, encoded by hupSL) that recaptures H2 and oxidize it, and a bidirectional hydrogenase (encoded by hoxEFUYH, forming an enzyme with a hydrogenase part, HoxYH, and an electron transfer partner protein, HoxEFU) that can both oxidize and produce H2, see Tamagnini et al 2007. The transcription of cyanobacterial hup and hox operons are known to be regulated. The maturation of hydrogenases into active enzymes is a complex process and eg a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded by either hupW or hoxW. We have addressed trancriptional regulation and hydrogenase maturation in a few selected strains. Methods: The unicellular cyanobacterium Synechocystis PCC 6803 contains a single bidirectional hydrogenase. The filamentous cyanobacteria Nostoc punctiforme and Nostoc PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase, respectively. Molecular experiments were performed as detailed elsewhere. Results: Generally, little is known about the transcriptional regulation of cyanobacterial uptake hydrogenases. We showed that NtcA has a specific affinity to a region of the hupSL promoter of Nostoc punctiforme. Truncated versions of the promoter region of the hupSL operon were fused to gfp. All constructs showed heterocyst specific expression (Holmqvist et al 2009). Unexpectedly the shortest promoter fragment, covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. The cyanobacterial hox genes have been characterised in some strains. LexA interacts with the promoter region of the hox operon in Synechocystis PCC 6803. The hox genes of Nostoc PCC 7120 are transcribed as two different clusters with LexA interacting with both promoter regions. DNA affinity assays also identified an AbrB-type DNA-binding protein as interacting directly with the promoter region of the hox operon in Synechocystis PCC 6803. This protein binds to the its own promoter region and the promoter region of the hox operon, suggested to function as a positive regulator of hox gene expression. The presence and expression of hyp-genes were examined in the N2-fixing cyanobacterium Nostoc PCC 7120. RT-PCRs demonstrated that the six hyp-genes may be transcribed as a single operon. TSPs were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively (Agervald et al 2008). Five upstream ORFs located in between hupSL and the hyp-operon, and two downstream ORFs from the hyp-genes were shown to be part of the same transcript unit. Transcriptional analyses were performed of hupW in Nostoc punctiforme, and hupW and hoxW in Nostoc PCC 7120. We identified numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW) (Devine et al 2009). A phylogenetic tree of hupW & hoxW showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Bioinformatic studies revealed a so called “HOXBOX”; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.
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Final Program and Abstracts Conclusions: Significant advances have been made in the understanding of both the transcriptional regulations and the maturation of cyanobacterial hydrogenases. References: Agervald Å, Stensjö K, Holmqvist M, Lindblad P. 2008. BMC Microbiol 8: 69 Devine E, Holmqvist M, Stensjö K, Lindblad P. 2009. BMC Microbiol 9: 53
either an integration or expression plasmid. The Rubrivivax transgenes are cooL, U, and H encoding the small and large subunits of a typical NiFe-hydrogenase as well as hypA, putatively involved in nickel insertion for hydrogenase maturation. However, no hydrogenase activity was detected in the recombinant Synechocystis strain, suggesting the need to transfer additional Rubrivivax hydrogenase genes to yield activity.
Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris D, Heidorn T & Lindblad P. 2007. FEMS Microbiol Rev 31: 692-720
Conclusions: Our approach to generate knockout mutants lacking the various Hox subunits led to the conclusion that (1) HoxYH alone constitutes in vitro hydrogenase activity and (2) HoxEFU are required for complex stability and for NAD(P)H-supported H2 production, in vivo. Work is underway to express additional Rubrivivax coo hydrogenase and its hyp maturation genes into Synechocystis, presumably needed to confer activity for sustained photobiological H2 production.
OC-18.2
OC-18.3
PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803.
ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATION OF THE BIDIRECTIONAL [NIFE]-HYDROGENASE FROM SYNECHOCYSTIS SP. PCC 6803.
Holmqvist M, Stensjö K, Oliveira P, Lindberg P, Lindblad P. 2009. BMC Microbiol 9: 54 Oliveira P & Lindblad P. 2008. J Bacteriol 190: 1011-1019
Carrie Eckert, Jianping Yu, and Pin-Ching Maness. National Renewable Energy Laboratory, Golden, CO, USA. Introduction: The photosynthetic cyanobacterium Synechocystis sp. PCC6803 has the capacity to integrate light harvesting with charge separation and transfer the electrons to a bidirectional NiFehydrogenase for the photoproduction of H2 from water. However, the H2-production reaction is short-lived in the presence of O2, the latter an inherent byproduct of oxygenic photosynthesis. We propose two approaches to surmount this challenge: (1) to gain more in-depth understanding of the Synechocystis Hox hydrogenase for its improvement; and (2) to genetically transfer and express a foreign O2tolerant hydrogenase into Synechocystis. Both approaches will guide the design of an O2-tolerant system in Synechocystis for sustained H2 production. Methods: The native Synechocystis Hox hydrogenase is a pentameric complex consisting of HoxEFUYH subunits. HoxYH are the catalytic subunits for H2 production, accepting electrons from NAD(P)H mediated by the HoxEFU diaphorase moiety. We are genetically dissecting the 5-subunit Hox complex by generating mutants lacking one or more of the subunits in order to probe their roles both in maintaining complex stability and in hydrogenase activity. Our second approach entails the genetic transfer and expression in Synechocystis a hexameric O2-tolerant NiFe-hydrogenase (CooMKLXUH) from the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus. The Coo hydrogenase is ferredoxin linked; its expression in Synechocystis may improve the overall efficiency of H2 production using a photo-reduced ferredoxin as the electron mediator. Results: We have either acquired or generated Synechocystis mutant lines lacking one or more of the Hox subunits. Western blots confirmed the deletion of the subunits using their respective antibodies. We determined that mutant containing only the HoxY and HoxH subunits retained hydrogenase activity in vitro, using methyl viologen as the redox mediator reduced by sodium dithionite. However, this mutant cannot produce H2 in vivo likely due to the lack of HoxEFU subunits, required for electron mediation from NAD(P)H. Moreover, mutants with deletions in HoxE, F, or U individually or in various combinations still retained 20 to 50% of its in vitro hydrogenase activity, with most loss observed with deletions of the HoxFU subunits. In the second approach, we have successfully transformed and expressed four Rubrivivax hydrogenase and its related genes into Synechocystis hosts lacking native hoxH, using 82
Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2, Rüdiger Schulz3 and Frauke Germer3. 1
School of Life Sciences, Arizona State University, Tempe, AZ, USA; Max-Volmer-Laboratorium, Technische Universität, Berlin, Germany; 3 Botanisches Institut, Universität Kiel, Kiel, Germany. 2
Introduction: In cyanobacteria two different types of hydrogenases are known. They both belong to the NiFe-hydrogenases and are called uptake and bidirectional due to their physiological characteristics. The cyanobacterial bidirectional hydrogenase is of particular interest, since it is rapidly reactivated in the absence of oxygen and its traits seem to be intermediate between the standard oxygen-sensitive and the oxygen-tolerant NiFe-hydrogenases. In addition, this hydrogenase is well suited for solar-driven biological hydrogen production in cyanobacteria. In this study we developed a rapid and gentle method to purify the bidirectional hydrogenase of Synechocystis sp. PCC 6803 for biochemical as well spectroscopical investigations to characterize its active site. Methods: To overcome the low expression level of the bidirectional hydrogenase and to simplify its isolation a strain containing the psbAII promoter upstream of the hox-operon was constructed. In the same strain the Strep tag II was fused to the C-terminus of hoxF encoding a subunit of the diaphorase part of the enzyme. In the same strain a hyp-gene cluster containing the genes necessary for the posttranslational processing of the hydrogenase was also overexpressed. After isolation, the hydrogenase was subjected to EPR (electron pulse resonance) and FT-IR (Fourier transform infrared) spectroscopy in different redox states. Results: The insertion of the psbAII promoter in conjunction with the Strep tag led to an increase of total hydrogenase activity of two fold. Overexpressing the hyp-genes led to an additional two fold increase. From these strains the hydrogenase could be isolated in a highly pure and active state after a single run of the affinity column. EPR measurements reveal the presence of at least two redox active FeS clusters, but no nickel specific signals. A paramagnetic Ni-species was absent under any conditions. This either indicates that the nickel stays in the diamagnetic Ni(II) state and the redox changes take place at another ligand or that its spin is coupled to a nearby paramagnetic center in the protein. August 9 to 14, 2009 • Montréal, QC, Canada
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In the FT-IR spectra three different signals could be discerned, that are attributed to two cyanides and one carbon monoxide coordinating the iron ion. The FT-IR spectra under different redox conditions are indicative of a NiFe-hydrogenase attaining a Ni-B like state not the Ni-A state when oxidatively inactivated even in the presence of oxygen. For other hydrogenases it is known that they can be rapidly activated when in the Ni-B state but need much more time when in the Ni-A state. Conclusions: The spectroscopic investigations of cyanobacterial bidirectional hydrogenase confirm its intermediary behavior between the anaerobic standard enzymes and the aerobic oxygen tolerant enzymes. With these characteristics it is very well adapted to rapidly changing redox conditions in oxygenic phototrophs. It will be quickly activated under anoxic conditions and able to produce hydrogen by fermentation. It will produce hydrogen as an overflow valve when photosynthesis resumes from oxygen free conditions, but it will also be very rapidly inactivated when photosynthetic oxygen production takes over so as not to waste energy.
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P.001 FUNCTIONAL ANALYSIS OF THE PUH PROTEINS IN RHODOSPIRILLUM RUBRUM. Caroline Autenrieth, Robin Ghosh. Dept. of Bioenergetics, University of Stuttgart, Stuttgart, Germany. Introduction: Our present view of the photosynthetic unit (PSU) of Rhodospirillum rubrum is that it contains a reaction centre (RC; H, L and M subunits) surrounded by a light-harvesting (LH) 1 complex, composed of alpha and beta subunits which aggregate to form a 16fold circular assembly. The polypeptides of the LH1 complex as well as the L and M subunits of the RC are encoded by the puf operon, whereas the H subunit is encoded by the puhA gene of the puh operon. The puh operon encodes a number other putative gene products (puhB, puhC, puhD and puhE) which have been implicated in PSU assembly though their precise role has been unclear so far. In this study we provide new biochemical, functional insight into the role of some of the puh proteins in PSU assembly. Methods and Results: Selected puh proteins were deleted from the R. rubrum chromosome and the phenotype examined. The phenotype, which is unique so far, was quantified functionally using spectroscopic and biochemical methods. The results point to an unsuspected role of the puh proteins in PSU assembly. Conclusions: We will present data as well as a structural hypothesis, that for R. rubrum, the structure of the PSU is more complex than previously thought. In particular, we present a new biochemical insight into the role of the proteins encoded by the puh operon. If our hypothesis is correct, our view of the PSU will have wide-reaching consequences for the interpretations of spectroscopic, cryoEM and AFM data in general.
P.002 THE ROLE OF CYTOCHROME C-554 IN THE ELECTRON TRANSFER PATHWAYS IN THE GREEN SULFUR BACTERIUM CHLOROBACULUM TEPIDUM. Chihiro Azai1, Yusuke Tsukatani2, Jiro Harada3, Ryo Miyamoto4, Toru Kondo4, Hiroumi Murakami4, Shigeru Itoh4, Hirozo Oh-oka1. 1
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043; 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki; 3Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga; 4Division of Material Science, Graduate School of Science, Nagoya University, Nagoya, Aichi; Japan. Introduction: In the green sulfur bacterium Chlorobaculum (Cba.) tepidum (syn. Chlorobium tepidum), the photo-oxidized primary electron donor, P840+, is rapidly rereduced by cytochrome (cyt) cz, one of subunits of the reaction center (RC) complex. Previous studies have revealed two different electron donors to the oxidized cyt cz. In the purified membranes, menaquinol:cyt c oxidoreductase function as the direct electron donor to cyt cz, while in the in vitro reconstitution experiment using the purified RC complex, water-soluble and low molecular-weight (approx. 10,000) cyt c-554 (CT0075) does. However, it has still remained unknown whether the electron transfer scheme from menaquinol:cyt c oxidoreductase to cyt cz via cyt c-554 is operative when all of them co-exist together in vivo. On the other hands, green sulfur bacteria use reduced sulfur compounds (sulfide, thiosulfate, polysulfide and elemental sulfur) as August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts the photosynthetic electron sources. Electrons obtained from the oxidations of these sulfur compounds are transferred to the RC through the photosynthetic electron transfer pathways. Although several in vitro reconstitution experiments carried out in 1970s demonstrated that cyt c-555 isolated from Cba. thiosulfatiphilum, which corresponds to cyt c-554 from Cba. tepidum, could accept electrons from sulfide/thiosulfate oxidation systems, there has been no experimental evidence to support this in vivo. In this study, we constructed three mutants of Cba. tepidum, which were devoid of cyt c-554 (cycA::aadA strain), SoxB (CT1021) (soxB::aacC1 strain), and cyt c-554 and SoxB (cycA::aadA/soxB::aacC1 strain), respectively, to investigate electron transfer pathways coupled with sulfide/thiosulfate oxidations. Results: When purified cyt c-554 was added to the membranes prepared from the cycA::aadA strain, the oxidized cyt cz, immediately formed after flash excitation, was rereduced by both cyt c-554 and quinol oxidoreductase independently; the contribution of the former became larger by increasing its concentration, indicating that the reaction between cyt c-554 and cyt cz obeyed the second-order reaction mode. Unlike the case of the electron transfer reaction from cyt bc1 complex to cyt c2 in purple bacteria, cyt c-554 in the Cba. tepidum never serves as a shuttle-like carrier between menaquinol:cyt c oxidoreductase and cyt cz. The cycA::aadA strain exhibited a decreased growth rate but normal growth yield when compared to the wild type. In accordance with this, the strain was found to oxidize thiosulfate more slowly than the wild type but completely to sulfate as the wild type. This indicates that cyt c-554 is not indispensable for thiosulfate oxidation itself. Furthermore, both the soxB::aacC1 and cycA::aadA/soxB::aacC1 strains did not grow at all in a medium containing only thiosulfate as an electron source. They also grew incompletely even in a medium containing only sulfide when compared to the wild type. This suggests that SoxB is not only essential for thiosulfate oxidation but also responsible for sulfide oxidation. Conclusions: In Cba. tepidum, cyt c-554 serves as an electron carrier between Sox system and cyt cz in the RC. But an alternative electron carrier or electron transfer path should be operating between them, judging from the complete oxidation of thiosulfate in the cycA::aadA strain. The Sox system also seems to be involved in the sulfide oxidation, whose issue would be a future work to be resolved.
P.003 EXPRESSION OF HYDSL HYDROGENASE FROM THIOCAPSA ROSEOPERSICINA IN ESCHERICHIA. COLI. Horcheska Batyrova, Anna Khusnutdinova, Galina Shirshikova, Olga Postnikova, Elena Patrusheva, Aleksander Boutanaev, Anatoly Tsygankov. Institute of Basic Biological Problems, Institutskaya, Pushchino, Moscow Region, Russia. Introduction: The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS contains several Ni-Fe hydrogenases. One of two membrane-bound hydrogenases appears to be very stable against temperature, CO, H2S and proteases. The Ni-Fe hydrogenase HydSL assembly includes the concordant expression of structural genes (hydS and hydL with two additional genes of unknown functional) as well as a number of accessory genes, participating in the maturation process of this enzyme. Structural genes have been cloned by the K.L. Kovacs team (Szeged, Hungary) earlier and their sequences are available in Genbank database. 85
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Final Program and Abstracts The hydSL genes are expressed on the low level in T. roseopersicina what makes difficult both basic and applied investigations of the protein. One possibility to increase HydSL yield might be heterologous expression in a well established system, for instance, such as Escherichia. coli. However, little is known about this possibility. From the other hand, unlike many other hydrogenases, accessory genes of HydSL are dispersed along the T. roseopersicina chromosome what makes difficult their complementation study in E. coli cells. The aim of this research is the expression of hydS and hydL in heterologous system. Methods: In this investigation we started from an attempt to express in E. coli two structural genes hydS and hydL, keeping in mind that some of E. coli accessory genes might complement the lack of their counterparts in T. roseopersicina, as well as the fact that high level of Ni in growth medium might compensate, at least partially, some of T. roseopersicina accessory genes. Two genes have been cloned by means of PCR and artificial operon has been constructed in E. coli pET22b expression vector. After that, all two genes have been expressed in E. coli. Results: An artificial operon from two genes (hydS, hydL) has been created to investigate expression of HydSL hydrogenase from Thiocapsa roseopersicina in E.coli. Proteins were overexpressed. However no hydrogenase activity was found. Detailed description of experiments will be presented. Conclusion: The construction with hydS and hydL was created, introduced in E.coli cells and HydS and HydL proteins were overexpressed.
P.004 THE RHODOBACTER SPHAEROIDES REACTION CENTER ASSEMBLED WITH ZINC BACTERIOCHLOROPHYLL YIELDS EVOLUTIONARY INSIGHTS. Su Lin*†, Paul R. Jaschke§, Haiyu Wang*, Mark Paddock¶, Aaron Tufts†, James P. Allen†, Federico I. Rosell||, A. Grant Mauk||, Neal W. Woodbury*†, J. Thomas Beatty§ *
The Biodesign Institute at Arizona State University, Arizona State University; †Department of Chemistry and Biochemistry, Arizona State University; §Department of Microbiology and Immunology, The University of British Columbia; ¶Department of Physics, University of California, San Diego; ||Department of Biochemistry and Molecular Biology, The University of British Columbia. Introduction: The purple bacterial reaction center RC binds 6 chlorin cofactors: the ‘special pair’ (P), a dimer of bacteriochlorophyll (BChl) a molecules on the periplasmic side of the RC; two monomeric BChls (BA and BB) present on either side of P; and two bacteriopheophytin (BPhe) molecules (HA and HB). When P is excited, an electron is transferred through BA to HA, and then to a quinone. Studies on a variety of species and mutants in which cofactors were changed indicated the importance of the energetics of the cofactors in influencing ET rates. The RC from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides was studied. In this RC, which we call the Zn-RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) binding sites contain Zn-BChl rather than BChl. We used spectroscopic measurements to fully elucidate the chlorin composition at the P, B and H sites, and to measure the efficiency of ET. Methods: Mutant and wild type RCs were isolated and the absorption spectra at room temperature and 10 K were measured, as well as the kinetics of ET in timescales ranging from fs to ms. The 86
electrochemical midpoint of P was determined by potentiometric titration and measurement of changes at the P absorption maximum. Results: Analysis of the room temperature and 10 K absorption spectra of the Zn-RC revealed that the P, B and H sites all contain ZnBChl. Furthermore, the absorption peaks in the visible region indicated that the metals of the Zn-BChls in the P and B sites are pentacoordinated, as is the Mg of BChl in these sites in the wild type RC, whereas the Zn-BChls in the H sites appear to be tetracoordinated. The midpoint potential of P in the Zn-RC was determined to be 515 ± 5 mV, similar to the value of 505 ± 5 mV for the wild type RC. Photobleaching experiments indicated that ET proceeds from P to the quinones, producing a long-lived charge-separated state, as in the wild type RC. The ultrafast kinetics of ET from P to HA in the Zn-RC were found to be very similar to those in the wild type RC, in contrast to an RC in which HA is BChl. Conclusions: The Zn-RC H sites contain Zn-BChl instead of BPhe, and so contains 6 identical chlorins. The Zn-BChl H-cofactor spectral properties in the visible region are proposed to be due to the absence of a 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions that significantly contribute to the redox midpoint potential of H, and the formation of the charge-separated state. In both the purple bacterial and the cyanobacterial PS2 RC, P is a (B)Chl dimer, B sites contain (B)Chl, and H sites contain (B)Phe. In contrast, the cyanobacterial RC in PS1 has six Mg-containing Chls in a similar spatial arrangement, analogous to the Zn-RC, with the A0 Chl in the PS1 RC equivalent to the HA BPhe in the WT RC. The Mg of the Chl electron acceptor A0 in the PS1 RC protein is weakly ligated by the sulfur of a Met side chain, in contrast to the usual His. We speculate that evolution has resulted in two strategies for a high rate of ET in RCs: 1) in the PS2-type of RCs, the primary electron acceptor is a (B)Phe surrounded by protein that excludes (B)Chl and water from the H sites; 2) in the PS1-type of RCs, the primary acceptor is a Chl (A0), but Chl functions well as the primary acceptor because the Mg in the A0 Chl is ligated weakly by a sulphur atom coming from the protein, endowing this Chl with tetracoordination-like electrochemical properties.
P.005 A NEW PROTEIN INVOLVED IN THE OCP-RELATED PHOTOPROTECTIVE MECHANISM IN CYANOBACTERIA. Clémence Boulay, Adjélé Wilson, Diana Kirilovsky. Commissariat à L’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTec-S) et Centre National de la Recherche Scientifique (CNRS) URA 2096, 91 191 Gif-sur-Yvette, France. Introduction: The most characterised photoprotective mechanism in cyanobacteria is named qEcya. Under high light conditions, it diminishes the formation of dangerous oxygen species by increasing energy dissipation into heat and thus, decreases the energy arriving at the reaction centers from the phycobilisomes (PBS, the cyanobacterial external antennae). The mechanism is induced by the absorption of blue-green light by the carotenoid of the soluble photoactive Orange Carotenoid Protein, OCP [1, 2]. The process is accompanied by a reversible decrease (quenching) of fluorescence. When the “quenched” cells are then transferred to darkness or low light intensities, the initial level of fluorescence is recovered within 15-20 minutes. The aim of this study was to discover other proteins involved in this photoprotective mechanism.
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13th International Symposium on Phototrophic Prokaryotes Methods: A mutant unable to recover the lost fluorescence along with several mutants which complemented it were constructed. Their phenotype, with respect to the photoprotective mechanism, was studied by measuring fluorescence kinetics in a pulse amplified modulated fluorimeter (PAM-fluorimeter). Co-purification and coimmunoprecipitation experiments were used to investigate the interaction of a new protein with the OCP. Results: We have demonstrated that a 13kDa membrane protein, which we have named Fluorescence Recovery Protein (FRP), is involved in the PBS fluorescence emission recovery after a strong blue-green light illumination. In the absence of this protein, there is no fluorescence recovery, while overexpression of the same protein accelerates the recovery. When the His-tagged FRP protein was isolated from cells overexpressing this protein, traces of the OCP were co-isolated. Moreover, OCP and FRP co-immunoprecipitated using an antibody against the His-tag present only in FRP. These results strongly suggested that OCP and FRP interact. Conclusion: We have discovered a new protein involved in the OCPrelated-photoprotective mechanism. This protein, named FRP, is essential for fluorescence recovery. [1] A. Wilson, G. Ajlani, J.M. Verbavatz, I. Vass, C.A. Kerfeld, D. Kirilovsky, A soluble carotenoid protein involved in phycobilisomerelated energy dissipation in cyanobacteria, Plant Cell 18 (2006) 992-1007. [2] A. Wilson, C. Punginelli, A. Gall, C. Bonetti, M. Alexandre, J.M. Routaboul, C.A. Kerfeld, R. van Grondelle, B. Robert, J.T. Kennis, D. Kirilovsky, A photoactive carotenoid protein acting as light intensity sensor, Proc Natl Acad Sci U S A 105 (2008) 12075-12080.
P.006 PROBING THE STRUCTURAL CHANGES INVOLVED IN D1 PROTEIN TURNOVER OF PHOTOSYSTEM II IN SYNECHOCYSIS SP. PCC6803. Aparna Nagarajan, Hong-Jin Hwang, Robert Burnap. Dept. Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK, USA. D1 protein subunit is deeply buried within the PSII complex and has a high turnover rate (approximately 30min., depending upon light intensity). The process of turnover involves an efficient process of damage recognition and repair, which is largely unknown. Rearrangements in the structure of the D1 protein and PSII complex are inevitable during this turnover process. We are adopting two technical approaches to detect the structural changes during this process. The first approach involves the use of reagents causing a more general modification of various residues that become exposed to the aqueous phase during the turnover process. Different chemical modification reagents (e.g. carbodiimides) are being explored to probe changes in conformation. These modified PSII subunits can be analyzed by mass spectrometry (MALDI-TOF). We have obtained good coverage for the core PSII proteins with 64% of D1, 57% of CP43 and 47% of MSP full-length sequence. The regions of coverage include the domains that are hypothesized undergo changes. A comparison of peptides before and after treatment could demonstrate the changes in the solvent exposure post modification. The alternative and more specific approach is to detect these structural changes using cysteine-scanning mutagenesis. Analysis of the structure of D1 has allowed us to select a few domains that might be involved in solvent exposure. Based on this structure, a set of mutants have been generated which have all the four native cysteines August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts removed and one cysteine substituted at a specific location. These cysteine residues are probed using thiol reactive reagents like 5iodoacetamido-fluorescein. A cysteine residue that is solvent exposed can react with the probe and can thus be hypothesized as the domain that undergoes a conformational change. These chemically modified domains of D1 protein can be detected by using gel electrophoresis. This system involves the use of PSII particles and will allow us to probe the solvent exposed residues both under native and damage conditions in vitro. Supported by National Science Foundation NSF MCB-0818371
P.007 KINETIC ANALYSIS OF CCMR AND CMPR (LYSR-TYPE TRANSCRIPTIONAL REGULATORS) WITHIN SYNECHOCYSTIS SP. PCC6803. Shawn M. E. Daley1, Marla Carrick1, Robert L. Burnap2. 1
Biochemistry & Molecular Biology; 2Dept. Microbiology & Molecular Genetics; Oklahoma State University, Stillwater, OK, USA.
Aquatic photosynthetic organisms are generally dependent upon active transport of inorganic carbon to supply the Calvin-BashamBenson carbon fixation process. This is an energetically expensive active transport uptake system termed the carbon concentration mechanism (CCM). The control of the high-affinity/low-flux inorganic CCM in cyanobacteria has been of great interest for many years. Two DNA-binding proteins, CcmR and CmpR, are responsible for the transcriptional control of the expression of transport genes for the CCM. CmpR has been shown to be an activator of cmpABCD (Takahashi et al 2004). CcmR has been shown to be a repressor in vivo as established by microarray analysis appearing to control several different genes/operons that are spread throughout the genome of Synechocystis sp. PCC6803 (Wang et al 2004). Therefore the genes/operons under the control of CcmR are referred to as the ‘CcmR Regulon’. This putative regulon contains 5 distinct genes/operons; ccmR, ndhF3 [operon], ndhD5 [operon], sbtA [operon] and ubiX. However, direct physical evidence for the interaction between CcmR and the proposed genes comprising this regulon is limited. This work aims at testing the hypothesis that CcmR indeed interacts with the DNA regulatory region of the genes previously identified by microarray analysis and to provide a more complete molecular characterization of the regulatory features of this putative regulon. For the first time in vitro data supporting direct binding of CcmR to all of the regulon components will be presented. These have been determined by electrophoretic mobility shift assay. The control of this energy intensive system of carbon sequestration has also been of great interest for many years. Recently Nishimura et al (2008) has determined the ligand molecule for CmpR within Synechococcus elongatus PCC7942. Efforts to determine the ligand molecule, using surface plasmon resonance, and its associated kinetic constants (ka, kd, kD) will be reported, for CcmR and CmpR within Synechocystis sp. PCC6803. Supported by US Department of Energy, Energy Biosciences Division DOE DE-FG02-08ER15968
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Final Program and Abstracts
P.008 SUPRAMOLECULAR ORGANIZATION OF PHYCOBILIPROTEINS IN THE CHLOROPHYLL D-CONTAINING CYANOBACTRIUM ACARYOCHLORIS MARINA. Min Chen1, Tom Bibby2, Matthias Floetenmeyer3. 1
School of Biological Sciences, University of Sydney, Australia, National Oceanography Centre, Southampton University, United Kingdom; 3Centre for Microscopy and Microanalysis, University of Queensland, Australia. 2
Acaryochloris marina MBIC11017 is an ecologically important, biochemically interesting and evolutionarily fascinating marine cyanobacterium using the unique chlorophyll, Chl d. In addition to the accessory Chl d-binding light-harvesting protein complexes as its major light harvesting system, which feeds energy to photosystems I and II, it possesses a primitive phycobiliprotein complex as an accessory peripheral antenna system. Using advanced cryo-electronic microscope technologies we have revealed the high resolution details of the organisation of phycobiliprotein structures of Acaryochloris MBIC11017. Interestingly, the new strain isolated from the Salton Sea ( Acaryochloris CCMEE5410) lacks phycobiliproteins and no such supramolecular structure of phycobiliproteins was observed. Cryo-electron transmission-microscopy on native cell-sections of Acaryochloris MBIC11017 shows extensive patches of near-crystalline phycobiliprotein rods that are associated the thylakoid membranes. They are organized as an array of rods along the stromal (or cytoplasmic) side of the thylakoid membranes and divide the thylakoid membranes into an uneven “ribbon” with about 25 nm spaces (for phycobiliprotein region) or a closely stacked region leaving no spaces (less than 10 nm, without phycobiliproteins between the membranes). The dimensions of the individual rods are 10 nm × 25 nm. Aligning the PSII supercomplexes to rod-arrays of phycobliprotein complexes, we suggest that one array of phycobiliprotein rod-complexes lines up with one array of PSII dimer-antenna supercomplexes. Additionally, Acaryochloris possesses a huge genome with size up to 8.3 Mb including nine plasmids from 2.1 kb to 370 kb. The genes encoding phycocyanin related peptides but allophycocyanin α/β subunits are located in the plasmid pREB3. The genes encoding allophycocyanin α/β subunits are located in the main chromosome, and are not clustered together. With the aid of SDS-PAGE analysis we have identified a number of phycobiliprotein polypeptides and the proposed model structure of phycobiliproteins in Acaryochloris agrees with the structure observed by using electronmicroscope. This supramoleuclar photosynthetic structure represents a novel mechanism of organizing the photosynthetic light harvesting machinery. The relationship among photosystems and antenna systems in Acaryochloris will be discussed.
P.009 CRYSTALLOGRAPHIC STRUCTURE OF A LIGHT AND REDOX SENSING PROTEIN MUTANT, AppA17-133 Q63E, FROM RHODOBACTER SPHAEROIDES LOCKED IN PSEUDO-SIGNALING STATE. Vladimira Dragnea, George Feldman, Arun Ignatius, David Giedroc, Carl Bauer. Indiana University, Bloomington, IN, USA. AppA is a light and redox sensing protein and an anti-repressor of photosynthetic gene transcription in Rb. sphaeroides. Structure of the light sensing domain (called Blue-Light- Using-FAD) from several 88
different species has been solved. AppA BLUF domain binds oxidized FAD and is able to undergo a slow photocycle, represented by a 10 nm red-shift in the absorption spectrum of the flavin. Despite the efforts of many groups, the exact mechanism of the photocycle and the structure of its signaling state is still a mystery. Several mutants of AppA were constructed and their influence on AppA photocycle is discussed. Particularly interesting is a Q63E mutant, which exhibits no photocycle, permanent 3 nm red-shift of the flavin absorption spectrum and permanent fluorescence quenching of flavin peak. Acrylamide quenching of tryptophan fluorescence indicated that this mutant is “locked” in the light (signaling) conformation. We have confirmed this conclusion further by recording 2D-NMR spectra of the Q63E mutant, which exhibits almost identical magnetic shifts as AppAlight state. We have solved the crystal structure of AppA17-133 Q63E mutant that represents “light” conformation of this clone.
P.010 HETEROLOGOUS EXPRESSION OF [FeFe]-HYDROGENASES IN ANABAENA SP. STRAIN PCC 7120 FOR IMPROVED HYDROGEN PRODUCTION. Katrin Gaertner1, Ivan Khudyakov2, Sigal Lechno-Yossef3, Hajime Masukawa4, C. Peter Wolk3, and Eric L. Hegg1. 1 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; 2All-Russia Research Institute for Agricultural Microbiology, Saint Petersburg - Pushkin, Russia 3MSUDOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA; 4Department of Microbiology and Molecular Genetics, Michigan State University, E. Lansing, MI; USA.
Introduction: Problems related to the massive use of fossil fuels are well known. H2 is a promising, clean fuel, but currently the vast majority of industrially produced H2 is generated from fossil fuels and is therefore neither environmentally friendly nor renewable. Some photosynthetic organisms produce H2 from sunlight and water, utilizing the reductant released during water oxidation to reduce protons. Although sunlight and water are abundant and inexpensive, the application of photosynthetic organisms to H2 production on an industrial scale is challenging. The major classes of enzymes capable of synthesizing H2 are nitrogenases (N2ases); [FeFe]-hydrogenases (H2ases); and [NiFe]-H2ases, such as uptake H2ase (Hup) and reversible H2ase (Hox). O2, produced by water-splitting photosynthesis, inhibits or inactivates all known N2ases and H2ases. Unlike N2ases, H2ases require no ATP. [NiFe]-H2ases are less O2-sensitive than [FeFe]-H2ases, but have lower specific activities and turnover rates. Anabaena spp. are filamentous N2-fixing cyanobacteria that contain differentiated, micro-oxic cells called heterocysts that protect N2ases and H2ases from O2. Our goal is to enhance H2 production by Anabaena by heterologously expressing [FeFe]-H2ases in the heterocysts. Methods: We are using standard genetic and molecular genetic techniques to target [FeFe]-H2ases, and the proteins needed for their maturation, to the heterocysts of Anabaena. To obtain optimal expression, these genes from various organisms are being driven by heterocyst-specific promoters (P) starting with Anabaena Pnif with and without amplification (Wolk et al., Mol Microbiol 7: 441, 1993), using integrating and replicating vectors, in Hup- and Hup- Hox- strains. Different combinations of [FeFe]-H2ases and maturation proteins will be tested, beginning with Chlamydomonas reinhardtii hydA and Clostridium acetobutylicum maturation genes hydE, hydF, and hydG (King et al., J Bacteriol 188: 2163, 2006). Results: We have integrated the gene encoding T7 RNA polymerase into the genome of Anabaena in the nif operon, eliminating N2ase August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes activity. The hyd genes will be expressed from PT7 on a replicating plasmid that has been transferred to Anabaena. All H2 production by our Hup- Hox- recipient strains will be due to heterologous expression of an [FeFe]-H2ase. Conclusion: Micro-oxic heterocysts provide a promising environment for the expression of O2-sensitive [FeFe]-H2ases under oxic external conditions. If strains expressing [FeFe]-H2ases in heterocysts produce more H2 than strains containing only the native N2ases, the former strains may provide a route to economically practicable production of H2 from renewable resources. P.011
Final Program and Abstracts mutant indicated a non-essential function. Spectral analysis by EPR indicates the existence of the redox cofactor heme ci in T. elongatus (as reported before for M. laminosus and C. reinhardtii). Due to its transformability, the purification of fully active dimeric cyt b6f complex from T. elongatus now enables an in-depth characterization including site-directed mutants. References: [1] Volkmer, T., Schneider, D., Bernát, G., Kirchhoff, H., Wenk, S-O., Rögner, M., J Biol Chem, 2007, 282, 3730-7 [2] Kurisu, G., Zhang, H., Smith, J.L., Cramer, W.A., Science, 2003, 302, 1009-14 [3] Stroebel, D., Choquet, Y., Popot, J-L., Picot D., Nature, 2003, 426, 413-18
CHARACTERIZATION OF AN ACTIVE CYTOCHROME B6F COMPLEX FROM THERMOSYNECHOCOCCUS ELONGATUS INCLUDING THE NEW SUBUNIT PETP.
[4] Baymann, F., Giusti, F., Picot, D., Nitschke, W., PNAS, 2007, 104, 519-24
Dorothea Gomolla1, Regina Oworah-Nkruma1, Corinna Lüer1, Meike Gendrullis1, Gábor Bernát1, Sascha Rexroth1, Frauke Baymann2, Matthias Rögner1.
P.012
Plant Biochemistry, Ruhr-University Bochum, 44801 Bochum,Germany; 2BIP/CNRS, 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France.
PROTEOMIC ANALYSIS OF KNOCKOUT MUTANTS FOR PLASTOCYANIN AND CYTOCHROME C6 IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803
Introduction: The cytochrome b6f (cyt b6f) complex of oxygenic photosynthesis from the thermophilic cyanobacterium Thermosynechococcus elongatus connects the electron transport between Photosystem 2 and Photosystem 1 (PS1). Each monomer of the dimeric integral membrane complex consists of four major subunits: cyt f (PetA, 33 kDa), cyt b6 (PetB, 24 kDa), the Rieske ironsulfur protein (PetC, 19 kDa) and subunit IV (PetD, 17 kDa). Additionally, there are four small subunits - i.e. PetG, PetL, PetM and PetN with molecular masses ranging from three to seven kDa. Recently, we identified an additional 7.2 kDa subunit of the cyt b6f complex of Synechocystis sp. PCC 6803 (S. 6803), which we named PetP [1].
Manuel Hervás, Ornella Castielli, José A. Navarro, Berta De la Cerda and Miguel A. De la Rosa.
1
Methods: In order to investigate structure-function relationships in a stable and fully active complex we developed a new strategy for the purification of the cyt b6f complex from T. elongatus, involving Histagged cyt f and two chromatographic steps. The purified dimeric complex was analyzed by MALDI-MS and ESI-MS, as well as EPR measurements. Results: The resulting dimeric complex contains all cyt b6f subunits and a 7.2 kDa protein. ESI-MS and MALDI-MS analysis revealed that this protein is encoded by the open reading frame tlr0524 – with high homology to PetP (ORF ssr2998) of S. 6803. Disruption of tlr0524 showed impaired growth especially under high light conditions and a slower electron donation to cyt f and PS1 according to cyt f and P700 absorption kinetics. Crystal structure analysis of cyt b6f from the thermophilic cyanobacterium Mastigocladus laminosus [2] and the green alga Chlamydomonas reinhardtii [3] showed an additional redox cofactor heme ci. This cofactor is located in the Qi pocket of the complex (close to heme bH) and covalently linked to a cystein residue of the cyt b6 protein via a single thioether. Preliminary EPR analysis of heme ci from T. elongatus in presence and absence of the inhibitor 2-n-nonyl-4hydroxyquinoline N-oxide showed similar spectra as obtained with the cyt b6f complex from C. reinhardtii [4]. Conclusions: In summary, we suggest that PetP, which is missing in the crystal structures of M. laminosus and C. reinhardtii, is a new – possibly regulatory – subunit of the cyt b6f complex. Similar to S. 6803 our results showed a direct association of PetP with the electron transport of the cyt b6f complex, although characterization of the PetP-less August 9 to 14, 2009 • Montréal, QC, Canada
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC. Centro Isla de la Cartuja, Américo Vespucio 49, E-41092 Sevilla, Spain. Introduction: Iron and copper participate as essential protein cofactors in photosynthesis and respiration. However, both metals at high concentration promote the generation of reactive oxygen species. Thus, metal homeostasis is crucial in maintaining the adequate concentration of metal ions inside the cell [1]. Plastocyanin (Pc) and cytochrome c6 (Cyt) are two alternative electron transport proteins that can replace each other depending on copper availability [2]. In this work, we describe a comparative proteomic analysis of Synechocystis wild-type (WT) and two knockout mutants for either Pc or Cyt growing with or without copper, thus yielding relevant information on metal homeostasis in cyanobacteria. Methods: Changes in the proteome of Synechocystis have been studied by comparing 2-DE gels of cytoplasmic soluble fractions when cultured with or without copper. Soluble proteins were first resolved by isoelectric focusing on a pH range from 4 to 7, followed by SDSPAGE. Qualitative differences, as well as quantitative comparison of spots, were analyzed using the PDQuest software package (Biorad). The selected spots were further identified by MALDI-TOF. Results: Due to the relevance of copper and iron homeostasis in cyanobacteria, we have studied the effect of copper deprivation on the proteome of Synechocystis [3,4]. Particular emphasis has been made on the combined effect of copper deficiency and deletion of the petE and petJ genes coding for Pc and Cyt, respectively, whose expression is regulated by this metal [2]. The differences in the proteome of WT cells reveal that copper induces not only the over-expression of 19 enzymes involved in the main metabolic pathways, but also the synthesis of the GroEL1 chaperone and of the ATP-dependent ClpP protease induced under stress. On the contrary, the lack of copper enhances the expression of an ABC transporter, which is the major contributor to ferric iron transport across the plasma membrane. When comparing the proteomes of WT and DpetE cells grown in the presence of copper, substantial expression changes are observed in 89
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Final Program and Abstracts 43 proteins involved in the main metabolic pathways. In particular, the changes observed in the mutant concern proteins involved in photosynthesis and enzymes related to regulation of phycobilisomes coupling to PSII. Similarly, the proteomes of WT and DpetJ cells grown in the absence of copper show different expression levels in 29 proteins involved in the main metabolic pathways. Actually, the expression level of proteins involved in carbohydrate metabolism, along with the antenna proteins and enzymes participating in the synthesis of pigments are significantly altered in the mutant cells. Moreover, Synechocystis cells grown with neither Pc nor Cyt suffer from redox stress due to the blocking of the electron transport chains. Accordingly, the over-expression of thioredoxins, peroxiredoxin, superoxide dismutase and the DnaK chaperone, among other proteins, can be observed. Conclusions: The proteomic analyses of Pc and Cyt deletion mutants of Synechocystis grown in the absence or presence of copper, as compared to WT cells, can be used as a powerful tool to understand metal homeostasis in cyanobacteria. References [1] Messerschmidt A et al (eds) (2001) Handbook of Metalloproteins. John Wiley & Sons, Chichester [2] Hervás M, Navarro JA, De la Rosa MA (2003) Accounts Chem Res 36, 798–805 [3] De la Cerda B et al (2008) Brief Funct Genomic Proteomic 6, 322– 329 [4] Castielli O et al (2009) FEBS Lett (in press)
Methods: A genomic library of Gloeothece sp. constructed in EMBL3 was used for screening and sequencing of nif-genes. The gene encoding NtcA (ntcA) from Gloeothece sp. was cloned into the expression vector, pET-28a (+). The vector was transformed into E. coli BL21 (DE3) and recombinant NtcA was purified using a His trap column. An electrophoretic mobility shift assay (EMSA) was performed to determine the NtcA binding site in the upstream region of the nifgenes. Results: The nif-genes were arranged as a continuous cluster spanning approximately 19 kb and organized into eight operons (in order: orf4orf5, nifVZT, nifP, nifBfdxnifSU, nifHDK, nifENX, orf2 and nifW). A typical NtcA-binding sequence was not found at an upstream region of each operon. However, sequences similar to the typical NtcAbinding site were found between nifP and nifBfdxnifSU (TGTN10ACA) that corresponds to the upstream regions of these operons and an upstream region of nifVZT (GTGN10ACA). EMSA showed NtcA bound only to the region between nifP and nifBfdxnifSU. The binding affinity of NtcA to the region between nifP and nifBfdxnifSU was similar to the typical NtcA-binding sequence present in an upstream region of the glnA in Gloeothece sp. The binding of NtcA to the region between nifP and nifBfdxnifSU was enhanced by the addition of 2-OG. Both nifP and nifBfdxnifSU were expressed under nitrogen-limiting conditions in Gloeothece sp. Conclusion: Transcriptional regulation of nifP and/or nifBfdxnifSU is thought to be mediated by NtcA. Enhancement of NtcA binding by adding 2-OG suggests regulation occurs in response to the N-status within the cells according to the level of 2-OG. The data suggests there may be other transcription factor(s) that regulate the transcription of nif-genes other than nifP and/or nifBfdxnifSU.
P.013 TRANSCRIPTIONAL REGULATION OF THE NITROGEN FIXATION RELATED GENES (NIF-GENES) BY NTCA IN A UNICELLULAR CYANOBACTERIUM GLOEOTHECE SP. 68DGA. Tadashi Kawabata1, Yukiko Taniuchi2, Shinya Yoshikawa1, Mitsunobu Kamiya1, Kaori Ohki1. 1
Faculty of Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan; 2Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan. Introduction: The gene set encoding proteins required for the diazotrophic cyanobacteria synthesis of functional nitrogenase (nifgenes) are clustered and organized as several transcriptional units. Expression of the nif-genes (including the structural genes for nitrogenase, nif H, D and K) occurs under nitrogen-limiting conditions. However, it is unclear whether expression of the nif-genes is controlled by a single transcriptional regulator as found in enteric bacteria. A possible candidate for a transcriptional regulator is NtcA that is known as a global nitrogen regulator in cyanobacteria. NtcA is thought to be activated by 2-oxoglutarate (2-OG) whose level increases during nitrogen depletion. A typical binding site for NtcA on the DNA was found to contain the palindromic sequence, GTAN8TAC. In the filamentous cyanobacteria, Anabaena sp. PCC7120, NtcA is required for heterocyst formation where the expression of nif-genes occurs in the late stage of heterocyst differentiation. However, direct interactions between NtcA and nif-genes have not been clear yet. To determine the role of NtcA in transcriptional regulation of nif-genes, we cloned the nif-genes from the unicellular diazotrophic cyanobacteria, Gloeothece sp. 68DGA (Gloeothece sp.); and characterized the binding of NtcA in the upstream region of the nifgenes.
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P.014 THE CBB3 OXIDASE FROM RUBRIVIVAX GELATINOSUS. Bahia Khalfaoui Hassani 2, Ileana Agalidis2, Chantal Astier2, Soufian Ouchane 2, Robert van Lis1, Wolfgang Nitschke1 and Barbara Schoepp-Cothenet1. 1
Laboratoire de Bioénergétique et Ingénierie des Protéines (UPR 9036), Institut de Microbiologie de la Méditerrannée, CNRS, Marseille; 2 CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; 2 Université Paris-Sud, Orsay; 2Université Pierre et Marie Curie, Paris; France. Introduction: Among the three groups of heme/copper O2 reductases, cbb3–type oxidases are the least well-understood. We observed that the β-proteobacterium Rubrivivax (R.) gelatinosus expresses high quantities of a cbb3–oxygen reductase when grown under oxygen-limited conditions. As a result of the high level of enzyme, this species thus lends itself both for the study of the enzyme in membranes and as starting material for biochemical purifications. Methods: The operon coding for the enzyme together with flanking regions containing genes involved in its regulation and maturation were sequenced and analysed. The enzyme was studied in membrane fragments and purified samples by UV/Vis and EPR (including studies on oriented material) spectroscopy and electrochemistry. The two extrinsic c-type cytochrome subunits CcoO and CcoP and sitedirected mutants thereof were heterologously expressed in E. coli. Attribution of EPR spectral features to heme cofactors in the whole enzyme was achieved via a CcoP null mutant. Results: EPR spectral characteristics, redox midpoint potentials and orientations with respect to the membrane of all heme components
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13th International Symposium on Phototrophic Prokaryotes present in the R. gelatinosus cbb3 oxidase were determined. The heme contained in CcoO was found to be spectrally and electrochemically heterogeneous. The spectroscopic results obtained on the product of the ccoO gene and site-directed mutants thereof suggest that during the enzyme’s catalytic cycle, CcoO switches between Met143 and His130 as axial 6th ligand rationalizing the observed redox heterogeneity. This ligand switch is furthermore in line with the absence of a well-defined orientation of the CcoO heme with respect to the membrane as opposed to the situation which we observe for all other hemes of the complex and which are in specific, well-determined orientations. Conclusions: The obtained results together with the observation that both Met143 and His130 are conserved in cbb3 oxidases indicate that the ligand switch is an essential part of the catalytic turnover of this group of O2 reductases. P.015 OPTICAL PROPERTIES OF LIGHT-HARVESTING COMPLEX 2 FROM THERMOPHILIC PURPLE SULFUR BACTERIUM, THERMOCHROMATIUM TEPIDUM.
Final Program and Abstracts concentration of LDAO. Since LH2 complex has the ring structure, the CD signals of B800 and B850 indicate two couplet type CD signals [4]. These results reflected the differences of interaction of BChls a in the B850 and the B800 and suggested that the interaction between BChls a in the B850 became weaker in high concentration LDAO. We also found the blue-shifted LH2 complex was returned to 855 nm by dilution of the concentration of LDAO to 0.05%. Sucrose density gradient of the blue-shifted LH2 complex did not show clearly band. The absorption spectra of the solution of upper and lower area showed blue-shifted LH2 complex and native like LH2 complex, respectively. This result suggested that the size of the blueshifted LH2 complex smaller than that of the native LH2 complex Conclusions: We found that the reversible spectral changes of the LH2 complex from Tch. tepidum were induced by the increasing of the concentrations of detergents. The blue-shifted LH2 complex was smaller than native LH2 complex. The interaction among BChls a of the B850 in blue-shifted LH2 complex were weaker than that in native LH2 complex. Reference [1] M. T. Madigan, Science, 225, 313-315(1985)
Masayuki Kobayashi, Hiroaki Suzuki, Zheng-Yu Wang, Tsunenori Nozawa.
[2] H.Suzuki, Biochim. Biophys. Acta 1767, 1057-1063(2007)
Ariake National College of Technology, Omuta, Fukuoka, Japan.
[4] S. Georgakopoulou et. al., Biophys. J. 82: 2184-2197(2002)
Introduction: In photosynthetic purple bacteria, light energy is absorbed efficiently by antenna complexes and the captured photons are transferred to photosynthetic reaction center. Purple sulfur bacteria have two types of antennas which are core light-harvesting complex 1 (LH1) and peripheral light-harvesting complex 2 (LH2). Thermochromatium (Tch.) tepidum belongs thermophilic purple sulfur bacterium and its optimum growth temperature is the highest of all purple bacteria [1]. In this study, we tried to isolate the LH2 complex from Tch. tepidum and found interested optical properties of that. Methods: Intracytoplasmic membranes (ICM’s) from Tch. tepidum cell were prepared by a sonication method [2]. LH2 complexes were isolated from the ICM’s by treatment with 0.35 % (w/v) lauryl-N,Ndimethyl amine-N-oxide (LDAO) at room temperature for 1 hour. Purification of the LH2 was carried out DEAE-anion exchanged column chromatography. Absorption and circular dichroism (CD) spectra were measured with Shimadzu U-3100 and Jasco J720-W spectrometers, respectively. Results: In near IR region, absorption spectrum of ICM’s from Tch. tepidum has three peaks at 917, 855 and 800 nm. These peaks has been assigned to B915 for LH1-RC complex and B850 and B800 for LH2 complex, respectively [3]. The profiles and peak position of absorption and CD spectra of the solubilized LH2 complex in 0.05% LDAO were very similar to those of native LH2 complex in ICM [3]. These suggest that the solubilized LH2 complex maintains native pigment-protein structure. We observed the changes of absorption and CD spectra at higher concentration of LDAO. The peak position of Qy transition of the B850 band was blue-shifted to 852 nm in 0.1% LDAO, 847 nm in 0.2% LDAO and 841 nm in 0.4% LDAO and the magnitude of that was decreasing according to increasing of the concentration of LDAO. The peak position of Qy transition of the B800 band was not shifted but the intensity of that was increasing by addition of LDAO. The B800 and B850 of LH2 from Tch. tepidum shows positive and negative CD signal from shorter wavelength, respectively [3]. Although the couplet type CD signal of the B850 was changed to one negative CD signal, the couplet type CD of the B800 was unchanged by increasing of the
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[3] T. Nozawa et. al., Biochim. Biophys. Acta 852, 191-197(1986)
P.016 PURIFICATION AND CHARACTERIZATION OF A PHYCOBILISOME SUBCOMPLEX CONTAINING THE LARGE ISOFORM OF FERREDOXIN:NADP OXIDOREDUCTASE. A. Korn, G. Ajlani, B. Lagoutte, A. Gall, P. Sétif. CEA, Institut de Biologie et de Technologies de Saclay, Gif sur Yvette, France The main role of the Ferredoxin:NADP oxidoreductase (FNR) in photoautotrophs is to provide the NADPH for CO2 reduction. In plantroot plastids, a distinct FNR isoform functions in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by carbohydrate oxidation. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene in most cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNRS specifically induced under heterotrophic conditions) is similar in size to the plant FNR (34 kDa) while the other one (FNRL 47 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome (PBS). Mutants have been constructed that contain only one of the two isoforms present in the wild type. The mutants that express exclusively the small isoform and the long isoform attached to the PBS are called FS and MI6, respectively. Following an NADP+/NADPH quantification, significant differences have been found for the NADP+/NADPH ratio in these mutants compared to the wild type. Using a genetically engineered strain of Synechocystis, we successfully purified a native complex composed of a phycocyanin hexamer, the rod-core linker and FNRL (FNRL-PC), in a 1:1:1 stoichiometry. We compared the NADPH-oxidase and NADP+-reductase activities of the two isoforms. Both FNRS and FNRL-PC exhibit similar oxidase activities when using potassium ferricyanide as an artificial electron acceptor, but differences in catalytic constants have been observed when analyzing the Fd mediated reduction of cyt c. This corresponds to a 40 % decrease in the affinity of FNRL-PC to Fd compared to FNRS. 91
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Final Program and Abstracts Furthermore, we found a significant decrease of the reaction rates of the first reduction of FNR by reduced Fd for the complex FNRL-PC compared to FNRS in the absence of NADP+. We also performed multiple catalytic turnover experiments and found that FNRS and FNRL-PC reductions are only limited by electron transfer from reduced Fd. Our results suggest a modified affinity for Fd in the purified FNRLPC complex compared to FNRS, which may be due to steric hindrance, by PC. These differences obtained in the catalytic activities do not explain the different phenotypes of the respective mutants that express only one FNR isoform. Therefore we propose that the activity must be determined by the differential localization of the FNR isoforms, which also probably determine their substrates availability, and their involvement in different pathways of electron flow. Further in vivo studies are underway to elucidate the function of each FNR isoform.
P.017 STRUCTURAL AND FUNCTIONAL ANALYSIS OF A CONSERVED TYROSINE TRIAD IN CYTOCHROME C1. J.A. Kyndt, J.C. Fitch, T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA. Introduction: Cytochrome bc1 (bc1), is a ubiquitous membrane protein found in all chloroplasts and mitochondria of eukaryotes and the cytoplasmic membrane of many respiratory and photosynthetic bacteria. Bc1 serves two functions—to transfer an electron to the soluble electron carrier cytochrome c, and to generate a proton gradient used in photosynthesis and oxidative phosphorylation. The c1 subunit forms the pivotal connection between the bc1 complex and its soluble partners. A triad of tyrosine residues in the c1 subunit of R. capsulatus bc1 (Y152-154) are ideally positioned to be involved in electron shuttling or correlate conformational communication between the Rieske protein, c1 and c2. The role of these residues was investigated by mutagenesis followed by spectral and kinetic analysis. Methods: R. capsulatus bc1 mutants and WT protein were produced in R. sphaeroides ∆fbc (gift from Prof. A. Crofts) with a c1 His tag. All cultures were grown photosynthetically, except for the Y154A complement which could only be grown aerobically in the dark. Proteins were purified on Ni-NTA and eluted in MOPS (pH 7.8) with 20% glycerol, 200mM His, 100mM NaCl, 1mM MgCl2, 0.01 % DM and 15 μg/ml PC. Decylubiquinol was used in the catalytic assays as substrate and the reduction of cytochrome c was measured by monitoring the absorbance change at 550 nm. Results: Mutation of Y153 to Q or A resulted in a spectrally altered cyt bc1. Both absolute and reduced minus oxidized spectra have the Soret peaks slightly blue shifted (by ~ 4 nm). Moreover, the intensities of the cyt c1 α and β peaks are significantly lower than in wild-type cyt c1 upon reduction. Measuring the redox midpoint potential (Em) for Y153Q showed that the Em for the c1 heme in this mutant was lowered by about 115 mV. Since this alone cannot explain the lower intensity of the c1 α and β peaks upon reduction with dithionite, and the fact that there is a slight Soret blue shift, we believe that the mutation of Y153 creates both redox and spin heterogeneity in cyt c1. In addition, the catalytic activity, measured in amount of cyt c reduction per second, is about 10 fold lower for the Y153Q mutant compared to WT. Since Y153 is positioned on the opposite side of the c1 hinge region as the c1 heme, it is likely that mutagenesis at that position creates steric clashes with the hinge region, which will affect the position of the sixth ligand. This was confirmed by structural homology modeling. Mutagenesis of Y to Q at position 152 did not change the spectral and
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electrochemical properties of c1, and showed WT enzymatic c2 reduction rates. However, changing Y154 resulted in very low yield and non-functional protein. Conclusion: Structural homology modeling and directed mutational analysis of these three tyrosines showed that mutations to Q and A at position 153 clearly affect the electrostatic properties of c1 and the electron transfer rate from quinol to cyt c, presumably through alteration the sixth heme ligand position. Therefore, a model where Y153 has a structural function is more likely than a role as an electron path. The fact that Y152Q has properties very similar to WT indicates that this mutation did not introduce major structural changes in c1 and does not create rate limiting changes in the activity of bc1. Mutagenesis of Y154 displayed a significant destabilization of c1, underscoring its structural importance in bc1.
P.018 EFFORTS TO ENHANCE SOLAR HYDROGEN PRODUCTION BY HETEROCYST-FORMING CYANOBACTERIA. Sigal Lechno-Yossef, C. Peter Wolk. Michigan State University, East Lansing, MI, USA. Introduction: The negative effects of fossil fuel use on our economy and environment make the utilization of sustainable energy sources desirable. A principal such source is sunlight. Its energy may be used by diverse microorganisms to generate H2 from water via photosynthesis by use of nitrogenases (N2ases) or hydrogenases (H2ases). Because these enzymes are oxygen(O2)-sensitive, processes of oxygenic photosynthesis and H2 production are normally separated temporally or spatially. Anabaena spp. and closely related cyanobacteria form specialized cells, heterocysts, in which N2ases and H2ases are protected from O2 by inactivation of O2-producing PSII, accelerated respiration, and a thick envelope of glycolipids and polysaccharide that impedes penetration of O2. Reductant required for N2 fixation and H2 production is generated by photosynthesis in vegetative cells and moves to heterocysts as sugar. We are trying, by modifying Anabaena metabolism, to increase H2 production to a commercially practicable level. In preliminary experiments, in which a short version of the nifH promoter was inserted before hoxYH in an uptake H2ase(Hup)-minus nifHD mutant of Anabaena variabilis strain ATCC 29413, no H2 was produced under oxic conditions. Methods: Our revised approach is to replace N2ase by a native, bidirectional H2ase (Hox) in a Hup-minus derivative of Anabaena sp. strain PCC 7120, in which Hox is encoded by the genes hoxE, F, U, Y, and H that (with other ORFs) are located in two independent transcriptional units separated by ca. 9 kb (1). We are generating a construct in which those two operons are contiguous, together replacing much of nifD, so as to over-express hox, rather than nifD, in heterocysts from the strong nif promoter. We retain nifH because it may provide part of the mechanism to reduce the concentration of O2 in heterocysts (2). The products of genes hydF, C, D, B, and A are required for synthesis of the NiFe cluster of Hox, and of gene hoxW is required for proteolytic processing of HoxH (1). These genes will be expressed on a replicating plasmid from the heterocyst-specific promoters of coxBII and patB (3,4). Abstracts by H. Masukawa, K. Gaertner, J.J. Park and co-authors describe complementary approaches to enhancement of H2 production: modifying N2ase such that it reduces more protons to H2 and fewer N2 to NH3, utilizing not Hox but exogenous Fe-only H2ases, and studying electron donors and pathways in heterocysts.
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13th International Symposium on Phototrophic Prokaryotes Results: To facilitate further genetic modification of the host strain, we have successfully modified a flp-FRT-based technique for removal of antibiotic markers from the genome (5) for use in Anabaena sp. strain PCC 7120. The constructs described above are initiated, and some are quite advanced. Conclusions: It remains to be determined whether the native hox genes, expressed in heterocysts in an oxic milieu, can serve practicably as a source of H2 at the level of a household or a transportation system. Although available strains of Anabaena are convenient experimental objects, it is likely that hitherto unstudied strains may produce H2 more abundantly. References: 1. Tamagnini et al., FEMS Microbiol Lett 31: 692, 2007; 2. Thorneley and Ashby, Biochem J 261: 181, 1989; 3. Valladares et al., Mol Microbiol 4&: 1239, 2003; 4. Wang and Xu, J Bacteriol 187: 8489, 2005; 5. Hoang et al., Gene 212: 77, 1998.
Final Program and Abstracts increased after dark incubation, and is even higher after dark anaerobic incubation. Although the numbers of hits were quite low, a significant increase of transcription of hydrogenase and hydrogenaserelated genes was also observed upon dark incubation. Conclusions: The data obtained in this study demonstrate that both 454 pyrosequencing and SOLiD sequencing of cDNA are appropriate techniques to monitor the transcriptome in Synechococcus sp. PCC 7002. A comparison of four different conditions revealed that under CO2 limitation the transcription of genes involved in CO2 fixation are strongly up-regulated. Transcription of genes coding for components of the photosystems and light harvesting complexes are downregulated upon dark incubation. An interesting finding is the up-regulation of transcription of two genes of pyruvate metabolism upon dark incubation. These transcriptome data provide valuable data for finding strongly regulated promoters for expression systems and give information about metabolic pathways, which is important for future metabolic engineering for biotechnological applications.
P.019 TRANSCRIPTIONAL ANALYSES IN SYNECHOCOCCUS SPECIES PCC 7002.
P.020
Marcus Ludwig, Zhenfeng Liu, Craig A. Praul, Lynn P. Tomsho, Stephan C. Schuster, Donald A. Bryant.
OCCURRENCE OF THE OXYGEN-BINDING SPHAEROIDES HEME PROTEIN AND DIHEME CYTOCHROME C IN RHODOBACTERACEAE.
Dept. of Biochemistry and Molecular Biology, University Park, PA, USA
T.E. Meyer, J.A. Kyndt, M.A. Cusanovich.
Introduction: The cyanobacterium Synechococcus sp. PCC 7002 grows in brackish and marine water, tolerates very high light intensities, and has an extremely short doubling time. Because Synechococcus sp. PCC 7002 is easily manipulated genetically, the organism is a good candidate for biotechnological applications. In this study, we performed transcriptional analyses in Synechococcus sp. PCC 7002 by mRNA/cDNA sequencing. We compared the transcriptomes of cells cultivated under four different conditions in order to find differentially transcribed genes.
Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
Methods: Cells used for transcriptional analyses were cultivated in mineral medium at 38oC, 250µmol photons*m-2*s-1 and continuous bubbling with air containing 1% CO2 (standard conditions). Growth under CO2 limitation was the same except that the air was not enriched with CO2. The cultures were grown to an OD730 of 0.7. In the case of cultures incubated in the dark, the cultures (at OD 0.7) were incubated in dark for 1h with either air or N2 containing 1% CO2 to produce anoxic conditions. cDNA libraries were synthesized after RNA isolation and subjected to 454 pyrosequencing or SOLiD sequencing. The obtained sequences were mapped against the Synechococcus sp. PCC 7002 genome using BLAST (maximum expect value 0.01, minimum identity 78%). The genes with the best hits were counted, and the counts were used for subsequent comparisons. Results: 454 sequencing resulted in about 100000 sequences per condition. Mapping against the genome of Synechococcus sp. PCC 7002 resulted in about 370000 hits in open reading frames and about 2000 in intergenic regions in total. The major fraction (87-96%) of the hits was identified as 16S and 23S rRNA, although procedures had been undertaken to deplete rRNA. Further analyses were only based on the mRNA sequences. The most abundant transcripts resulted from genes coding for the photosystems and light-harvesting complexes. After dark incubation (both aerobic and anaerobic) the transcription level of these genes is significantly lowered. Genes of the CO2 concentration and fixation mechanism are also quite abundant. The transcription level of these genes decreased upon dark incubation, whereas under CO2 limitation these genes showed an increase of transcription. Interestingly, transcription of pyruvate:ferredoxin oxidoreductase and phosphoenolpyruvate synthase is strongly
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Introduction: The oxygen-binding Sphaeroides Heme Protein (SHP) and soluble Diheme Cytochrome c (sDHC) were originally characterized from Rhodobacter sphaeroides, but have since been identified in more than 36 proteobacterial genomes. They usually occur in a three-gene operon along with a membrane-spanning cytochrome b (CytB), which in Rb. sphaeroides is fused with a duplicated mDHC domain (CytB/mDHC). In most species, the SHP operon includes regulatory genes. However, not all strains of Rb. sphaeroides have the SHP operon, it is not associated with regulatory genes, and has not been reported in other species of Rhodobacteraceae. We therefore set out to analyze a variety of Rhodobacter strains for the presence of the SHP operon and determine whether the SHP genes could have been acquired through random gene transfer or are functionally related. Methods: Rhodobacter species were obtained from our own collection, from the DSMZ, Braunschweig Germany, CV Ramana, Hyderabad India, and DK Newmann, Pasadena California and grown photosynthetically on the recommended media. PCR primers were designed based upon the conserved heme binding sites in SHP and DHC. Universal primers were also used to sequence 16S rRNA as a quality control. Results: We found that SHP and DHC are present in 9 out of 10 strains of Rb. sphaeroides, the only negative strain was 2.4.3. We were unable to detect any differences between our own isolates, strains TJ4 and JB15, but they differ from strains 2.4.1 and 2.4.9 by about 30 bases in the 2kb operon. Strain FY differs by about 41 bases and strain KD131 by 56 bases. Strain SCJ differs by about 87 bases. Strains 2.4.18 and IL106 differ from one another by only 5 bases, but are 365 bases distant from strain 2.4.1. As might be expected, there are also significant differences in the translated sequences of the 3 proteins from these two strains, which are 76-82% identical to those of strain 2.4.1, which is about the same extent as observed for distinct species. The SHP operon was also found in Rhodobacter sp. SW2, Rb. changlensis, Rhodovulum adriaticum, Rv. marinum, and Rv. robiginosum. PCR was negative for Rb. capsulatus strains SB1003, 2.3.1, and SP108, Rhodobacter sp. TJ12, Rb. blasticus, Rb. veldkampii, 93
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Final Program and Abstracts Rv. imhoffii, and Rv. sulfidophilum W4 and BSW8, however this does not prove that the genes are absent.
for twitching motility were also present. There are 26 histidine kinases, 43 response regulators, and 40 diguanylate cyclases.
Conclusions: The SHP operon is present in 9 out of 10 R. sphaeroides strains studied and was found in five related species of Rhodobacteraceae. Its occurrence in this family is therefore not due to random gene transfer but must provide a selective advantage. Furthermore, duplication of DHC and formation of the chimera occurred before speciation. SHP is postulated to function as an oxygenase and to share the same role and substrate specificity throughout the Rhodobacteraceae. The function is likely to be different in the related Rhodospirillaceae and in bacteria that have operons with associated sensor kinases and response regulators.
Conclusions: It is now possible to obtain high quality draft genome sequence data at an affordable cost in less than a month and it does not require any special expertise. Most of the expense of genome sequencing comes at the finishing stage, although there is no compelling reason to determine complete sequences. Draft sequences provide most information required and any additional sequence can be readily obtained by walking PCR.
P.021 DRAFT GENOME SEQUENCE OF THE PHOTOSYNTHETIC PURPLE SULFUR BACTERIUM, ECTOTHIORHODOSPIRA VACUOLATA. T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA. Introduction: There are now more than 700 complete bacterial genome sequences, but only five species of non-sulfur purple bacteria and one purple sulfur species completed. By way of contrast, there are more than a dozen green sulfur bacterial genomes and more than 20 distinct cyanobacterial genome sequences. There are also more than 20 species of aerobic anoxygenic phototrophic species that have been sequenced. Although there is tremendous diversity within the purple bacteria, they have been neglected to date with only a handful of species in the pipeline. We have thus initiated sequence determination of the purple sulfur bacterium, Ectothiorhodospira vacuolata (aka Ect. shaposhnikovii). The Ectothiorhodospiraceae are normally found in desert soda lakes that are characterized by alkaline pH, high carbonate coupled with low calcium and magnesium concentrations, and high productivity. When oxidizing sulfur compounds such as sulfide and thiosulfate, elemental sulfur is deposited outside the cells. Ect. vacuolata is unique as the only species in the family to produce gas vesicles. Methods: Ect. vacuolata strain DSM2111 was obtained from DSMZ, Braunschweig Germany and grown photosynthetically on the recommended medium. DNA was extracted with the Qiagen DNeasy kit, sonically sheared (250 base average size), and fragments sequenced in a single run on the Roche 454 sequencer. Sequences were assembled into contigs using Newbler and annotated with Rast. Results: 51 Mb high quality DNA sequence data were obtained and assembled into 121 contigs greater than 500 bases resulting in 3.3 Mb unique sequence (15-fold coverage). The CG content was found to be 63%. Automatic annotation resulted in 2900 coding sequences. The 16S rRNA sequence was in agreement with the published results and all 24 genes previously identified in Ect. vacuolata and Ect. shaposhnikovii were found to be in complete agreement except for the fccB gene for sulfide dehydrogenase which differed by 50%. It is likely that there are two genes for FCSD, only one of which was recovered. The alternative sulfide dehydrogenases, SoxEF and SQR are apparently absent. In the sulfur oxidation pathway, the sulfur carrier SoxYZ and the hydrolase SoxB were clustered, but the SoxAX cytochrome was located on a separate contig. The only possible sulfite dehydrogenase we found was a homolog of YedYZ. A close relative of Ect. vacuolata from Mono Lake was reported to photosynthetically oxidize arsenite to arsenate, but we could only find genes for arsenic resistance, ArrAB and ArsDABC. The GvpANFGSKJ genes for gas vesicle synthesis were recovered. There were 4 clusters of chemotaxis genes and 22 MCPs. The PilABCD and PilGHIJL genes 94
P.022 OPERATION OF THE CYTOCHROME BC1 COMPLEX OF RHODOBACTER CAPSULATUS AT HIGH PH VALUES. Katrin Jahns1, Natalia E. Voskoboynikova1, Maria A. Kozlova2, Armen Y. Mulkidjanian1,2. 1 2
School of Physics, University of Osnabrück, Osnabrück, Germany; Moscow State University, Moscow, Russia.
Introduction: In a dimeric cytochrome bc1 complex an ubiquinol molecule is oxidized in one of the two catalytic centers P and the two released electrons go to different acceptors. One is taken by the mobile domain of the [2Fe–2S] iron–sulphur Rieske protein to be passed further to the c-type cytochromes. The other electron crosses the membrane, via the low- and high-potential hemes of cytochrome b, to reduce a stable semiquinone molecule in one of the two centers N from the opposite membrane side (see refs. [1-3] for reviews). At neutral pH values, a single Zn2+ ion, which can bind close to the center P [4], not only retarded the proton release from this center and the movement of the FeS domain towards cytochrome c1, but also slowed down the oxidation of heme bh and the formation of ubiquinol in center N [5]. This correlation was attributed to the mechanistic coupling between the two quinone-binding centers [1-3,5,6]. Methods: The flash-induced generation of membrane voltage was traced, via spectral shifts of native carotenoid pigments, in vesicular preparations of the inner cellular membranes (chromatophores) of phototrophic α-proteobacteria Rhodobacter capsulatus and correlated with the optically monitored redox changes of the cytochrome hemes as described in ref. [5]. Results: The aforementioned transmembrane coupling between the quinone-binding centers implies that the retardation of events in center N should, reciprocally, affect the events in center P. To check this prediction, we have measured the pH-dependence of flashinduced reactions in the cytochrome bc1 complex, both in the absence and in presence of Zn2+ ions. The difference between kinetic traces, as obtained in the absence and presence of Zn2+ ions, respectively, diminished both under acidic and at alkaline conditions. At acidic pH values, the impact from the addition of Zn2+ ions diminished; most likely, the binding site became protonated with apparent pK of ca. 7.0 and could not bind Zn2+ ions anymore. Under alkaline conditions, in contrast, the oxidation of heme bh slowed down even in the absence of Zn2+ ions. At pH of ca. 9.0, the kinetics of reactions in the cytochrome bc1 complex resembled those measured in the presence of Zn2+ ions at neutral pH. Conclusions: The same kinetic behaviour of the cytochrome bc1 complex could be achieved either by blocking the proton release from center P by Zn2+ ions or via preventing the protonation of ubiquinone in center N by high pH. These observations support our suggestion of a cross-membrane mechanistic/thermodynamic coupling between the quinone-binding sites of this enzyme [1-3, 5, 6].
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13th International Symposium on Phototrophic Prokaryotes References: [1] Mulkidjanian A.Y. (2005) Biochim Biophys Acta 1709, 5-34. [2] Mulkidjanian A.Y. (2006) Biochim Biophys Acta 1757, 415-427. [3] Mulkidjanian A.Y. (2007) Photochem Photobiol Sci 6, 19-34. [4] Giachini L. et al. (2007) Biophys J. 93, 2934-2951. [5] Klishin, S.S., W. Junge, and A.Y. Mulkidjanian (2002) Biochim Biophys Acta 1553, 177-182. [6] Mulkidjanian, A.Y. and W. Junge (1995) in: Photosynthesis: from Light to Biosphere: (P. Mathis, Ed.), Kluwer Academic Publishers, Dordrecht, Vol.II, pp. 547-550.
Final Program and Abstracts and intracellular accumulation of CO2 / HCO3- was comparable to wild-type levels, indicating a capacity for photosynthetic electron transport. Conclusions: Microarray and proteomic analysis indicates CrhR RNA helicase interacts with transcripts involved with maintenance of photosynthetic capacity and protein synthesis. The inability of the crhR mutant to utilize the intracellular CO2 / HCO3- pool implies a defect in photosynthetic carbon assimilation. This is the first time that a RNA helicase has been associated with carbon assimilation in photosynthetic organisms.
P.025 P.023 CYANOBACTERIAL RNA HELICASES: A NOVEL ROLE IN REGULATING CARBON ASSIMILATION. Laura Patterson-Fortin1, Lige Wu1, Meghana Ventakesh2, George S. Espie3, George W. Owttrim1. 1 Department of Biological Sciences, University of Alberta, Edmonton, AB; 2Department of Biology, University of Toronto, Mississauga, ON,; 3 Department of Cell and Systems Biology, University of Toronto, Mississauga, ON; Canada.
Introduction: RNA participates actively in the regulation of gene expression in all organisms. To be functional, RNA molecules must fold into the appropriate secondary structure. RNA helicases are enzymes that alter RNA or RNP structure, thereby participating in the posttranscriptional regulation of gene expression. We have utilized a multidisciplinary approach to study the mechanisms by which alteration of RNA secondary structure by RNA helicases influences gene expression in cyanobacteria. Expression of the helicase, crhR (cyanobacterial RNA helicase Redox), is regulated by the redox potential of the electron transport chain and is therefore enhanced by low temperature (20ºC). Biochemically, CrhR rearranges RNA secondary structure by both unwinding dsRNA and annealing complementary ssRNA. Here we explore the broad ranging effects of a crhR null mutation in the cyanobacterium Synechocystis sp. PCC 6803. Methods: CO2 / HCO3- transport and accumulation was measured using an aqueous inlet mass spectrometer. Photosynthetic O2 evolution was determined with a Clarke electrode. For microarray analysis of RNAs interacting with CrhR, CrhR was immunoprecipated from Synechocystis cells grown at 20ºC for 3 hours. After phenolchloroform extraction the RNA was labeled and hybridized to a Synechocystis microarry consisting of three copies of all identified ORFs. Transcripts detected in all 9 replicates from 3 biological samples were considered positive as potential RNAs interacting with CrhR in vivo. Proteomic analysis was performed using 2-D gels followed by mass spectrophotometric identification of proteins whose levels altered between wild type and crhR mutant cells. Results: Microarray analysis indicates CrhR interacts with a limited number of transcripts encoding genes involved with photosynthetic electron transport, energy metabolism, protein synthesis/degradation and unexpectedly transponsons. Initial proteomic analysis indicates that crhR mutants lack CcmK, a major component of the carboxysome shell. Phenotypically, the crhR mutant grows at near-normal wild-type rates at 30ºC, but not at 20ºC. The reduced rate of growth at 20ºC was correlated with a 4-fold reduction in the rate of photosynthetic oxygen evolution. Interestingly, after brief exposures to 20ºC, photosynthetic capability was restored upon return to 30ºC. In shortterm experiments, mass spectrometric analysis revealed that transport August 9 to 14, 2009 • Montréal, QC, Canada
IDENTIFICATION AND CHARACTERIZATION OF HIGHLY CONSERVED PROTEINS IN THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER SPHAEROIDES. Aaron Setterdahl, Allan Huth, Olga Simmons, Wes Halfacre. Indiana University Southeast, New Albany, IN, USA. Introduction: Genome sequencing of many organisms in recent years has revealed an enormous database of gene sequences that code for many previously unknown proteins (Zhou, Kvikstad et al. 2003). A number of these proteins are highly conserved between many diverse organisms. Sequence analysis of the purple photosynthetic bacterium Rhodobacter sphaeroides reveals several protein sequences that are uncharacterized and are highly conserved among bacteria such as Escherichia coli, Staphylococcus aureus, Xanthomonas axonopodis, Bradyrhizobium japonicum, Rhodopirellula baltica, Gluconobacter oxydans, Pseudomonas syringae, Shigella sonnei, Tenacibaculum, Flavobacterium, Blastopirellula marina, Rickettsiella grylli, Mesorhizobium, Streptomyces ambofaciens, Enterobacter spp., Planctomyces maris, and Klebsiella pneumoniae. Even with detailed annotation of genomes in which sequences of DNA from one organism are compared to other DNA sequences of different organisms, not all genes and their respective gene products can be correctly identified. Numerous instances exist where annotation reveals many genes to be “unknown function” or “hypothetical protein.” Recent studies used microarray analysis to determine the expression of hundreds genes in response to anaerobic-light to aerobic-dark conditions (Arai, Roh et al. 2008). Of the genes analyzed in that study at least 89 genes code for hypothetical proteins. Many of the genes that show sequence similarity and have been assigned a function by sequence comparison have been previously uncharacterized in R. sphaeroides or any other photosynthetic bacteria. Methods: The program Artemis was used to scan the annotated genomic DNA database of R. sphaeroides. Gene sequences that were labeled as unknown function or uncharacterized protein, were BLAST using the non-redundant NCBI database. Conserved protein sequences were targeted for PCR. Genomic DNA of R. sphaeroides is purified and used as a template for PCR. Primers were designed with a specific sequence that will amplify each several genes. Results: PCR has confirmed the presence of the gene sequences of the Iron-containing alcohol dehydrogenase, Putative FAD-dependent glycerol-3-phosphate dehydrogenase, a conserved hypothetical protein, and a Probable FAD oxidoreductase. Gene sequences that were targeted but were unconfirmed by PCR were a putative copper binding protein, a conserved hypothetical protein, probable bacterioferritin, putative iron-regulated protein, and a probable c-type cytochrome.
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Final Program and Abstracts Conclusions: PCR has confirmed the presence of the gene sequences, but more work needs to be done to confirm or discover the function of the gene products. This is a first step in the elucidation of the gene functions. The next step is to systematically clone, express and purify these and many other gene products. The confirmation of these gene identities is paramount to our understanding of these organisms. Many genes have proposed functions, yet still need to be systematically characterized in order to confirm or discover their proper function within R. sphaeroides. References: Arai, H., J. H. Roh, et al. (2008). “Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4.1.” J Bacteriol 190(1): 286-99. Zhou, S., E. Kvikstad, et al. (2003). “Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.” Genome Res 13(9): 2142-51.
P.026 THE INVESTIGATION OF THE ACSF PROTEIN AND PIGMENT BIOSYNTHESIS IN CHLOROFLEXUS AURANTIACUS. Kuo-Hsiang Tang, Jianzhong Wen, Xianglu Li, Robert E. Blankenship. Departments of Biology and Chemistry, Campus Box 1137, Washington University, St. Louis, MO, USA. Introduction: The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have primarily a structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophyll (BChl) c, d and e is not well understood. Methods: In this report, we used spectroscopic, proteomic and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus (C. aurantiacus). Results: We characterized the chlorosome proteins, CsmM, CsmN, and unexpectedly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, under anaerobic growth conditions for C. aurantiacus. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestions. Additionally, realtime (RT)-qualitative PCR studies showed that the gene expression of csmM, csmN, and bchE is up-regulated under anaerobic conditions, but that the transcript level of the acsF gene is not lower in anaerobic growth than in semi-aerobic growth (Tang, K.-H. et al. J. Bacteriol. in press). Finally, we have investigated on the location of AcsF in C. aurantiacus and other photosynthetic bacteria, as well as probed on the biosynthesis of the isocyclic ring formation in BChl a and BChl c. Conclusion: Together, it is clear that even for the most investigated FAP bacterium, C. aurantiacus, the role(s) of AcsF is far away from being understood. Additionally, since no studies have been reported previously for AcsF and BchE in any green bacteria, which have a specialized photosynthetic compartment— the chlorosome, caution is needed to compare the properties and mechanism of action of AcsF and BchE in GSB and FAP with the studies of higher plant, algae, and other bacteria. We are currently investigating these two enzymes with both in vitro and in vivo studies.
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P.027 PURIFICATION AND CHARACTERIZATION OF THE FMO PROTEIN FROM THE NEWLY DISCOVERED AEROBIC PHOTOTROPH, CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUM. Yusuke Tsukatani, Donald A. Bryant. The Pennsylvania State University, PA, USA. Introduction: We have discovered a new thermophilic, aerobic phototroph from the phylum Acidobacteria which had not included photosynthetic members. The discovery of the new acidobacterium Candidatus Chloracidobacterium (Cab.) thermophilum enables us to explore new mechanisms of photosynthesis and may provide new insights into evolution of photosynthesis. Genomic analysis of Cab. thermophilum revealed that this organism possesses genes encoding a reaction center core subunit (pscA), a baseplate protein of chlorosomes (csmA), and a light-harvesting Fenna-Matthews-Olson, so-called FMO protein (fmoA). Thus, Cab. thermophilum appears to do type-1 reaction center-based photosynthesis in the presence of oxygen, whereas green sulfur bacteria and heliobacteria that also have type-1 reaction centers are all strictly anaerobic phototrophs. Here we report the first isolation of the FMO protein from Cab. thermophilum. The FMO protein shows different absorption and fluorescence properties compared to that from green sulfur bacteria. Methods: Membranes prepared from photosynthetically grown cells of Cab. thermophilum were incubated with sodium carbonate and then a supernatant was collected by ultracentrifugation. After dialysis, the supernatant was subjected to anion-exchange DEAE column chromatography. The blue-colored FMO protein was eluted by 500 mM NaCl. Results: The purified FMO protein appeared as a single band upon Coomassie blue-stained SDS-PAGE. The molecular mass predicted from SDS-PAGE was about 44 kDa, which corresponds to the mass predicted by the amino acid sequence of the FMO protein (40.5 kDa). The 44-kDa protein band was confirmed as the FMO protein by peptide mass fingerprinting analysis. Absorption spectrum of pigments extracted from the FMO protein showed that the protein binds bacteriochlorophyll a. The FMO protein showed a large absorption peak at 797 nm and shoulder peaks at around 810 and 824 nm at room temperature. The fluorescence emission spectrum of the FMO protein showed two peaks at around 814 and 827 nm at room temperature. On the other hand, the FMO protein of green sulfur bacteria is known to have a single fluorescence peak at 818 nm at room temperature and the peak is sharpened and shifted to 831 nm at 77K. We will report the low-temperature spectroscopic properties of the FMO protein. We are also characterizing components in membrane preparations and attempting to isolate an active reaction center. Conclusions: We successfully purified the FMO protein from the new aerobic phototroph Cab. thermophilum. The FMO protein was a bacteriochlorophyll a-containing protein like that of green sulfur bacteria. However the FMO protein of Cab. thermophilum had distinctive spectroscopic properties compared to that of green sulfur bacteria. The results suggest that the energy transfer scheme inside the FMO protein of Cab. thermophilum may be significantly different from that of green sulfur bacteria.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
P.028 GLOBAL PROTEOMICS REVEAL NOVEL NITROGEN UTILIZATION MECHANISM UNDER NUTRITIONAL STRESS BY SYNECHOCYSTIS 6803, A MODEL PHOTOTROPH. Kimberly M. Wegener1, Jon M. Jacobs2, Abhay K. Singh1, Thanura Elvitigala3, Eric A.Welsh1, 4, Nir Keren1, 5, Marina A. Gritsenko2, Bijoy K. Ghosh1, 6, Richard D. Smith2, and Himadri B. Pakrasi1. 1
Department of Biology, Washington University, St. Louis, MO, USA; Pacific Northwest National Laboratory, Richland, WA, USA; 3 Department of Electrical and Systems Engineering, Washington University, St. Louis, MO, USA; 4Pfizer, Inc., St. Louis, MO, USA; 5Dept. of Plant and Environmental Sciences, Silberman Institute of Life Science, The Hebrew University of Jerusalem, erusalem, Israel; 6 Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, USA. 2
Introduction: Cyanobacteria, oxygenic phototrophic prokaryotes, are the progenitors of the modern chloroplast. They are crucial to global oxygen production and worldwide carbon and nitrogen cycles. Synechocystis sp. PCC 6803 has long been a model cyanobacterium for photosynthesis research. Here we present the first global proteome analysis of an autotrophic photosynthetic bacteria and the most complete coverage of a photosynthetic prokaryotic proteome to date. Methods: Synechocystis 6803 cells were subjected to iron, phosphate, nitrogen, or sulfate depletion and subsequent repletion. Cells were also subjected to environmental stresses including heat shock, cold shock, salt stress, high CO2 (3%) or growth under photoheterotrophic conditions.. Samples were collected at a total of 33 time points across conditions, fractionated, and subjected to proteomic analysis using the Accurate Mass and Time (AMT) tag approach. These results were analyzed statistically to combine technical replicates. Protein fold changes were calculated by comparing levels of an individual protein in a stress condition to those in cells grown in complete BG11. Results: The resulting proteome dataset consists of 22,318 unique peptides, corresponding to 2,369 unique proteins, covering 65% of the predicted proteins. These proteins uniformly represent all functional categories and include proteins encoded by the Synechocystis 6803 chromosome as well as the plasmids pSYSM, pSYSX, pSYSA, and pSYSG (Cyanobase). In contrast, previous proteome studies of Synechocystis 6803 have identified just 1,099 proteins (30% of the predicted proteome). 1,359 of the proteins identified in our study were novel proteomic identifications while 1,010 proteins were common proteins in our study and previously published proteomes. Additionally, we have identified 758 proteins that are currently annotated as hypothetical. These proteins can now be reclassified as unknown proteins, reducing the number of hypothetical proteins in Synechocystis 6803 by over one half. Importantly, quantitative analysis of protein abundance ratio in cultures grown under nutritional stresses revealed that Synechocystis 6803 resorts to a universal mechanism for nitrogen utilization under phosphate, sulfate, iron, and nitrogen depletion. Of the 2,369 proteins identified, 1,233 proteins were differentially regulated under environmental stress conditions. Conclusions: Global proteomics offers a detailed picture into the state of an organism that reveals insight that cannot be garnered from transcriptomic studies alone. Here we present the first global proteome analysis of an autotrophic photosynthetic prokaryote to date. This study offers the most complete coverage of a photosynthetic prokaryotic proteome with quantitative data as to protein response under various critical nutritional stresses. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Acknowledgements: This work was supported by National Science Foundation Grants FIBR 0425749 and MCB 0745611 to H. B. P. This work was additionally supported as part of the Membrane Biology Scientific Grand Challenge project at the W. R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research program (Pacific Northwest National Laboratory).
P.029 INFLUENCE OF THE CAROTENOID NATURE AND OF THE AMINOACIDS AROUND THE CAROTENOID IN THE ORANGE CAROTENOID PROTEIN ACTIVITY IN PHOTOPROTECTION. Adjélé Wilson, Claire Punginelli, Jean-Marc Routaboul, Diana Kirilovsky. School of Biological Sciences (A08), University of Sydney, New South Wales, Australia. Introduction: In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. We studied the effects on the activity of the OCP of changes in the nature of the bound carotenoid and of modifications of the 100% conserved amino-acids surrounding it. Methods: Synechocystis PCC6803 mutants overexpressing the OCP and containing a mutated OCPs, or lacking the crtO gene were constructed. The existence of the photoprotective process was tested by fluorescence measurements using a PAM fluorometer. The OCP protein was isolated and its photoactivity tested.
Results and Conclusions: In the ∆crtO mutant, lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results confirmed that the red OCP is the active form and strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity. Trp288 and Tyr201 forming a hydrogen bond with the carbonyl of the carotenoid are essential for OCP photoactivity and induction of the photoprotective mechanism. They cannot be replaced by other amino-acid without loose of activity. While Trp110 and Tyr 44 can be changed by a Phe without loose activity, they cannot be replaced by Ser suggesting that the interaction of the carotenoid hydroxyl ring with aromatic groups is important for the formation and/or stability of the red form. Trp 277 also is involved in the conversion and /or stabilization of the red form. Finally, while Arg155 is essential for the induction of the photoprotective mechanism is not involved in the light-induced orange to red form conversion.
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P.030
of inactivation by oxygen and that the oxygen-sensitive DPOR, especially L-protein, is protected by some molecular mechanisms in oxygenic cyanobacterial cells.
CHARACTERIZATION OF A NITROGENASE-LIKE PROTOCHLOROPHYLLIDE REDUCTASE IN THE CYANOBACTERIUM LEPTOLYNGBYA BORYANA.
P.031
Haruki Yamamoto, Shohei Kurumiya, Rie Ohashi, Yuichi Fujita.
PPSR AS A HEMIN SENSOR.
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Indiana University, Bloomington, IN, USA.
Introduction: ark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), the determinant enzyme for the greening in the dark, catalyzes the stereo-specific D-ring reduction of Pchlide to convert porphyrin ring to chlorin ring in the bacteriochlorophyll/chlorophyll biosynthesis. DPOR is a three-subunit enzyme of BchL/ChlL, BchN/ChlN and BchB/ChlB, and their amino acid sequences show significant similarity to the nitrogenase subunits NifH, NifD, and NifK, respectively. A series of initial biochemical analysis of DPOR from the anoxygenic photosynthetic bacterium Rhodobacter capsulatus confirmed the nitrogenase-like features. DPOR is distributed widely not only among anoxygenic photosynthetic bacteria but also among oxygenic phototrophs such as cyanobacteria, green algae and gymnosperms, which raises a question how the nitrogenase-like DPOR operates in oxygenic photosynthetic cells. Here we report in-vivo complementation of cyanobacterial DPOR mutants by the expression vectors, reconstitution of DPOR with two components purified from cyanobacterial cells, and the oxygen sensitivity of the DPOR components. Methods: Two mutants YFC2 (∆chlL) and YFB14 (∆chlB) of the cyanobacterium Leptolyngbya boryana strain dg5 were used as the host cells to overexpress the DPOR subunits, ChlL and ChlN-ChlB. We constructed two shuttle vectors for overexpression of Strep-tagged ChlL (pHBL2) and Strep-tagged ChlN-ChlB (pHBNB2) in L. boryana. YFC2 and YFB14 were transformed with pHBL2 and pHBNB2, respectively, by electroporation. These transformants were cultivated in BG-11 supplemented with 20 mM HEPES-KOH; pH7.4, 30 mM glucose and 10 µg ml-1 chloramphenicol. Pigments of cells were extracted in 90 % methanol and the Chl concentration was spectroscopically determined. Crude extracts of these transformants were prepared by sonication in an anaerobic chamber. Strep-tagged ChlL and ChlN protein were purified by Strep-Tactin Sepharose columns. DPOR assays were carried out as described for DPOR from R. capsulatus. Purified components were exposed to air followed by the standard DPOR assay. Results: The ability of chlorophyll biosynthesis in the dark was restored in both transformants, suggesting that both DPOR components are expressed as active forms. Strep-tagged ChlL and ChlN proteins were purified from the crude extracts of the transformants. ChlL protein was purified as a single band. ChlN was co-purified with a protein that was cross-reacted with an anti-ChlB antibody, indicating that ChlN forms a stable complex with ChlB as shown in NB-protein from R. capsulatus. DPOR assay was carried out with the purified proteins. When both proteins, L-protein and NBprotein, were added to the reaction mixture, a marked chlorophyllide (Chlide) formation was observed, while there was no detectable conversion of Pchlide to Chlide in the reaction mixtures containing only one of the purified components. Then we evaluated the oxygen sensitivity of L-protein and NB-protein. While the activity of NBprotein maintained more than 60% of the original level upon 30-min exposure to air, and the L-protein activity rapidly disappeared with a half-life of about less than 3 min upon the exposure to air. Conclusion: The results suggested that L-protein is the primary target 98
Liang Yin, Vladimira Dragnea, Carl Bauer.
PpsR and AppA coordinate light and redox control of photosynthesis gene expression in R. sphaeroides. PpsR is a DNA-binding transcription factor that contains two PAS domains followed by a carboxyl terminal DNA binding domain. Under aerobic conditions, PpsR blocks the transcription of tetrapyrrole biosynthesis and photosynthetic genes that are responsible for biosynthesis of the pigments and the light harvesting II complex (1). AppA is flavin containing photoreceptor that functions as an antirepressor of PpsR by forming an inactive PpsR2-AppA complex (1). In the past year, our results indicate that hemin provides another level of control in this system, showing the first direct link between the biosynthesis of heme with that of the photosystem. In this study we demonstrate that PpsR is a hemin-binding protein containing a 1:1 stoichoimetry of [PpsR]/[hemin]. The presence of hemin significantly inhibited the DNA-binding ability of PpsR, while other tetrapyrrole products did not show any inhibition. Experiments with truncated PpsR suggest that hemin binds to the C-terminal region. Our results suggest that PpsR is likely a hemin sensor with heme controlling the DNA binding activity or PpsR. Most of well characterized hemin-binding proteins have tightly-coordinated hemin to perform many different tasks, such as electron transfer, oxygen storage and gas molecule sensing (2). However in recent years several heme sensors, have been identified from bacteria (3) to mammals (4) that are able to sense the free hemin in vivo. Our study of heme binding by PpsR will provide new insights about hemin sensors. 1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623 2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L., Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073 3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A 96, 13056-13061 4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc Natl Acad Sci U S A 105, 76-81
P.032 EXCITATION ENERGY TRANSFER IN FILAMENTS AND HETEROCYSTS FROM NOSTOC PUNCTIFORME. Tanai Cardona, Ann Magnuson. Department of Photochemistry and Molecular Science, The Ångström Laboratories, Uppsala University, Uppsala, Sweden. The filamentous cyanobacterium Nostoc punctiforme sp. ATCC 29133 is able to differentiate a type of cell specialized in N2 fixation, a heterocyst. Heterocysts undergo dramatic changes associated with nitrogen starvation and the protection of the oxygen sensitive nitrogenase enzyme, like the partial degradation of phycobilisomes and the inactivation of Photosystem II. It is poorly understood whether heterocysts are capable or not, to control the distribution of excitation energy to Photosystem I and maximize N2 fixation under different light conditions. Using fluorescence spectroscopy, we compared the responsiveness of filaments and isolated heterocysts to green and red August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes light illumination. It was found in both cell types that 5 minutes green illumination triggered the quenching of a 680 nm emission peak corresponding to fluorescence from the terminal emitter of the phycobilisome and the appearance of a fluorescence peak at 750 nm corresponding to the trimerization of Photosystem I. We concluded that the quenching of the terminal emitter might be due to direct energy transfer from the phycobilisome core to the Photosystem I trimer. It is suggested that heterocysts possess a functional, possibly modified phycobilisome capable of distributing excitation energy to and away from Photosystem I to optimize cyclic photophosphorilation.
P.033 TRANSCRIPTION OF A “SILENT” CYANOBACTERIAL PSBA GENE IS INDUCED BY MICROAEROBIC CONDITIONS. Cosmin Ionel Sicora1,2, Felix M. Ho3, Tiina Salminen4, Stenbjörn Styring3, Eva-Mari Aro1. 1
Department of Biology, University of Turku, FI-20014, Finland; Biological Research Center Jibou, 455200, Romania; 3Molecular Biomimetics, Department of Photochemistry and Molecular Science, Uppsala University, SE-75120 Uppsala, Sweden; 4Department of Biochemistry and Pharmacy, Åbo Academy University, Finland. 2
Introduction: Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Methods: The main investigative methode used was Real-time RT-PCR for the estimation of expression of each individual gene in the family using specific primers. We also used molecular modeling to compare the structural differences between different forms of D1 and flash fluorescence to estimate the functional changes between isoforms. Results: Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities
Final Program and Abstracts Conclusion: The presence of an anaerobically induced gene in three unrelated cyanobacteria, makes us to conclude that a new form of D1 is present and important to cellular adaptation to specific conditions.
P.034 IDENTIFICATION AND CHARACTERIZATION OF BILIN LYASES FOR PHYCOERYTHRIN I BIOSYNTHESIS IN MARINE SYNECHOCOCCUS. Yasmin M. Vasquez, Avijit Biswas, and Wendy M. Schluchter. Department of Biological Sciences, University of New Orleans, New Orleans LA, USA. Introduction: Cyanobacteria have evolved photosynthetic lightharvesting complexes called phycobilisomes. The main component of the phycobilisome is phycobiliproteins. In marine Synechococcus, one of the most abundant phycobiliproteins is phycoerythrin (PE). PE is composed of α and β subunits that have the linear tetrapyrrole phycoerythrobilin (PEB) and/or phycourobilin (PUB) attached to specific cysteine residues via thioether linkages. We are interested in identifying bilin lyases involved in attaching the PEB chromophores to PE I subunits in marine Synechococcus. Methods: Putative bilin lyases from Synechococcus sp. WH8020 (CpeU, CpeY, CpeZ) or from Synechococcus sp. WH8102 (CpeS) were amplified by PCR and individually cloned in different Duet vectors. Bilin biosynthetic genes for making PEB from heme (pebS and ho1) were cloned together in pACYC (kind gift of Dr. Nicole FrankenbergDinkel). The cpeA and cpeB genes were cloned into the pMAL vector (fusing each to the gene encoding the maltose binding protein). The cultures were initially grown at 37ºC until the OD at 600 nm was between 0.5-0.6, then they were cooled down to 18ºC and induced with 0.5 mM IPTG for 15-18 hours for protein expression. The purification of MBP-CpeA and MBP-CpeB was accomplished using an amylose resin affinity column. Results: We achieved successful cloning of all the genes, which was confirmed by sequencing. Bilin lyase as well as phycobiliprotein subunit expression was found to occur optimally at 18 ºC with IPTG for a period of 15 hours. Soluble subunits CpeA and CpeB were purified with amylose resin column. SDS-PAGE confirmed bands at 57.1 kD corresponding to CpeA with maltose binding protein tag and a band at 59.1 kD corresponding to CpeB with the maltose binding protein tag. SDS-PAGE also confirmed bands for CpeZ, CpeY, CpeS and CpeU at 20.6 kD, 47.4 kD, 20.8 kD and 22.7 kD respectively. Solubility of these bilin lyases is currently being analyzed. Once conditions are optimized to obtain soluble lyases, these will be tested for bilin addition activity in in vitro reactions with PEB. Conclusions: CpeZ, CpeY, CpeS and CpeU bilin lyases are hypothesized to participate in PEB addition to the α and β phycobiliprotein subunits for PE I. Further work will focus on cloning the suspected lyases with a histidine tag in pCDF vector. The histidine tag will allow for further purification of the lyases using a nickelnitrilotriacetic acid (Ni-NTA) affinity column. Purified proteins will be used to carry out in vitro reactions to confirm the specificity of the bilin lyase for specific cysteine residues on the α and β PEI subunits.
between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also presented.
August 9 to 14, 2009 • Montréal, QC, Canada
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P.035
P.036
USE OF BARLEY STRAW FOR THE CONTROL FRESHWATER CYANOBACTERIAL BLOOMS. Rob Iredale, Japareng Lalung, David Adams.
DIFFERENTIAL RESPONSES OF EPIPHYTIC AND PLANKTONIC TOXIC CYANOBACTERIA TO ALLELOAPTHIC ACTIVITY OF THE SUBMERGED MACROPHYTES STRATIOTES ALOIDES.
University of Leeds, Leeds, West Yorkshire, United Kingdom.
Abdulrahman M. Al-Shehri, Zakaria Mohamed.
Introduction: The potential of barley straw to control nuisance growths of cyanobacteria in freshwater has been well documented in a number of laboratory and field studies, but its mode of action is still unclear. In light of this, a study was designed to investigate the effect of straw preparation and decomposition conditions on treatment efficacy, in the hope that the collected data would allow us to comment on current theory. Additionally, the relative straw-sensitivity of different cyanobacteria, some of which were freshly isolated, was determined, and we attempted to tentatively identify some of the compounds leached from straw during the early stages of decomposition.
Department of Biological Sciences, College of Science, King Khalid University, Abha, Saudi Arabia.
Methods: A specially constructed decomposition chamber containing a combination of natural spectrum and UV fluorescent tubes was used to vary the light conditions under which straw was decomposed. Laboratory bioassays were used as a means of testing organism response to the presence of straw and straw liquor, with all assays conducted in triplicate. Solid phase extraction and reverse-phase HPLC were used in order to identify compounds in straw leachate, and flow cytometry was employed as a means of estimating chlorophyll a per cell for a strain of Microcystis. Results: We found varying sensitivity of different cyanobacteria strains to the effects of straw, consistent with previous studies. Fine chopping of fresh straw resulted in instant activity even at low concentrations, whereas fresh, whole straw exhibited little activity. We have shown that microbial activity is essential for straw to become active, and that straw decomposed under UV light appeared to be more active than batches decomposed under natural spectrum light alone. The addition of catalase to UV-decomposed straw bioassays reduced the inhibitory action of straw, suggesting hydrogen peroxide may play a role in the activity of straw. Flow cytometry data showed that Microcystis cells exposed to straw for 48 hours contained around 35% of the chlorophyll a content of cells not exposed to straw. HPLC spiking experiments suggested that a mixture of low molecular weight aromatic compounds were leached from the straw in the first week of decomposition. Conclusions: It is currently accepted that the activity of straw is at least partly attributed to the solubilisation of lignin by microorganisms, possibly yielding a cocktail of various smaller aromatic compounds. Our findings support this theory, both in terms of our HPLC detection of compounds which could have originated from lignin, and the fact that fresh, finely chopped straw was inhibitory to growth, whereas fresh whole straw was not. This implies that the breakdown of lignin might be the rate-limiting step determining the onset of activity, which can be enhanced by the combination of lignin disruption and increased surface area resulting from fine chopping of the straw. The possibility of hydrogen peroxide playing a role in the activity of straw has been previously hypothesised, but this study has provided the first experimental data that this may be the case. Data from this study have also highlighted the potential use of fresh, finely chopped straw as a cyanobacterial growth control agent, and larger scale studies, possibly involving the use of chopped and whole straw in combination should be considered in the future. Although many questions remain regarding its exact mode of action, barley straw remains the leading candidate for environmentally sound control of cyanobacteria in freshwater. 100
Introduction: Cyanobacteria are prevalent bloom-forming algae, commonly occurring in most water bodies, such as lakes and reservoirs. The cyanobacterial bloom can lead to the deterioration of water quality through production of toxins that pose a risk to animal and human health. Therefore, control and prevention of the growth of such harmful organisms is a significant goal in environmental science. Bioremediation using macrophyte allelochemicals for algal bloom control has been widely accepted because of its higher environmental safety and the ability of macrophytes to restore and renew the damaged aquatic ecosystem. The present study investigates whether epiphytic and planktonic toxic cyanobacteria respond similarly to allelopathic activity of the submerged macrophyte stratiotes aloides. Methods: Changes in growth, toxin production, and activities of alkaline phosphatase (APA) and antioxidative enzymes (Glutathione Stransferase (GST) & Glutathione peroidase (GPX) of toxic epiphytic (Merismopedia tenuissima & Leptolyngbya boryana) and planktonic (Anabaena variabilis) of cyanobacteria were studied in a batch experiment over a 15-day exposure to different concentrations of the 50 % aqueous acetone extract of the submerged macrophyte Stratiotes aloides. Results: The results showed that epiphytic and planktonic species of cyanobacteria exhibited differential responses to the allelopathic activity of this macrophyte. Stratiotes aloides extract reduced the growth, alkaline phosphatase activity and toxin production of planktonic cyanobacterim, with an increase in lipid peroxidation, glutathione and activities of antioxidative enzymes (GST & GPX) of this alga. In contrast, this extract increased the growth and toxin production in epiphytic cyanobacteria, but it did not significantly affect lipid peroixdation or the activities of APA and antioxidative enzymes. Conclusions: The macrophyte Stratiotes aloides has an allelopathic inhibition on planktonic cyanobacteria only, but can stimulate the growth and toxin production of epiphytic cyanobacteria attached to its surface. Therefore, this study suggests that macrophytes stimulating the growth of toxic epiphytic cyanobacteria should be avoided as a biological control of harmful cyanobacteria in water resources.
P.037 LEAD(II) UPTAKE BY THE CYANOBACTERIUM SYNECHOCYSTIS SP. STRAIN PCC 6803 IMMOBILIZED IN CALCIUM-ALGINATE BEADS. L.F. Allison, J-H. Yau, N. T. Flynn, M.M. Allen. Wellesley College, Wellesley, MA, USA. The presence of unsafe levels of lead(II) in water systems is a problem worldwide as lead is known to cause irreversible health problems. Bioremediation, the use of living organisms to remove pollutants, has emerged as an efficient and less expensive method for environmental decontamination. The cyanobacterium Synechocystis sp. strain PCC 6803 was immobilized in calcium-alginate beads in order to enable easy removal of contaminated bacteria from the environment. Alginate is a polymer derived from brown algae, which has shown potential to bind cations. Beads were prepared by dropping varying concentrations of alginate and cells through a syringe needle into 0.1 August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes M calcium chloride. Lead accumulation experiments were conducted for 4 hours in 200 mL of 500 ppm lead; the concentration of lead in solution was monitored with an ion-selective lead electrode. The amount of lead removed from the system was measured relative to the dry weight of the beads and reported as mg Pb2+ per gram dry weight of alginate. In order to examine the effect of immobilization on the cyanobacteria, 5% EDTA (pH 9) was used to degrade the calciumalginate. Cells were immobilized in beads and grown in BG-11 medium with 100 ppm lead; the calcium-alginate was then degraded and optical density at 750 nm was measured. It was demonstrated that the cells exposed to lead in beads grew at the same rate as free cells in BG-11 medium with no lead. The cells were observed to remain in clusters after the alginate was degraded. Rapid aggregation was also observed for free cells inoculated into a 500 ppm lead solution. These results suggest the cyanobacteria may be forming a biofilm when stressed in the calcium-alginate beads and when exposed to lead. The ability of calcium-alginate to absorb lead from solution was evaluated; beads prepared with 4% (w/v) alginate and without cells were capable of removing an average of 340±30 mg Pb2+ per gram. When Synechocystis sp. strain PCC 6803 cells were embedded in the beads, the maximum accumulation of lead for all cell samples measured was 479 mg Pb2+ per gram; this sorption was produced by cells with an optical density at 750 nm of 1.0 in the exponential phase of growth. Stationary phase cells at the same optical density were found to remove 466 mg Pb2+ per gram. However, cells in the exponential and stationary phases of growth, when tested at higher optical densities of 2.0 and 4.0, had decreased lead uptake by 110 to 170 mg Pb2+ per gram. Beads embedded with killed cyanobacteria were able to absorb lead at levels approximately equal to the 4% beads without cells. The average uptake for the dead cells in 4% alginate was 330±15 mg Pb2+ per gram. Thus, cyanobacteria have been shown to remove lead from solution at levels significantly higher than calcium-alginate beads without cells. Both the growth phase and density of the cells at the time of uptake appear to significantly affect the amount of lead the cells are capable of removing from solution.
P.038 USED OF ARBUSCULAR MYCORRHIZAL FUNGI AND PSEUDOMONAS PUTIDA IN PHYTOTOXICITY TESTS OF SEWAGED SOIL. M. Attia, Namet M. Awad, Azza Sh. Turky. Agricultural Microbiology Dep. National Research Centre, Dokki, Cairo, Egypt. A pot experiment, with tomato plants, was carried out to judge the use of bio-indicators for phytotoxicity in sewaged soil. A sandy soil heavily fertilized with compost was used for comparison. Results show that mycorrhizal root colonization of tomato plants and total mycorrhizal spore numbers were significantly low in sewaged soil compared to organic fertilizer amended soil. More dense population of Pseudomonas spp., bacterial counts as well as high dehydrogenase activity was retrieved in the rhizosphere of tomato plants grown in soil amended with organic fertilizers compared to those grown in sewaged soil. Relatively different CO2 release potentials were showed among different treated soils (from about 15 to about 72 mg CO2 100g-1 within 7 days). Soils biofertilized with both microorganisms liberated more CO2 than non-biofertilized ones. Microbial respiration looks as though unaffected even at heavy metal concentrations. AM fungi and Ps. putida elevated soil bio-activity regime as a result of improving soil quality, as indicated by the dehydrogenase activity, microbial density, CO2 evolution, as well as percentage of mycorrhizal infection and spore numbers. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts
P.039 USING METABOLIC NETWORKS TO EVALUATE PATHWAY CONSTRAINTS OF BIOENERGY PRODUCTS IN SYNECHOCYSTIS SP. PCC 6803. K.L. Tibbles, S. Bryan, N.J. Burroughs. Warwick Systems Biology Centre, Coventry, United Kingdom. A stoichiometric metabolic network of Synechocystis sp. PCC 6803 was constructed from genomic data and coupled with models for respiration, photosystem electron transport and bioenergy product generation- hydrogen, ethanol and bio-diesel, building on the previous models of Shastri & Morgan, 2005 and Fu, 2008. By determining the elementary models of the system we examined the optimal pathways for growth and bioenergy product generation. This allowed determination of essential genes for production of these products and their commonality with requirements for growth, thereby ascertaining the biotechnological constraints of synechocystis for bioenergy utilisation. Further, we determined which of these 3 products is theoretically the most efficient as regards light energy capture.
P.040 NOVEL PIGMENT PROFILES OF AEROBIC ANOXYGENIC PHOTOTROPHS INDUCED BY HEAVY METAL STRESS. Julius T. Csotonyi, Vladimir Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. Aerobic anoxygenic phototrophs (AAP) are obligately aerobic bacteria that can nonetheless supplement their energy requirements with light, using the pigment BChl a, which is functionally incorporated into a reaction center and light harvesting complexes. Many AAP possess constitutive high level resistance to several toxic oxyanions of heavy metals or metalloids – collectively, metal(loid)s – such as Se, Te or V. The reason for their high level resistance is incompletely understood, but the involvement of photosynthetic pigments has been hypothesized. To gain an understanding of the response of their pigment systems to heavy metal duress, AAP from hypersaline supralitoral marine microbial mats, hypersaline springs and hydrothermal systems were cultured in the presence of high concentrations of K2TeO3 (up to 1000 µg/ml), Na2SeO3 (up to 1000 µg/ml) and NaVO3 (up to 10000 µg/ml). Spectrophotometry was performed on pigments extracted with acetone/methanol (7:2, v/v). From Erythrobacter litoralis strain T4, extracted pigments were further purified by thin layer chromatography for identification. AAP expressed a diversity of altered pigment profiles under heavy metal stress, including decreased or enhanced BChl and carotenoid synthesis, and the appearance of novel pigments not expressed in metal-free cultures. In the presence of tellurite, Erythrobacter litoralis strain T4 accumulated a putative precursor to BChl a, tentatively identified by spectrophotometric analysis as Mg-protoporphyrin IX or its monomethyl ester. Identification of accumulated intermediates of pigment synthesis pathways helps to reveal the nature of the response of AAP to heavy metal stress. Elucidation of their molecular defense strategies will be invaluable to the development of waste management techniques in heavy metal industries.
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P.041 HYDROGEN PHOTO-PRODUCTION FROM VARIOUS DARK FERMENTATION EFFLUENTS BY TWO RHODOPSEUDOMONAS PALUSTRIS STRAINS. Alessandra Adessi, Lucia Bianchi, Luca Collina, Michela Palomba, Roberto De Philippis. Department of Agricultural Biotechnology, University of Florence, Piazzale Cascine 24, I 50144 Firenze, Italy. Introduction: Among the biological H2 production processes so far tested or still under study, the sequential fermentations carried out by chemo- and photo-heterotrophic bacteria seems to be the most promising one, owing to the possibility given by the combination of the two types of fermentation to approach the theoretical maximum yield of 12 mol of H2 produced per mol of glucose consumed. However, in spite of the theoretical advantages of the integration of the two processes, only few experimental data are available on the use of the combined H2 production system. The aim of this work was to test the H2 photo-evolution performances of two Rhodopseudomonas palustris strains growing on three different spent media deriving from dark fermentations carried out by chemoheterotrophs. Methods: Rp. palustris strains 42OL and AV33 were grown in 250 ml bioreactors under anaerobic conditions, 30°C, light intensity of 200 µmol (photons) m-2 sec-1. The media utilized for photo-fermentation were: (1) TDF, constituted by the effluent of the dark fermentation of glucose carried out by the thermophilic bacterium Thermotoga neapolitana DSM 4359 (2.1 g L-1 acetate and 0.3 g L-1 lactate). (2) MDF, constituted by the effluent of the dark fermentation of glucose carried out by the mesophilic bacterium Paenibacillus polimixa ISSDS-851 (0.8 g L-1 acetate and 2.7 g L-1 lactate). (3 VDF, constituted by the effluent of the dark fermentation of vegetable residues carried out by the autochthonous microflora present on the residues (2.1 g L-1 acetate and 7.8 g L-1 lactate). Results: A first set of experiments showed that the addition of ferric citrate to the effluents is necessary in order to have H2 production. Both R. palustris strains showed good performances with VDF: a mean H2 production rate of 15.6±1.0 ml H2 (L*h)-1, with a substrate conversion of 43%, and a mean H2 production rate of 10.7±2.6 ml H2 (L*h)-1, with a substrate conversion of 44%, were obtained with 42OL and AV33, respectively. Photo fermentation of the other two effluents showed a lower degree of substrate conversion in H2 as a consequence of the considerable growth observed with both substrates. Indeed, with TDF the highest H2 production rate, obtained with strain 42OL, was about 4 ml H2(L*h)-1, with a substrate conversion of 16.5%, while with MDF the highest H2 production rate, obtained with strain AV33, was about 2 ml H2(L*h)-1, with a substrate conversion of 7.1%. NMR analyses of TDF and MDF substrates showed the presence of large amounts of N-containing compounds, which caused the utilization of fatty acids as carbon sources for cell growth instead of as electron donors for the synthesis of H2. Conclusions: Photo-fermentation of vegetable residues or of effluents derived from previous dark fermentations is a promising process, giving the possibility to recover energy from organic wastes by coupling the production of H2 with waste disposal, but needs to be optimized, in particular in the presence of N-containing compounds which cause the shift of the microbial metabolism from H2 production to cell growth. 102
Acknowledgments The Authors would like to thank the Italian Ministry of University and Research (MIUR) (FISR Project IDROBIO) and Ente Cassa di Risparmio di Firenze (Firenze Hydrolab Project), which partially supported this research, and Dr Agata Gambacorta, ICB-CNR, Pozzuoli, Italy, and Dr Giulio Izzo, ENEA - Casaccia, Roma , Italy, who kindly provided the spent media of Thermotoga neapolitana and Paenibacillus polimixa, respectively.
P.042 NEW MOLECULAR MARKERS FOR QUICK IDENTIFICATION OF THE HEPATOTOXIN-PRODUCING CYANOBACTERIAL STRAINS BELONGING TO GENUS MICROCYSTIS. Bogdan Drug 1,2, Martin Welker3, Bogdan Fren iu1, Adriana Bica1,2, Cristian Coman1,2, Nicolae Drago 1,2. 1
Babe -Bolyai University, Cluj-Napoca, Romania; 2Institute of Biological Research, Cluj-Napoca, Romania; 3Technische Universität, Berlin.
Introduction The cyanobacteria belonging to genus Microcystis are well-known for their capacity to produce hepatotoxins, also known as “microcystins”. These are secondary metabolites of peptide nature, and they are nonribosomally synthesized by a multi-enzymatic complex, consisting of peptide synthetases and poliketide synthases. The microcystins characteristics make their identification and characterization very difficult, and thus, the cyanobacterial blooms may be extremely dangerous for the humans or animals that interact with the water in which these blooms are developed. The cells destruction by chemical means does not represent a proper solution, as long as the toxins are usually found only inside of the cells, thus being released if the cells are lysed. That is why the most suitable solution to this problem is to find ways to identify the toxic cyanobacterial blooms during their initial phases, in order to limit the risk of intoxication. The purpose of this study was to find suitable molecular markers able to provide the strict and quick detection of the cyanobacterial strains with toxic potential of genus Microcystis. Methods The hepatotoxic potential of the strains was analyzed by PCR amplification of certain genomic regions considered responsible for the synthesis of the enzymes involved in toxin production. The results achieved by this molecular method were verified through certain toxicity tests that consisted in the intraperitoneal injection of mice with cyanobacterial suspension concentrated by centrifugation. The strains toxicity was also verified by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization) using a mass spectrometer. In order to achieve a correlation between PCR results and toxicity tests we tried to discriminate the amplicons by DGGE (Denaturing Gradient Gel Electrophoresis). Eventually, we have sequenced the PCR amplicons, and based on the sequences multiple alignments we have designed new primer pairs able to generate amplicons only in those strains which produce hepatotoxins. Results The intraperitoneal injection of mice with cyanobacteria has emphasized the presence of toxicity in 5 of the 24 analyzed strains. The MALDI-TOF MS spectra have also displayed the presence of microcystins in these 5 strains, while cyanopeptolins and other secondary metabolites were detected in other strains. The primers used for the amplification of certain genomic regions correlated with the toxic potential were found in several articles, but during PCR they have generated amplicons in 7 strains, not only in the 5 toxic ones. The DGGE profiles could not differentiate these 7 amplicons according to the strain toxicity. The sequences multiple alignments allowed us to design new primer pairs, which were specific for the 5 toxic strains.
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Conclusions During this study we have observed that the oligonucleotide primers found in various articles did not generate amplicons during PCR in our toxic strains, as expected, but also in other two non-toxic strains. The DGGE technique has not proved to be proper to discriminate between amplicons according to strains toxicity.
design and construction of functional modules that can be assembled into circuits with predictable behaviour. The final goal is the construction of the cyanobacterial chassis accommodating the characterized synthetic modules will allow a sustainable photobiological H2 production, and the parts and devices produced will become available for other biotechnological applications.
Based on the sequences multiple alignments, we have obtained new primer pairs which are able to amplify certain regions of the gene cluster responsible for toxicity, these being highly specific for the toxic strains. One of these 5 strains belongs to species Microcystis wesenbergii, which was thought to be the only species of genus Microcystis which does not produce microcystins.
References:
P.043
1,2
1,3
P.044 CHANGES IN MICROCYSTINS AND OTHER OLIGOPEPTIDES PROFILES IN THE CYANOBACTERIUM RADIOCYSTIS FERNANDOI EXPOSED AT THREE DIFFERENT LIGHT INTENSITIES. Alessandra Giani, Daniel de Albuquerque Pereira.
PARTS AND MODULES FOR H2 PRODUCTION AND CONSTRUCTION OF A CYANOBACTERIAL CHASSIS. 1,2
[1] http://www.biomodularh2.org
1
Daniela Ferreira , Filipe Pinto , Catarina C. Pacheco , Miguel Lopo , Pedro Moradas-Ferreira1,3 & Paula Tamagnini1,2. 1
IBMC - Instituto de Biologia Molecular e Celular; 2Faculdade de Ciências, Departamento de Botânica; 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto, Portugal. Introduction: The BioModularH2 project (FP6, NEST-2005-Path-SYN, Contract 043340) aims at designing reusable, standardized molecular building blocks that integrated into a chassis will result in a photosynthetic bacterium containing engineered chemical pathways for competitive, clean and sustainable hydrogen production [1]. The chassis is being constructed using as basis the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synthetic parts and modules related to oxygen consumption and hydrogen production were designed, and are being constructed/synthesized. Methods: Design and construction of standardized vectors compatible with the BioBrick™ format. The vectors are being used to remove redundant parts (e.g. genes) from the bacterial genome and/or to integrate synthetic parts/devices. Generation of mutants through double homologous recombination, using the constructed standard vectors. Mapping of the Synechocystis chromosomal neutral sites using bioinformatics tools and RT-PCRs. Design and construction of synthetic parts and assembly of modules following the BioBrick™ system philosophy. Characterization of parts and devices in the cyanobacterial chassis, using reporter genes and measuring enzyme activities. Results: The chassis preparation started with the removal of redundant parts, like the deletion of genes encoding the Synechocystis native bidirectional hydrogenase. In addition, the Synechocystis genome was screened for putative neutral sites, which will facilitate the integration of the synthetic parts/devices (e.g. an efficient heterologous hydrogenase). The construction of optimized synthetic modules/devices requires the preliminary characterization of parts, such as promoters, transcription factors, RBSs and terminators, which is currently being performed. Furthermore, several oxygen consuming devices were designed and synthesized, and are in the process of being tested. One of these devices will be chosen to provide a microaerobic/anaerobic environment required for the optimal expression of a heterologous hydrogenase.
Department of Botany, Institute of Biological Sciences, Universidade Fedearl de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. Introduction: Cyanobacteria are known to produce various kinds of bioactive oligopeptides. The most common peptides are microcystins, cyanopeptolins, anabaenopeptins and microviridins. There are several hypotheses about the functions of these metabolites, which includes protection against grazing, allelophatic effects, quorum sensing and internal metabolism, but some of these functions might be only of secondary importance. The comprehension of the factors affecting the production of these secondary metabolites may give some clues about the ecological and physiological functions of these compounds. Methods: In this study, the influence of light intensity on the pattern of oligopeptide production of a Radiocystis fernandoi strain was tested. The strain used in this work was isolated from a reservoir located in the southeastern region of Brazil. This species usually forms blooms in eutrophic aquatic systems. The experiments were done in triplicates and consisted of three treatments, each one at a different light intensity: 25 µmol.m-2.s-1, 65 µmol.m-2.s-1 and 95 µmol.m-2.s-1. Experiments lasted 10 days and growth conditions were 12h light: 12h dark photoperiod at 20°C. At the end of the experimental time, cultures were freeze dried and the peptides extracted with 75% methanol and purified with C18 SPE cartridges. The peptides were quantified by HPLC (Waters Alliance) and the main peaks were collected and analyzed in a MALDI-TOF Bruker Autoflex III mass spectrometer. Results: Seven different peptides were identified, 1 cyanopeptolin (Cy-1071), 1 microviridin (Mv-1709) and 5 microcystins (Mc-RR, dMcYR, Mc-YR, Mc-FR and Mc-WR). The production of Mc-YR, Mc-FR, Mc-WR and the sum of all five microcystins were highest at 25 µmol.m2 -1 .s , lowest at 65 µmol.m-2.s-1 and intermediate at 95 µmol.m-2.s-1. The production of Mc-RR was intermediate at 25 µmol.m-2.s-1, higher in the 65 µmol.m-2.s-1 treatment and lower at 95 µmol.m-2.s-1. The Mv-1709 was produced in the greatest amount at 65 µmol.m-2.s-1 and in lower amounts in the other two treatments. The production of the seven peptides together was highest in the 25 µmol.m-2.s-1 treatment and lower in the other two treatments. The production of Cy-1071 and the dMc-YR showed no significant differences among the treatments. Conclusions: It is possible that differences observed between the MCRR and other microcystins could be explained by genetic regulation and/or aminoacids and enzyme activities modulated by different light intesities. The fact that the different classes of peptides do not present similar responses to environmental factors, such as light, suggests that these compounds may have different functions.
Conclusions: The characterization of the different parts will allow the August 9 to 14, 2009 • Montréal, QC, Canada
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P.045 ENGINEERING BIODEGRADABLE PLASTICS USING EXTRACELLULAR MATRIX IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE.
blended control, indicating that the composite has the strength for practical usages as bioplastics. The composites have water-absorbing capacity and can be used as a container for either fresh vegetables or dry medicament. N. commune EPSs enhance biodegradability of the composites in soil and it maybe an advantage for the disposal of the plastics.
K. Inoue-Sakamoto1, T. Ogawa2, T. Sakamoto3. 1 Department of Applied Bioscience, College of Biotechnology and Chemistry, Kanazawa Institute of Technology (K.I.T.), Ohgigaoka, Nonoichi; 2Department of Applied Chemistry, College of Biotechnology and Chemistry, K.I.T., Ohgigaoka, Nonoichi; 3Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa; Japan.
Introduction: The terrestrial cyanobacterium Nostoc commune is widely distributed on earth and is adapted to various external conditions by acquiring tolerance to desiccation, ultraviolet light and extreme temperatures. N. commune forms colonies in which the filamentous cells are embedded in extracellular matrix. The major component of extracellular matrix in N. commune is extracellular polysaccharides (EPSs). EPSs absorb large amount of water rapidly and help the cells to maintain biological activities including photosynthesis and respiration. The hydroscopic or absorbent ability of EPSs can be applied to non-hydroscopic materials so the composites are used as a package to regulate moisture level inside the package. Alternatively, the composites can be used for agricultural soil cover sheet as a biodegradable sheet to enhance degradation of the composite by microorganisms in soil. In this study, colonies of N. commune or isolated EPSs were blended with plastics to test the capacity of water in the composites, and the physical properties of the composites were investigated. Methods: The colonies of N. commune naturally grown in the field were collected, washed and air-dried. The dried colonies were crushed in a kitchen mixer and then powdered in a frozen pulverizer. The powder with diameter less than 100 µm were selected with a sieve and blended directly with pellets of polybuthylene succinate (PBS) or low density polyethylene (LDPE) in weight ratio of 10:90, 15:85 and 20:80 by using a compression molding machine and an extruder in combination. EPSs were prepared from N. commune colonies by extraction with acetone and diluted acetic acid and precipitation by ethanol. EPSs were freeze-dried, powdered and blended with PBS or LDPE as described above. The sample sheets of the composite were used for the analyses. For mechanical properties, tensile stress and tensile strain at break were measured by a tensile tester. For the water absorption capacity, the samples were immersed in deionized water and the increase in weight of the samples was measured. For the biodegradability assay, the samples were buried in wet soil and their surfaces were periodically observed by scanning electron microscopy.
P.046 ONE-STEP PLASMID CONSTRUCTION FOR GENERATION OF KNOCK-OUT MUTANTS IN CYANOBACTERIA. J.H. Jacobsen1, D. Paiva2, N.-U. Frigaard1, Y. Sakuragi2. 1
Department of Biology, Faculty of Science, University of Copenhagen, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark. 2
Introduction: Cyanobacteria include some of the most efficient phototrophic organisms known, featuring doubling times of less than 4 hours at exponential growth for certain strains. They are therefore ideal for biomass and bioenergy production through metabolic engineering, including gain-of-function and loss-of-function mutagenesis. The recently reported uracil-specific excision reagent (USER) cloning method1, 2, previously applied on plants and fungi, allows highly efficient ligase- and restriction-enzyme-independent construction of knock-out vectors. In the present work we adapted the “four fragment USER-cloning method”1, for utilization in cyanobacteria in order to streamline the construction of vectors for loss-of-function mutagenesis. Methods: Synthetic oligonucleotides encoding the USER-cloning cassette, comprising a BamHI restriction site, flanked by PacI and Nt.BbvCI restriction sites, was inserted into pUC19. Antibiotic resistance cassettes suitable for selection in cyanobacteria were inserted at this unique BamHI restriction site within the USER-cloning cassette. Digestion of the resulting USER-cloning vectors with PacI and Nt.BbvCI generates two fragments, the vector backbone and a fragment containing the antibiotic resistance gene, respectively. Regions flanking the genes encoding glycogen phosphorylase (glgP) and photomixotrophic growth related protein (pmgA) from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 were amplified with uracil-containing oligonucleotides and annealed with the digested vector and directly used for Escherichia coli transformation. Constructs were confirmed by PCR and restriction map analysis. The final constructs were used to transform Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803.
Results: The surface of sheets made from plastic composites with N. commune powders were rougher than that of control, showing that adhesion between plastics and N. commune powder is poor and the two polymers are incompatible. The tensile stress of pure LDPE polymer was about 10 MPa, whereas the stresses of LDPE composites were 6-9 MPa. The composites absorbed 1.2-1.5% (w/w) of water and the tensile stresses of the water-absorbed composites were 4-5 MPa, which was approximately 50% of control. The surface of the composite sheets kept in soil for several months had more irregular structure than that of untreated control, suggesting that the polymer from N. commune absorb water and enhance degrability of the composites.
Results: A set of eight different plasmids for “four fragment USERcloning” has been constructed, each construct containing one of the antibiotic resistance conferring genes aadA (SmR,SpR), aacC1 (Gmr), ermC-cat (Emr,Cmr), nptII (Kmr,Neor), inserted in either parallel or antiparallel orientation. Knock-out vectors for insertional deletion of glgP or pmgA in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 have been constructed in the plasmid vectors containing aacC1 and aadA for glgP and pmgA, respectively. Restriction digestion patterns of selected clones confirmed the presence of the correct inserts in all the clones, demonstrating the high efficiency of the cloning strategy. Transformants from cyanobacterial transformation with glgP and pmgA deletion constructs have been obtained, and homozygous mutants are currently being selected. The results from analysis of glycogen contents, thus biomass capacity, of the mutants will be presented.
Conclusions: The composites were successfully made from plastic pellets and powders of N. commune colonies or EPSs. The plastic composites still remained over 50% of the tensile stress of non-
Conclusions: The USER-cloning method provides a fast and reliable strategy for generation of knock-out mutants in the cyanobacterial species Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803
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13th International Symposium on Phototrophic Prokaryotes through single step construction of knock-out vectors. References: 1 Frandsen, R.J., Andersson, J.A., Kristensen, M.B., Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Molecular Biology (2008), 9. 70 2 Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H., Advancing uracilexcision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Research (2006), 34. e122
P.047 MAXIMIZING HYDROGEN PRODUCTION IN RUBISCOCOMPROMISED MUTANTS OF NONSULFUR PURPLE PHOTOSYNTHETIC BACTERIA. Rick A. Laguna, F. Robert Tabita. Department of Microbiology, The Ohio State University, Columbus, OH, USA. Introduction: The Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway allows for the reduction of CO2 into cellular carbon under aerobic chemoautotrophic and anaerobic photoautotrophic growth conditions. By contrast to its use as a major carbon source, during photoheterotrophic growt CO2 is primarily used as an electron acceptor. The CBB cycle thus performs the important function of maintaining the redox balance of the cell under these growth conditions. Ribulose 1,5-bisphosphate carboxylase-oxygenase is the key enzyme of the CBB pathway that catalyzes the reduction of CO2. Over the years we have shown that nonsulfur purple (NSP) photosynthetic bacteria possess an array of metabolic and regulatory capabilities that allow for the utilization of alternative redox sinks when the primary electron sink, CO2, is nullified via the inactivation or deletion of the RubisCO genes. For example, in many instances the derepression of nitrogenase synthesis occurs under normal repressive conditions. Such gain-of-function adaptive mutant strains have been obtained from Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, and Rhodopseudomonas palustris, whereby such strains balance their redox potential via nitrogenase-catalyzed reduction of protons to hydrogen gas. Over the years we have shown that nitrogenase-derepressed mutant strains produce copious quantities of hydrogen gas by virtue of using the nitrogenase enzyme complex exclusively as a hydrogenase. NifA is the transcriptional activator of the nif (nitrogen fixation) operons. In general nifA expression is regulated by the cascade of proteins that encompass the nitrogen fixation regulon (Ntr), while post-translational regulation of NifA occurs through protein-protein or protein-metabolite mediated contact. Method: To determine if a mutation occurred in NifA of RubisCOdeletion nitrogenase derepressed mutant strains, the nifA genes were sequenced. Next, the mutant NifA protein was inserted into a wildtype background to determine if derepression of the nitrogenase complex occurred. Hydrogen production values were recorded from the RubisCO-deletion nitrogenase derepressed mutant strains. Results: A single point mutation in the NifA protein was identified in the RubisCO-deletion nitrogenase derepressed mutant strains of R. capsulatus, R. sphaeroides, and R. palustris. A mutant NifA protein appeared to be responsible for derepression of the nitrogenase complex in R. palustris. In R. sphaeroides an additional unidentified mutation appears to be involved in the derepression of the nitrogenase complex. Interestingly, no such nifA mutation was found in a nitrogenase-derepressed strain of a RubisCO knockout strain of R.
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Final Program and Abstracts rubrum. The RubisCO-deletion strain of R. palustris produced the most hydrogen compared to the other mutant strains. Conclusion: These results suggest that some NSP bacteria utilize different mechanisms to derepress nitrogenase synthesis in CBBcompromised mutant strains. Inactivating competing redox balancing processes, such as the CBB pathway, plays a crucial role in maximizing hydrogen production in nitrogenase-derepressed strains. Further studies are directed at understanding the molecular mechanisms that allows the cbb and nif systems to compete for reducing equivalents and become subject to reciprocal control.
P.048 SCREENING OF SOIL CYANOBACTERIA FROM VIETNAM FOR ANTIBACTERIAL ACTIVITY. Le Thi Anh Tuyet1, Sabine Mundt2. 1
Department of Natural Sciences, Hong Duc University, Thanh Hoa, Vietnam; 2Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Germany. Introduction: A large number of interesting metabolites of wide chemical diversity has been isolated from cyanobacteria and compounds with antibacterial, antiviral, antifungal and cytotoxic activity have been detected. Currently we are investigating biologically active chemical substances from cyanobacteria with the objective of finding new antibacterial compounds which could be a source of new lead structures for development of antibacterial drugs. As only a few cyanobacterial species from Vietnam have been screened for antibacterial activity , in the present study 11 cyanobacterial strains isolated from acidic soils of paddy and cotton fields in Vietnam belonging to the genera Nostoc, Anabaena, Calothrix, Scytonema, Oscillatoria and Westiellopsis were cultured and tested. Methods: All the organisms were grown under controlled laboratory conditions in Greifswald University, Germany. The biomasses were lyophilised and extracted with n-hexane, methanol, and water, respectively. Culture media were extracted with ethyl acetate. All crude extracts were tested for antibacterial activity against Bacillus subtilic ATCC 6051, Staphylococcus aureus ATCC 6538, E.coli ATCC11229, and Pseudomonas aeruginosa ATCC 22853 in the agar diffusion test system using 6mm diameter paper disc containing 2 mg extract per disc. An inhibition zone of 8 mm or more was considered as good antibacterial activity. Ampicilline was used as control. Results: Antibacterial evaluation demonstrated that 91 % of methanol extracts exhibited activity to Bacillus subtilis and Staphylococcus aureus, whereas the maximum inhibition zones of 19mm were presented by methanol extracts of Westiellopsis sp. VN and Calothrix elenkinii. 45 % of the methanol extracts inhibited the growth of E.coli, while 18 % of methanol extracts possessed antibacterial activity against Pseudomonas aeruginosa. 17 % of n-hexane extracts inhibited the growth of Gram-positive bacteria and 6 % of n- hexane extracts exhibited activity against Gram- negative bacteria. No aqueous extract showed antibacterial activity. 75% of the ethyl acetate extracts of culture medium exhibited significant activity against Gram-positive bacteria (a maximum inhibition zone of 25mm was exhibited by Westiellopsis sp. VN) and 50 % of ethyl acetate extracts inhibited growth of Gram-negative bacteria. Of the 11 cyanobacteria investigated, at least one of the prepared extracts from biomass or cultivation medium showed antibacterial activity. Among the active extracts, the best antibacterial activity was detected for methanol and ethyl acetate extracts of Westiellopsis sp. VN and methanol extract of Calothrix elenkinii. 105
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Final Program and Abstracts Conclusions: This screening proves that Vietnamese cyanobacteria are a promising source of new antibacterial substances. Based on these results Westiellopsis sp.VN and Calothrix elenkinii were selected for bioassay-guided isolation of the active compounds. Structural elucidation of the substances is in progress.
these mutants exhibited significantly higher H2 production rates than did the parental ∆hupL strain.
P.049
P.050
SITE-DIRECTED MUTAGENESIS OF ANABAENA SP. PCC 7120 NITROGENASE TO INCREASE PHOTOBIOLOGICAL H2 PRODUCTION.
CYANOBACTERIA - A PROLIFIC SOURCE OF NEW PHARMACEUTICALS.
Hajime Masukawa1, Kazuhito Inoue2,3, Hidehiro Sakurai3,4, and Robert P. Hausinger1,5. 1
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA; 2Department of Biological Sciences and 3Research Institute for Photobiological Hydrogen Production, Kanagawa University, Hiratsuka, Kanagawa, Japan; 4Department of Biology, School of Education, Waseda University, Shinjuku, Tokyo, Japan; 5Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA. Introduction: Hydrogen gas (H2) produced photobiologically by cyanobacteria that use sunlight as the sole energy source and water as the ultimate electron donor has the potential to become a large-scale, environmentally-friendly, renewable energy commodity. H2 is generated by hydrogenases and, as an inevitable by-product of N2 fixation, by nitrogenases. The best studied nitrogenase consists of two separable metal-containing proteins, the Fe protein (dinitrogenase reductase encoded by nifH) and the MoFe protein (dinitrogenase encoded by nifDK). The latter contains the FeMo cofactor (FeMoco) at which substrate is bound and reduced. Mutagenesis of Azotobacter vinelandii MoFe protein has shown that selected amino acid substitutions in the vicinity of FeMoco allow for effective proton reduction, while eliminating or greatly diminishing N2 fixation. We have examined whether modifications of residues near FeMoco of the cyanobacterial MoFe protein would allow electron flow through nitrogenase to be directed preferentially toward proton reduction to increase the rate of photobiological H2 production. Methods: The nifHDK genes, with the insertion element within nifD excised, were obtained from Anabaena sp. (sometimes called Nostoc sp.) PCC 7120 genome and a non-replicating vector expressing nifHDK was constructed. Site-directed mutations were introduced into nifD. To express the mutated version of nifHDK and eliminate the wild-type version, the nifHDK genes were deleted within a ∆hupL, uptake hydrogenase-negative, variant of Anabaena sp. PCC 7120 as the parental strain and the vector was integrated into the nifHDK deletion mutant chromosome by homologous recombination. Results: We performed site-directed mutation to replace six target residues, all predicted to lie within 5 of FeMoco, with different residues for a total of 49 variants. H2 production and nitrogenase (acetylene reduction) activities of the site-directed mutants were determined and several mutants were shown to exhibit significantly higher H2 production rates in the presence of N2 than did the parental ∆hupL strain. Additional targets for site-directed mutagenesis were based on the proposed hydrophobic substrate channel that connects FeMoco to the protein surface in the X-ray crystal structure of A. vinelandii MoFe protein. Corresponding residues were predicted in the cyanobacterial enzyme for this region. Four hydrophobic residues in the putative channel were replaced with bulky or charged residues in an attempt to inhibit access of N2 to FeMoco. Thirteen single and six double site-directed mutants were constructed; however, none of 106
Conclusion: Particular mutations of certain residues close to the FeMoco prosthetic group were shown to lead to increased partitioning of electron flow toward proton reduction relative to acetylene reduction.
Sabine Mundt1,2, Elmi Zainuddin1, Thi Ngoc Ha Bui1, Susann Kreitlow1, GeroldLukowski2, Rolf Jansen 3, Victor Wray3, Manfred Nimtz3. 1
Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald; Institute of Marine Biotechnology e.V., Greifswald; 3Helmholtz Centre for Infection Research, Braunschweig; Germany.
2
Introduction: In the last decades screening programs have revealed that cyanobacteria are potential sources of new active substances, but the rich resources of German lakes, the Baltic Sea and Asian countries are hardly screened so far. Due to recent development in bacterial and viral resistance and the increase in incidence of tumour diseases worldwide we focused on screening of antibiotic and cytostatic/cytotoxic activity of such hardly tested cyanobacterial strains and isolation and structural elucidation of the active substances. For practical use of biomass or isolated substances the form of application is very important. Methods: Laboratory cultures were established and biomass and cultivation medium were extracted with solvents of different polarity. Lipophilic and hydrophilic extracts have been tested in agar-plate diffusion test for antibacterial and antifungal activity, for antiviral activity against Influenza virus A and Herpes virus Typ1 and for cytotoxic/cytostatic activity against MCF7 (breast cancer cell line), 5637 (urinary bladder) and FL (amnion) cell lines. Bioassay-guided isolation of the active compounds was done by column chromatography including HPLC. Structure was elucidated by analysis of ESI-MS-MS, ESI-TOF-MS, 1D (1H and 13C) and 2D (COSY, TOCSY, ROESY, NOESY, HMQC and HMBC) NMR spectra and amino acid analyses. The preparation of micro- and nanoparticles was done according to PCT/DE 03/00747. A pre-suspension of the biomass and a surfactant-water mixture was produced by stirring followed by high pressure homogenization. Results: Separation of the n-hexane extract of Limnothrix redekei HUB 051 (Müggelsee, Germany) resulted in the identification of three unsaturated fatty acids, α-linolenic acid, coriolic acid and αdimorphecolic acid showing antimicrobial activity in vitro. From the methanol extract of the filamentous Nostoc strain CAVN 10 from a soil sample collected in North Vietnam four new compounds, carbamidocyclophanes, with cytotoxic activity against tumor cell lines have been isolated. Separation of the methanol extract of a Microcystis strain (Plön, Germany) led to identification of two new cyclic peptides with activity against Influenza virus A. Application of micro- or nanoparticles prepared from the biomass of an Anabaena strain Bio 33, isolated from the Baltic Sea, prevents the dermal colonisation of MRSA. Conclusions: Our first results show that so far hardly investigated cyanobacteria from the Baltic Sea, German lakes and from Asian countries are sources of structural new active compounds with potential therapeutically value. Especially for external application micro- and nanoparticles are a promising application form.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
P.051
P.052
GENES PUTATIVELY INVOLVED IN THE SYNTHESIS OF CYANOBACTERIAL EXOPOLYSACCHARIDES.
IDENTIFICATION OF PROTEINS INVOLVED IN THE CHROMIUM REDUCTION IN SYNECHOCYSTIS DERIVATIVE MK-TR OBTAINED BY GAMMA IRRADIATION.
Sara Pereira1,2, Ângela Brito1,2, Andrea Zille1, Pedro Moradas-Ferreira1,3 & Paula Tamagnini1,2. 1
2
IBMC - Instituto de Biologia Molecular e Celular; Faculdade de Ciências, Departamento de Botânica; 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto, Portugal Introduction: Many cyanobacterial strains possess outside their outer membrane, additional surface structures, mainly of polysaccharidic nature, that are usually referred to as exopolysaccharides (EPS). Even though EPS are common in cyanobacteria, their biosynthetic pathways and the factors that regulate these processes are far from being fully understood. However, the mechanisms involved in the synthesis of EPS are relatively conserved throughout bacteria, involving three classes of proteins: (i) enzymes involved in the biosynthetic pathways of nucleotide sugars (ii) glycosyltransferases, and (iii) those required for the oligosaccharide or polysaccharide extension and processing. The sugar activation/modification enzymes and the glycosyltransferases are strain dependent, whereas the proteins involved in the polymerization, chain length control and export are generally conserved among bacteria [1]. Methods: Genome sequences were retrieved from GenBank and computer-assisted sequence comparisons were performed using BLASTp, cDART (NCBI), ClustalW (EMBL-EBI), and SMART. Transcription profiles of the genes were evaluated by RT-PCR using Cyanothece sp. CCY 0110 cells grown under different physiological conditions that are known to affect EPS production: presence/absence of combined nitrogen and different light regiments. Carbohydrate content of the cultures was measured using the Dubois method [5]. Results: An in silico analysis of genome sequences performed in three morphological distinct types of cyanobacteria (unicellular, filamentous and filamentous heterocystous) revealed the presence of genes encoding proteins that, in other organisms, are involved in the last steps of EPS production. In cyanobacteria those genes, namely wza, wzb, wzc, wzx and wzy are frequently clustered and often occurring as multiple copies. Using this information a model was proposed for the polymerization and export of cyanobacterial EPS. Additionally, preliminary results revealed that the transcripts levels of wzb (encoding a tyrosine phosphatase that putatively regulates polysaccharide chain-length by dephosphorylation of Wzc) and wzy (encoding a polymerase) are higher in cells grown in absence of combined nitrogen. Conclusions: Although several genes encoding proteins putatively involved in the EPS polymerization and export were identified in cyanobacterial genomes, their physical organization differs from what is observed in other microorganisms, where the genes are frequently clustered and transcribed as one or two operons [2,3] suggesting that the regulation of the synthesis and export of EPS is more complex in cyanobacteria. References: [1] Pereira et al (2009) FEMS Microbiol Rev (accepted for publication) [1] De Vuyst L et al (2001) Int Dairy J 11: 687-707 [1] Whitfield C (2006) Annu Rev Biochem 75: 39-68
August 9 to 14, 2009 • Montréal, QC, Canada
Saiqa Razi, Shahida Hasnain, Byung-Gee Kim. University Of The Punjab, Lahore, Punjab, Pakistan. Introduction: Synechocystis species, AHZ-HB-MK (DQ 381960) isolated from the local chromium contaminated industrial wastewater, able to reduce 68% toxic hexavalent Chromium (Cr VI), was used in this study. Cultural characteristics of this strain have been determined (Hameed and Hasnain, 2005) and the plant-microbe interaction experiments with this strain proved that it is important in the bioremediation of toxic Cr (VI) from the soil and in the improvement of growth parameters of Triticum aestivum grown under Cr (VI) stress (Faisal et al., 2005). To produce mutants of this strain with better chromate reduction abilities, the strain was irradiated with a 60Co source in a dose range of 0.5, 1, 2, 5, 10 and 20 Grays (Gy) at different stages of growth i.e. after 5, 15 and 30 days of incubation. Derivatives reducing < 90% Cr VI were obtained with low dose irradiation in cultures irradiated at earlier growth stage (Razi and Hasnain, 2006). Biochemical analysis including the estimation of soluble proteins, peroxidases, carotenoids and auxin showed that low radiation also caused stimulatory effects on all of these parameters at earlier stage of growth (Razi and Hasnain, unpublished data). Maximum reduction potential i.e. 94.93% was obtained at dose 2 Gy in cultures irradiated after 5 days of incubation. This derivative was labeled as Synechocystis MK-TR. In the present study, proteomic study of this selected derivative was done in order to identify the proteins involved in the chromium reduction. Methods: After three days of Cr VI exposure, crude extracts from treated and untreated cells were compared at the proteome level using one-dimensional SDS-polyacryalmide gel electrophoretic approach. Proteins were extracted from 100 ml of cultures by repeated freeze-thawing, sonication and vortex mixing (Nayak et al., 2007). From cell extracts, the concentration of proteins was measured by the Coomassie Blue protein assay at 595 nm utilizing bovine serum albumin as a standard (Bradford, 1976). Ten out of total twenty protein bands were upregulated in this derivative under Cr VI stress. These ten protein bands were cut from 1D-SDS polyacryalmide gels and were subjected to in-gel trypsin digestion (Shevchenko et al., 1996). The eluted peptides were dissolved in 0.1% formic acid and loaded on C18 columns. Samples were analyzed by a combination of a nano-HPLC/microelectrospray ionization on a LCQ Deca mass spectrometer (ThermoFinnigan, San Jose, CA). MS-MS spectra were matched with the genome sequence of the model strain Synechocystis PCC 6803 CyanoBase http://bacteria.kazusa.or.jp/cyanobase/Synechocystis/index.html (Kaneko et al., 1996), Results: Seventy-four peptides were identified from these ten selected protein bands. In the long list of these identified peptides we found sll0170, a heat shock protein, slr2076 a 60kD chaperonin and sll0416 identified as 60kD chaperonin 2 and GroEL2. Whereas sll0170 is also a heat shock protein 70, slr1198 was identified to be an antioxidant protein and slr1516 was annotated as superoxide dismutase. Conclusion: This can be assumed from these findings that these proteins were induced due to radiation-stress and are associated with General Stress-proteins family including Heat-shock proteins (HSP). They helped this strain to cope with the osmotic and heat stress when treated with gamma radiation. When this strain was subjected to 107
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Final Program and Abstracts chromium stress these proteins gave stronger expression and again helped the strain to overcome the osmotic stress in this case induced by chromium. The radiation dose of 2 Gy might have caused mutations in the genes of this derivative resulting in the overproduction of these stress proteins.
P.053 ENHANCEMENT OF GROWTH AND YIELD OF TOMATO BY APPLICATION OF PURPLE NONSULFUR BACTERIA. Hong-Gyu Song, Kang-Hyeong Lee. Kangwon National University, Chuncheon, South Korea. Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strain Rhodopseudomonas sp. KL9 maximally produced 5.56 mM/min/mg protein and 67.2 μM/min/mg protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. Germination percentage of tomato seed, total length and dry weight of germinated tomato seedling increased by 30.2%, 71.1% and 270.8%, respectively compared to those of the uninoculated control 7 days after inoculation of strain KL9. A greenhouse test was also carried out to examine the effects on tomato growth of application of purple nonsulfur bacterium Rhodopseudomonas sp. The shoot length of tomato plant inoculated by Rhodopseudomonas sp. KL9 increased by 34.6% compared to that of control in 8 weeks of cultivation. During the same period, this strain increased 120.6 and 78.6% of dry weight of shoot and root of tomato plants, respectively. The formation ratio of tomato fruit from flower was also raised by inoculation of KL9. In addition, Rhodopseudomonas sp. KL9 treatment enhanced the fresh weight and lycopene content in the harvested tomato fruits by 98.3 and 48.3%, respectively compared to those of the uninoculated control. When the effect on the indigenous bacterial community and fate of the inoculated Rhodopseudomonas sp. KL9 were monitored by denaturing gradient gel electrophoresis analysis, its application did not affect the native bacterial community in tomato rhizosphere soil, but should be repeated to maintain its population size. This bacterial capability may be applied as an environment-friendly biofertilizer to cultivation of high quality tomato and other crops including lycopenecontaining vegetables and fruits.
P.054 UNUSUAL CAROTENOIDS FROM SOME NEWLY DESCRIBED PURPLE BACTERIA. 1
2
3
Shinichi Takaichi , Ch. Sasikala , Ch. V. Ramana . 1
Department of Biology, Nippon Medical School; 2Center for Environment, Institute of Science and Technology, J. N. T. University; 3 Department of Plant Sciences, School of Life Sciences, University of Hyderabad. Introduction: Anoxgenic phototrophic purple bacteria belong to the Proteobacteria, and more than 170 species have been described. These bacteria produce many different carotenoids, which are essential for photoprotection and light-harvesting. Base on the structures of the carotenoids and the characteristics of the carotenogenesis enzymes, the normal pathway of carotenogenesis is the normal spirilloxanthin pathway, and is found from around half species. When one enzyme of the normal spirilloxanthin pathway is lacking or is present with reduced activity, the carotenoid composition of the bacterium will be expected to change (unusual spirilloxanthin, spheroidene, and carotenal pathways) (Takaichi (2009) Distribution and 108
Biosynthesis of Carotenoids, In The Purple Phototrophic Bacteria, pp.97-117). Further, Ramana group have recently found and described more than 20 species of purple bacteria. We found some unusual carotenoids from these newly described purple bacteria. Methods: New purple bacteria were described in Int. J. Syst. Evol. Microbiol., and cultured anaerobically under light. Carotenoids were extracted from the lyophilized cells and purified. They were identified based on absorption spectra, retention time on C18-HPLC, MS, and 1HNMR analyses. Results: Phaeospirillum chandramohanii JA145 (Anil Kumar et al. IJSEM in press) contained 10% (mol% of total carotenoids) lycopene, 77% hydroxylycopene, 11% hydroxylycopene glucoside and 2% dihydroxylycopene diglucoside; “Phs. oryzae” JA317 contained 12% lycopene, 81% hydroxylycopene and 7% hydroxylycopene glucoside; Phs. molischianum DSM120 contained 17% lycopene, 77% hydroxylycopene, 5% hydroxylycopene glucoside and 1% dihydroxylycopene glucoside; Phs. fuluvum contained 11% lycopene, 84% hydroxylycopene, 5% hydroxylycopene glucoside and 1% dihydroxylycopene glucoside. Carotenoid glycosides in purple bacteria are rare, and dihydroxylycopene diglucoside is only found from Halorhodospira. This may be due to no activity of CrtD, and additionally these have glucosyl transferase. Roseospira visakhapatnamensis JA131 (Chakravarthy et al. (2007) IJSEM 57: 2453-2457) contained 3% lycopene, 82% 3,4dehydrorhodopin, 1% anhydrorhodovibrin, 4% hydroxyspirilloxanthin and 10% spirilloxanthin. This may be due to low activity of CrtF. Since all of other four species of Roseospira showed almost compatible absorption spectra of the cells, the major carotenoid may also be 3,4dehydrorhodopin. Conclusion: The main carotenogenesis pathway of purple bacteria is the normal spirilloxanthin pathway, and unusual spirilloxanthin pathways are also found. We found genus specific unusual carotenoid compositions, and this might be due to absence or low activity and presence of certain novel enzyme.
P.055 HYDROGEN PHOTOPRODUCTION BY SUSPENSION OR IMMOBILIZED PURPLE PHOTOSYNTHETIC BACTERIAL CULTURES USING PRODUCTS OF DARK FERMENTATION OF STARCH AND POTATO. Darya N. Tekucheva1,3, Tatyana V. Laurinavichene1, Maria L. Ghirardi2, Michael Seibert2, Anatoly A. Tsygankov1. 1
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia; 2National Renewable Energy Laboratory, Chemical and Biosciences Center, Golden, CO, USA; 3 South Federal University, Rostov-on-Don, Russia. Introduction. Transformation of organic wastes to pure water and H2 is an important societal goal. This project is a preliminary study of the steps required to integrate dark fermentative waste treatment with light-dependent H2 production into a single process. Methods. Fermentative effluents (FEs) after starch and potato treatment in the dark were characterized and tested as substrates for subsequent light-dependent H2 production. Before testing, the FEs were neutralized, centrifuged and sterilized. H2 photoproduction and the efficiency of volatile fatty acid (VFA) utilization by the purple bacteria (PB) were characterized in suspension and immobilized cultures using different FE dilutions. Results. Acetate and butyrate were the predominant products of both starch and potato fermentations. Concentrated FEs inhibited both August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes growth and H2 production of PB due mainly to the high VFA content. FEs after potato fermentation contained a variable amount of NH4+, which is detrimental for short-term H2 photoproduction mediated by the nitrogenase enzyme. Furthermore, during long-term experiments in the presence of N compounds, a portion of the VFAs is converted to biomass, thus lowering the efficiency of H2 production. Moreover, at ≥2.5% FE, light limitations occur due to high biomass concentration promoted by the availability of N compounds in the FE. In thin-layer photobioreactors (PhBR) with higher irradiation, a higher percentage of the FE could be treated, and 10% FE provided a volumetric H2 output of approx 2.7 L L-1 PhBR, which corresponded to a potential value of 27 L L-1 FE. PhBRs containing PB immobilized on a glass-fiber matrix can also avoid light limitation. The operational characteristics of such a PhBR under a continuous H2-production regime were examined using artificial media with different concentrations of N-sources and electron donors. (lactate, acetate, and mixed VFAs) or real FEs (at different dilutions). H2 photoproduction was stable for ~3.5 months. The FEs obtained from the fermentation of 400 g L-1 of potato biomass (total VFA ~400 mM) maintained H2 photoproduction at ~60 ml h-1 L-1 of PhBR volume even at concentrations of 1.25% FE in water. The potential total volumetric H2 output recalculated for 100% FE was ~36 L L-1 FE. If the dark fermentations were performed at low substrate concentration (i.e., at 5% starch) and the accumulated VFA concentration was not high (~27 mM total), the photofermentation stage could be successfully operated without preliminary FE dilution. Conclusions. Our results indicate that the integration of dark fermentative treatment of starch or potato biomass and light dependent H2 production by PB using FEs is possible in principle. However, some critical problems will need to be solved. This work was supported by the Russian Program of Basic Research RAS, RFBR, and the US DOE Hydrogen, Fuel Cell, & Infrastructure Technologies Program.
P.056 Metabolic Engineering of Glycogen Metabolism to Enhance the Production of Glucose Derivatives in a Cyanobacterium Synechococcus sp. PCC 7002. Yu Xu, Donald A. Bryant. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. Introduction: Glycogen is a major storage form for carbon and reducing power in cyanobacteria. Slower or interrupted glycogen assimilation could provide more glucose or glucose derivatives in an industrially appealing way for biofuel production. Accumulation of these substances could also facilitate the biosynthesis of some valuable metabolites such as sucrose, lactate, H2 etc., by either converting the carbon product directly or using the reducing equivalents of glucose, or by both ways. Another promising application is to co-cultivate a cyanobacterium with a heterotrophic bacterium, in which system the cyanobacterium provides feedstock for the heterotroph and the latter produces a valuable end product(s). In this report, we use a marine-type cyanobacterium Synechococcus sp. PCC 7002 as the model system to achieve the goals above, by interrupting several key enzymes involved in glycogen biosynthesis. Methods: Linearized DNA fragments that contain a suitable antibiotic resistance gene marker flanked by upstream and downstream homologous regions of the targeted gene were constructed and used August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts to transform WT cells. Desired deletion mutants were selected and segregated by selection with specific antibiotics. Glucose concentrations in different cell fractions were measured by a glucose assay kit provided by Sigma. Photosynthetic O2 evolution rates were measured with a Clark-type electrode. Cell ultrastructure was observed by TEM. Results: Two glycogen synthase genes glgA1 and glgA2, a 1,4-alphaglucan branching enzyme gene glgB, and a glucose-1-phosphate adenylyltransferase gene glgC were successfully deleted in Synechococcus sp. PCC 7002 to create the following mutants: glgA1, glgA2, glgB, glgC, glgA1 glgA2, glgA1 glgB, glgA2 glgB and glgA1 glgA2 glgB. All mutant alleles segregated completely. High-light adapted (HLA) strains of each mutant are also selected. Preliminary data show no obvious difference in O2 evolution rates in those HLA mutants compared to HLA WT cells. HLA mutants of glgA1 glgA2, glgA1 glgA2 glgB, and particularly glgC, were found to have less insoluble glycans. They excrete more glucose into the growth medium than wild type, although excretion levels were still low. The ultrastructure of HLA glgA1 glgA2 shows noticeably altered ultrastructure, which could result from high levels of glucose derivatives in cells. Conclusion: These mutants should have comparable photosynthesis rates as the WT, indicating a stable carbon and energy input in these mutants. However, they are defective in depositing carbon and reducing equivalents into glycogen. Therefore, more glucose or glucose derivatives should accumulate in cells or be excreted. Further analyses are in progress to identify these glucose derivatives and quantify their amounts compared to the WT cells.
P.057 PHOSPHOLIPID FATTY ACID ANALYSIS OF THE EUPHOTIC MICROBIAL COMMUNITY OF LAKE TUSCALOOSA. P.O. Akinwole, E. Lefevre, M.J. Powell, R.H. Findlay. Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, USA. Introduction: Euphotic microbial communities are metabolically important in aquatic system, since they carry out primary production and are critical to the cycling of both organic and inorganic carbon. We studied the seasonal variation of a euphotic microbial community to determine the factors related to its structural dynamics. Method: Integrated water samples were collected fortnightly from the euphotic zone of Lake Tuscaloosa; a man-made system in the westcentral Alabama, southeastern USA, during the period February 2008 through March 2009. Euphotic microbial biomass and community structure were determined using the total phospholipid phosphate (PLP) and fatty acid techniques respectively. Results: Lake Tuscaloosa is a warm dystrophic monomictic lake that was strongly stratified from April to November 2008. Secchi disk transparency depth was inversely correlated with total euphotic microbial biomass (r2= 0.6). Euphotic microbial biomass, measured as PLP, ranged from 5.63 (during the period of warm water) to 20.18 nmol PLP L−1 (during the period of cold water). Phospholipid fatty acids (PLFA) profiles were dominated by polyenoic fatty acids. Principal component analysis of PLFA profiles showed seasonal pattern of change in the euphotic microbial community. There was a significant shift from aerobic bacterial and phototrophic microeucaryotes during period of cold water (Winter mixing) to anaerobic bacteria and heterotrophic microeucaryotes during the period of warm water when the lake was thermally stratified. Maximum
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Final Program and Abstracts contribution to total microbial biomass by microeucaryotes occurred during the period of cold water while maximum contribution by prokaryotes occurred during the period of warm water and during Winter flood events. Conclusions: This study suggests that euphotic planktonic microbial communities show regular variations in structure that are similar to those previously observed in shallow-water marine and freshwater sediment communities.
paper contributes to outlining the ecostrategies of different species, and probes below the species level to show that we have been attempting to understand the dynamics of a “strain flock” rather than an individual genotypic organism. Genetic tracing has provided us insights that were not accessable by microscope alone.
P.059
P.058
ADAPTATION REACTIONS OF SIDEROPHILIC CYANOBACTERIA TO HIGH AND LOW LEVELS OF ENVIRONMENTAL IRON: IMPLICATIONS FOR BIOSPHERE HISTORY.
GENE TRACING OF SEASONAL SUCCESSION OF CYANOPROKARYOTES IN MISSISQUOI BAY, LAKE CHAMPLAIN.
I.I. Brown1, D. A. Bryant2, K.L. Thomas-Keprta1, S.A. Sarkisova1, E.E. Foraker1, W.J. Clarke1, G. Shen2, D.H. Garrison1, D.S. McKay1.
Hongmei Jing, David Bird, Nathalie Fortin, Rocio Aranda-Rodriguez, Charles Greer, Serge Paquet, Catalina Gonzalez Rueda.
1
Université du Québec à Montréal, Montreal, Quebec, Canada.
Introduction: Of all extant environments, iron-depositing hot springs may constitute the most appropriate natural models (Pierson and Parenteau, 2000) for analyzing the ecophysiology of ancient cyanobacteria (CB) that may have emerged in association with hydrothermal activity (Brown et al., 2007) and elevated levels of environmental Fe (Rouxel et al., 2005). Elevated environmental Fe2+ posed a significant challenge to the first oxygenic phototrophs because reduced Fe2+ induces toxic Fenton reactions (Wiedenheft et al., 2005). Ancient CB could have also experienced stress during occasional migrations from the Fe2+-rich ocean to the basaltic land that was almost devoid of dissolved Fe2+; therefore, the study of adaptation reactions of siderophilic CB, which inhabit iron-depositing hot springs, to varying levels of environmental Fe may shed light on the paleophysiology of ancient oxygenic prokaryotes.
Introduction: Shallow Missisquoi Bay, in the northern part of Lake Champlain, suffers from serious cyanobacteria blooms. For several years now, the public has been advised to stay away from recreational use of the bay for most of the summer. Following suggestions from the literature, we looked for taxonomic and genotypic changes that we could associate with toxin presence, in order to understand successional events and identify driving factors. We followed the taxonomic and genotypic dynamics, along with changes in nutrient concentrations and weather variations, to look for linkages. Methods: Two stations, one pelagic and the other littoral and downwind, were sampled weekly over the growing season from May to November in 2006 and 2007. The seasonal dynamics of cyanobacterial populations were followed by taxonomic microscopy and denaturing gradient gel electrophoresis (DGGE). Bands were excised and sequenced. Two primers were used, one (CYA) that detects most Cyanobacteria, and another (MIC) that is more restrictive and detects mainly toxic species, such as Anabaena, Aphanizomenon and Microcystis. Results: There was generally a close correspondence between taxonomic species richness in the Cyanobacteria, and DGGE band richness with the general cyanobacterial primer. DGGE band cluster analysis revealed two very distinct cyanobacterial communities, one associated with summer months (July to September) and the other developing in fall and reappearing the following spring. We suggest that this pattern is indicative of 1) niche dynamics and 2) two ecological stategies: species that are constantly present at a low and stable level, and species that appear in summer, bloom and become dominant, then disappear. Both microscopy and DGGE sequencing showed that strains of Microcystis were dominant during the summer bloom period in 2006. The greatest number of Microcystis genotypes and morphotypes were detected during the annual radiation maximum at the summer equinox, before the biomass maximum of the bloom was reached. Higher intrageneric diversity of Microcystis was detected by DGGE than by microscopy. Major storm events were always associated with important changes in community makeup in this large shallow lake Although more genotypes of M. elabens, M. viridis and M. wesenbergii were present than were genotypes of the dominant M. aeruginosa, the succession of two major M. aeruginosa genotypes accounted for the observed Microcystis biomass dynamics. The potential toxic Microcystis genotype was present throughout the whole growing season. Conclusions: Phytoplankton ecology is in its infancy. Little to nothing is known of the factors that lead to cyanobacterial dominance in a given lake, nor especially to the dynamics of individual species. This
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NASA Johnson Space Center, Houston, TX; 2The Pennsylvania State University, University Park, PA; USA.
Methods: Siderophilic CB (Brown et al., 2007) were cultivated in media with different concentrations of added Fe3+. Basaltic rocks were used as a source of Fe and trace elements in some cases. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive spectrometry (EDS) techniques were used for studying Fe mineralization and rock dissolution processes. Fluorescence spectroscopy was used to estimate the contents of chlorophyll-protein complexes. Results: Five siderophilic isolates (Chroogloeocystis siderophila, JSC1, JSC-3, JSC-11, and JSC-12) precipitated Fe-bearing phases on the exopolymeric sheaths of their cells when Fe3+ concentration was ~ 400 – 600 µM (high Fe). The same Fe3+ concentration was optimal for the culture proliferation rate (Brown et al., 2005; Brown et al., 2007). Higher concentrations of Fe3+ repressed the growth of some siderophilic CB (Brown et al., 2005). No mineralized Fe3+ was observed on the sheath of freshwater isolates Synechocystis sp. PCC 6803 and Phormidium aa strain. Scanning TEM and thin-window EDS revealed intracellular Fe-rich phases within isolates JSC-1, JSC-3, and JSC-11. The elemental composition of the Fe-rich precipitates indicates P, Fe, and O as the major elements with minor amounts of Al and Ca. The PSI/PSII ratio is higher in JSC-1 and JSC-3 isolates than in CB without any detectable ability to mineralize Fe. SEM-EDS studies of the interaction of siderophilic CB with Fe-rich minerals and rocks revealed the ability to leach ilmenite, olivine, TiO, FeS, , and ferrosilicates, perhaps because the CB studied can secrete 2-oxo-glutarate and malate that possess chelating properties. The 7.8-Mb draft genome sequence of Cyanobacterium sp. JSC-1 has been determined and is currently being closed and polished; these data will hopefully reveal the molecular mechanisms of Fe mineralization and Fe-rich minerals by siderophilic CB. Conclusions: The results suggest that colloidal Fe3+ is transported in August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes the CB cytoplasm most likely through an ABC-type Fe3+ transport system (Braun et al., 2004). The prevalence of PSI over PSII in some species of siderophilic CB as well as the ability of siderophilic CB to mineralize Fe within their cytoplasm may indirectly support the hypothesis that PSI in CB can be involved in Fe2+ oxidation (Cohen, 1984; 1989) and could be considered a protective mechanism against oxidative stress induced by high levels of environmental Fe2+ and ultraviolet radiation. The ability to leach Fe-rich minerals could have supported the expansion of ancient CB onto basaltic land.
P.060 A CLONAL POPULATION OF CHLOROBIUM DOMINATES THE ANOXIC BACTERIAL COMMUNITY IN A SWISS MOUNTAIN LAKE. Raymond P. Cox1, Lea H. Gregersen2, Donald E. Canfield3, Kirsten Habicht3, Mette Miller1 and Niels-Ulrik Frigaard2. 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 2Department of Biology, University of Copenhagen, Copenhagen, Denmark; 3Institute of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense, Denmark.
Introduction: Lake Cadagno is a meromictic lake in the Swiss Alps. The anoxic bottom water contains about 2 mM sulfate and the sulfide produced by bacterial activity supports a large population of phototrophic sulfur bacteria at and below the chemocline. Historically the dominant anoxygenic phototrophs were purple sulfur bacteria but since 2000 these have been replaced by green sulfur bacteria (GSB). We have investigated this GSB population using a multigene sequencing approach. Methods: DNA was extracted from filtered water samples from different depths and partial gene sequences obtained by PCR, cloning and sequencing. The water samples were also used for turbidity measurements and pigment analysis by HPLC with diode-array detection. Results: Analysis of a 16S rRNA clone library obtained with general bacterial primers showed that at least 50% of the recovered sequences, and more than 99% of the GSB sequences, were essentially identical and also identical with 16S rRNA from the sequenced genome of Chlorobium clathratiforme DSM 5477. The three 16S rRNA genes on this genome show a 2:1 variation in one position, and a similar variation was observed in the recovered sequences. The clonal nature of the GSB population was confirmed by partial sequences from a protein-coding gene (fmoA) and from the csmCA region containing both coding and non-coding regions. The sequence differences which were observed (less than 1 base variation per 1000 bases) were within the range expected due to sequencing artefacts from cloning of PCR products.. The clonal population was present both at the chemocline and deeper in the water column where shading by GSB in the upper layers precludes significant phototrophic growth. Conclusions: We conclude that the currently dominating GSB has arisen since 2000 from a single cell or a small clonal population. The change might have been due to mutation, invasion or a change in the environmental conditions.
Final Program and Abstracts
P.061 CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUM REVEALS A NEW NICHE FOR CHLOROSOME-CONTAINING PHOTOTROPHS. Amaya M. Garcia Costas1, Zhenfeng Liu1, Christopher Klatt2, Lynn Tomsho1, Stephan C. Schuster1, David M. Ward2, Donald A. Bryant1. 1 Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2Dept. of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT; USA.
Introduction: Candidatus Chloracidobacterium (Cab.) thermophilum is a thermophilic heterophototroph isolated from the phototrophic mats of Octopus Spring in Yellowstone National Park. It is the only known phototroph in the bacterial phylum Acidobacteria, and it has not yet been isolated from any other environment. The light-harvesting apparatus of Cab. thermophilum resembles that of green sulfur bacteria, and it consists of the vesicle-like chlorosomes as antennae structures as well as the Fenna-Matthews-Olson (FMO) protein and a type 1, homodimeric reaction center. Unlike the strictly anaerobic green sulfur bacteria, Cab. thermophilum grows under oxic conditions. In order to further our understanding of the metabolic capabilities of Cab. thermophilum, as well as the distribution and role of this organism in natural environments, we have conducted biochemical analyses of its chlorosomes and have complemented these studies with genomic, metagenomic and metatranscriptomic analyses. Methods: Chlorosomes of Cab. thermophilum were isolated on sucrose gradients and analyzed by high pressure liquid chromatography (HPLC), absorption and fluorescence spectroscopy, electron microscopy and SDS-PAGE. The Cab. thermophilum genome was sequenced using 454 technology. Gap closing was done by Sanger sequencing of PCR products, and contigs were assembled with Phred/Phrap/Consed. For metatranscriptome analyses, total RNA was extracted from mat samples that had been collected at four different times during a diel cycle, converted into cDNA, and sequenced by pyrosequencing. RNAs were identified by blastN analyses, initially using the genomes of seven organisms known to occur in the mats and subsequently the GenBank NR database. Results: The chlorosomes of Cab. thermophilum contain unique pigments and proteins not present in other chlorosomes. The bacteriochlorophyll (BChl) c is methylated at the C-8 and C-12 positions as occurs in the green sulfur bacteria, but the BChl c is esterified with a mixture of alcohols, of which the major species is octadecanol. Interestingly, the chlorosomes of Cab. thermophilum exhibit redox-dependent fluorescence emission which suggested the presence of quinones in the chlorosomes. We have identified menaquinone-8 as the major quinone in chlorosomes. The Cab. thermophilum genome consists of two circular chromosomes, 2.7 and 1.1 Mb, respectively, and contains genes involved in phototrophy, photoprotection, and other Acidobacteria-specific genes. The genome encodes a complete aerobic respiratory chain, and genes involved in carbon fixation appear to be missing. This gene composition suggests that Cab. thermophilum is a photoheterotroph. Preliminary metatranscriptome analyses have revealed that the genes for the photosynthetic apparatus are highly expressed at dusk and dawn. Conclusions: Based on our combined biochemical and genomic sequencing studies we propose that Cab. thermophilum has a unique niche in the environment defined by aerophilic/microaerophilic conditions, tolerance of high light, and ability to harvest light in low light environments. Traditionally, chlorosomes have been associated
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Final Program and Abstracts with low-light, anoxic conditions. However, Cab. thermophilum appears to occupy a temporally defined, low-light niche in an otherwise highly oxic and highly illuminated environment.
P.063
P.062
ON THE OCCURRENCE OF MICROCYSTIN PRODUCING CYANOBACTERIA IN MURCHISON BAY OF LAKE VICTORIA, UGANDA.
PH-DEPENDENT HYDROGEN SULFIDE SPECIES MAY CONTROL COMPOSITION OF HOT SPRING MICROBIAL MATS.
Sigrid Haande, Thomas Rohrlack, Pål Brettum, Robert Ptacnik, Bente Edvardsen, Anne Lyche-Solheim, Unn-Hilde Refseth, Petter Larsson.
Zorigto Namsaraev1,2, Bair Namsaraev3,4, Vladimir Gorlenko1.
NIVA, Oslo, Norway.
1
2
Winogradsky Institute of Microbiology RAS, Moscow, Russia; Center for Protein Engineering, University of Liège, Liège, Belgium; 3Institute of General and Experimental Biology Siberian Branch RAS, Ulan-Ude, Russia; 4Buryat State University, Ulan-Ude, Buryat Republic, Russia. Introduction. Increasingly more data suggest that early life lived at an environmental temperature similar to those of today’s hot springs. At temperatures lower 73-77 °C and pH higher 4.5 hydrotherms are dominated by microbial mats formed by phototrophic bacteria. Castenholz have shown that with the increase of temperature above ~55 °C natural populations of cyanobacteria cannot tolerate high sulfide concentrations. It was concluded that at these temperatures and concentration of sulfide higher 1 mg/L mats are formed exclusively by anoxygenic phototrophic bacteria (APB). We studied alkaline (pH up to 9.8) non-mineralized (salinity less 1 g/L) hydrotherms of Baikal rift zone (Southern Siberia, Russia) and found that cyanobacteria could develop at much higher concentrations of sulfide than it was thought before (up to 5.9 mg/L at 62 °C). Methods. Microscopy, spectrophotometry, radioisotopy, cultivation and cultivation independent methods. Results. Cyanobacteria belonging to genus Phormidium were dominant and formed so called “translucent” mats up to 13 cm thick. The productivity of mats was relatively high: maximal content of chlorophyll a is 892 mg m–2, maximal rate of oxygenic photosynthesis 3.5 g C m–2d–1. APB Chloroflexus aurantiacus was found and isolated from mats, but it was not dominant. This fact can be explained by a decrease of sulfide toxicity in increase of pH. Other sulfidic hydrotherms worldwide follow the same rule. For example, at 60 °C and pH lower than 7 the majority of H2S molecules are not dissociated and are capable of penetrating easily through a cell membrane. The community dominated by APB Chloroflexus aurantiacus develops (Iceland, Italy and Yellowstone). Less toxic HSion starts to dominate at pH higher than 7 and cyanobacteria appear in the mat. They form a layer under top layer of APB Chloroflexus aurantiacus which protects cyanobacteria from the influence of sulfide dissolved in water above the mat (Iceland and Kamchatka). At pH higher than 8.5 all sulfide molecules are transformed to a hydrosulfide ion and, as it have been shown by us, cyanobacteria dominate in the microbial community. Conclusions. Our results show that distribution of different types of phototrophic communities in sulfidic hydrotherms can be explained by pH-dependent hydrogen sulfide species.
Introduction: One of the major threats to Lake Victoria is eutrophication and an increasing proliferation of cyanobacteria. The main concern is that many cyanobacterial species have the ability to produce toxic compounds, among them the commonly reported hepatotoxic microcystins, which can cause considerable hazards for animal and human health. Murchison Bay is a 30 km long embayment in the north western part of Lake Victoria, and the inner part of the bay serves as drinking water supply for the nearby city Kampala, the capital of Uganda. The bay is also the recipient of both industrial and municipal wastes, sewage effluents and surface runoff from the city, and is heavily eutrophicated. This study aimed to examine the abundance of cyanobacteria and microcystins in Murchison Bay and to identify the main microcystin producing species, as well as to test for possible correlation between microcystin concentration and environmental factors. Methods: Sampling (n=25) of physical, chemical and biological parameters in Murchison Bay was done at two stations, representing the semi-enclosed innermost part of the bay and the wider outer part of the bay, every second week in the period from April 2003-March 2004. Vertical profiles of water temperature, oxygen and electrical conductivity were measured, and the recorded secchi depths were used to estimate euphotic depth. The nutrients analysed were total phosphorous, phosphate, total nitrogen, nitrate, total organic carbon and silicon. Clorophyll-a was analysed and phytoplankton countings were performed. The presence of microcystins was analysed by Liquid chromatography (LC-MS), and the identification of the microcystin producing strains was performed by real-time PCR, using species specific primers. Correlations between microcystin concentration and various environmental parameters were analysed by Spearman’s rank correlations. Results: There was a considerable loading of nutrients to Murchison Bay with high concentrations of total phosphorous (>90µg/L) and total nitrogen (>1100µg/L) in the inner part of the bay. There was a rapid decrease in conductivity and nutrient concentrations from the innermost part of the bay to the outer part of the bay. The phytoplankton community was dominated by a number of cyanobacteria, among them known microcystin producing species of the genera Microcystis, Anabaena and Anabaenopsis. The proportion of N-fixing species like Anabaena sp. was higher in the outer part of the bay whereas Microcystis sp. was more abundant in the inner part of the bay. Microcystins (MC-RR, -LR, -YR) were detected during the whole study period, on average 1.1 µg L-1 in the inner part of the bay and 0.6 µg L-1 in the outer part of the bay. Based on qPCR analysis, Microcystis aeruginosa was identified as the main microcystin producer in Murchison Bay. Nutrient availability and light climate seemed to be the most probable influencing environmental factors influencing the microcystin production in Murchison Bay, but the microcystin concentrations could not be predicted by any given environmental factor alone, by the biovolume of cyanobacteria or of a certain cyanobacterial species. Conclusions: This study showed that Murchison Bay was heavily eutrophicated and that there was a presence of microcystin producing
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13th International Symposium on Phototrophic Prokaryotes cyanobacteria in the bay. The microcystin concentrations in Murchison Bay were at times higher than the WHO recommended limit of 1 µg L-1 MC for drinking water, possessing a health risk for lake water users.
P.064 DIVERSITY AND NITROGENASE GENE EXPRESSION OF DIAZOTROPHIC CYANOBACTERIA IN THE BALTIC SEA. Henning Johansen, Kirsten Isensee, Maren Voss, Klaus Jürgens. Leibniz Institute for Baltic Sea Research, Rostock, Germany. Introduction: Low phosphorous concentrations in the water column are important for the development of heterocystous cyanobacteria blooms recurring in summer in the Baltic Sea, a brackish water body in Northern Europe. These blooms significantly contribute to the nitrogen input into the system and consist mainly of the genera Aphanizomenon and Nodularia. While nitrogen fixation rate measurements indicate a distinct diel cycle, there are contradicting findings from the concentration of the nitrogenase enzyme during day. However, there is a general lack of knowledge about nitrogenase gene expression (nifH) and the coupling between gene activity and nitrogen fixing activity in the Baltic Sea so far. Here we present data on both gene expression and rate measurements, giving a comprehensive insight into regulation of nitrogen fixation of filamentous cyanobacteria. Methods: Surface water samples (3 m) were taken at a LaGrange drift station in the Bornholm Basin, Baltic Sea, in July 2008. The diversity of the nitrogen fixing community was studied using both cell counts and fingerprint analyses (Denaturing Gradient Gel Electrophoreses) of the nifH gene and its transcripts. Concentrations of gene copies and transcripts were determined by a quantitative PCR assay using primers specific for heterocystous cyanobacteria. Rate measurements of nitrogen fixation and primary production were done using stable isotope labelling (15N2 and 13C). Results: Sequencing of excised bands from nifH fingerprint analyses showed highest identity to strains Nodularia sp. KAC13, Aphanizomenon sp. KAC15 and Anabaena sp. PCC7120, being in accordance with microscopic cell counts. The analyses of cyanobacterial nifH transcripts indicated the presence and activity of the genera Anabaena, Aphanizomenon and Nodularia during a complete diel cycle. This is in accordance with the nitrogen fixation rate measurements showing a lowered rate but uptake of molecular nitrogen in darkness whereas the pattern of carbon fixation is following the solar radiation pattern. Results of the nifH gene expression measurements indicate an uncoupling of maximum nitrogen fixation rates and maximum transcripts. Conclusions: This study is the first to show nifH gene expression in natural bloom conditions in the Baltic Sea throughout the whole diel cycle. Therefore, an uncoupling of nitrogen fixation rate measurements and gene expression can be suggested. A tentative explanation is the enhanced regeneration of the nitrogenase enzyme during the night, while nitrogen fixation activity is more closely coupled to light energy during the day. In case our further studies confirm these findings the gene expression may not directly be linked to nitrogen fixation.
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Final Program and Abstracts
P.065 MAKING SENSE OF MARINE SYNECHOCOCCUS DIVERSITY, THE KEY TO UNDERSTANDING THEIR UBIQUITOUS ECOLOGICAL DISTRIBUTION. Sophie Mazard, Martin Ostrowski, Dave Scanlan. Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom. Introduction: At the global scale the marine photosynthetic community is dominated by two closely related genera of cyanobacteria, Prochlorococcus and Synechococcus. Their success is underpinned by a high degree of genetic and genomic variation, such that at least 16 different evolutionary lineages of Synechococcus have been identified, each occupying distinct spatial and temporal niches in the ocean. Our overall aim is to understand what factors define particular niches (e.g. light, temperature, nutrients, metals) and to correlate these factors with community structure. One way to achieve this is by monitoring how the genetic composition of photosynthetic communities changes in different ocean regimes linked with targeted metagenomics using high-throughput sequencing of DNA and RNA extracted from natural populations. Analysis of preliminary results revealed that our ability to interpret the data is limited by the low taxonomic resolution and low throughput of community analyses based on standard markers (16S rRNA and ITS). Methods: In this work, we describe the development of a fast, reliable and efficient method of assessing community composition at high taxonomic resolution using the petB locus. In preparation, we designed a Multi Locus Sequence Analysis (MLSA) scheme targeting 7 housekeeping genes for marine Synechococcus based on fully sequenced genomes. Metagenomics and transcriptomics studies were carried out on flow-sorted Synechococcus from contrasting stations on oceanographic cruises of the Atlantic (AMT18) and the Mediterranean Sea (BOUM). Results: A high resolution taxonomic framework for marine Synechococcus was produced with our MLSA scheme on more than 120 cultured isolates maintained in the Warwick culture collection. Phylogenetic reconstructions based on single and concatenated loci were congruent and comparable with those obtained with general phylogenetic makers (16S rDNA, 16S-23S ITS). However, sequencing of the single locus petB allowed us to obtain a resolution differentiating up to 55 coherent taxonomic units as compared to a maximum of 16 for 16S rDNA and ITS. Furthermore, the reliability of the petB alignment (cf. ITS) makes this locus suitable for assessing community structure with high-throughput and rapid analyses of environmental libraries. Indeed, preliminary results from environmental libraries highlighted ecologically important clades and sub-groups at a much higher resolution than previous studies. Conclusions: Earlier work performed using classical markers emphasized broad trends in the ecological distribution of marine Synechococcus. The establishment of a high resolution phylogenetic framework helps us to fine tune the geographical partitioning of this genus in situ, and will facilitate the uncovering of specific adaptation mechanisms of the numerous phylotypes observed. In concert with indepth genomic study of this genus, this might prove to be the key to understanding its complex but successful ecological distribution throughout the world’s oceans.
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P.066 ALGAECIDAL EFFECT OF RHODOCOCCUS ON MICROCYSTIS. Chi-Yong Ahn, Young-Ki Lee, Song-Gun Kim, Hee-Sik Kim, Hee-Mock Oh. Environmental Biotechnology Research Center, KRIBB, Daejeon, Republic of Korea. Rhodococcus sp. is one of the most common bacteria in the reservoirs of Korea. Rhodococcus sp. is also frequently found with Microcystis, a major bloom-forming cyanobacterium. Two strains of Microcystis were isolated in Daechung and Wangsong reservoir, Korea, during blooming season (from July to October, 2007). Eleven strains of coexiting bacteria were also isolated from mucilage of cultivated Microcystis strains. They were identified by 16S rRNA gene sequences. Two of them were designated as Rhodococcus sp. KWBRH2 and KWB-RH5. Rhodococcus strains exerted an inhibitory effect on the growth of Microcystis aeruginosa NIES 843, UTEX 2388 and isolated M. aeruginosa. The cell-free culture supernatant of Rhodococcus strains also showed prominent algaecidal activity. To identify the causal compounds, supernatants of Rhodococcus strains were dialyzed with a MW 8,000 and MW 2,000 membranes. The suspected extracellular substances had molecular weights below 2,000 Da. The small molecules were further analyzed by HPLC, LC-MS and enzyme treatments. The extracellular material from Rhodococcus strains could be a more effective bloom extinguisher, compared to generally known algaecidal bacteria, because it does not require bacterial cell itself for bloom control.
P.067 CHARACTERISATION OF CYANOPHAGE RESISTANTANCE IN THE MARINE CYANOBACTERIUM SYNECHOCOCCUS SP. WH7803. Edward Spence, Nicholas Mann. University of Warwick, Coventry, United Kingdom. Introduction: Cyanophage are viruses which specifically infect cyanobacteria. This project focuses on the interaction of the cyanophages S-PM2 and S-RSM42 with their host, the marine cyanobacterium Synechococcus sp. WH7803. The main aim of this project is to identify the host receptor(s) and characterise naturally occurring phage resistant mutants of Synechococcus sp. WH7803 which have been isolated. The phage resistant mutants have been characterised in order to determine what physical changes have occurred to their cell surface, in order to provide resistance and what impact this has on their fitness. Previous research with the freshwater cyanobacterium Anacystis nidulans has shown that the lipopolysaccharide (LPS) component is necessary for the adsorption of the cyanophage AS-1. Methodology: Spontaneous resistant mutants were isolated from regrowth of survivors from completely lysed cultures, following cyanophage infection. Single colonies were isolated from pour pates and maintained in liquid culture under selection with the addition of cyanophage lysate. The outer membrane protein component was isolated by Triton X-100 precipitation and profiled by gradient SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide was isolated using hot phenol/water extractions and profiles were obtained with SDS-PAGE using isolated LPS or using whole cell digests with protease K, visualised with silver staining. Results: The results indicated that the LPS component of the resistant mutants had an altered profile suggesting changes to the oligosaccharide or variable region. The outer membrane protein 114
components remained unchanged in the resistant mutants. The resistant mutants also had a reduction in their growth rates, in comparison to the wild type under standard conditions. An altered pigment content was also observed suggesting alteration to the photosynthetic components particularly to the phycobilisome. Conclusion: It seems likely that the LPS is the major receptor molecule for the cyanophages S-PM2 and S-RSM42 adhesion to their host Synechococcus WH7803. Outer membrane proteins do not seem to have a role in these cases for cyanophage attachment. There is also a cost of resistance relating to the growth rate of the resistant mutants and changes to the pigment content. This suggests that genetic changes resulting in the alteration of the cell surface receptor may be as a result to changes of a regulatory component. This may explain additional pleiotrophic phenotypic changes observed, relating to pigment composition and a reduction in the growth rate in the cyanophage resistant mutants.
P.068 THE ROLE OF PHOTOTROPHIC MICROORGANISMS IN BIOLOGICAL SOIL CRUSTS FROM THE MIDTRE LOVÉN GLACIER FORELAND. Laura Tiano1,2, Luigi D’Acqui1, Claudio Sili1, Marc Staal3, Silvia Turicchia4, Matthias Zielke5, Stefano Ventura1. 1
CNR–Institute of Ecosystem Study, Sesto Fiorentino, Italy; 2University of Florence, Master in Environmental Biology, Italy; 3Department of Biology, University of Copenhagen, Denmark; 4University of Siena, PhD School in Polar Science, Siena, Italy; 5Department of Marine Biotechnology, Norway College of Fishery Science, University of Tromsø, Norway. Introduction: Global change models predict that the speed and magnitude of climate warming will be greatest at the highest latitudes, with raised mean temperatures, particularly during winter, and altered patterns of precipitation (IPCC 2001). These predicted changes are under way in many parts of the high Arctic, as it can be seen from the decreasing extent and thickness of the sea ice and the process of glacial ‘retreat’. Glacial regression makes new ground surface available to biotic colonization. Low temperatures, short growing seasons, the effects of permafrost, limited moisture and nutrients availability limit the growth of the vegetation, leading to different successions from those observed elsewhere. In these polar environments, pioneering organisms such as cyanobacteria, green algae, lichens, mosses and heterotrophic bacteria are typically the first to colonize the substrate as biological soil crusts (BSCs). Cyanobacteria are often found in great variety in all the high arctic zones, and in several ecosystems they are the dominant microorganisms in terms of biomass and productivity (Vincent, 2000). Nitrogen fixation by cyanobacteria compensates for the lack of nitrogen in arctic soils (Lennihan et al., 1994) and facilitates the subsequent colonization of these habitats by other microorganisms and plants (Bliss and Gold, 1994). This study describes community assembly on typical high Arctic glacier forelands, with a focus on the phototrophic components of the BSCs, to gain a broader understanding about dynamics and development of these fragile systems and their rates of recovery following disturbance. Methods: Sampling sites were located close to Ny-Ålesund, Svalbard, on the foreland of the Midtre Lovén glacier. Seven locations with different types of crust were chosen, and an integrated approach was August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes applied, carrying out both morphological and molecular analysis of environmental samples. A general insight into the phototrophic microorganisms colonizing the crusts was gained with microscopy observations; a deeper understanding of the communities’ structure is expected with molecular fingerprints (ARISA, T-RFLP). Also physical, chemical and granulometric characteristics of soil were analyzed to investigate the influence of those parameters on the development of the crusts. Furthermore, respiration rate, nitrogen fixation and production were measured, thus adding more information on the main activities performed by the primary colonizers of these surfaces.
Final Program and Abstracts sp., Phormidium autumnale, Phormidium priestleyi, Stigonema sp. Cultivation studies showed the importance of the incubation temperature for the composition of enrichment cultures, with an increased proportion of unicellular cyanobacteria at lower temperatures. Conclusion: Our first results show that the Western part of the Sör Rondane Moutains harbour a high and unique diversity of cyanobacteria. Precautions should be taken to avoid introduction of non-native species in the area of the research station.
P.070 SYNTHESIS OF BACTERIOCHLOROPHYLL-F.
Results: The molecular characterization of the soil crusts is still in progress, the morphological analysis by microscopy allowed us to identify several species of cyanobacteria and algae, as Nostoc sp., Phormidium sp., Leptolyngbya sp., several diatoms, etc.
Hitoshi Tamiaki, Jun Komada, Michio Kunieda, Kazuhiro Fukai, Taichi Yoshitomi, Jiro Harada, Tadashi Mizoguchi.
Nitrogen fixation rate, C/N concentration and granulometric characteristics of soil, confirmed the low nutrients availability of the high arctic ground and the extremely slow pedogenesis of these soils.
Introduction: Green sulfur bacteria are a family of anaerobic photoautotrophic bacteria living. These bacteria have elliptic shaped light-harvesting apparatus, called chlorosomes, that are attached with the inner side of their cytoplasmic membrane. Chlorosomes are unique photosynthetic antennae, where characteristic chlorophyllous pigments, bacteriochlorophyll(BChl)s-c, d and e self-aggregated to form large oligomers without any assistance of peptides. In contrast, the other antennae are prepared by complexation of pigments with peptides. In chlorosomal aggregates, specific interaction between the 31-OH, Mg and 13-C=O is observed. All the chlorosomal chlorophylls have such functional groups and three BChls mentioned above are distinguishable from substituents on the C7- and C20-positions. BChlc has methyl groups at the C7- and C20-positions, and BChl-d is the 20-demethyl form of BChl-c. On the other hand, BChl-e has a formyl group at the C7-position and a methyl group at the C20-position. The 20-demethylated form of BChl-e is named as BChl-f, but it has not been detected in any natural bacteria yet. Here we report the synthesis of BChl-f and its physical properties comparing with BChls-c, d and e extracted from natural photosynthetic green bacteria.
Conclusions: We expect to draw conclusions about the role of phototrophic microorganisms in these high arctic biological soil crusts as soon as all the results, collected from the different type of analysis, will be matched together and organized under a statistical approach.
P.069 CYANOBACTERIAL DIVERSITY OF THE SÖR RONDANE MOUNTAINS NEAR THE NEW “PRINCESS ELISABETH” STATION (ANTARCTICA). Zorigto Namsaraev1,2, Rafael Fernandez1, Marie-José Mano1, Patricia Simon1, Annick Wilmotte1. 1
Centre for Protein Engineering, University of Liège, Liège, Belgium; Winogradsky Institute of Microbiology RAS, Moscow, Russia.
2
Introduction: During austral summer 2008, the new Belgian “Princess Elisabeth” research station has been constructed on a granite ridge near the Utsteinen nunatak in the Western part of the Sör Rondane Mountains. This pristine area has not yet been studied at the biological level, and exhaustive baseline data are needed to obtain information about the initial state of the environment. Moreover, the Sör Rondane Mountains could be a possible refuge during the Last Glacial Maximum. Cyanobacterial communities in this area could harbour species that survived the population bottleneck of glaciations. Methods. Samples were taken in several locations in the austral summers of 2007 (before the construction start) and of 2009 (BELSPO projects ANTAR-IMPACT and BELDIVA). A polyphasic approach was used to study of cyanobacterial diversity. It included a molecular study using 16S rRNA gene sequences, an isolation of cyanobacteria in culture and a microscopic observation of environmental samples and isolated cultures. Results: Preliminary results showed a relatively high biodiversity in the studied area. Cyanobacteria were mostly associated with gravel and in some cases with rocks, whereas lichens were dominant on rock surfaces. Molecular studies showed a quite high degree of endemism and genotypes never recorded before. 92% of the studied samples shared at least one OTU, concordant with a mobility of species between nearby habitats. Microscopical observations showed the presence of at least 11 morphotypes of cyanobacteria: Asterocapsa sp., Aphanocapsa sp., Chroococcus sp., Coleodesmium sp., Cyanothece aeruginosa, Leptolyngbya sp., Nostoc commune, Nostoc August 9 to 14, 2009 • Montréal, QC, Canada
Department of Bioscience and Biotechnology, Kusatsu, Shiga, Japan.
Methods: Naturally occurring chlorophyll(Chl)-b was extracted from spinach with organic solvents. By modifying Chl-b, methyl bacteriopheophorbide-f was obtained according to the previous report [1]. The methyl ester was transferred to the farnesyl ester and successive magnesium-metallation gave BChl-f possessing 8-ethyl and 12-methyl groups. BChls-c, d and e were extracted from cultured green bacteria, Chlorobium tepidum, vibrioforme and phaeovibrioides, respectively. BChls-c, d, e and f were analyzed by reverse phase high performance liquid chromatography (RP-HPLC). Results: RP-HPLC analysis of farnesylated BChls-c, d, e and f indicated that they were eluted in the order of BChl-f < BChl-e < BChl-d < BChlc (31R, 8-Et, 12-Me = R[EM]): ODS column and aqueous MeOH. The 20-methylation and 7-reduction retarded their elution. Their visible spectra showed that the 20-methylation red-shifted both Qy and Soret peaks in their monomeric states and that the 7-oxidation induced the Qy and Soret peaks to move to shorter and longer wavelengths, respectively. Moreover, the ratio of Qy/Soret peak intensities decreased to less than half by the oxidation. Conclusion: We first synthesized BChl-f from easily available Chl-b. Farnesylated R[EM]BChl-f was eluted more quickly than the corresponding BChls-e (20-methylation) and d (7-reduction) in RPHPLC. Based on the analysis, the HPLC peaks of BChl-f were searched in the extracts from cultured green bacteria, but we could not detect it. [1] H. Tamiaki, M. Kubo and T. Oba, Tetrahedron, 56, 6245-6257 (2000). 115
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Final Program and Abstracts
P.071 QUORUM SENSING IN CYANOBACTERIAL BLOOM. Young-Ki Lee, Chi-Yong Ahn, Hee-Sik Kim, Jung-Kee Lee, Won-Gon Kim, Hee-Mock Oh. Environmental Biotechnology Research Center, KRIBB, Daejeon, Republic of Korea. Microcystis aeruginosa is a ubiquitous cyanobacterium that causes ecological and economic damage to freshwater ecosystems by bloom formation. Cyanobacterial blooms are visually unpleasant and they produce malodorous compounds (geosmin, MIB) and toxic metabolites (microcystin), which pose more serious problems for animals and humans. Recent studies focused on cell to cell communications in ecogenomics. Cell density-dependent communication has been known as “quorum sensing”. To investigate the relationship between quorum sensing and bloom, we isolated Microcystis sp. Mi0601 and Microcystis sp. KW from surface scums during bloom season (from July to October, 2007) in Daechung reservoir and Wangsong reservoir, Korea. Both of them were identified as M. aeruginosa by 16S rRNA gene and cpc-IGS region. Concentrated cell-free extracts were bioassayed with Agrobacterium tumefaciens NT1 (traR, tra::lacZ749) and separated by thin-layer chromatography. Two signal molecules were detected in TLC plate but they were not general signal molecules, such as C4-, C6-, C10homoserine lactones. Extracted signal molecules were analyzed by LC-MS and NMR. The signal molecule from each strain exhibited the same molecular weight. NMR analysis indicated that they were composed of two unknown materials. A strong correlation was found between Microcystis growth and accumulation of signal molecule. This implies that quorum sensing plays an important role in the onset of cyanobacterial bloom.
Methods: Nine collection strains including Allochromatium vinosum (strain DSMZ 183 and DSMZ 185), Lamprocystis roseopersicina (DSMZ 229), Thiocapsa roseopersicina (DSMZ 217), Marichromatium purpuratum (DSMZ 1591), Thiocystis violaceae (DSMZ 207), Thiocystis minor (DSMZ 178) Chlorobium limicola f. thiosulfatophilum (DSMZ 249), were used to test a basal medium specific for purple and green sulfur bacteria proposed by Pfennig and Trüper (1981) and changing the electron donors like (alone and mixed): a) S2-, b) S2-+S2O32-+SA, c) Sx2- d) Sx2-+SA e) cysteine+SA, f) methionine+SA, g) SA h) cysteine and i) methionine. Liquid culture media (Na2S as electron donor) specific for phototrophic sulfur bacteria was enriched with water samples from Tampachachoco Lagoon and positive cultures were used to inoculate Petri dishes containing the next electron donors: a) S2-, b) S2-+S2O32+SA, c) Sx2- d) Sx2-+SA. In all cases, anaerobic conditions were gotten using gas pack bags and the cultures were incubated at room temperature and light as energy source (incandescent=IL and fluorescent=FL). Red colonies were isolated in cultures growing in medium d (Sx2- and SA). Pure culture of these bacteria were verified by optical microscopy and their phonotipic identity was done by the use of pigment and fatty acid composition (by gas-chromatography) as well as cellular morphological properties.
P.072
Results: All the collection strains grew well in the medium d (S2+S2O32- ) specially on IL (incandescent light) but DSMZ 249 only grew with FL (fluorescent light) and both strains were able to grow. Acetate alone and mixed with cysteine were used only for strain DSMZ 235 and DSMZ 183 (IL and FL); But DSMZ 1591, all purple sulfur bacteria assayed used Sx2- (IL). Except DSM235 (IL) and DSMZ 185 (IL and FL) all the strains were not able to use acetate as electron donator. It was possible to obtain five pure cultures of phototrophic purple sulfur bacteria using the media d (Sx2-+SA). The strains were also able to grow well in media b (S2-+S2O32-+SA) and d ( Sx2-+SA. Sulfur globules into the cells, the presence of Bchl a, as well as the octadecenoic (C18:1), hexadecenoic (C16:1) and hexadecanoic (C16:0) as major fatty acids, related all the strains isolated as members of the Chromatiacea family.
ISOLATION OF ANOXYGENIC PHOTOTROPHIC SULFUR BACTERIA FROM TAMPAMACHOCO LAGOON (VERACRUZ, MÉXICO) USING DIFFERENT ELECTRON DONORS AND AGAR PLATE CULTURES.
Conclusions: A combination of electron donors could be an alternative to isolate and to obtain pure cultures of marine phototrophic sulfur bacteria in agar plate cultures incubated in gas pack bags.
María Teresa Núñez-Cardona1, Jordi Mas2, Marina Luquin3, Martha Signoret4.
P.073
1,4
Universidad Autónoma Metropolitana-Xochimilco, 1Laboratory of Ecología Microbiana; 4Plancton y Bioenergética, Distrito Federal, México; 2,3Departament de Genetica i Microbiologia, Universidad Autónoma de Barcelona, Spain. Introduction: Phototrohic sulfur bacteria (PSB) usually live in aquatic systems. Actually there is a limited number of strains described if we compare with the chemoorganoheterotrophic bacteria. Maybe it is because PSB are anaerobic microorganisms, and the use of organic compounds as carbon and electron donors, for growing is limited. It has been shown that PSB are able to use different containing sulfur compounds as electron donors like H2S (in laboratory cultures as Na2S=S2-), polysulfides (Sx2-), thiosulfate (S2O32-), cysteine, methionine, and acetate (sodium acetate=SA) and also that is possible to incubate them in gas pack bags. With the aim to isolate and getting pure cultures of anoxygenic phototrophic sulfur bacteria it was assayed a basal medium adding different electron donors alone and mixed in agar plate cultures. Fatty acids and pigment composition as well as morphological properties were used to characterize the strains isolated from water samples collected at Tampamachoco Lagoon (Veracruz, México) 116
TOWARDS TO INTEGRATION OF BACTERIAL LIGHT-DEPENDENT HYDROGEN PRODUCTION AND ELECTRICITY GENERATION. Evgeny Shastik1,3, Tatyana Laurinavichene1, Evgeny Minakov1, Oleg Voronin2, Nikolay Zorin1, Arkady Karyakin2, Anatoly Tsygankov1. 1 Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region; 2Chemical Faculty, Lomonosov Moscow State University, Moscow; 3South Federal University, Rostov-on-Don, Russia.
Introduction. For future H2-based economy many issues should be addressed. Human-friendly and cheap H2 production and transformation of H2 energy into electricity are among them. Purple bacteria are promising H2 producers since they use sunlight and simple organic compounds to evolve H2 gas with high rates. A fuel cell (FC) application to transform hydrogen energy to electrical one seems to be perspective. Existing FC’s with electrodes based on noble metal catalysts (e.g. platinum) are not able to work in microbiological media, and an enzymatic catalyst is the best alternative for biological systems. Particularly, hydrogenase (H2ase) from Thiocapsa roseopersicina was shown to be stable at high temperature, and in the presence of CO, August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes H2S, and proteases. A direct H2ase bioelectrocatalysis on technological carbon materials used in energetic would be optimal and profitable. The aim of this study was to explore a possibility to combine bacterial H2 production with hydrogen electrode in one space. Methods. Reactors with H2 producing bacteria (including immobilized purple bacteria), hydrogen enzyme electrodes (carbon textile with polymerized different promoters) with immobilized H2ases from T. roseopersicina and D. baculatum, and combination of them were used in this study. Results. Before the integration of hydrogen producing reactors with bacteria and hydrogen enzyme electrode, some preliminary research on bacterial reactors and H2 electrode have been done. It was shown that the photobioreactor with immobilized purple bacteria (Rhodobacter capsulatus and Rba. sphaeroides) under continuous synthetic medium flow stably produced H2 with average rate app. 1.5 ml h-1 ml-1 matrix for more than 5000 hours. Operational stability of H2 electrodes in K-P buffer saturated by H2 was studied for more than 500 h. pH-dependence of H2 electrode with different H2ases was studied. Temperature profiles of H2 electrodes with different H2ases with polyviologen and neutral red as promoters was studied. It was shown that the temperature dependence of the current was dependent on the voltage applied to the electrode. At low (5-20 mV) overvoltage the temperature dependence was maximal whereas at high (150-200 mV) overvoltage the temperature dependence was very low. The dependence of activation energy on the overvoltage of the electrode will be discussed. The stability of H2 electrode in the medium going from the bioreactor was shown to be the same as in KP buffer. Finally, the stability of H2 electrode integrated into the bioreactors with H2 producing bacteria was studied, detailed results will be presented, and problems appeared during the integration of bacterial H2 production with H2 enzyme electrodes will be discussed. Conclusion. The integration of H2 producing bacteria with H2 enzyme electrode in one space is possible. However, many problems should be solved before this system shows its practical efficiency. This work was supported by the Russian Program of Basic Research RAS
Final Program and Abstracts Methods: In order to study functions of the subdomains in SID of Hik33 firstly we attempted to replace SID of a phosphate-deficient sensor SphS with the SID from Hik33 to express chimera sensor containing SID of Hik33 and transmitter domain of SphS (Hik33nSphSc). SphS regulates expression of proteins for the phosphate-acquisition, including an alkaline phosphatase and highaffinity phosphate transporters. When SphS was replaced with the chimeric sensor Hik33n-SphSc, it altered expression pattern of the SphS-regulon under the environmental conditions, which might be perceived by the Hik33 but not under the phosphate-deficiency. Then, we deleted the subdomains in the SID of Hik33n-SphSc and examined effects on the expression of the phoA gene for alkaline phosphatase to identify the important subdomain to perceive the stimuli. Results: SphS is activated under the phosphate-deficient condition and upregulates expression of the phoA gene. However, the strain expressing Hik33n-SphSc expressed the phoA gene under the standard growth condition and expression levels of phoA were repressed under high salt and cold stress conditions, which might be perceived by Hik33. These results indicated that Hik33n-SphSc might be active under the standard conditions and once the signals were accepted, it might become inactive. Deletion of the periplasmic loop did not affect the ability of Hik33n-SphSc, i.e., the phoA gene was expressed under the standard conditions and repressed under the stressed conditions. However, deletion of PAS domain lost the responsiveness to the stress, i.e., the phoA gene was constitutively expressed. And simultaneous deletion of HAMP and PAS domains lost activity of expression of the phoA gene regardless of the growth conditions, suggesting that the HAMP domain might be essential for the kinase activity. Conclusions: A chimeric sensor Hik33n-SphSc indicated that SID of Hik33 might activate the transmitter domain under the normal growth conditions. The PAS domain in the SID might play important roles in perception of the stimuli.
P.075 RNASE P FROM THE CHROMATOPHORE OF PAULINELLA CHROMATOPHORA. Agustin Vioque, Pilar Bernal, Leonor Puerto-Galan.
P.074 FUNCTIONAL ANALYSIS OF DOMAINS FOR THE SIGNAL PERCEPTION OF HIK33 IN SYNECHOCYSTIS SP. PCC 6803. Yohei Shimura, Satoshi Kimura, Yoshihiro Shiraiwa, Iwane Suzuki. Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan. Introduction: Two-component systems, which generally consist of a sensory histidine kinase and a response regulator, are major signaling pathways in most of bacteria. In general, histidine kinases possess a conserved transmitter domain associating kinase activity at the Cterminus and a unique signal input domain (SID) at the N-terminus. The SIDs in the histidine kinases are thought to play important roles in perception of the specific stimuli and regulation of the kinase activity of the transmitter domain, however, the actual molecular mechanisms of signal perception are not clarified yet. Hik33, namely NblS ortholog in the cyanobacterium Synechocystis sp. PCC 6803, regulates gene expression under cold, high light, oxidative, high salt and hyperosmotic stress conditions. It has two transmembrane helices, a periplasmic loop, a HAMP and a PAS domain in its SID. In this study, we examined functions of the subdomains in SID of Hik33 in perception of the stimuli. August 9 to 14, 2009 • Montréal, QC, Canada
Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla and CSIC, Sevilla, Spain. Introduction: Ribonuclease P (RNase P) is required for the generation of the mature 5’ end of tRNAs from precursors. RNase P is present in all domains of life as a ribonucleoprotein with a catalytic RNA subunit (P RNA). The only exceptions found so far are in human mitochondria and higher plant chloroplasts, where RNase P seems to be a protein enzyme lacking an RNA subunit. In several algae the plastid genome contains a gene homologous to the bacterial P RNA gene (rnpB). In contrast with the bacterial P RNA, plastids P RNA has no significant activity in vitro in the absence of protein, and only very weak activity could be reconstituted with a bacterial protein subunit (P protein) and the P RNA from Cyanophora paradoxa. Plastid P RNA seems to be unable to fold into a functional structure for pre-tRNA substrate binding. Paulinella chromatophora is a protist that contains an obligate cyanobacterium endosymbiont (chromatophore) whose genome has been recently sequenced. This genome is highly reduced compared with free-living cyanobacteria and could represent an early step in the integration of a cyanobacterium into a eukaryotic cell, a process that is at the origin of chloroplasts. Therefore, we have characterized the chromatophore RNase P, and compared its structure and function with 117
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Final Program and Abstracts both cyanobacterial and plastid RNase P, under the hypothesis that its properties could illustrate about the process of P RNA loss of function in plastid evolution. Methods: We have identified in the chromatophore genome the rnpA (encoding the P protein) and the rnpB genes and have undertaken their functional and structural analysis. The rnpB gene has been cloned and the P RNA has been obtained by in vitro transcription. Its RNase P activity has been analyzed either in the absence or presence of a cyanobacterial P protein. The structure and folding of the P RNAs has been analyzed by native gel electrophoresis and sensitivity to lead-induced cleavage. Results: Chromatophore P RNA has a predicted secondary structure similar to other cyanobacterial P RNAs. Chromatophore P RNA has reduced activity in the absence of protein, but it can reconstitute a fully functional holoenzyme with cyanobacterial P protein. Comparison of reaction rates in the presence or absence of the protein subunit indicate that chromatophore RNase P is more dependent on the protein subunit for activity than free living cyanobacterial RNase P. The pattern of lead induced cleavage of chromatophore P RNA is similar to the pattern observed with Anabaena 7120 P RNA, but the mobility of the P RNA on native gel electrophoresis indicates that this RNA has a reduced ability to fold into a functional native compact structure. Conclusions: The chromatophore P RNA has functional and structural properties that suggest that it might represent an early step in the process of increased dependence on protein for folding and activity prevalent in present day plastids.
P.076 THE GENOME SEQUENCE OF THIODICTYON SP. AND ITS SIGNIFICANCE FOR THE CAROTENOGENESIS OF OKENONE. Kajetan Vogl, Niels-Ulrik Frigaard, Mauro Tonolla, Lynn Thomso, Qi Ji, Stephan C. Schuster,Donald A. Bryant.
proximity to the gene cluster. Conclusions: The carotenogenesis gene cluster revealed a so far unknown duplication of the crtD gene. Since this duplication was not observed in other phototrophic bacteria, it seems to be possible that the second crtD gene has another function than desaturating the 3,4 and/or 3’,4’ bond (Albrecht et al. 1997), and its function is correlated with okenone production. In the past the function of certain carotenoid genes were successfully examined by heterologous expression in Eschericha coli (Maresca et al. 2007). In combination with the approach of heterologous gene expression in E. coli and Chlorobaculum tepidum, the genome sequence of Thiocystis sp. will allow the elucidation of the carotenogenesis pathway for okenone. References Albrecht M, Ruther A and Sandmann G (1997) Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides. J Bacteriol 179:7462-7467 Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W and Lipman DJ (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res 25:33893402 Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA and Bowden SA (2005) Biomarker evidence for green and purple sulphur bacteria in a stratified Palaeoproterozoic sea. Nature 437:866-870 Madigan MT and Jung DO (2009) An Overview of purple bacteria: systematics, physiology, and habitats. In: Hunter CN, Daldal F, Thurnauer MC and Beatty JT (eds) The Purple Phototrophic Bacteria, pp 1-15. Springer Netherlands Maresca JA, Graham JE, Wu M, Eisen J and Bryant DA (2007) Identification of a fourth type of carotenoid cyclase in photosynthetic organisms. Proc Natl Acad Sci USA 104:11784-11789
Penn State University, University Park, PA, USA.
P.077
Introduction: Anoxygenic phototrophic purple bacteria are a major group of phototrophic microorganisms which occur mainly in aquatic environments. The group of purple bacteria consists of purple nonsulfur bacteria (alpha- or beta- proteobacteria) and purple sulfur bacteria (gammaproteobacteria). Within the group of purple sulfur bacteria, over 25 genera are recognized (Madigan and Jung 2009). These bacteria have two pathways for carotenogenesis, the spirilloxanthin pathway and the okenone pathway. The carotenoid okenone is of special interest, because okenone and its derivative okenane serve as the only known hydrocarbon biomarker for purple sulfur bacteria (Chromatiaceae) used to reconstruct geologic history (Brocks et al. 2005). However, so far no genome sequence of an okenone-producing purple sulfur bacterium is available and the biosynthetic pathway of okenone is not understood in detail.
THYLAKOID MEMBRANE REDUCTION AFFECTS THE PHOTOSYSTEM STOICHIOMETRY IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Methods: Thiodictyon sp. strain Cad 16 genomic DNA was isolated using standard protocols. Then 454 pyrosequencing was performed and contigs harboring carotenoid genes were identified with BLASTN (Altschul et al. 1997) and annotated manually with MacVector. Results: The following homologs of carotenogenesis genes are present in the okenone producing purple sulfur bacterium: geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI), hydroxyneurosporene synthase (crtC), two gene copies of C-3’,4’ desaturase (crtD), hydroxyneurosporene-O-methyltransferase (crtF), gamma-carotene desaturase (crtU) and lycopene cyclase (crtY), The crtY, crtU, crtD1, crtD2 and crtC are clustered together. CrtE, crtI and crtB are in close 118
Eva Fuhrmann, Dirk Schneider. University Freiburg - Department of Biochemistry and Molecular Biology, Freiburg, Baden-Wurttemberg, Germany. Introduction: Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is incompletely understood today. The “vesicle inducing protein in plastids 1” (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Since the exact physiological function of VIPP1 is still mysterious, we generated a Synechocystis sp. PCC 6803 Vipp1 depletion strain to characterize the physiological effects of the Vipp1 depletion in cyanobacteria more in detail. Methods: To inactivate the Vipp1 gene a kanamycin resistance cassette was introduced into the vipp1 gene (sll0617) from Synechocystis sp. PCC 6803. Wild type (wt) and mutant cells were analyzed by electron microscopy as well as by absorbance and 77 K fluorescence emission spectroscopy. Photosystem (PS) II and PS I activity was determined by oxygen evolution and consumption measurements, respectively. Membrane protein complexes were characterized by sucrose density gradient centrifugation as well as by blue native polyacrylamid gelelectrophoresis (BN-PAGE). Results: Although the vipp1 gene was disrupted in a few chromosomal copies, no completely segregated strain obtained even August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes after two years on selective medium. Nevertheless, based on immunoblot analyses the total Vipp1 content in the mutant strain was significantly decreased when compared to wt cells. Electron microscopy revealed an average of four to six layers of thylakoid membranes present in wt cells whereas in the mutant strain only two to three layers were observed. Under increasing light intensities the chlorophyll content per cell decreases, and, furthermore, it is always lower in the merodiploid cells at any light intensity compared to wt cells. Sucrose density gradient centrifugation and BN-PAGE analyses revealed that the amount of PS I trimers is significantly reduced in membranes isolated from the mutant cells. Furthermore, the cellular concentration of chlorophyll binding proteins is significantly lowered in the merodiploid cells. By 77 K and UV/VIS spectroscopy we observed that the ratio of PS I to PS II has changed in the mutant strain. While the PS II activity is not significantly altered the activity of PS I was determined to be decreased in the mutant relative to the wt strain. However, the mutant cells still appear to be able to operate the electron transfer chain at wt rates. Conclusion: We show that in the cyanobacterium Synechocystis sp. PCC 6803 the synthesis of active PS I depends on the amount of thylakoid membranes present per cell. Down-regulation of the amount of active PS I appears to be a general adaptation mechanism in cyanobacteria and Vipp1 could be directly involved in such mechanisms, resulting in changes of the PS I to PS II ratio in response to e.g. increasing light intensities. The depletion of the vipp1 gene product resulted in a specific decrease of functional PS I in Synechocystis, whereas the amount of functional PS II was not significantly altered. In contrast, a Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain1. Therefore, it has been suggested that depletion of Vipp1 in Arabidopsis affects thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes2. 1
Kroll et al (2001) Proc Natl Acad Sci USA 98: 4238–4242
2
Aseeva et al (2007) Plant Physiol Biochem 45: 119–128
P.078 THE EVOLUTIONARY PATH TO TERMINAL DIFFERENTIATION AND DIVISION OF LABOR IN CYANOBACTERIA. Valentina Rossetti, Bettina E Schirrmeister, Marco V. Bernasconi, Homayoun C Bagheri. University of Zurich, Zurich, Zurich, Switzerland. Introduction: The transition from unicellular to multicellular organisms is not a well understood process in evolution. A common trait often associated with multicellularity is cellular differentiation, which is a separation of tasks through the division of labor. However, the division of labor does not necessarily have to be constrained to a multicellular setting. In this study, we focus on the possible evolutionary paths leading to terminal differentiation in cyanobacteria. Methods: We develop mathematical models for two developmental strategies. One, of populations of terminally differentiated single cells surviving by the exchange of common goods. Second, of populations exhibiting terminal differentiation in a multicellular setting. We test the evolutionary stability of the two strategies with respect to resistance against disruptive mutations (i.e. “cheater” mutants). We also assess the effects of selection on the optimization of the ratio of vegetative August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts (carbon fixing) to heterocystous (nitrogen fixing) cells, which in turn leads to the maximization of the carrying capacity for the population density. In addition we compare the performance of differentiated populations to undifferentiated ones that temporally separate tasks in accordance to a day/night cycle. We then compare some predictions of our model with phylogenetic relationships derived from analyzing 16S rDNA sequences of different cyanobacterial strains. Results: In line with studies indicating that group or spatial structure are ways to evolve cooperation and protect against the spread of cheaters, our work provides theoretical and phylogenetic evidence that differentiated single-celled populations of cyanobacteria are not stable. The compartmentalization afforded by multicellularity is required to maintain the vegetative/heterocyst division and for selection to optimize the carrying capacity. Conclusions: In cyanobacteria, multicellularity is a necessary condition for both the evolutionary stability of terminal differentiation and for the optimization of the division of labor. Furthermore, in regimes of long daylight periods, terminally differentiated species perform better than undifferentiated species that follow the day/night cycle; indicating that terminal differentiation can be an evolutionary advantage in high irradiance regions. Conversely, undifferentiated species have an advantage in regimes of short daylight periods.
P.079 DIVERSITY OF PHOTOTROPHIC BACTERIA IN MICROBIAL MATS OF LAGUNA CHAXA AND LAGUNA TEBENQUICHE AT THE SALAR DE ATACAMA, CHILE. Cristina Dorador, Andrea Gärtner, Yaneisi Vazquez, Carolina Cubillos, Vera Thiel, Johannes F. Imhoff. Departamento de Acuicultura, Universidad de Antofagasta, Avenida Universidad de Antofagasta, Antofagasta, Chile. Introduction: The Salar de Atacama is located in the Atacama Desert (Chile) at an altitude of 2300 m above sea level. In the interior of the salar there are several small shallow ponds that exhibit salt concentrations >100 mS cm-1. Intensive solar radiation and fluctuating daily temperatures between day and night are typical features of these environments. It has been proposed that phototrophic bacteria should play an important role in the primary production of saline lakes in the Atacama Desert; however their diversity and functionality are under investigated. The diversity of phototrophic bacteria was focused in two main lakes of the Atacama Desert which exhibit microbial mats on their shore. Methods: We investigated the diversity of specific phototrophic bacterial in samples collected from Laguna Chaxa and Laguna Tebenquiche at the Salar de Atacama. DNA was extracted from homogenized samples of microbial mats and was amplified using described specific 16S rRNA gene primers for the groups Chromatiaceae, Chlorobi, Cyanobacteria and Roseobacter clade. Microbial diversity was examined through DGGE and clone libraries of the 16S rRNA gene. Results: Microbial mats were found at the shore of Laguna Tebenquiche and Laguna Chaxa. Mats from Laguna Tebenquiche exhibited 1-3 layers compared with mats of Laguna Chaxa which had between 3-4 layers. The target groups were recorded from all samples analysed. Analyses of DGGE band patterns and clone libraries revealed that the lakes supported different microbial communities. Conclusions: Phototrophic bacteria occupied an important part of the microbial diversity of inland lakes of Salar de Atacama. The Roseobacter clade, initially described from marine systems, 119
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Final Program and Abstracts represented an important component of the diversity of phototrophic bacteria from inland lakes of the Salar de Atacama. Future studies will include activity assays to determine the role of the phototrophic bacteria in the primary productivity of evaporitic basins in northern Chile.
P.080 CYANOBACTERIA IN THE PCC: WHO IS RELATED TO WHOM? Muriel Gugger1, Alexandra Calteau2, Rosmarie Rippka1, Thérèse Coursin1, Thierry Laurent1, Jennifer Tambosco1, Corinne Cruaud2, Frederik Gavory2, Jean Weissenbach2, Nicole Tandeau de Marsac1. 1
Unité des Cyanobactéries, Institut Pasteur, Paris ; 2CEA / Institut de Génomique / Génoscope / Laboratoire de Génomique Comparative, Evry, France. Introduction: Cyanobacteria are oxygenic phototrophic prokaryotes that exhibit a wide range of morphological and physiological features and colonize strikingly different ecosystems. The global picture of cyanobacterial interrelationships is mainly based on complete or partial 16S rDNA sequences of non-axenic isolates for which little information may be available, or on environmental samples representative of very specific ecosystems. Recently, the evolution of this phylum has been re-examined based on cyanobacterial genome sequences, but so far is highly biased towards marine picocyanobacteria (50 % of the genomes). Methods: The PCC (Pasteur Culture Collection of Cyanobacteria) presently includes almost 800 axenic strains originating from diverse ecosystems and representing about 60 major morphotypes. They exhibit widely differing physiological characteristics, and some produce hepato- or neurotoxins. To overcome the paucity of information associated with many of the available 16S rDNA sequences, either with respect to knowledge of the organisms or because of insufficient sequence length, the 16S rRNA genes of 709 PCC strains were sequenced over their full length to establish more precisely their phylogenetic relationships. Results: The sequence analyses confirmed the monophyly of the heterocystous cyanobacteria, and the intermixed relationships of the other morphotypes. Although many genera are polyphyletic, several sustained genetic clusters were recognized that contain PCC reference strains, previously proposed based on mean DNA base composition and phenotypic characters. Moreover, PCC strains hitherto insufficiently characterized can now be assigned to appropriate genetic clusters within the cyanobacterial phylum. In addition, relatives from other culture collections or those only known from environmental sequences could be recognized. Conclusions: The new 16S rDNA sequence database of the PCC provides a more complete reference frame for the genetic diversity and interrelationships of cultured and uncultured cyanobacteria, and should help to place the phenotypic properties of the organisms into an evolutionary context.
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P.081 MOLECULAR BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF ODOR PRODUCING CYANOBACTERIA FROM DRINKING WATER RESERVOIRS. Frank Ludwig, Sabine Hacker, Andy Weiss, Hetvi R. Gandhi. Isolde Röske Institute of Microbiology, Dresden University of Technology, Dresden, Germany. Introduction: Recently, taste and odor problems (TO problems) could be observed frequently. In the most cases the odor is described as an earthy-musty smell, which may be caused by the volatile sesquiterpene geosmin, which has a very low odor threshold concentration of 4 ng/l (Young et al. 1996). Besides actinomycetes, cyanobacteria are mostly responsible for the release of odorous substances as geosmin and 2-methylisoborneol (2-mib) into the water. Because the drinking water should be free from flavor and smell, TO problems lead to higher costs. Therefore, we have investigated the occuring cyanobacteria from three different drinking water reservoirs. Methods: Material from the phytobenthic mats of the Cranzahl, Klingenberg and Saidenbach (all in Saxony, Germany) drinking water reservoirs was used to prepare single trichomes of cyanobacteria. From the obtained cultures the isolated dna were used to amplify genes like the rbcL and geoA. These partial gene sequences and furthermore the sequence area between the 16S rDNA and 23S rDNA could be used for the characterization of the isolates. The development of degenerated primer pairs for the amplification of the cyanobacterial genes like rbcL and the geoA were the basis for the successful molecular biological determination of the isolated strains. Supplementary we have conducted biochemical analyses with selected cultures for the confirmation that these isolates are capable to produce odorous substances as geosmin or/and 2-mib. Finally phylogenetic trees were established with the obtained nucleotide sequences for the depiction of the existing diversity. Results: Through the successful development of a new method to isolate pure cultures of cyanobacteria from very heterogeneous environmental samples we were able to obtain a lot of taxonomically very different cyanobacterial cultures. This could be affirmed by the nucleotide sequences of the 16S-23S rDNA, rbcL and the geoA genes. The biochemical analyses shows also the important potential of cyanobacteria to produce and to release odor to the ambient biocenosis. Experiments with different light and nutrient conditions have demonstrated that the pattern of the produced odor is variable. Conclusions: The achieved results show the great diversity of the occurring cyanobacteria in different drinking water reservoirs. Through the biochemical analyses it can be confirmed that cyanobacteria are a major originator of TO problems. It is very interesting that especially the light quality has an influence of the synthesized odorous substances. Because it is very difficult to cultivate cyanobacteria and to identify factors which affect the odor production, the management of the drinking water reservoirs is very interested to find out parameters which can indicate that odorous substances will be released into the water at a great scale.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
P.082
P.083
THE TWIN ARGININE TRANSLOCATION SYSTEM IN SYNECHOCYSTIS PCC6803.
POLYFACETIC STUDY OF NON PURPLE SULFUR BACTERIA ISOLATED FROM THE SOUTHERN GULF OF MEXICO WATER SAMPLES.
Anja Nenninger1,3, Gemma Warren2, Conrad W. Mullineaux1 and Colin Robinson3. 1
Queen Mary University of London, London; 2MOAC, Doctoral Training Centre, University of Warwick, Coventry; 3University of Warwick, Coventry; United Kingdom Introduction: Targeting and translocation of newly synthesised proteins is a crucial process in living calls, using a range of mechanism at different membrane systems. The transport mechanism studied here is the twin arginine translocation (Tat) system. Discovered in the early 1990s in plant thylakoids it has since been shown to operate in the plasma membrane of a wide variety bacteria. Cyanobacteria represent a unique subject for Tat studies. They are considered to be the progenitors of plant chloroplasts with an oxygenevolving photosynthetic apparatus very similar to higher plants at an abundant internal network of thylakoid membranes; and as Gramnegative bacteria they have a cell envelope consisting of an outer membrane, a periplasmic compartment, and a plasma membrane. Sequencing of the Synechocystis PCC6803 genome has shown that a single set of tat genes is present, with a homologue of cptatC/tatC and two paralogues of tha4/tatA. Methods: The plasmid described in Spence et al. (2003) was used as template for QuikChange® Site-directed Mutagenesis and the following mutants of the twin arginine motif were obtained: RK, KR, KK. Translocation of GFP in Synechocystis was observed using a confocal microscopy. (Spence E. et al., 2003, Mol. Micro. 48, 1481-89) Results: In Synechocystis GFP tagged with a Tat signal sequence (from E. coli TMAO reductase; torA-GFP) is found exclusively in the periplasm. GFP fluorescence can be seen contiguous to the thylakoid membrane system corresponding with exported periplasmic GFP. Disrupting the twin arginine motif leads to accumulation of clearly visible GFP aggregates inside the plasma membrane. The analysis of numerous cells for the different mutations has shown that these aggregates are primarily located in the thylakoid region, but they can as well be found in the cytoplasm. Conclusions: Proteomic studies on Synechocystis identified several potential Tat substrates. The twin arginine is part of the signal sequence in most Tat substrates in plants and bacteria. However, the importance of this motif varies between the two systems. In plants substitution of either arginine led to a complete block in translocation, whilst bacterial Tat system only shows a full block in translocation when both arginine residues were replaced. Substitution of the arginine residues in Synechocystis shows an extreme level of stringency very similar to the situation observed in plants. This absolute requirement for an intact twin arginine motif is unique among bacteria studied to date. With the endosymbiotic theory now widely accepted for the origin of plastids in higher plants and phylogenetic studies clustering the cyanobacterial tatC (sll0194) with the chloroplast cptatC the results described here emphasise the relationship of cyanobacteria and plant chloroplast on a functional level for the first time.
August 9 to 14, 2009 • Montréal, QC, Canada
María Teresa Núñez-Cardona1, Verónica Pacheco-González2, Jaime García-Mena3, Martha Signoret4. Universidad Autónoma Metropolitana-Xochimilco, 1,2Laboratory of Ecología Microbiana; 4Plancton and Bioenergética; 3Centro de Investigación y de Estudios Avanzados-Zacatenco, Departamento de Genética y Biología Molecular, Distrito Federal, México. Introduction: Studies about diversity of phototrophic bacteria (PB) using laboratory cultures are scarce if we compared with the application of molecular techniques like nucleic acids hybridization in situ and the gene bank. These molecular tools have lead to the conclusion that the most abundant marine microbial groups are as yet uncultivated and probably they play a significant role in marine biogeochemical cycling. Nevertheless pure laboratory cultures are used as reference and as models too. The goal of this investigation was to isolate and to identify nonpurple sulfur bacteria from a 100 m depth water sample collected in the Gulf of Mexico. Methods: Conventional and molecular analyses were used to characterize and to identify the isolated strains. Water samples were collected at 100 m depth and were used to enrich glass vials containing van Niel medium, specific for growth of nonpurple sulfur bacteria. Strains were purified by the agar shake technique. All the cultures were incubated at room temperature (20-28 °C) and exposed to light (1.3-1.8 Klux). Morphological, pigment composition (in vivo analysis) and the ability to use different chemical compounds as electron donors in van Niel basal medium, were used to characterize the isolated strains. They were identified by the analysis of the 16S rDNA. This one was achieved and amplified by PCR technique and the use of Unifor and Unirev primers. Results were analyzed by electrophoresis in an agarose gel (1.0%) and the DNA sequences were gotten. Results: Pure cultures (13 strains) of nonsulfur bacteria were obtained after serial dilutions on semisolid agar. Cell morphology of these corresponded to small budding Gram negative rods, with a little difference in size (2-3 μm). In vivo pigments analyses revealed the presence of bacteriochlorophyll a and spirilloxanthin as major accessory pigment in all wild strains. It was assayed 20 different compounds to know the capability of the strains to use them as electron donors. Sodium and magnesium acetate as well as glycerol have been used for all the strains assayed, 12 used yeast extract and eleven to the pyruvate and propionic acid, sucrose, maltose, lactose, by other hand, succinate. methionin, mannitol and glycine were used only by 5, 6 and 7, strains, respectively. A BLAST and phylogenetic analyses based on 16S rDNA gene sequence revealed that four strains were related with Rhodobium bactotapetarum, three to Rhodopseudomonas spp and other three were identified as members of Rhodopseudomonas julia. It was observed differences in the use of the electron donors among the strains of the same specie. Conclusions: With the enrichment technique used it was possible to detect, isolate and to get pure cultures of phototrophic bacteria from marine water samples. The presence of bacteriocholorophyll a and the 16S rDNA analysis identify them as members of purple non sulfur bacteria
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P.084 DETECTION OF CYANOBACTERIA WITH CLASSICAL MOLECULAR METHODS IN THE SEDIMENT OF A DRINKING WATER RESERVOIR. Susanne Schumann1, Heidemarie Horn2, Tim Köhler3, Frank Ludwig1, Isolde Röske1, Kerstin Röske2. 1
Institute of Microbiology, Dresden University of Technology, Dresden; Taxony Academy of Sciences, Leipzig; 3Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, Marburg; Germany.
genera of purple sulphur bacteria (Chromatiaceae). Allochromatium was the most common genus 161 (50%) followed by Thiocystis 50 (15.53%), Thiocapsa 40 (12.42%), Thiodictyon 38 (11.8%) and Lamprocystis 33 (I0.25%) from the total number of isolates during the whole period of study. These genera represented by six speces Allochromatium vinosum and A. warmingii; Thiocystis violacea; Thiocapsa roseopersicina; Thiodictyon elegans and Lamprocystis roseopersicina. Physico–chemical parameters of water samples were determined.
2
Introduction: Cyanobacteria were able to colonize almost all habitats and are special important in the water because they have the ability to do photosynthesis. So they make a great contribution to primary production (Martínez-Alonso et al., 2004). Through formation of mats on the water surface, cyanobacteria can be able to reduce the development of organisms in deeper water layers. Methods: Sediment samples of different sampling sites of the dinking water reservoir Saidenbach (Saxony, Germany) and the pre-reservoir Forchheim were examined to follow up the deposition of cyanobacteria. To analyze the seasonal changes of the cyanobacteria composition in this habitat cultivation methods as well as PCR with established primer sets for cyanobacteria were applied. These partly well-known primer were combined with Cya564F, a new one to sequence the 16S rDNA to 23S rDNA of the cyanobacteria cultures which provides the basis of an alignment and cluster analysis. This results were compared with a phylogenetic tree which was established on the basis of the sequencing of the cyanobacterial phycocyanine gene (cpc). In addition a new primer system specific for species of the genus Synechococcus was developed and tested by PCR and cloning. Results: With the applied methods it was possible to detect differences and parallels in the emerging of cyanobacteria during the examination period. The amplification with the new primer system designated ProCR demonstrated clear variation in the occurrence of species of the genus Synechococcus. In spring, all samples were positive but in the further examination period it showed partly negative results. These findings could be confirmed by microscopy and cultivation. The alignment of the whole 16S rDNA of about 30 cyanobacterial pure cultures corroborates the belief that there is an increase of differences in the rear sequence sections. Conclusions: The received results show seasonal changes in the deposition of cyanobacteria of the examined drinking water reservoir. Furthermore differences between the sampling sites Saidenbach and Forchheim could be demonstrated. The first molecular biological analysis show the necessity of the development of new primer sets to sequence the whole 16S rDNA. So a reliable analysis of the isolated cyanobacterial cultures becomes possible.
P.085 ECOLOGICAL AND PHYSIOLOGICAL STUDIES ON PURPLE SULFUR BACTERIA (CHROMATIACEAE) AT ASWAN HIGH DAM LAKE. Mohamed S. A. Shabeb 1, Nahla S. Abd-El Azim1, Ahmed A. M. A. Shoreit 2. 1
Botany Department, Faculty of Science, Aswan South Valley University, Aswan, Egypt; 2Botany Department, Faculty of Science, Assiut University, Assiut, Egypt 322 isolates of purple sulfur bacteria were isolated from different localities of study in Aswan High Dam Lake. These comprise five 122
P.086 FUNCTIONAL GENE APPROACH ON COMMUNITIES OF PHOTOTROPHIC PROKARYOTES IN HIGH ALTITUDE WETLANDS IN NORTHERN CHILE. Vera Thiel, Marcus Tank, Cristina Dorador, Johannes F. Imhoff. IFM-GEOMAR, Leibniz Institute of Marine Sciences (at the University of Kiel), Kiel, Schlewig-Holstein, Germany. Introduction: The Chilean Altiplano located 2300 m above sea level harbours a set of extreme habitats characterized by high UV-radiation, partly extreme saline conditions and high diurnal temperature variations. These unexpectedly quite productive habitats are dominated by microbial life and phototrophic bacteria are assumed to have a major impact. However, key processes of primary production have not yet been identified and little is known about the diversity and composition of the phototrophic prokaryotic communities. To gain insight into the diversity of assumed key players involved in primary production in Chilean Altiplano salares, we studied the phototrophic bacterial communities of different locations around the Salar de Atacama in the Chilean Altiplano characterized by salinities varying from 54 mS cm-1 to 261 mS cm-1. Due to the polyphyletic character of the physiological groups of phototrophic bacteria, 16S rRNA gene based molecular studies often fail to cover the diversity of the phototrophic community. Thus, in order to specifically study the phototrophic prokaryotes of the habitats, we used molecular genetic analyses with group specific primers for functional genes in addition to isolation methods. Methods: We sampled several colourful microbial mats along two lakes of the Salar de Atacama (Laguna Chaxa and Laguna Tebenquiche). Different liquid media were inoculated for enrichment and isolation of phototrophic bacteria. Pure cultures were obtained by serial dilution in agar shakes and identified by 16S rRNA gene sequencing. For the molecular approach genomic DNA was extracted and the specific functional genes fmoA, pufLM and bchY amplified using PCR method. Clone libraries were constructed and the community composition was established on the basis of the functional gene sequences. Results: Enrichment and purification in different media lead to the isolation and identification of representatives of the green sulfur and purple sulfur bacteria as well as purple nonsulfur bacteria. Using functional genes members of different phototrophic bacterial groups were detected in all samples. The clone libraries lead to an estimate of the diversity of phototrophic prokaryotes in the different samples. The diversity and composition of the phototrophic prokaryotic communities varied between the different samples and between the two lakes. Conclusion: This study is the first report on the diversity of phototrophic prokaryotes in the Chilean Altiplano salares determined by functional gene approach and isolation methods. We demonstrate the suitability of the used functional genes, fmoA, pufLM and bchY for August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes studying phototrophic communities as well as the unique phototrophic communities of the different habitats around the Salar de Atacama.
P.087 EVOLUTION OF THE CYANOBACTERIUM NOSTOC –PLANT SYMBIOSES. Akiko Tomitani. Institute of Biogeosciences, Japan Agency for Marine-Earth Science & Technology, Yokosuka, Kanagawa, Japan. Cyanobacteria are oxygenic photosynthetic bacteria, many of which have ability to fix nitrogen. Filamentous cyanobacteria of the genus Nostoc produce differentiated cells such as heterocysts (specialized cells for nitrogen fixation) and hormogonia (transient motile filements), and many of them are known to establish symbiotic association with various plants (bryophytes, pteridophytes, gymnosperms, angiosperms) and algae and supply fixed nitrogen to the hosts. To investigate how Nostoc has evolved the capability to infect a wide range of plants, molecular-phylogenetic analyses together with coculturing experiments were performed. Phylogenetic analyses of free-living and symbiotic Nostoc species suggest that symbiotic competency may have evoloved polyphyletically within the genus. Co-culturing assays using 10 Nostoc strains and a host bryophyte indicate that their ability of hormogonia formation may not correspond to plant-infection efficiency. Continued molecular-biological and physiological study of diverse Nostoc species will provide a clue to understand mechanisms and evolution of the cyanobacterium-plant symbioses.
Final Program and Abstracts endemic to Antarctica and three others constitute a previously undiscovered diversity. A multivariate analysis run with data from various samples revealed that the OTU composition is geographically structured as each region has its more or less unique flora. Moreover, salinity seems to play an important role on the composition of the cyanobacterial communities in these samples. Conclusions: The three new OTUs included sequences from the Belgian Base, Transantarctic Mountains and East Antarctica which suggests a flux of microorganisms between these regions. On the other hand, differences in cyanobacterial composition between geographically close lakes were observed. These might be underlain by several reasons, such as differences in limnological properties (inferring different histories and ecological processes between regions), or rather the result from dispersal limitation among cyanobacteria. Also, samples originating from coastal lakes seem to be more diverse (average number of OTUs) than others, this may be due to the difference in the meteorological conditions (oceanic versus continental climates). More detailed studies and refined molecular analysis (Real Time Quantitative PCR, clone libraries) should allow us to assess the real cyanobacterial diversity in antarctic lakes and the importance of geographical and environmental factors shaping the microbial communities.
P.089 IN THE FRAME OF THE BELSPO PROJECT B-BLOOMS2, WE HAVE OBSERVED THE PRESENCE OF MICROCYSTINS BY ELISA IN BLOOMS OF WORONICHINIA IN ONE BRUSSELS POND. Annick Wilmotte, Yannick Lara, Alexandre Lambion, Anatoly Peretyatko, Ludwig Triest, Geoffrey Codd.
P.088
University of Liege, Liege, Belgium.
BIOGEOGRAPHICAL DISTRIBUTION AND DIVERSITY OF CYANOBACTERIA IN ANTARCTICA, A POLYPHASIC APPROACH.
So far, Woronichinia naegliana was isolated only two times according to the literature (Rajaniemi et al., 2005; Willame et al., 2006) and the isolates were not toxic. However, the cultures were quickly lost, illustrating the difficulties in obtaining and keeping strains of this genus. Therefore, there is an important lack of knowledge and molecular data. As an alternative, we propose to analyze genotypes of environmental single colonies.
Annick Wilmotte1, Pedro De Caralho Maalouf1, Frédéric Zakhia1, Arnaud Taton1, Rafael Fernandez-Carazo1, Aaike De Wever2, Elie Verleyen2. 1
Centre d’Ingénierie des Protéines, B6, Université de Liège, Liège; Department of Biology, Research group of Protistology and Aquatic Ecology (PAE), Gent University, Gent; Belgium. 2
Introduction: In high latitude ecosystems, cyanobacteria are of particular interest because they often represent the predominant phototrophs. The cyanobacterial diversity from microbial mats in antarctic lakes was investigated using microscopic and molecular approaches. This enables us to assess the relative importance of ecological versus historical factors in explaining the geographical distribution of cyanobacteria in Antarctica. Methods: Samples from a wide geographic scale were chosen in order to represent a range of environmental conditions and to evaluate the influence of lake characteristics on the cyanobacterial diversity. In the framework of the AMBIO project (“Antarctic Microbial BIOdiversity: the importance of geographical and ecological factors”, www.ambio.ulg.ac.be), microscopic identification and morphological characterization were complemented with molecular tools (Polymerase Chain Reaction, Denaturating Gradient Gel Electrophoresis, sequencing, clone libraries). This polyphasic approach aims to reveal the cultivated and non-cultivated diversity of microorganisms.
Methods: Woronichinia were directly isolated under a binocular from a fresh sample. As the DNA content of one single colony limits the number of PCR reactions that can be carried out, we have developed a new approach using Whole Genome Amplification with Phi29 polymerase to allow for the Multi Locus Sequences Analysis of a single colony. Subsequent PCR reactions were performed with cyanbacterial specific primers. Results: For the first time, we have obtained the sequences of rpoC1, rbcLX and rRNA-ITS from 4 single colonies of the genus Woronichinia (identified by microscopy). About 12 PCR reactions were successfully performed on one single colony. mcyE genes were not detected by PCR. Conclusion: This approach allows to work with a small amount of DNA, and represents a concrete answer to the lack of molecular data on non-cultivable or difficult to isolate cyanobacteria.
Results: Preliminary results from the 16S rDNA sequence analysis allowed the construction of phylogenetic tree and revealed the presence of 23 Operational Taxonomic Units (OTU). Five OTUs are August 9 to 14, 2009 • Montréal, QC, Canada
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P.090 HOW DOES CYANOBACTERIAL PHOTOSYNTHESIS CHANGE WITH SALINITY AND TEMPERATURE IN HYPERSALINE MICROBIAL MATS LOCATED AT DIFFERENT INTERTIDAL POSITIONS? Raeid M. M. Abed. College of Science, Biology Department, Sultan Qaboos University, Al Khoud, Sultanate of Oman. Intertidal cyanobacterial mats of the Arabian Gulf are exposed to continuous fluctuation in temperature and salinity, daily and seasonally. The temperature ranges between 18-55°C and salinity varies between 4-25% depending on the mat’s tidal position. Using microsensors, we studied the short-term temperature effects and the effects of salinity fluctuation on gross photosynthesis (GP), net photosynthesis (NP) and light respiration (LR) in three submerged mats from a transect on an intertidal flat. Areal rates of GP and NP increased with temperature and maximum rates were detected at 45°C. The photosynthetic zone decreased from 3mm to 1.75mm with increasing temperature. Above 50°C, photosynthesis was completely inhibited, probably due to high sulfide concentrations. The areal LR rates between 25°C and 45°C did not change significantly but showed a decreasing trend in the photosynthetic zone. This trend suggested that the coupling between photosynthesis and LR was apparently broken. The response of GP, NP and LR in lower, middle and upper tidal mats to various salinities (65, 100, 150 and 200‰) was compared. GP and LR at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP and LR rates at salinities >100‰. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different temperatures and salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.
P.091 DISCOVERING THE METABOLIC BOTTLENECKS OF FUEL PRODUCTION: LIQUID CHROMATOGRAPHY MASS SPECTROMETRY (LC-MS) ANALYSIS OF THE FERMENTATIVE METABOLOME OF THE HYDROGEN-EVOLVING CYANOBACTRIUM SYNECHOCOCCUS SP. PCC 7002.
the KEGG pathway database (www.genome.jp/kegg/pathway.html) and NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). Analysis of autofermentation of Synechococcus was conducted by re-suspension of cells in the appropriate medium, sealing to light & air, purging with argon gas, and performing an optimized chemical extraction method on samples in triplicate at selecting time points post-anaerobisis. A chemical assay for total internal reducing sugars was performed on remaining cell material, and extracellular nitrate and metabolic end products were measured by chemical assay and NMR of the fermentation medium, respectively. Analysis of metabolite extracts was conducted using reversed-phase ion-pairing chromatography coupled directly to multiple-reaction monitoring (MRM) detection using an Agilent 6410 Triple Quadrupole mass spectrometer. Results: A hypothetical fermentative reaction network was constructed for Synechococcus based on whole genome information. An analysis method for these individual metabolites was developed using online chromatography coupled to mass-spectrometry, in which compoundspecific mass decomposition channels were optimized for each target metabolite and chromatography examined for optimal resolution and sensitivity of the suite of target compounds. Using this method, an anaerobic extraction protocol for Synechococcous samples employing fast cell filtration and cold chemical extraction was optimized for the target metabolome. This chemical/analytical procedure was applied to monitor the metabolic response of Synechococcous during autofermentation. As predicted, direct LC-MS detection of pyridine nucleotides and adenosine phosphates indicate increased redox poise and decreased cellular energy charge during fermentation. The differential effect of nitrate availability on metabolite pools during auto-fermentation was subsequently tested, and the roles of nitrogen as a both an electron sink (and competitor with hydrogenase) as well as a precursor to biosynthesis examined. Conclusions: A robust method for the metabolomic analysis of cyanobacterial fermentation has been developed using the model organism Synechococcus sp. PCC 7002. This approach has been employed to quantitatively monitor the intracellular response of this phototroph to auto-fermentative conditions, as well as the specific effect of nitrate on hydrogen evolution and fermentative metabolism.
P.092 EXAMINATION OF ALLOPHYCOCYANIN AND PHYCOERYTHRIN SUBUNIT BIOSYNTHESIS.
Nick Bennette, Kelsey McNeely, John Eng, G. Charles Dismukes.
Avijit Biswas, Shervonda Williams, Monica Kronfel, Yasmin Vasquez, Richard Alvey, Donald Bryant, Wendy Schluchter.
Princeton University, Princeton, NJ, USA.
Univeristy of New Orleans, New Orleans, Louisiana, USA.
Introduction: Many cyanobacteria possess the capability to evolve hydrogen fermentatively, deriving the requisite protons and electrons from water in a potential method of clean and renewable energy production. Unfortunately, much of the research regarding the production of hydrogen from cyanobacteria has dealt with internal cellular metabolism as a “black box” rather than a complex network of individual biochemical reactions in which electron and proton flux to hydrogen production may be bottlenecked at many different points. In order to resolve the internal cellular dynamics leading to hydrogen evolution, detection of the involved metabolite pools as well as resolution of their intervening fluxes is required. The objective of this research is to quantify the time-dependent intracellular metabolic response of a model hydrogen-evolving cyanobacterium, Synechococcus Sp PCC 7002, to effectors of H2 rate and yield, specifically the effect of extracellular nitrate.
Introduction: Cyanobacterial phycobilisomes are composed of the brilliantly-colored, water-soluble, highly fluorescent phycobiliproteins in addition to the linker proteins which hold the structure together. Each phycobiliprotein is composed of two subunits, α and β, and each subunit has between one and three linear tetrapyrrole prosthetic group(s) called bilin(s) attached at Cys residues via a thioether bond. In cyanobacteria, there are four different naturally occurring bilins with the most prevalent being phycocyanobilin (PCB) and phycoerythrobilin (PEB). For most phycobiliproteins, enzymes called bilin lyases are responsible for catalyzing the attachment of the bilin to the appropriate Cys residue. This study determined the bilin lyases involved in allophycocyanin subunit biosynthesis, evaluated the substrate specificity of two different types of bilin lyase; CpcE/F and CpcSU toward addition of PEB instead of PCB, and expressed putative bilin lyases to be tested for attachment of bilins to Phycoerythrin II (PEII) subunits.
Methods: Genetic analysis of Synechococcus was performed using 124
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13th International Symposium on Phototrophic Prokaryotes Methods: An in vivo heterologous expression system using E. coli was developed by co-expression of genes encoding the phycobiliprotein substrate, the biosynthetic enzymes of the prosthetic group, and the enzymes which catalyze the attachment of bilin. The phycobiliprotein subunit genes were fused downstream of a gene encoding a tag (e.g. Histidine tag or Glutathione S-transferase tag). The purified products were evaluated by absorbance and fluorescence spectroscopy, and the protein and bilin content were assessed after SDS-PAGE. For some experiments, in vitro assays were used to test activity where all recombinant proteins were expressed separately. Results: A multi-plasmid heterologous system was developed and used to create holo-allophycocyanin in E. coli cells. We also used this system to show that the heterodimeric CpcS/CpcU from Synechococcus sp. PCC 7002 is required for attachment of PCB to the allophycocyanin subunits ApcF and ApcD. For the ApcE linker protein (LCM99), we show that the amino-terminal allophycocyanin-like domain has autocatalytic bilin lyase activity and is also similar in sequence to CpcS and CpcU proteins. We also show that the CpcEF bilin lyase can efficiently attach PEB to CpcA using the in vivo heterologous system, enabling us to create unique phycobiliproteins. The CpcSU bilin lyase was not as efficient at attaching PEB over PCB to CpcB in this system. We have successfully overproduced soluble Synechococcus WH8020 HT-MpeB (β-phycoerythrin II) and HT-MpeA (α-phycoerythrin II) by coexpression with the E. coli GroES chaperone. The putative bilin lyase subunits MpeU, MpeV, MpeY, CpeU, and CpeS have been overproduced separately. Most of them are insoluble, but we are attempting to renature them from inclusion bodies. Results from these studies will be discussed. Conclusions: This in vivo heterologous system was developed in order to facilitate to test the activity of many different bilin lyases, to test the ability of known bilin lyases to attach different chromophores to create unique phycobiliproteins, and to produce large amounts of holophycobiliproteins. For some bilin lyases that are insoluble in E. coli, we may be able to renature them and test for bilin lyase activity in vitro.
P.093 GENETIC ANALYSIS OF CHLOROBACULUM TEPIDUM SULFUR ISLANDS. Ernest O. Bonsu1,2, Lisa Waidner2, Thomas E. Hanson1,2,3 . Department of Biological Sciences1, Delaware Biotechnology Institute2 and College of Marine and Earth Studies3, University of Delaware, Newark, DE, USA. Introduction: Chlorobaculum tepidum is an anaerobic phototrophic bacterium that utilizes reduced sulfur compounds such as sulfide, elemental sulfur and thiosulfate as electron donors during anoxygenic photosynthesis. Chlorobaculum tepidum is a model system for the study of anaerobic sulfur oxidation pathways as it grows rapidly, is genetically amenable, and its genome has been completely sequenced. In the C. tepidum genome sequence many predicted sulfur oxidation genes are grouped in clusters called Sulfur Islands. The overarching goal of this project is to refine models of anaerobic sulfur oxidation in C. tepidum by the genetic analysis of the Sulfur Islands. The analysis of two specific mutant strains will be discussed. Methods: In-vitro transposon mutagenesis (IVTM) was used to generate mutations in regions of the C. tepidum chromosome containing the sulfur islands. Mutant C. tepidum strains showing distinct growth or sulfur oxidation phenotypes were then characterized in detail to determine what genes were affected by the transposon insertion. Pulsed field gel electrophoresis, PCR, RT-PCR, DNA August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts sequencing and rescue cloning of DNA flanking transposon insertions have all been utilized to define the genotypes of interesting mutant strains. Further characterization of the genes affected is often necessary by creating mutant strains carrying a single gene inactivation. Results: Two strains are the focus of the current study. Mutant strain C5 was incapable of growth with thiosulfate as the sole electron donor. The genotype of strain C5 (∆CT0867CT0876::TnOGm) is complex, a deletion-insertion event that eliminated all or part of nine genes in a section of Sulfur Island I. By comparison of the deleted region with other anaerobic sulfur oxidizers, gene CT0872 was hypothesized to be responsible for the thiosulfate growth defect. The phenotype of strain CT0872::TnOGm is consistent with this prediction while the inactivation of CT0873 and CT0874 had no significant effect. Strain C3 grows slowly under all conditions tested and accumulates extremely high levels of extracellular sulfur globules during growth. PFGE data indicated that mutant strain C3 carries two TnOGm insertions localized in two distinct regions of the chromosome that are consistent with insertions in Sulfur Islands I and II, both of which carry copies of the dsr genes that are crucial for elemental sulfur oxidation in purple sulfur phototrophic bacteria. Conclusion: Detailed analysis of genes deleted in strain C5 has implicated the CT0872 gene product in thiosulfate oxidation. The identification of genes inactivated in strain C3 will provide important clues to the specific gene products required for elemental sulfur oxidation in C. tepidum.
P.094 THE STOMATIN, PROHIBITIN, FLOTILIN AND HFLK/C DOMAIN HOMOLOGUE SLR1768 IS REQUIRED FOR THYLAKOID BIOGENESIS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Samantha Bryan, Edward Spence, Conrad Mullineaux. Queen Mary, University of London, London, United Kingdom. Introduction: Nearly all cyanobacteria possess thylakoid membranes, the single known exception being the atypical cyanobacterium Gloeobacter, which houses all its photosynthetic complexes in the plasma membrane. Thylakoids are intricate internal membrane systems, where all the photosynthetic complexes that catalyse the light induced oxidation of water to molecular oxygen are contained. Biogenesis and regulation of the thylakoids is still an open problem, with only two known regulators, Vipp1 and Alb3 indentified to date. Methods: A slr1768 knockout was generated using the pGEM T-easy vector and REDIRECT technology. Whole cells were scanned for absorption in a UV 500 spectrophotometer. Photochemical efficiency was determined using 77K fluorescence emission spectra, and chlorophyll a fluorescence. Maximum photochemical efficiency was calculated as (FM`dark – F0)/ FM`dark. Both wild-type Synechocystis sp. PCC 6803 and ∆slr1768 strains were collected and fixed, ultrathin sections were then negative stained and viewed using a JEOL 1220 transmission electron microscope. Results: By comparing growth and the photosynthetic efficiency of the slr1768 mutant with the wild-type, we found that slr1768 has a conditional phenotype; specifically under highlight conditions (130 µmol m-2 s-1) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density. The chlorophyll a pigment content decreases with the loss of thylakoids, although the photosynthetic efficiency is unaffected.
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Final Program and Abstracts Conclusions: This is the first example of a gene that affects thylakoid biogenesis with a phenotype conditional on light intensity. Our results demonstrate that Slr768 has a leading role in acclimatisation, linking light damage with maintenance and biogenesis of the thylakoids.
P.095 LIGHT VARIABILITY ILLUMINATES NICHE-PARTITIONING AMONG MARINE PICOCYANOBACTERIA. Douglas Campbell, Zoe Finkel, Andrew Irwin, Christophe Six. Mount Allison University, Sackville, NB, Canada. Prochlorococcus and Synechococcus picocyanobacteria are dominant contributors to marine primary production over large areas of the ocean. Phytoplankton cells are entrained in the water column and are thus often exposed to rapid changes in irradiance within the upper mixed layer of the ocean. An upward fluctuation in irradiance can result in photosystem II photoinactivation exceeding counteracting repair rates through protein turnover, thereby leading to net photoinhibition of primary productivity, and potentially to cell death. We show that the effective cross-section for photosystem II photoinactivation is conserved across the picocyanobacteria, but that their photosystem II repair capacity and protein-specific photosystem II light capture are negatively correlated, and vary widely across the strains. The differences in repair rate correspond to the light and nutrient conditions that characterize the site of origin of the Prochlorococcus and Synechococcus isolates, and determine the upward fluctuation in irradiance they can tolerate, indicating that photoinhibition due to transient high-light exposure influences their distribution in the ocean.
P.096 CHROMATIC PHOTOACCLIMATION EXTENDS UTILISABLE PHOTOSYNTHETICALLY ACTIVE RADIATION IN THE CHLOROPHYLL D-CONTAINING CYANOBACTERIUM, ACARYOCHLORIS MARINA. Zane Duxbury, Martin Schliep, Raymond J. Ritchie, Anthony W. D. Larkum, Min Chen. School of Biological Sciences, University of Sydney, Australia. Chromatic photoacclimation and photosynthesis were examined in two strains of Acaryochloris marina (MBIC11017 and CCMEE5410) and in Synechococcus PCC7942. Acaryochloris contains Chl d, which has an absorption peak at ca 710 nm in vivo. Cultures were grown in one of three wavelengths (525 nm, 625 nm and 720 nm) of light from narrow-band photodiodes to determine the effects on pigment composition, growth rate and photosynthesis. No growth occurred in 525 nm light. Synechococcus did not grow in 720 nm light because Chl a does not absorb effectively at this long wavelength. Acaryochloris did grow in 720 nm light, although strain MBIC11017 showed a decrease in phycobilins over time. Synechococcus and Acaryochloris MBIC11017 showed a dramatic increase in phycobilins when grown in 625 nm light. The various photopigments were acclimated in response to the light spectral conditions. Photoacclimation and the Qy peak of Chl d can be understood in terms of the ecological niche of Acaryochloris, which grows in habitats enriched in near infra-red radiation.
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P.097 ROLE OF RUBISCO AND RUBISCO LIKE PROTEINS IN SULFUR METABOLISM IN RHODOSPIRULLUM RUBRUM. Swati Dey, Jaya Singh, F. Robert Tabita. Departments of Microbiology, Environmental Sciences Graduate Program and the Plant Cellular Molecular Biology, The Ohio State University, Columbus, Ohio, USA. Introduction: With the discovery of the type IV RubisCOs or the RubisCO- like proteins (RLP), a new family of proteins was discovered that was found to be structurally similar to the large subunit of RubisCO, though RLP was unable to catalyze CO2 fixation like RubisCO. RLPs from organisms like Bacillus subtilis, Geobacillus kaustophilus and Microcystis aeruginosa have been shown to be involved in an enolase reaction of a methionine sulfur salvage pathway. We have been studying the functional role of RLP in various organisms such as Chlorobium tepidum, Rhodopseudomonas palustris, and Rhodospriullum rubrum and its relation to RubisCO. It was shown that RLP from R. rubrum utilizes methylthioadenosine (MTA, an intermediate of known methionine salvage pathways) as sole sulfur source. The RLP from R. rubrum catalyzes a novel isomerization reaction distinct from that catalyzed by the B. subtilis-type RLP of the methionine salvage pathway. Interestingly R. rubrum RubisCO was also shown to weakly catalyze an enolase reaction similar to the RLP of B. subtilis. In the present study, we found that R. rubrum RubisCO is involved in the sulfur salvage pathways under anaerobic condition apart from its normal CO2 fixing ability. Methods: Using MTA as a sole source of sulfur, growth experiments were performed under both aerobic and anaerobic conditions to illustrate the function of RLP and the dual function of RubisCO in R. rubrum after inactivation of the RLP gene. Complementation studies were used to confirm these results. In addition, RubisCO activity assays and western immunoblots also confirmed the presence of RubisCO in mutant strains lacking RLP that were capable of simultaneously fixing CO2 and using MTA as a sole source of sulfur. Results: Studies show that R. rubrum strains with an inactivated RLP gene were able to grow with MTA as a sole source of sulfur under anaerobic conditions. R. rubrum RLP gene deletion strains were unable to grow under aerobic conditions. Complementation of RLP gene supported aerobic growth in these strains. Western blots showed the expression of RLP under aerobic conditions and RubisCO under anaerobic conditions in various mutant strains. Conclusion: These studies showed that under aerobic growth conditions RLP is involved in the salvage of sulfur in R. rubrum. A different and novel substrate is utilized by RLP under aerobic growth conditions when MTA is used as a sulfur source for growth. However, under anaerobic conditions, using MTA as sole sulfur source, it is not RLP, but RubisCO that catalyzes a B. subtilis type reaction using 2,3diketo-5-methylthiopentanyl -1- phosphate as substrate. In addition, this work illustrates the dual role of RubisCO as an enzyme required for CO2 fixation as well as sulfur salvage. Hence, the RubisCO family not only is involved in carbon metabolism but this enzyme may be used simultaneously to catalyze an important reaction of sulfur metabolism in vivo.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
P.098
P.099
CHARACTERIZATION OF BILIN LYASES FOR R-PHYCOCYANIN IN SYNECHOCOCCUS SP. WH8020
MOLECULAR COMMUNICATION BETWEEN CYANOBACTERIA AND THEIR HOST PLANTS: ROLE OF CHEMOTAXIS IN SYMBIOTIC COMPETENCY.
Tierna Dragomani, Avijit Biswas, Wendy M. Schluchter. Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA.
Paula Duggan, Dave Adams.
Introduction: Cyanobacteria utilize phycobilisomes composed primarily of phycobiliproteins to maximize capture of light energy. In marine Synechococcus, various strains exhibit distinctive phycobiliprotein compositions adapted to capitalize on the wavelengths of available light. Specific attachment of phycobilins endows each phycobiliprotein with characteristic capacities for light absorption and emission. Rarely, this attachment may be autocatalytic, but more often it is catalyzed by bilin lyases that attach phycobilins at conserved cysteine residues on the phycobiliprotein subunit. The E/F type bilin lyase was the first one characterized; each CpcE and CpcF subunit contains conserved “E-Z” motifs, and together, the heterodimer catalyzes bilin attachment at Cys-84 of the α-subunit of the phycobiliprotein, phycocyanin (PC). The rpcE and rpcF genes are similar to cpcE and cpcF, respectively and are located downstream of the rpcA gene in Synechococcus sp. WH8020. Recently, RpcG from Synechococcus sp. WH8102, a fusion of RpcE and RpcF, was shown to have phycoerythrobilin (PEB) lyase and isomerase activity (Blot et al., 2009; J. Biol. Chem. 284: 9290-9298). The purpose of this study is to test whether rpcE and rpcF in strain WH8020 encode an E/F heterodimeric lyase that catalyzes attachment of PEB to Cys-84 of α-Rphycocyanin (R-PC).
Introduction: Cyanobacteria, such as Nostoc punctiforme, are able to live in symbiotic association with a wide range of host plants. Prerequisites for successful infection of the host tissues include the formation of motile filaments known as hormogonia and the production of a plant-derived chemical(s) that attracts these filaments towards the sites of infection within the host plant. There is evidence of two-way communication between plants and their prokaryotic partners regulating the infection process and the events involved in the establishment of a functional dintrogen-fixing association. Moreover there is increasing evidence to suggest that chemotaxis-like signal transduction systems may be central to these processes. The genome of N. punctiforme contains five loci of genes encoding elements resembling chemotaxis genes of other bacteria. We have focused our attention on one locus, which appears to have been acquired from a horizontal transfer event and is unique amongst the other clusters in that it contains a putative CheR methyltransferase and CheB methylesterase, which are central to chemotactic adaptation in other bacterial systems and enable the bacterium to respond to new stimuli in a background containing constant levels of chemoattractants and/or chemorepellents.
Methods: In Synechococcus sp. WH8020, the cpe/ mpe operons encoding phycoerythrin and R-Phycocyanin genes have been sequenced (Wilbanks and Glazer, 1993; J. Biol. Chem. 268:12261235). Oligonucleotide primers were developed to amplify the rpcA gene (α-R-PC) from WH8020 chromosomal DNA, and this product was ligated into the pET Duet vector so that its sequence was fused to the reading frame encoding a Hexa-histidine tag. The rpcE and rpcF genes were cloned into the pCDF Duet vector (individually and together). A pACYC Duet plasmid contained genes (ho1, pebS) encoding enzymes known to convert heme to biliverdin and ultimately to PEB (generous gift of Dr. Frankenberg-Dinkel). Plasmids will be introduced into chemically competent E. coli strain BL21(DE3) and plated onto Luria-Brutani agar containing the appropriate antibiotics. Transformants will be cultured and protein production will be induced with IPTG. Each plasmid will be tested individually in cells for protein production to determine optimal growth temperature conditions for each set of proteins. Results: The rpcA plasmid construct resulted in the production of soluble His-tagged RpcA which was purified by metal affinity chromatography and verified by SDS-PAGE. We have successfully demonstrated that CpcE and CpcF can attach PEB to HT-RpcA. However, we are currently testing conditions for expression of soluble RpcE and RpcF. Results from these studies will be presented. In addition, we will test whether RpcE and RpcF form a heterodimer.
Faculty of Biological Sciences, Leeds, Yorkshire, United Kingdom.
Methods : Nostoc genes NpR0244 (cheB) and NpR0248 (cheR) were inactivated by insertional mutagenesis and recombinant strains were characterised in terms of chemotactic motility and their ability to infect the host bryophyte Blasia pusilla. Electron microscopy was used to examine the levels of cell surface piliation of hormogonia differentiated by the mutant strains compared with those of the wildtype. Results: Both the NpR0244 (cheB) and NpR0248 (cheR) mutants failed to infect the host plant even after protracted periods of co-culture with the host B. pusilla. Chemotaxis assays revealed that the mutant strains lack the wild-type positive chemotactic behaviour towards exudates released from nitrogen-starved B. pusilla tissue. Wild-type piliation phenotypes were observed with both the cheB-like NpR0244 and the cheR-like NpR0248 mutants implying that, unlike some other bacterial taxis systems, these genes are not involved in the biosynthesis of the pilus-like appendages present on the surface of hormogonia. Conclusions: Our data provide molecular evidence that the chemotaxis-like elements studied here function in the establishment of the Nostoc-Blasia symbiosis, most probably through the disruption of the normal chemotaxis responses that occur towards host-derived signals and that cyanobacteria, like other bacteria, also exhibit an adaptation response.
Conclusions: Soluble expression of RpcA was achieved, and it was a suitable substrate for bilin addition by another closely related bilin lyase CpcE/CpcF (from Synechocystis sp. PCC 6803). Assays with RpcA, RpcE and RpcF will be performed if soluble protein can be obtained.
August 9 to 14, 2009 • Montréal, QC, Canada
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P.100 RESPONSES OF CHLOROBACULUM TEPIDUM TO ELEVATED SULFIDE. Brian J. Eddie, Leong-Keat Chan and Thomas E. Hanson. College of Marine and Earth Studies and DBI, Newark, DE, USA. Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is a green sulfur bacterium (GSB) that has become a model system for phototrophic sulfur oxidation due to the relative ease of culture and genetic manipulation. While it preferentially uses sulfide as an electron donor, C. tepidum is inhibited by sulfide concentrations in excess of 8 mM, a much higher sulfide tolerance than originally reported for this strain. To understand mechanisms of sulfide tolerance, we seek to identify sulfide regulated genes and assess their role(s) in C. tepidum‘s physiology Methods: C. tepidum was grown either on thiosulfate as the sole electron donor or with varying concentrations of sulfide followed by a comparison of cellular properties and the proteome of cultures by 1D SDS-PAGE. In separate experiments, C. tepidum actively growing on thiosulfate as the sole electron donor was provided with varying doses of sulfide and the adaptation of the cultures to this shock was monitored by quantifying sulfur compound turnover, cellular properties and changes in the proteome. In addition, samples were taken for subsequent analysis by a modified version of the mRNA-Seq protocol normally applied to larger sized eukaryotic genomes. This protocol has been developed to allow inexpensive multiplexing of samples from prokaryotic genomes to scale the power of next generation sequencing techniques to smaller genome sizes. Results: C. tepidum cultures grown with varying sulfide concentrations showed a clear alteration in the major in vivo absorption peak of Bchl c and several polypeptides were significantly more abundant in high sulfide cultures. A high concentration sulfide shock (8 mM) resulted in complete growth inhibition, but it is not known whether this was due to sulfide poisoning or pH inhibition. Milder doses of neutralized sulfide (1.6mM) did not result in growth inhibition or cause significant alterations in the protein profile of the culture. Two independent samples from this experiment were analyzed by the mRNA-seq protocol and produced millions of sequences that could be aligned to the C. tepidum TLS genome. Analysis of the reproducibility of the modified mRNA-seq protocol and multiplexing technique will be presented as will cell property and protein data from additional sulfide shock experiments. Conclusions: This study is the first to investigate the response of C. tepidum TLS to sulfide and is one of the first to use mRNA-seq to investigate a prokaryotic transcriptome. Preliminary results indicate that the modified mRNA-seq protocol applied to C. tepidum identifies actively transcribed regions of the genome in a less annotation dependent fashion than can be achieved with current microarray technology. This included the identification of non-coding RNA’s, which are receiving increased attention as regulatory elements in prokaryotes.
P.101 IN VITRO ASSEMBLY OF THE CARBOXYSOMAL BICARBONATE DEHYDRATION COMPLEX. Charlotte De Araujo, Swan Cot, George Espie. University of Toronto, Mississauga, ON, Canada. Introduction: Cyanobacteria possess a carbon dioxide concentrating 128
mechanism (CCM) that greatly enhances photosynthetic efficiency. This adaptive mechanism minimally consists of multiple membrane transport systems for inorganic carbon and intracellular inclusions known as carboxysomes, where Rubisco is located. The structural and functional basis through which carboxysomes enhance CO2 fixation remains unclear. Based on yeast two-hybrid analysis Cot et al. (J. Bacteriol. 2008,190: 936) hypothesized that the proteins, CcaA, CcmM and CcmN interact to form a multiprotein bicarbonate dehydration complex (BDC) within the carboxysome which vectorially channels CO2 to Rubisco, thereby enhancing fixation. Methods: ccaA, ccmM and ccmN (slr1347, sll1031 & slr1032) from Synechocystis sp. PCC6803 were cloned and over-expressed using standard techniques. Carbonic anhydrase activity was determined by mass spectrometry. Results: Mass spectrometric assays revealed that neither recombinant CcmM nor CcmN possessed HCO3- dehydration activity alone. Overexpression of CcaA using the pET-15b E.coli expression system produced active protein capable of the catalytic interconversion of HCO3- and CO2. To examine the role of the BDC in the supply of CO2, attempts were made to reconstitute it in vitro using affinity purification techniques. We found that N-terminal 6xHis-tag-CcaA retained catalytic activity when bound alone to an affinity matrix. In contrast, a complex of matrix-bound T7-tag-CcmM + 6x-His-CcaA was catalytically inactive as was the T7-tag-CcmM + flag-CcmN + 6x-HisCcaA. Western blot analysis verified the presence of all 3 proteins within the affinity matrix. However, matrix-bound 6x His-CcaA + T7CcmM complexes and His-CcaA + T7-CcmM + flag-CcmN exhibited CA activity, suggesting that the orientation of CcaA was an important determinant in maintaining catalytic activity. In buffered solution at pH 8, CcaA activity was reduced by 20 % in the presence of CcmM, but not BSA. Conclusions: Initial results show that CcmM-CcmN-CcaA complexes are formed by affinity tag binding to a matrix and subsequent protein/protein interactions. The orientations of the proteins in relationship to one another are critical in maintaining CA activity. When the CcaA active site at the N-terminus of the protein is exposed to CcmM, activity was eliminated, suggesting that the binding of CcmM modulates CcaA activity. Potentially, small differences in the nature of the CcmM – CcaA interaction within carboxysomes may play a role in controlling the supply of HCO3- to CcaA and in regulating CcaA dehydration activity.
P.102 THE CYANOBACTERIAL RIBOSOMAL-ASSOCIATED PROTEIN LrtA MODULATES TRANSLATION IN SYNECHOCYSTIS SP. PCC 6803. Carla V. Galmozzi, Francisco J. Florencio Bellido, M. Isabel Muro Pastor. Instituto De Bioquímica Vegetal Y Fotosíntesis. Universidad De SevillaCsic, Seville, Seville, Spain. LrtA is a protein encoded by a light-repressed transcript in cyanobacteria. The gene coding for LrtA was identified in Synechococcus sp. PCC 7002 and showed expression dependent on darkness (Samartzidou y Widger, 1998; Tan et al., 1994). LrtA is related to a family of proteins present in several bacteria. In Escherichia coli two proteins belonging to this family, termed YfiA and YhbH, have been shown to be associated with the 30S ribosome subunit (Agafonov et al., 1999; Maki et al., 2000). YfiA has also been shown to inhibit translation in vitro (Agafonov et al., 2001) suggesting a role of this family of proteins in translation control related to stress adaptation. However, in vivo data that demonstrate a function of these August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes proteins in translation have not yet been reported. The lrtA gene product also displays sequence similarity to a spinach plastid-specific ribosomal protein (PSRP-1), which is present in the chloroplast stroma either unbound or associated with the 30S ribosomal subunit (Yamaguchi et al., 2000). Genes homologous to lrtA are present in all the cyanobacterial sequenced genomes, but their function remains unknown. The analysis of the Synechocystis sp. PCC 6803 genome showed the existence of an open reading frame (sll0947), the product of which displayed strong similarity to the previously identified Synechococcus sp. LrtA protein. In Synechocystis it has been shown that the amount of SigB sigma factor increased in darkness and contributes significantly to the up-regulation of lrtA in these conditions (Imamura et al., 2003). In addition, proteomic analysis of salt-stress proteins in the plasma membrane of Synechocystis resulted in the identification of LrtA protein induced under these conditions (Huang et al., 2006). We analyzed the transcript levels of the Synechocystis lrtA gene and the LrtA protein. In agreement with the Synechococcus homologous gene, the level of the Synechocystis lrtA transcript is up-regulated under dark conditions. However, the level of LrtA protein remains constant upon light-dark transitions. Using extracts from Synechocystis fractionated by sucrose gradient we show that LrtA is a ribosome associated protein present in 30S and 70S ribosomal particles, suggesting a role of this protein in translation control. We have constructed an lrtA-deleted Synechocystis strain, which was shown to be viable in laboratory conditions, indicating that lrtA is a dispensable gene in this cyanobacterium. Furthermore, lrtA mutants present decreased sensitivity to tylosin and erythromycin, two inhibitors of protein synthesis, again connecting the role of LrtA with translation. In addition, we analyzed in vivo incorporation of [35S]methionine into proteins in both wild-type and ∆lrtA strains. We found that at least two proteins were diferentially synthesized in WT versus ∆lrtA cells. We will carry out proteomic analysis comparing wild-type and ∆lrtA strains, in different culture conditions or stresses, to identify changes provoked by the absence of the LrtA protein. We expect to identify proteins whose synthesis is in some way regulated by LrtA. These results may allow us to establish a role of this protein in translational adaptation. References: Agafonov, D.E., Kolb, V.A., Nazimov, I.V., y Spirin, A.S. (1999) A protein residing at the subunit interface of the bacterial ribosome. Proc Natl Acad Sci U S A 96: 12345-12349. Agafonov, D.E., Kolb, V.A., y Spirin, A.S. (2001) Ribosomeassociated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep 2: 399-402. Huang, F., Fulda, S., Hagemann, M., y Norling, B. (2006) Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803. Proteomics 6: 910-920. Imamura, S., Asayama, M., Takahashi, H., Tanaka, K., Takahashi, H., y Shirai, M. (2003) Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria. FEBS Lett 554: 357-362. Maki, Y., Yoshida, H., y Wada, A. (2000) Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli. Genes Cells 5: 965-974. Samartzidou, H., y Widger, W.R. (1998) Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002. Plant Physiol 117: 225-234. Tan, X., Varughese, M., y Widger, W.R. (1994) A light-repressed transcript found in Synechococcus PCC 7002 is similar to a chloroplast-specific small subunit ribosomal protein and to a August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts transcription modulator protein associated with sigma 54. J Biol Chem 269: 20905-20912. Yamaguchi, K., von Knoblauch, K., y Subramanian, A.R. (2000) The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast). J Biol Chem 275: 28455-28465.
P.103 INVESTIGATING THE MOLECULAR BASIS FOR CYANOBACTERIAL EXCAVATION IN A BORING CYANOBACTERIUM (MASTIGOCOLEUS/FISCHERELLA). Q. Gao, E. Ramirez-Reinat, F. Garcia-Pichel. School of Life Sciences, Arizona State University, Tempe, AZ, USA. Introduction: Microorganisms are the most common, widespread and environmentally significant among carbonate borers. Boring cyanobacteria, as photosynthetic microbes, have been a part of the geological reworking of carbonates since at least 1500 Myr ago. Their action has played important roles in geological cycles, and they are important to the fisheries industry, as they can be a pest of mullusks. Despite this, the boring mechanism of any of these cyanobacteria remains unknown. Concurrent physiological experiments in our laboratory have recently shown that active pumping of calcium ions (through cellular uptake, trans-cellular transport and extrusion processes) is at the base of the boring ability, likely involving P-type ATPases. To rigorously identify the putative cellular mechanism for this unique phenomenon, we attempted to find genes encoding for calcium-transporting P-type ATPases in a model cyanobacterium (Mastigocoleus/ Fischerella), so that expression analyses could be carried out. Our study provides the first glance at the molecular mechanism for microbial excavation. Methods: The genes sequences for calcium related P-type ATPases from a variety of cyanobacteria were retrieved from the NCBI database and Cyanobase. 40 such putative calcium-related P-type ATPase genes were identified as having both a calcium binding motif and an ATP-binding motif, and classified into two groups according to independently obtained consensus phylogeny of the cyanobacteria from which the genes were obtained. Group-specific sequence alignments were used to design degenerate primers matching conserved sequence regions corresponding to the phosphorylating and ATP-binding motifs. These primers were used to clone calciumtransporting P-type ATPases from Fischerella/Mastigocoleus genome through PCR amplification of genomic DNA. The Purified PCR products were cloned into pCR®4 vector through invitrogen TOPO TA Cloning system. Six individual clones were sequenced to verify each PCR product. The corresponding, deduced protein sequences were compared with known proteins in the databases by available blast methods. Results: Using genomic DNA from two known calcium P-type ATPasecontaining strains, Synechocystis sp. PCC 6803 and Nostoc punctiforme, we could show that our primers amplify P-type ATPases from cyanobacteria, and were specific for P-type ATPase genes, since they yielded a single product of the predicted size. Using genomic DNA of boring Mastigocoleus/Fischerella as template, each primer pair yielded product of the expected size, about 155 bp and 1098 bp, resepctively. Cloning and sequencing of the two products, proved that they belonged to two distinct P-type ATPase genes, one resembling most that of Nostoc punctiforme and another one most closey related to one from Synechocystis sp. The sequence analysis of the two PCR products indicates there exist at least two putative calcium-binding P-type ATPases in Mastigocladus/Fischerella genome. 129
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Final Program and Abstracts Conclusions: Our experiments confirmed the feasibility of the genomic scanning by PCR method in the detection of calcium related P-type ATPases genes. This result, more importantly, provides molecular evidence for the existence of multiple putative calcium transporter genes in Fischerella, which could support our proposed mechanism for boring activity. Although this experiment is a first step in our exploration, cloning of calcium P-type ATPase paves the way for the further investigation of the molecular mechanism of cyanobacterial excavation. Ongoing studies attempt to address the expression of each of these genes under boring vs. free-living conditions, and the localization of this activity in these morphologically complex cyanobacteria.
P.104 THE GLOBAL RESPONSE TO UVA STRESS IN NOSTOC PUNCTIFORME ATCC 29133 ASSESSED BY A TEMPORAL DNA MICROARRAY STUDY. F. Garcia-Pichel1, Q. Gao1, J. C. Meeks2, V. Stout1, T. Soule1,3. 1
School of Life Sciences, Arizona State University, Tempe, AZ; 2Section of Microbiology, University of California, Davis, CA; 3Environmental Biotechnology, Savannah River National Laboratory, Aiken, SC; USA. Introduction: UVA radiation (320-400 nm) is an important component of the solar spectrum reaching the ground. Unlike short-wavelength ultraviolet, UVA does not directly damage biological materials. Its harmful effects come about through photosensitizers, molecules that create reactive compounds upon photons absorption and in the presence of oxygen. Cyanobacteria, because they contain high level of photosensitizing pigments, and are exposed to internally generated oxygen, are particularly prone to UVA-mediated damage. Despite this, gene expression studies have focused in the response to UVA have not been presented. To identify some of the cellular mechanisms used to cope with this part of solar radiation, we examined the temporal global stress response to an up-shift in UVA the cyanobacterium Nostoc punctiforme ATCC 29133 as a model organism using whole genome DNA microarrays. Our study provides the first global UVA stress response analysis for any phototroph. Methods: Nostoc cultures grown under white light were split into 2 series, one kept under white light, and the second exposed to supplemental UVA radiation, over the course of four days. At 24 hr intervals, triplicate cultures were harvested from each treatment. Total RNA was extracted, reverse-transcribed, and labeled with fluorescent dyes. Labeled cDNAs were hybridized to microarray slides and raw images were processed for analysis. Genes with at least a statistically significant 2-fold differential expression level between the UVAtreated and untreated samples were considered responsive to UVA and were used to construct gene lists for analysis. Results: The study detected statistically robust signals for differential regulation in response to UVA in 573 out of the 6903 genes probed on the array. Most of the up-regulated genes, 299, were uncategorized, and 8% (82) of all of the genes known to be dedicated to adaptive metabolism were upregulated. The most common category of downregulated genes was those associated with light-harvesting pigment biosynthesis, likely to minimize photosensitizer density. Among the expected responses, we could detect the up-regulation of several antioxidant enzymes, likely to help the cells cope with reactive oxygen species, and the up-regulation of genes for the biosynthetic pathway of the UVA sunscreen, scytonemin. Most intriguingly, we found that by far, the strongest and most sustained up-regulation involved a group of contiguous genes of unassigned metabolism, but widely distributed among cyanobacteria, that on plasmid A. 130
Conclusions: Our experiments confirmed the importance of several well known adaptive strategies to cope with UV: decrease in photosensitizers, increase in antioxidant systems, and synthesis of UVA sunscreens. It also revealed that we know virtually nothing about the vast majority of genes involved in this response. Overall, our results suggest that UVA plays an important role in the physiology of Nostoc, but also that the UVA cyanobacterial stress response is obviously far from being well understood. Some of the genes identified here provide new avenues for exploration.
P.105 THE BRANCHPOINT FOR BACTERIOPHEOPHYTIN BIOSYNTHESIS IN RHODOSPIRILLUM RUBRUM. Robin Ghosh1, Hartmut Grammel2, Jörg Hammel1, Rudolf Saegesser1, and Khaled Abou-Aisha1. 1 Dept. of Bioenergetics, University of Stuttgart, Pfaffenwaldring 57, D70550 Stuttgart, Germany, 2Max-Planck Institute for the Dynamics of Complex Technical Systems, Magdeburg, Germany.
Introduction: The pathway for bacteriochlorophyll a (BChla) biosynthesis has now been well-characterized in purple photosynthetic bacteria, particularly in the species Rhodobacter capsulatus and Rhodobacter sphaeroides. By contrast, the biosynthesis of bacteriopheophytin a (BPha) appears to have been largely ignored. In R. rubrum, the chemical structure of BPha is useful for tracing the pathway of biosynthesis, as BChla contains a geranyl-geranyl chain, wheras BPha contains a phytyl chain. In this study, by a combination of organic separation and spectroscopy we have deduced the probably branch point of BPha biosynthesis from the main BChla pathway. Methods and Results: A number of photosynthetically incompetent mutants in genes for pigment synthesis in R. rubrum were generated by both random as well as Tn5 mutagenesis. The lesions were identified by both Southern hybridization as well as complementation analysis. Cells were grown semi-aerobically in the dark using a unique medium (M2SF) which allows up-regulation of photosynthetic genes to levels normally only observed under low light photoheterotrophic conditions. Growth in this medium causes R. rubrum to produce very large quantities of pigments, thereby simplifying subsequent chemical analysis. The pigments were separated in organic solvents and analyzed by absorption, fluorescence and mass spectroscopy. This analysis allowed us to deduce the probable locus of the BPha shunt. Conclusions: The removal of Mg2+ from the BChla-precursor occurs at an unexpected locus.
P.106 HGDD, A TOLC-LIKE OMF INVOLVED IN HETEROCYST DEVELOPMENT. Alexander Hahn, Peter Staron, Iris Maldener, Enrico Schleiff. Goethe Universitat, Cluster of Excellence, Center of Membrane Proteomics, Frankfurt, Hessen, Germany. Protein secretion of the type I system and multi-drug efflux in Gramnegative bacteria are based on a tripartite secretion complex. This complex usually contains an inner membrane ABC-transporter or a proton driven efflux pump, a membrane fusion protein (MFP) and an outer membrane factor (OMF) of the TolC-family. Together they form a single channel tunnel to secret proteins and toxic substances from the cytoplasm or the inter membrane space. The cyanobacterium Anabaena sp. PCC 7120 has only one TolC–like protein. We identified its expression in all cells of the filament, but it was found to be August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes indispensable only for heterocyst development. The phenotype of the deletion strain parallels that of the knock out of the devBCA operon encoding for an ABC-transporter. Hence, it is concluded that DevBCA together with the identified TolC-like protein is involved in the assembly of the heterocyst specific glycolipid-layer (HGL-layer) and is therefore termed HgdD. As HgdD is the only TolC present in the outer membrane of Anabaena sp. PCC 7120 an interaction with the multitude of inner membrane located ABC-transporter is expected, a hypothesis which is currently investigated.
P.107 CYANOBACTERIOCHROME CCAS REGULATES PHYCOERYTHRIN ACCUMULATION IN NOSTOC PUNCTIFORME THAT PERFORMS GROUP II CHROMATIC ADAPTATION. Yuu Hirose1, Mitsunori Katayama2, Rei Narikawa3, Masahiko Ikeuchi1,3. 1
Department of Biological Science, Graduate School of Sciences, the University of Tokyo; 2College of Industrial Technology, Nihon University; 3Department of Life Sciences (Biology), the University of Tokyo; Japan. Introduction: Cyanobacteriochromes are unique phytochrome-related photoreceptors that undergoes reversible conversion between violet/yellow, blue/green or green/red absorbing forms with photoisomerization of a covalently bound linear tetrapyrrole (Ikeuchi and Ishizuka, 2008). Cyanobacteria utilize phycobilisome (PBS) as light harvesting antenna. Typical light harvesting proteins of PBS are phycoerythrin (PE) and phycocyanin (PC), which absorb green light (GL) and red light (RL), respectively. Certain cyanobacterial species are able to raise PE/PC ratio under GL and reduce under RL, which has been called “complementary chromatic adaptation”. Some acclimate only PE accumulation (group II) while the others acclimate both PE and PC accumulation (group III). In Fremyella diplosiphon that performs group III chromatic adaptation, a cyanobacteriochrome gene, rcaE, is suggested to induce the expression of PC genes under RL, although another GL receptor was implicated to regulate the expression of PE genes (Kehoe and Gutu, 2006). Our recent studies suggested that another cyanobacteriochrome CcaS regulates the expression of PBS linker gene, cpcG2, in Synechocystis sp. PCC 6803 that possesses PC but not PE. CcaS shows reversible photoconversion between GL and RL-absorbing forms and GL-activated phosphorylation to the cognate response regulator CcaR (Hirose et al, 2008). Interestingly, CcaS and CcaR orthologs are clustered with PE genes (cpeC, cpcG2 and cpeR) in the genome of Nostoc punctiforme ATCC 29133 that performs group II chromatic adaptation. In this study, we disrupted ccaS, ccaR and the clustered PE genes in N. punctiforme and examined their response to GL and RL. Material and methods: N. punctiforme cells were grown in liquid BG11 medium under 20 μE m-2s-1 of GL (peaked at 530 nm) or RL (650 nm). Absorption spectra of fully acclimated cells were measured by spectrophotometer with an end-on photomultiplier. To create ccaS, ccaR and PE gene mutants, each gene was replaced with a neomycin cassette from pSCR9 as described (Cohen and Meeks, 1997). RNA was extracted by acidic phenol. Northern blotting analysis was performed according to standard protocol using radio-labeled probes. Results: We found that PE was hardly detected in wild type cells grown under RL, while PE content was high in cells grown under GL. In a ∆ccaS mutant, some amounts of PE accumulated under both GL and RL, suggesting that ccaS is essential not only for PE accumulation under GL but also for repression under RL. In a ∆ccaR mutant, PE hardly accumulated under both GL and RL, suggesting that CcaR is transcriptional activator for PE accumulation. Northern blotting August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts analysis indicated that the clustered PE genes, cpeC, cpcG2, and cpeR, are co-transcribed under GL but not RL in wild type. ∆cpeC/∆cpeG2/∆cpeR triple mutant was also defective in PE accumulation under both GL and RL, suggesting that the induction of these genes are essential for PE accumulation. From these results, we conclude that CcaS regulates PE accumulation by the transcriptional regulation of cpeC, cpcG2, and cpeR, via GL-activated phosphorylation and probably RL-activated dephosphorylation of CcaR. Conclusion: In complementary chromatic adaptation, the green light receptor for PE accumulation has not yet been identified. In this study, we found that cyanobacteriochrome CcaS and response regulator CcaR regulates PE accumulation via the transcriptional regulation of the clustered PE genes in N. punctiforme.
P.108 CIRCADIAN TRANSCRIPTIONAL REGULATION IN THE DARK WITHOUT CYCLIC KAI GENE EXPRESSION IN SYNECHOCOCCUS. N. Hosokawa1, T. Hatakeyama1, H. Iwasaki1,2. 1 Department of Electrical Engineering and Bioscience, Waseda University; 2PRESTO, Japan Science and Technology Agency, Wakamatsu, Shinjuku, Tokyo; Japan.
Most of organisms exhibit daily cycles, driven by endogenous circadian clocks. The unicellular cyanobacterium, Synechococcus elongatus PCC7942 is an obligate photoautotroph and known as the simplest model organism for circadian biology. In S.elongatus, most of genes exhibit circadian expression rhythms, which are regulated by three clock genes, kaiA, kaiB and kaiC, under continuous light (LL) conditions. Interestingly, when cells are transferred to continuous dark (DD) conditions from hour 12 in the light, kaiA and kaiBC genes are rapidly downregulated to the zero level, while the KaiC phosphorylation cycle persists in the dark even in the presence of excess transcription/translation inhibitors. Thus, the basic oscillation is generated via post-translational process (Tomita et al., 2005). When we performed DNA microarray analysis, expression of most of genes on the genome was also dramatically and rapidly suppressed in the dark, whereas a minor subset of genes was upregulated. These dark-induced genes required de novo transcription under DD. Then, we examined if such gene expression profiles under DD were affected by the Kai-based clock even in the absence of de novo kai-gene expression. We found the magnitude of dark-induction in some genes was dependent on time when the cells were transferred from light to DD. Moreover, expression profiles of such genes in DD were dramatically altered in the kaiABC-null mutant strain. Thus, in contrast to our previous model, the Synechococcus clock regulates transcriptional outputs even in the dark.
P.109 CHARACTERIZATION OF UNIQUE PHOTOCHEMICAL PROPERTIES OF CYANOBACTERIOCHROME TePixJ. T. Ishizuka1, A. Kamiya2, H. Suzuki3, T. Noguchi3, T. Kohchi4, K. Inomata2, M. Ikeuchi1. 1
Department of Life Sciences (Biology), The university of Tokyo; Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University; 3Institute of Materials Science, University of Tsukuba; 4Graduate school of Biostudies, Kyoto University; Japan. 2
Introduction:Cyanobacteria harbor many putative GAF-containing 131
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Final Program and Abstracts photoreceptors that may bind a linear tetrapyrrole as a chromophore (cyanobacteriochromes) in addition to typical phytochromes. One of them, TePixJ of Thermosynechococcus elongatus BP-1 is essential for phototaxis. Previously, we reported novel properties of the GAF domain of TePixJ (denoted TePixJ_GAF) that was expressed in Synechocystis PCC 6803. Purified TePixJ_GAF showed reversible photoconversion between the 433nm (Pb form) and 531nm-absorbing forms (Pg form). In contrast with Pr and Pfr forms of Synechocystis phytochrome Cph1 carrying phycocyanobilin (PCB), Pb and Pg of TePixJ_GAF are extremely blue-shifted (approximately 150~200nm). Here, we studied the unique photochemical properties of TePixJ. Methods: in vitro reconstitution: synthetic PCB was added to a reconstitution buffer containing TePixJ_GAF apoprotein prepared from E. coli. The mixture was incubated in the dark at 50ºC, which is close to the optimum growth temperature of T. elongatus. in vivo coexpression: TePixJ_GAF apoprotein and two enzymes (Ho1, PcyA), which synthesize PCB from heme, were co-expressed in E. coli cells to allow in vivo assembly of the holoprotein. Denaturation analysis: The Pb*, Pb, Pg* and Pg forms of TePixJ_GAF and Pr and Pfr of Cph1 were denatured with 8 M urea/HCl pH 2.0 at room temperature in the dark. Native holo-TePixJ_GAF (~20 mg/ml) was subjected to FTIR analysis. Results: Previously, we reported that molecular mass of the chromophore of TePixJ was identical to PCB but was different in spectral properties. Moreover, we carefully compared TePixJ with Cph1 after denaturation with acidic urea. The spectral properties of TePixJ chromophore were clearly different from those of Cph1 PCB, but were very similar to those of phycoviolobilin (PVB), an isomer of PCB. In the early phase of in vitro reconstitution, free PCB was covalently incorporated with concomitant accumulation of photoconvertible holoprotein between Pb-like blue-absorbing form (Pb* form) and Pg-like green-absorbing form (Pg* form). Further incubation at 50ºC resulted in conversion to spectral properties similar to the native TePixJ. Concomitantly, PCB was isomerized to PVB as revealed by denaturation analysis. The holocomplex prepared from in vivo co-expression included both PCB and PVB as a chromophore. When this holocomplex was further incubated, additional isomerization from PCB to PVB was detected. There is conserved cysteine residue (Cys494: TePixJ numbering) among blue-green photoreversible cyanobacteriochromes. Mutation of Cys494 resulted in assembly of a red-absorbing form which did not exhibit photoconversion. Moreover, FTIR spectroscopy of the native TePixJ_GAF revealed green light-induced crosslinking of a free SH group. These results suggest that the light-induced crosslinking of a cycteine residue (probably Cys494) to the PVB takes place in the assembly of Pb that is extremely blue-shifted compared with the typical phytochrome. Conclusions: As a first step, TePixJ apoprotein and PCB form an intermediary photoactive Pb*. Secondly, subsequent isomerization from PCB to PVB proceeds during in vitro reconstitution. The extreme blue shift of TePixJ can be explained by reversible adduct formation between the chromophore and Cys494. Thus, we can conclude that the GAF domain of TePixJ is sufficient for both lyase, isomerase and photoconversion activities.
P.110 CELL LINEAGE ANALYSIS ON HETEROCYST PATTERN FORMATION IN ANABAENA SP. PCC 7120. S. Iwamori1, H. Asai1, K. Kawai1, S. Shoji1, H. Iwasaki1,2. 1
Department of Electrical Engineering and Bioscience, Waseda University, Japan; 2PRESTO, Japan Science and Technology Agency, 132
Wakamatsu, Shinjuku, Tokyo; Japan. hetR and patS genes have been reported as an activator and a repressor of heterocyst formation, respectively, in the filamentous cyanobacterium, Anabaena sp. PCC 7120. It is proposed that hetR activates its own transcription and patS gene expression, while the PatS peptide inhibits the HetR function. This type of combination of the negative and positive feedback loops with a possible diffusible inhibitor is reminiscent of the Turing instability dynamics, while its possibility has not yet been well validated experimentally. For better understanding of spatio-temporal dynamics underlying the heterocyst patterning, we have developed a monitoring/culturing system that enabled us to observe morphological changes, hetR expression profile, and chlorophyll fluorescence from individual filaments during the course of heterocyst development. Moreover, micro-liquid chamber arrays have been developed to analyze multiple Anabaena filaments simultaneously. Cell lineage analyses demonstrated that initial distributions of hetR::gfp signals and chlorophyll activity signals at nitrogen-step-down were not correlated to the resulting distribution of developed heterocysts, supporting a random and non-deterministic selection of initial heterocyst positions. We also observed cells that differentiated into heterocysts without cell division after nitrogen-stepdown, suggesting that cell division of mother cells is not an essential requirement for heterocyst differentiation. We will also report that the hetR expression profile exhibits a complicated, transient and possibly competing dynamics even before determination of initial heterocyst positions.
P.111 THE PHYSIOLOGICAL ROLE OF THE CBBRRS TWO – COMPONENT SYSTEM IN RHODOPSEUDOMONAS PALUSTRIS. Gauri. S. Joshi, F. Robert Tabita. Department of Microbiology, The Ohio State University, Columbus, OH, USA. Introduction: Rhodopseudomonas palustris is a metabolically versatile nonsulfur purple photosynthetic bacterium. The unique feature of the R. palustris cbbI CO2 fixation regulon is the presence of a twocomponent system (referred to as the CbbbRRS system) between the master transcriptional regulator CbbR and genes encoding form I RubisCO (cbbLS). CbbR belongs to the LysR family of regulators. The CbbRRS system is an atypical two-component system consisting of a sensor kinase and two response regulators with no apparent DNA binding domains on any of these proteins. Recent physiological studies have shown that the CbbRRS system plays a regulatory role in modulating the expression and activity of form I RubisCO only during photoautotrophic (CO2) growth and not during photoheterotrophic (benzoate) growth. The distinctive presence of this two component system and its role in modulating form I RubisCO expression is unique to R. palustris as similar systems have not been described in closely related NSP bacteria, Rhodobacter spaheroides, Rhodobacter capsulatus, and Rhodospirillum rubrum. The observations stemming from the physiological studies with photoautotrophically grown cells led to the hypothesis that the CbbRRS proteins (especially the response regulators CbbRR1 and CbbRR2) influence the interactions of CbbR at the cbbI promoter and thereby influence form I RubisCO expression. Methods: A bacterial two hybrid analysis (qualitative and quantitative) was performed to test whether the response regulators (CbbRR1 and CbbRR2) interact with CbbR in vivo. In this Escherichia.coli-based system, interaction between a pair of proteins results in transcriptional activation of a selectable (His3- aadA)/quantitative (lacZ) reporter gene August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes by recruitment of RNA polymerase at the promoter. The strength of the interaction between the protein pair determines the magnitude of transcriptional activation. In vitro gel shift assays and chemical crosslinking are additional approaches adopted to demonstrate the DNA – protein and protein – protein interactions. Results: The bacterial two hybrid system enabled the identification of a protein – protein interaction between the transcriptional regulator CbbR and CbbRR1, response regulator 1 of the CbbRRS system. Site directed mutagenesis of the phosphoacceptor residues of CbbRR1 (D54N and H171D) did not affect its interaction with CbbR significantly suggesting that phosphorylation of CbbRR1 may not be important for the interaction. Gel mobility shift analyses revealed that the affinity of CbbR for the cbbI promoter was specifically enhanced in the presence of CbbRR1, while the presence of CbbRR2 in the complex decreased the mobility of the DNA – protein complex. Chemical crosslinking of CbbR with the response regulators CbbRR1 and CbbRR2 is underway to further support the in vivo protein – protein interaction data. Conclusions: The results of the bacterial two hybrid analysis indicate that the CbbRRS two component system potentially exerts its regulatory effect on the cbbI promoter (form I RubisCO expression) by interacting with the master transcriptional regulator CbbR. The binding of CbbR to the cbbI promoter is stabilized in the presence of CbbRR1 as observed in gel shift assays, further strengthening the in vivo data. The interactions of the response regulators with CbbR represent additional transcriptional control beyond that provided by CbbR alone, for fine tuning the expression of form I RubisCO in R. palustris.
P.112 PHOTOTROPISM OF CYANOBACTERIA. Mitsunori Katayama1, Mari Kobayashi2, Masahiko Ikeuchi3. 1
Department of Liberal Arts and Basic Sciences, Nihon University; 2 Tokyo Institute of Technology, School and Graduate School of 3 Bioscience and Biotechnology; Department of Life Sciences (Biology), The University of Tokyo; Japan. Introduction. Phototropism is directional growth in response to directional light, which is widely observed in eukaryotes such as fungi, algae, and higher plants. In eukaryotes, blue light commonly induces phototropic response. On the contrary, phototropism was barely reported for prokaryotes. We recently found out phototropic response in filamentous cyanobacterium Rivularia sp. IAM M-261 (Rivularia M261). To characterize phototropism in cyanobacteria, its spectral dependence and the distribution among cyanobacteria were investigated. Methods. Microscopic examination of filamentous cyanobacteria collected from environmental samples and culture collections was made to survey species exhibiting phototropsm. Spectral dependence of the phototropic response was examined in the three Calothrix species and in the one Fischerella species by irradiation with series of monochromatic light (400 nm to 750 nm) provided by light emitting diode. Phototropic response was quantified by measurement of average angles of curvature of multicellular filaments. Results. Phototropism was observed in some species that belong to Calothrix, Tolypothrix, Scytonema and Fischerella. Rivularia M-261 was thought to be classified into Calothrix based on the 16S rDNA sequence. As for spectral dependence of the phototropic response, Rivularia M-261, Calothrix sp. PCC 7715 and Calothrix sp. PCC 7102 exhibited positive phototropic response to blue light (460 nm). On the other hand, one Fischerella sp. exhibited positive phototropic
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Final Program and Abstracts response to blue light (460 nm) and far-red light (750 nm). Conclusions. Cyanobacteria belongs to diverse group (subsection IV and subsection V) were shown to exhibit phototropic response. Blue light commonly induced phototropic response in Calothrix and Fischerella likewise eukaryotes. In addition to this, one Fischerella species exhibited phototropic response to far-red light. Most of species exhibiting phototropism grow on surface of soil or rock suggesting that phototropism is advantageous for terrestrial growth in cyanobacteria.
P.113 EFFECTS OF GROWTH CONDITIONS AND THE COMPOSITION OF GAS PHASE ON PHOTOBIOLOGICAL HYDROGEN ACCUMULATION IN THE HYDROGENASE MUTANT OF NOSTOC SP. PCC 7422. Masaharu Kitashima, Hajime Masukawa, Hidehiro Sakurai, Kazuhito Inoue. Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, Japan. Introduction: We are aiming at developing nitrogenase-based photobiological hydrogen production utilizing cyanobacteria with water as the terminal electron donor. In the previous report (Yoshino et al., 2007), we reported that the uptake hydrogenase gene disrupted (∆hup) mutant of Nostoc sp. PCC 7422 had high hydrogen production activity and accumulated hydrogen to about 30% (v/v) under a starting gas phase of Ar + 5% CO2. In order to attain a higher level of accumulation of hydrogen concentration, we have studied the effects of nitrogen and CO2 gas concentration of the first (heterocyst induction) and the second (hydrogen production period) gas phase on hydrogen accumulation. Methods: The culture media were either BG11 or BG110 (NaNO3 omitted) medium. The starting culture of the ∆hup mutant was grown photoautotrophically at 26°C by bubbling air under the 12 hour light12 hour dark cycle. In the light period, cultures were illuminated by fluorescent light at photosynthetically active radiation (PAR) of about 100 µmol photons m-2 s-1. The cells in the exponential growth phase were washed with and suspended in BG110 medium at a chlorophyll α concentration of about 3 µg ml-1, and 15 ml samples were transferred into 25-ml Fernbach flasks equipped with butyl rubber stoppers. Gas composition of the first phase was Ar + 5% CO2 + varied concentration of N2 (1 - 80%). After photoautotrophically cultivating for 2 to 4 days, the gas phase was changed to Ar + 5% CO2 + varied concentration of N2 (0.5 - 80%) for the second phase. For determination of accumulated hydrogen and oxygen, 50 µl each of gas sample was withdrawn from flasks at about the middle of the light phase and analyzed by gas chromatography (Molecular Sieve 13X column). Nitrogenase activity was assayed by reduction of acetylene to ethylene. Results: When the duration of in the first phase was two days, the nitrogen concentration at 1-20% did not greatly affect the hydrogen accumulation in the second phase, but 80% nitrogen significantly decreased the accumulation. With the first phase of three days, the hydrogen accumulation by 5% nitrogen culture was lower than that by 1% nitrogen culture. After two days under 1% nitrogen, the nitrogen concentration was changed (the second phase). As for the hydrogen accumulation of the culture (0-13 days), 0.5% nitrogen gave slightly higher concentration than 1%. At 5% and 20% nitrogen, the accumulation was much lower than the formers. After about 8 days under 0.5% nitrogen, the hydrogen concentration became about 50% (v/v) with oxygen concentration of about 20-25%. The profiles of 133
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Final Program and Abstracts nitrogenase activity could largely account for the hydrogen accumulation profiles. These results indicate that a large part of hydrogen was produced with water as the terminal electron donor under the experimental conditions used. Conclusions: By optimizing the gas composition of the culture, the genetically improved mutant strain of cyanobacterium cells are able to accumulate hydrogen to about 50% catalyzed by nitrogenase reaction, mostly using water as the terminal electron donor. Reference: F. Yoshino, H. Ikeda, H. Masukawa, H. Sakurai, High photobiological hydrogen production activity of a Nostoc sp. PCC 7422 uptake hydrogenase-deficient mutant with high nitrogenase activity, Mar. Biotechnol., 9: 101-112 (2007)
metabolism. Our results will be supplemented with and tested against high-quality data, aiming to iteratively improve and refine the current annotation. Our stoichiometric model is intended to serve as a first step towards a comprehensive computational description of cellular metabolism in unicellular autotrophs and can be extended to a genome-scale level in further studies.
P.115 CIRCADIAN SYSTEM IN THE HETEROCYST-FORMING CYANOBACTERIUM, ANABAENA SP. PCC 7120. H. Kushige1, M. Matsuoka1, H. Iwasaki1,2.
P.114 CHARACTERIZING THE PRIMARY METABOLISM OF SYNECHOCYSTIS SP. PCC6803: NETWORK RECONSTRUCTION, THERMODYNAMIC CONSTRAINTS AND FLUX-BALANCE ANALYSIS. H. Knoop, Y. Zilliges, W. Lockau and R. Steuer. Institut fuer Biologie, Humboldt Universitaet Berlin, Germany. Introduction: Cyanobacteria, are the only prokaryotes which possess the capability to carry out photosynthesis, similar to higher plants. Therefor it is not surprising, that cyanobacteria attract growing attention in various areas of research and for economic purposes. Among the diverse strains, Synechocystis sp. PCC6803 is a widely used model organism for the analysis of regulatory networks, metabolic pathways as well as photosynthetic processes. With a rich compendium of genomic, biochemical and physiological data available, the cyanobacterium Synechocystis sp. Strain PCC 6803 can serve as an ideal example for the reconstruction of an integrative computational model that describes the relevant metabolic processes in the cell. Methods and Results: We present a draft reconstruction of the primary metabolism of Synechocystis sp. PCC6803. Our reconstruction is based on multiple data sources and extensive manual curation. Integrating information from the genome database (CyanoBase), several pathway databases, as well as information from the primary biochemical literature. Our reconstruction comprises the main pathways of central and intermediate metabolism, including the Calvin cycle, glycolysis, the TCA cycle, the pentose phosphate pathway, oxidative phosphorylation and amino-acid synthesis. Based on the reconstruction, for which also thermodynamic constraints were taken into account, we systematically evaluate and describe the metabolic capabilities of Synechocystis sp. PCC6803 during phototrophic growth. In more detail, we used flux balance analysis to calculate the robustness of the model and to check the sensitivity of specific pathways. Additionally essential genes, correlated reaction sets and potential metabolic bottlenecks were identified, as well as several gaps and inconsistencies in current annotations. In addition, kinetic parameters were added to the model at interesting metabolic branch points, which could play a key role for biotechnological engineering. Conclusion: Owing to the complexity of even comparatively simple metabolic maps, a detailed and systematic evaluation of the properties and metabolic capabilities of organisms necessitates a computational approach. Our reconstruction of the primary metabolism of Synechocystis sp. PCC6803 allows for such a systematic computational evaluation of metabolic routes. The reconstruction significantly improves upon the current genome annotation and provides a resource for targeted manipulation of
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Department of Electrical Engineering and Bioscience, Waseda University, Japan; 2PRESTO, Japan Science and Technology Agency, 22 Wakamatsu, Shinjuku, Tokyo, Japan. Cyanobacteria are the simplest organisms to exhibit circadian rhythms, while studies on the mechanism and physical properties of the clock regulation have been exclusively performed in the unicellular Synechococcus. Since Anabaena is one of the simplest multicellular organisms which harbor both pattern formation with cell differentiation and circadian rhythms, it provides an excellent model system to analyze circadian functions and mechanisms which cannot be addressed in unicellular species. i) Is there any difference in the kaiABC clock gene functions between Synechococcus and Anabaena? Note that KaiA homologs in filamentous species lack two-third of the protein that are well conserved among unicellular species. ii) Is there any difference between the clock systems in vegetative cells and heterocysts? iii) Are clocks in neighboring cells synchronized to each other? Note that in unicellular Synechococcus cell-cell communication has been validated to be negligible in terms of circadian synchronization. iv) Is heterocyst differentiation/patterning modified by the circadian clock? As an initial step to address these questions, we performed DNA microarray analysis to reveal the genome-wide circadian expression profile. Surprisingly, none of Anabaena kai genes show significant expression rhythms, while we found ~600 clockcontrolled genes. In contrast, in Synechococcus the kaiBC operon expression shows the highest amplitude cycle. Moreover, although clock-controlled genes in Synechococcus peaked exclusively at subjective dawn or dusk, that in Anabaena peaked more widely throughout the circadian cycle. Thus, there is striking difference in circadian outputs as well as the clock gene expression profile between the two species. For better understanding the clock function, we have disrupted the kai genes in Anabaena. Transcriptomic and physiological properties in the mutants will be also reported.
P.116 EXAMINING THE ROLE OF THE CBBX PROTEIN IN RHODOBACTER SPHAEROIDES. Amanda K. Luther, Rick A. Laguna, F. R. Tabita. Department of Microbiology, The Ohio State University, Columbus, OH; USA. Introduction: Carbon dioxide is fixed into organic carbon via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway during photoautotrophic and chemoautotrophic growth of nonsulfur purple photosynthetic bacteria. However during photoheterotrophic growth, the primary function of CO2 fixation is to maintain cellular redox balance. Rhodobacter sphaeroides belongs to the alpha subdivision of proteobacteria. R. sphaeroides possesses two distinct cbb operons, cbbI and cbbII. Each operon encodes enzymes for the August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes CBB cycle, including ribulose 1,5-bisphosphate carboxylaseoxygenase (RubisCO), the enzyme that catalyzes the reduction of CO2. These operons are located on two distinct genetic elements and the regulation of the cbbI and cbbII operon differs upon carbon availability. During photoautotrophic growth, both operons are maximally expressed, although the cbbI operon attains a greater level of expression as compared to the cbbII operon. When conditions are changed to photoheterotrophic growth, down-regulation of each operon occurs. However, under this condition cbbII expression is greater than cbbI expression. In R. sphaeroides the cbbXYZ operon is located immediately downstream of the cbbI operon. With the exception of CbbZ (which functions as a phosphoglycolate phosphatase), the physiological functions of proteins encoded by genes of this operon have yet to be determined in R. sphaeroides. However, a deletion of the cbbX gene resulted in a long lag during photoautotrophic growth conditions compared to wild-type. It was recently shown that Cyanidioschyzon merolae, a unicellular red alga, encodes a CbbX protein that functions as a transcriptional regulator of the cbbLS gene that encodes RubisCO. We are currently examining whether the CbbX protein encoded by R. sphaeroides possesses such function. Methods: To gain a greater understanding of CbbX protein function in R. sphaeroides, the following experiments were performed: We compared the R. sphaeroides CbbX protein in terms of genomic context, primary amino acid sequence, and domain architecture to the CbbX protein of C. merolae. We examined the RubisCO activity and expression of the cbbI and cbbII operons in the cbbX gene deletion strain of R. sphaeroides as compared to the wild-type strain under different growth conditions and carbon sources. RubisCO activity was determined by a standard RubisCO assay, and cbbI and cbbII expression were determined by immunoblot analysis of the RubisCO proteins encoded by each operon. Results: The R sphaeroides cbbX gene deletion strain appeared to increase RubisCO activity as compared to the wild-type strain under photoautotrophic growth conditions. The increased activity appeared to be a result of increased RubisCO protein as determined by immunoblot analysis. Under photoheterotrophic growth conditions with malate as the carbon source, RubisCO activity was the same as the wild-type. However, when acetate was used as the carbon source, there was an apparent loss of regulation of the cbbII operon in the cbbX gene deletion strain, as compared to the wild-type. Conclusion: Based on the results thus far attained, it appears that the CbbX protein in R. sphaeroides may function as a regulator of one or both of the cbb operons. Further experiments are under way to determine the exact role that this protein may play in regulating these operons in R. sphaeroides.
P.117 CHARACTERIZATION OF CAROTENOID CLEAVAGE DIOXYGENASE IN ANOXYGENIC PHOTOTROPHIC BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS. Isamu Maeda, Atsushi Inaba, Kazuyuki Yoshida. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan. Introduction: Carotenoid oxygenases catalyze formation of apocarotenoids such as retinoids and the precursors of some phytohormones through oxidative cleavage at specific double bonds of carotenoids. Carotenoid cleavage dioxygenases (CCDs) belong to a subgroup of carotenoid oxygenases, and have been found in several higher plants and two cyanobacterial species. Although their substrate
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Final Program and Abstracts specificities and cleavage patterns have been characterized in detail, none of CCDs in anoxygenic phototrophic bacteria have been experimentally characterized. In this study, therefore, Rhodopseudomonas palustris CCD (RpsCCD) was chosen to analyze the activity of anoxygenic phototrophic bacterial enzyme. The in vivo and in vitro cleavages of carotenoids by RpsCCD were analyzed using lycopene, produced in a recombinant E. coli strain, and b-apo-8’carotenal as the substrate, respectively. Methods: Pantoea ananatis crtE, crtB, and Pantoea agglomerans crtI were introduced to E. coli JM109, resulting in lycopene-producing E. coli (pHYcrtEIB). Rps. palustris ccd was inserted in pMG103 to construct pMG-Rpsccd. Apocarotenoid production was performed with cultivation of E. coli (pHYcrtEIB, pMG-Rpsccd) and E. coli (pHYcrtEIB, pMG103) for 24 hours. RpsCCD produced in E. coli BL21 (DE3, pMG-Rpsccd) was used in the in vitro analysis. The crude protein extract was incubated with all-trans-b-apo-8’-carotenal (96% purity, Fluka) in the presence of 0.1% (v/v) Triton X-100. Apocarotenoids were directly extracted with hexane/diethyl ether (1:4, v/v). The organic phase was analyzed with GC-MS. Results: A specific peak was detected in the chromatogram of E. coli (pHYcrtEIB, pMG-Rpsccd) extract. The mass spectrum showed the parent-ion mass of 154, and corresponded to that of citrol, the reduced form of citral. The data suggests that citral is produced through the 7,8 (7’,8’) double bond cleavage of lycopene by RpsCCD and then reduced to citrol in E. coli cells. The in vitro analysis using b-apo-8’carotenal showed two specific peaks with the parent-ion masses of 284 and 164. The mass spectra corresponded to those of retinal (m/z 284) and apo-8’,15’-apocarotene-dial (m/z 164). The data demonstrates the 15,15’ double bond cleavage of bapo-8’-carotenal by RpsCCD. Conclusions: Dioxygenase activity was confirmed using recombinant RpsCCD protein. This is the first report that has identified cleavage patterns of an anoxygenic phototrophic bacterial enzyme.
P.118 ANABAENA SP. STRAIN PCC 7120 GENE ALL0187 IS DEVELOPMENTALLY REGULATED AND ESSENTIAL FOR DIAZOTROPHIC GROWTH AND HETEROCYST MORPHOGENESIS. Rodrigo A. Mella-Herrera*, M. Ramona Neunuebel, James W. Golden* Department of Biology, Texas A&M University, College Station, TX; *Current address: Division of Biological Sciences, University of California, San Diego, CA, USA. Introduction: Under diazotrophic growth conditions, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 undergoes development to form a pattern of about one heterocyst every ten to twenty vegetative cells along filaments. The heterocyst differentiation process involves genetic, physiological, and morphological changes to produce the micro-oxic environment necessary for nitrogen fixation. These changes include remodeling of the cell wall and deposition of two heterocyst-specific extracellular envelope layers. The all0187 gene is predicted to encode a protein containing a LytR domain, which is known to be associated with regulation of cell wall maintenance. We investigated all0187 to determine if it is involved in heterocyst differentiation or in diazotrophic growth.
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Methods: Anabaena (Nostoc) sp. strain PCC 7120 and its derivatives were grown in BG-11 or BG-110 (lacking sodium nitrate) medium at 30°C with white-light illumination of approximately 75 µM photons m−2 s−1. Standard protocols were used for cloning, E. coli transformation, PCR, and northern RNA blot analysis. Microarrays experiments were performed using Anabaena PCC 7120 RNA samples hybridized to a custom microarray slide containing 768 probes. Light and fluorescence microscopy were performed on a Zeiss Axioplan II microscope with a 40´ objective and green fluorescent protein (GFP)specific emission (518 ± 13 nm) filter sets. Nitrogenase activity was measured with an acetylene reduction assay. Results: Insertional inactivation of all0187 caused a partial cell division defect of vegetative cells grown in nitrate-containing medium. In nitrogen-free medium, mutant filaments formed abnormally long heterocysts and were unable to grow diazotrophically, but vegetative cell septation appeared normal. Septum formation between heterocysts and their flanking vegetative cells was incomplete, leaving one or both poles of the heterocysts partially opened. We investigated whether these morphological defects led to inactivation of nitrogenase due to lessened protection against oxygen. Acetylene reduction assays for nitrogenase activity showed that the mutant strain retained approximately seventy percent of the wild-type activity; this observation shows that heterocysts of the all0187 mutant strain are partially functional. Northern RNA blot analysis showed that all0187 expression was upregulated by 8 h after nitrogen step-down and fluorescence microscopy of a Pall0187-gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts by 9 h after nitrogen step-down, whereas vegetative cells maintained a lower level of fluorescence. Conclusions: We hypothesize that the diazotrophic growth defect of the all0187 mutant is caused by the inability of the heterocysts to transport fixed nitrogen to the neighboring vegetative cells.
P.119 FUNCTIONAL ANALYSIS OF THE ISCR-LIKE TRANSCRIPTION FACTOR, INVOLVED IN EXPRESSION OF THE PSAAB TRANSCRIPT IN SYNECHOCYSTIS SP. PCC 6803. T. Midorikawa1, K. Matsumoto1, R. Narikawa2, M. Ikeuchi1,2. 1
Department of Biological Science, Graduate School of Sciences; Graduate School of Arts and Sciences; University of Tokyo, Japan.
2
Introduction: Since the light environment varies depending on location, time, and weather, it is important for the oxygenic phototrophs to acclimate to these environmental changes. It has been established that the regulated accumulation of photosystem I (PSI) is critical for long term acclimation to the light conditions in cyanobacteria. To this end, expression of psaA and psaB, which encode the PSI reaction center subunits, is tightly regulated in the acclimation processes. In the course of our screening of transcriptional regulators in Synechocystis, we found that an iscR-like regulator Slr0846 is critical for maintenance of normal chlorophyll accumulation. Here we studied the target genes of Slr0846 by DNA microarray analysis, gel shift assay and primer extension analysis. We also studied PSI/PSII ratio of the slr0846 disruptant under various light conditions.
with CyanoCHIP version 1.6. The N-terminal poly histidine-tagged Slr0846 prepared from E. coli was used for gel shift assay.
Result: Full segregation of the ∆slr0846 mutant was achieved. It is suggested that Slr0846 is not essential for this strain. DNA microarray analysis revealed that the expression level of psaAB was downshifted in the ∆slr0846 mutant, which was grown under the standard light conditions. Direct binding of Slr0846 protein to a promoter of psaAB was studied by gel shift assay. Further dissection revealed that Slr0846 bound to a far upstream region in the promoter. Primer extension analysis showed that transcription from the two known start sites was almost equally affected in the mutant. These results suggest that Slr0846 is a transcriptional activator for psaAB. The ∆slr0846 mutant exhibited much lower chlorophyll contents and PSI/PSII ratio than wild type. The mutant also showed the growth sensitive to high light. Notably, the difference in the PSI/PSII ratio between wild type and the mutant was attenuated under the PSI light conditions. These findings suggest that the activity of Slr0846 may respond to various light conditions. Conclusion: In this study, we demonstrated that the Slr0846 binds to a far upstream region in a promoter of psaAB and acts as a transcriptional activator. It may contribute to the optimal regulation of the psaAB expression under various light conditions.
P.120 ALR2269 – THE OMP85 IN ANABAENA SP. PCC 7120. Kerstin Nicolaisen, Constance Vollmer, Iwo Tews, Enrique Flores, Enrico Schleiff. Goethe University Frankfurt, Frankfurt, Germany. The outer membrane of gram-negative bacteria protects the organism against environmental influences and allows interactions with its surrounding. Proteins of the Omp85 class embedded in the outer membrane function as “chaperones” for the insertion of the outer membrane proteome required for the function of this barrier. Omp85 are polypeptide transporting β-barrel proteins (1), which are also found to transport polypeptides across the outer envelope of chloroplasts (1). It is thereby an essential class of proteins in endosymbiotically derived organelles and bacteria. In Anabaena sp. PCC 7120, the closest relative to the ancestor of chloroplasts (2), Alr2269 functions as Omp85-like protein (3). Therefore we studied its role as transporter (3), analyzed its properties in protein recognition (5) and are investigating the possibilities to complement other proteins of the Omp85 class with Alr2269 and vice versa (6). References: 1. Schleiff, E., Soll, J. (2005) EMBO Rep. 6, 1023-1027 2. Bredemeier, R. et al. (2007) J.Biol.Chem. 282,1882-1890 3. Ertel, F. et al. (2005) J.Biol.Chem. 31, 28281-28289 4. Nicolaisen, K. et al. (2009) Mol. Microbiol. submitted 5. Wunder, T. et al. (2007) BMC Evol. Biol. 7: 236 6. Wunder, T. et al. (2009) Endocyt. Cell Res.
Methods: The original motile strain of Synechocystis sp. PCC 6803 showing positive phototaxis was used as wild type. The slr0846 gene was disrupted by insertion of the chloramphenicol resistance gene. The low temperature chlorophyll fluorescence at 77 K was monitored for estimation of the PSI/PSII ratio. DNA microarray analysis was done
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P.121 ANALYSIS OF BIOSYNTHETIC PATHWAY OF CHL A ESTRIFIED WITH ∆2,6-PHYTADIENOL IN CHLOROBIUM TEPIDUM BY CONSTRUCTING DISRUPTION MUTANTS OF CT1232 AND CT2256 GENES. JiroHarada1,SyoheiMiyago2,TadashiMizoguchi1,HitoshiTamiaki1, HirozoOh-oka2. 1
Final Program and Abstracts [2]T.Mizoguchi,J.HaradaandH.Tamiaki,FEBS Lett.,580:6644-6648 (2006). [3]J.Harada,S.Miyago,T.Mizoguchi,C.Azai,K.Inoue,H.Tamiaki andH.Oh-oka,Photochem. Photobiol. Sci. 7:1179-1187(2008). [4]T.Mizoguchi,M.Isaji,J.Harada,K.WatabeandH.Tamiaki,J. Porphyr. Phthal. (in press).
P.122
DepartmentofBioscienceandBiotechnology,FacultyofScienceand Engineering, RitsumeikanUniversity;2DepartmentofBiological Sciences,GraduateSchoolofScience,OsakaUniversity;Japan.
ACID STRESS RESPONSIVE GENES, SLR0967 AND SLL0939, ARE DIRECTLY INVOLVED IN LOW-PH TOLERANCE OF CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Thegreensulfurbacteria contain threekindsof(bacterio)chlorophyll [(B)Chl]pigments,BChl aP,ChlaPD andBChlcF.BChlscF arelocatedin largelight-harvestingapparatuses,chlorosomes,wherelightenergyis capturedandmigratedtoFMOproteinviabaseplateandfinally convergedonP840,aspecialdimerofBChlaP,inthereactioncenter. BChlsaP alsoserveasantennapigmentsassociatedwithCsmAprotein inbaseplate,FMOproteinandthereactioncentercomplex.Within thereactioncentercomplex,ChlsaPD aresupposedtofunctionasa primaryelectronacceptoraswellasaccessorypigments.ChlaPD has thesamechlorinπ-system asChlaP inhigherplantsandcyanobacteria exceptforalonghydrocarbonchainattheC-17position; theformer isesterifiedwith∆2,6-phytadienolandthelatteriswithphytol[1].A precursorofphytolisgeranylgeraniol,whichisstepwiselyreducedby geranylgeranylreductase(GGR)[2].However,thereactionprocessto produce∆2,6-phytadienylesterisstillunknown.Inthegenomeof Chlorobium tepidum,twoparalogousgenes,CT1232 andCT2256, exhibitsequencesimilaritytoGGRgenesofcyanobacteriaand/or purplebacteria,andarepresumedtobeinvolvedinhydrogenationof geranylgeranylgroup.
HisatakaOhta1,2,AtsushiMoriyama1,YukoKubo1,YousukeShibata1, YouheiHaseyama1,YukaYoshino1,TakehiroSuzuki1,MasahikoIkeuchi3, IsaoEnami1,ShuseiSato4,YasukazuNakamura4,SatoshiTabata4.
Inordertoclarifythebiosyntheticprocess of∆2,6-phytadienylester, weconstructeddisruptionmutantsof thesegenesusing an insertional inactivationmethodandanalyzedtheirpigmentcompositions and photosyntheticcompetences [3,4].TheCT2256 mutantaccumulated ChlaGG andBChlaGG esterifiedwithgeranylgeraniol,indicatingthat CT2256was involvedintheproduction ofboth ∆2,6-phytadienyland phytylgroups.Themutantstillgrewphotosyntheticallyalthoughits growthratedrasticallydecreasedtolessthanahalfcomparedtothat ofthewildtype.Thiscouldbeexplainedbysomehindranceofthe energytransferfromchlorosomestothereactioncentercomplex, becausetherelativelyhighfluorescenceemissionfromchlorosomesin themutantwasobserved.However,theCT1232 mutantshowedno apparentphenotype comparedtothewild type.The purple bacteriumRhodobacter capsulatus mutantdefectiveinthebchP gene waspartiallycomplementedwiththeCT2256 gene byaconventional conjugationmethod; BChlaP wassynthesizedinthemutantin additiontoaccumulatingotherintermediates. Furthermore, cyanobacterialandpurplebacterialGGRgenes,whicharechlP and bchP genes,respectively,wereincorporatedintotheCT2256 mutant genomebyreplacingthedisruptedsiteoftherelevantgene. Thetwo strains,thusobtained,partiallyrestoredareductionactivityto producephytolandotherintermediatesfromgeranylgeraniol.Allof theabovedatasuggestthattheCT2256mightdiscriminatethe differenceofringstructuresbetweenchlorin andbacteriochlorin and produceBChlaP andChlaPD fromBChlaGG andChlaGG,respectively. References [1]M.Kobayashi,H.Oh-oka,S.Akutus,M.Akiyama,K.Tominaga,H. Kise,F.Nishida,T.Watanabe,J.Amesz,M.Koizumi,N.IshidaandH. Kano,Photosynth. Res.,63:269-280(2000).
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1 Department ofBiology,Faculty ofScience,TokyoUniversity of Science, Shinjuku; 2TissueEngineeringResearchCenter,Tokyo UniversityofScience,Noda;3Department ofLifeScience,Universityof Tokyo,Meguro,Tokyo;4KazusaDNAResearchInstitute,Kisarazu, Chiba;Japan.
Introduction: DatingfromthePrecambrianera,cyanobacteriahavea longhistoryofadaptingtotheEarth’senvironment.Byevolving oxygenviaphotosyntheticreactionssimilartothoseofplantsand greenalgae,theseprokaryoteswereessentialtotheevolutionofthe presentbiosphere. Theycontinuetomakealargecontributiontothe equilibriumoftheEarth’satmospherebyproducingoxygenand removingcarbondioxide. Tosurviveinextremeorvariable environments,cyanobacteriahavedevelopedspecificregulatory systems,inadditiontomoregeneralmechanismsequivalenttothose ofotherprokaryotesorphotosynthesisbacteria (1). Severalspeciesof cyanobacteriaserveasmodelorganismsforelucidatingboth functionalandregulatoryaspectsofphotosynthesis (2). Aboveall, Synechocystis sp.PCC6803wasthefirstphotosyntheticorganismfor whichacompletegenomesequencebecameavailable(3), andDNA microarrayshavebeenusedtoexaminegeneexpressioninresponse tovariouskindsofstresssuchasosmotic,salinity, andhighlightstress (4-7). Acidrainis oneofthemostseriousofenvironmentalstresses.It causesacidificationoflakesandstreamsandcontributestodamage ofplants, algae, andcyanobacteria inmanypartsoftheworld. Rhizotoxicityinacidsoil,whichinvolvestheactionofAl3+ hasbeen wellinvestigated(8). Nevertheless, littlehasbeendonetoelucidate thebasicsetofadaptationsnecessaryforacidtoleranceinplants, algae, orcyanobacteria. DNAmicroarrayanalysisofSynechocystis sp.PCC6803 cells revealed that acidstressinducedtheexpressionof putativestress-related proteins,suchaschaperones (slr0093[dnaJ],sll1514[hspA],and sll0170[dnaK)]), regulatoryfactors(sll0306[sigB] andsll2012 [sigD)]), andproteinsof unknownfunction(9).Amongtheup-regulatedgenes withunknownfunction,slr0967 andsll0939 continuouslyincrease7 and16-foldafter4hofacidstressandareupregulated byosmotic andsaltstresses(6).Interestingly,thesetwogenesarelocated adjacentlyontheSynechocystis sp. PCC6803 genome(Fig.1). Inthisstudy,weexamined thephysiologicalfunctionofcyanobacteria genes using mutant cellsinwhicheachgenewasdisruptedbya kanamycin-resistance cartridgegene. Basedonphenotypes ofthe mutantsandreal-time quantitativeRTPCR analysis ofthetranscripts of thetwogenes, theexpressionprofileoftheslr0967 deletionmutant onacidstresswascomparedwiththatofwild-typecells.
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Final Program and Abstracts Methods: Culture conditions- Synechocystis sp.PCC6803was cultivatedin BG-11medium(10) bufferedwith10mMTESNaOH,pH 8.0, orin10mMMESNaOHpH6.0(long-termacidstresscondition), at30°C. Cultureswerebubbledwith3%CO2-containingair and illuminatedwith30µmolphotonsm−2 s−1 fromincandescent bulbs. CellgrowthwasmeasuredusingaPharmaciaspectrophotometerat 730nm (AmershamPharmaciaBiotech,Piscataway,NJ,USA). Generation of insertion mutants- Mutantsimpaired inselectedgenes weregeneratedbyreverse genetics.Thecodingsequencesand neighboring sequenceswereamplifiedbyPCR.Approximately 2kbof PCRproductswereclonedintopUC19 (Toyobo,Osaka,Japan).The primersforamplificationwere designedusingthecompletegenome sequenceof Synechocystis (3).Sequencesthat containedappropriate restrictionsiteswereselected toimprovecloningof fragments. The kanamycin(Km) resistance gene(kmr)isolatedfrom plasmidpUC4K (AmershamPharmacia)wasinsertedinto uniquerestrictionsitesofthe encodingsequences. Transformantswereinitiallyselectedona medium containing10μgKmmL−1 (WakoPureChemical,Osaka, Japan),whereasthe segregationofcloneswasperformedby restreaking(atleastthreetransfers)ofprimary clonesonplates supplementedwith50μgKm mL−1. Duringthecultivationofmutants, Kmwasaddedtotheliquidmedia. Generation of overexpressing mutants-Overexpressingmutantstrcslr0967 andtrc-sll0939 ofSynechocystis 6803 weregeneratedusing pTrc-slr0967andpTrc-sll0939 plasmids.Theplasmids wereintroduced intothe cmr gene from pLysS (Novagen,Gibbstown,NJ,USA) and the trcpromoterwasintroducedfrompTrcHisB (Invitrogen,Carlsbad, CA,USA) intotheupstreamoftherespectivegenesasdescribedby Kameietal.(11). Short-term acid stress conditions-Exponentially-growing cellswere acid-stressedbycentrifugingthecell culturesandresuspendingthe cellpelletsinapH adjusted BG-11mediumusing10mMglycine(pH 3.0)buffers insteadofTESbuffer for8h. Serialdilutionsofthecells werespotted ontonormalBG-11 plates aftertreatment andcultured for7 days at30°C.Experimentswere performedinduplicateatleast threetimes. RNA isolation and quantitative real-time RTPCR-TotalRNAwas isolatedusingtheRNeasy Minikit(Qiagen,Hilden,Germany)as described byHiharaetal. (7).Forthereversetranscriptase (RT) reaction,100ngRNAwasincubatedwitha mixtureofPCRreverse primersfor10minat70°C priortoadding100USuperscriptIIRT (Gibco-BRL,Carlsbad,CA,USA). TheRTreactionwasperformedat 42°Cfor1hand terminatedbyincubatingthecellsat72°Cfor10 min. APerfectRealTimekit(TakaraBio,Otsu,Shiga,Japan)wasused according tothemanufacturer’sinstructions. DNA microarray analysis- Cellswereharvestedfrom10mLofculture bycentrifugationat4,000×g for5minat25°Candthenbroken immediatelywithaMini-BeadBeater(Biospec,Bartlesville,OK,USA). TotalRNAwasisolatedusingtheRNeasyMinikit(Qiagen)as describedby Hiharaetal. (7). ASynechocystis DNAmicroarray (CyanoCHIP)wasobtainedfromTakara(Kyoto,Japan).Thismicroarray covered3,079ofthe3,168openreadingframes(ORFs) of Synechocystis,excludingORFstransposases.Conditionsforthe synthesisofCy3-labeledandCy5-labeledcDNAs,hybridization,and washingwereaspreviouslydescribed(Hiharaetal.2001). Image acquisitionwithaScanArray4000(GSILumonics, Watertown,MA, USA)wasperformedwiththeautobalance-autorange feature. With thisfeature,thesensitivityoftheinstrument canbeautomatically adjustedbychangingthelaserpowerand photomultipliergain settingssothatthesignaliswithin90% ofmaximumtoprevent saturation.Therawdataobtainedwith theScanArray4000were 138
13th International Symposium on Phototrophic Prokaryotes analyzedwithQuantArrayversion2.0 software(GSILumonics,Tokyo, Japan). Thefluorescenceintensityofeach spotforbothCy3andCy5 imageswasquantified,andlocalbackground fluorescencelevelswere subtracted. Cy3andCy5imageswere normalizedbyadjustingthe totalsignalintensitiesofthe twoimages. Theresultsshownare averagesof4–6 biologicallyindependentexperiments. Results: Characterization of the deletion mutants of slr0967 and sll0939 Wild-typeSynechocystis sp.PCC6803wastransformed with slr0967 andsll0939 that hadbeeninterruptedwithcassetteconferring resistance tokanamycin. Toexaminesegregation ofthesegenes withintheSynechocystis genome, we performedaPCRanalysisusing DNAofwild-typeand the∆slr0967 and∆sll0939 mutantcells.PCR with genomic DNAofwild-typecells asatemplateamplified respective 0.4 kbDNAfragmentsforslr0967 andsll0939,whereas PCRwithDNAfrom∆slr0967 and∆sll0939 mutantcellsyieldeda fragmentof 1.6 kb(results notshown). Theseresultsindicatedthat slr0967 andsll0939 genesin∆slr0967 and∆sll0939 mutantcellshad beendisruptedbytheinsertion ofthekmr gene. InnormalBG-11mediumatpH8.0,allstrainsexhibitedasimilar photoautotrophicdoublingtime(Fig.2A),suggesting thatdeletionof thesegenesdidnotaffect theirgrowthinnormalconditions. In contrast, intheacidstressed conditionatpH6.0,thegrowthofall mutant cells was slightlybutsignificantlyinhibited comparedwiththat ofwild-typecells(Fig.2Band2C). Thesll0939 andthedoubleslr0967 andsll0939 mutants were moresensitive toacidstressthanthe ∆slr0967 cells(Fig.2B) indicatingthatslr0967 andsll0939 areinvolved inacidtoleranceofSynechocystis cells. Overexpression of slr0967 and sll0939 Overexpressionofthesegenes failedtoenhancetheacidtoleranceafter culturingatpH6.0for7 days(Fig. 3A).Wethencharacterized theexpressionofthesetwo genesatthelevelofthetranscriptbyquantitativeRT-PCR usingtotal RNAisolatedfromacidtreatedwild-typecellsandtrc-slr0967andtrcsll0939 mutantcells. Therelativeexpressionof slr0967 and sll0939 promotedbytrc waslowerthanthatofwild-typecellsduringa 4h acidstressbuthigherthanthatfromnon-treatedwild-typecells(Fig. 3CandD).Accordingly,survivability ofthemutantsbyacidstress treatmentatpH3.0for1–12hwastested (datanotshown).Asa result,wefounda significantdifferencein survivalbetweenwild-type andtrc-mutantsthat weretreatedfor 8h(Fig. 3B).Thetrc-mutants became moretoleranttoacidstressthanwild-typecells. Quantitative RT-PCR analysis of slr0967 and sll0939 in mutual deletion mutants. Thesetwogenes arelocatedadjacent tothe operonandare codedbyopposite strands ontheSynechocystis genome.Toexamine theregulatoryrelationshipbetweenthesetwogenes whosedeletion mutants weresensitivetoacidstresscomparedwiththewild-type,the abundanceofmutual genes inthedeletionmutants was measured in normalgrowthandacidstress conditions by culturing thematpH 3.0 for30min. Asshown inFig. 3,therewasincreasedexpressionof slr0967 andsll0939 inwild-typecellsbyacidstressafter30 min; however,therewas nosignificant alterationinslr0967 expression evidentinthe sll0939 mutant.Therewasaslightup-regulationof sll0939 in the normalgrowthcondition,but deletionofsll0939 didnot affectslr0967 expressioninthe acidstressed condition.Incontrast, sll0939 expressionwasconsiderably affectedbydeletionofslr0967 in bothnormalandacidstressed conditions.Therelativelevelsofthe sll0939 transcriptwereonly3%and4%ofwild-typecells, respectively. Theseresultsindicatethatslr0967 regulatessll0939 expressionin Synechocystis inbothnormalandacidstressed conditions.
DNA micro array analysis of ∆slr0967 mutant. Weexaminedthe genome-wideexpressionofgenesin∆slr0967 mutantcellsusingDNA microarraystodeterminewhether slr0967 isinvolved intheexpression August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes ofotheracidstressresponsivegenes. Table1showsgenes thatwere 3-foldup-regulatedbyslr0967 deletion comparedto wild-typecells. Thesegenescanbeclassifiedinto twogroups.1) Genesthatwerenot repressed inmutantcellsbutwere3-foldormorerepressed inwildtypecellsunder acidstress,and2)Genesthatwereup-regulated morethan3-foldinmutantcellsbutnotinwild-typecells. Thefirst groupcontainsgenesforproteins involvedinCO2 uptake, CO2 fixation, andbicarbonatetransport(sll1732 [ndhF3],sll1733 [ndhD3], sll1734 [cupA],slr0040 [cmpA], slr0041 [cmpB], slr0042, slr0043, slr0044, andslr1512 [sbtA]) (12-16). Aspreviouslydescribed(9),the bicarbonatetransportsystemmighthavebecomeredundantforwildtypecellsunderacidstressbecausemostofthebicarbonateexistsin theformofCO2 thatdiffusesthroughthecellmembranesfreely. It maybethatthesegeneswerenotup-regulatedinmutantcellsfor survivalbuttherepressionmachineryofthesegeneswasvirtually haltedduetolackofslr0967. Thesecondgroupcontainsgenes encodingstressregulatedproteins(ssl0452 [nblA],sll0247 [isiA], slr0373,slr0374, slr0376, and slr0228 [ftsH]).Degradationofthe cyanobacterialphycobilisomelight-harvestingantennaisageneral acclimationresponsethatisobservedundervariousstressconditions suchasN-,S-,andP-starvation(17-20). IntheSynechocystis strain PCC6803genome,twotandemcopiesofnblA arepresent.The expressionofssl0453 (nblA2), whichislocateddownstreamofssl0452 (nblA1),wasnotaffectedbyslr0967 deletion(datanotshown). The isiA geneencodesaproteinthatissimilartothephotosystemII chlorophyll-bindingproteinCP43andisinducedunderiron-stressed conditions(21,22).Theslr0373,slr0374, and slr0376 genesconstitute an operon thatisresponsivetovariousenvironmentalstressesbut theirfunctionsareunknown(23,24).AnFtsH proteaseplaysan essentialroleintheturnoverofthereactioncenterD1proteinin Synechocystis PCC6803underheatstress aswellaslightstress conditions(25,26).SLR0967 deletionincreasesdeathduetoacid stress,resultinginincreaseinthenumberofstressresponsivegenes beingup-regulatedinmutantcells.Genes whoseexpressionwere upregulatedinwild-typecellbyacidstresswerealmostalldown regulated bydeletionofslr0967 (datanotshown). Table2showsgenesthatwere3-foldormorerepressedbyslr0967 deletionthanthoseinwild-typecells. Thesegeneswerealsodivided intotwogroups:1) Geneswhoserepressionwasobserved inmutant cellsbutnotinwild-typecellsbyacidstress,and2)Geneswhoseupregulationdecreasedinmutantcellsbut were 3-foldormore up-regulatedinwild-typecells.Genesencodingthephycobilisome proteins(sll1577 [cpcB], sll1578 [cpcA], sll1579 [cpcC2], sll1580 [cpcC1], slr0335 [apcE], slr1986 [apcB], slr2067 [apcA], slr2051 [cpcG1]),photosystemIsubunits(slr1834 [psaA], ssl0563 [psaC], slr0737 [psaD], sll0819 [psaF], and slr1655 [psaL]) andchaperone proteins(slr2075 [GroES] andslr2076 [GroEL1]) thatwererepressedby deletionofslr0967.Allakhverdievetal.(27,28).demonstratedthat saltstressandhyperosmoticstresshaveinhibitoryeffectsonthe electron-transferactivityofphotosystemI.Repressedexpressionof genesforphotosystemIandphycobilisomesmightbeimportantfor maintenanceofacertainlevelofphotosyntheticactivity.Moreover,it seemspossiblethat decreasedabundanceofFe-requiring photosystemIcomponentsisaccompaniedbyirondeficiency,which maybecausedbyacidstress.Itisunknownwhychaperoneexpression wasrepressesedasthesegeneswereonlymarginally up-regulatedby acidstressinwild-typecells. High-lightinducibleproteinssr2595 (HilB) (29)andthetwocomponentresponseregulatorslr1214 (rre15) showed remarkablydecreasedexpressionfollowing slr0967deletion. Conclusions: Inthisstudy,wefoundthatacidresponsiblegenes slr0967 andsll0939 aredirectlyinvolvedinacidtoleranceofthe cyanobacteriaSynechocystis sp.PCC 6803, andslr0967 mayregulate August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts theexpressionofgenesthatrespondtoenvironmentalstresses. The physiologicalfunctionsofthesegeneshavestillnotbeenidentified. Sequenceanalysisrevealedthatsll0939hastwotransmembrane segmentsandslr0967hastowpentapeptidesequences sequences. Interestingly,preliminarydatafromatwohybridyeastanalysis revealedthatsll0939interactswithslr1596(pxcA), whichisaproteinin thecytoplasmicmembraneinvolvedinlight-inducedprotonextrusion (datanotshown).Thedeletionmutantofslr1596 isunabletogrowat acidicpH(30). Thesedatasuggestthatsll0939 mayalsofunctionwith slr1596forlight-inducedprotonextrusion.However,deletionof sll0939 andslr0967 isnotaffectedbylight-inducedprotonextrusion (datanotshown). Up-regulationofslr1214 (rre15) considerably decreased by deletionofslr0967. Itwasthought thatslr1214 affected acidstresstolerance,butdeletionofthegenedid notaffectgrowthat pH6.0(31).Therefore,wearenowintensivelystudyingthe relationshipoftheseproteins. Tofurtherunderstandthefunctionof slr0967andsll0939,wehaveinvestigated theproteinexpressionof slr0967 andsll0939 inE. coli cellsusingpETvectors,buttheirfunction isstillunknown.IPTGinductionofthesegenesinE. coli causecell lysesandthecellsareunabletorecovertheseproteins.Weare interestedintheregulatorymechanismsofacid-responsiblegenes; therefore,analysisofthepromoterregionofslr0967 andsll0939 shouldbeinvestigatedintensively.
P.123 CELL-SPECIFIC TRANSCRIPTOME ANALYSIS IN ANABAENA VARIABILIS ATCC 29413 GROWN PHOTOTROPHICALLY, PHOTOHETEROTROPHICALLY, AND HETEROTROPHICALLY. Jeong-JinPark1,SigalLechno-Yossef2,HajimeMasukawa1, C.PeterWolk2,3,ClaireVieille1. 1
DepartmentofMicrobiologyandMolecularGenetics;2MSU-DOE PlantResearchLaboratory;3DepartmentofPlantBiology,Michigan StateUniversity,EastLansing,MI,USA. Introduction: Anabaena variabilis ATCC29413isapotential candidatefortheproductionofhydrogen(H2)asacleanrenewable energycommodity.A. variabilis filamentsarecomposedofvegetative cellsthatperformoxygenicphotosynthesis,andofheterocyststhatfix nitrogen(N2)inanoxicenvironment.A. variabilis producesH2 asabyproductofN2 fixation.Tounderstandhowelectronsarechanneledto H2 productioninA. variabilis underdifferentgrowthconditions,we arecomparinggeneexpressioninphototrophic(inthepresenceof light),photoheterotrophic(inthepresenceoflightplusfructose),and heterotrophic(withfructoseinthedark)culturesusingRNAextracted separatelyfromvegetativecells,heterocysts,andwholefilaments. ThesestudiesshouldhelpusidentifygeneswhoserolesinH2 productionareaffectedbycarbonandenergysources,andregulatory mechanismsthatinter-relatethosegenes. Methods: A.variabilis ATCC29413wasgrowninAA/8mediumat 30°CinFernbachflasksunderwhitefluorescentlightorinthedark. Heterotrophicculturesweresupplementedwith5mMfructose. Heterocystsandvegetativecellswereisolatedasdescribed(Peterson andWolk,PNAS,1978,75:6271),withvariations.RNAwasextracted fromeachtypeofcellusingAmbionRiboPure”!-Bacteriakits.RT-qPCR reactionswereperformedwithprimersspecifictotheA. variabilis 16s rRNA,rnpB,rbcL, ftsZ, nifK,andfdxN genes,using16srRNAorrnpB asinternalcontrolsfordatanormalization.cDNAwaspreparedby usingSuperscriptIIreversetranscriptaseandrandomprimers.RTqPCRwasperformedusingSYBRgreen(DuandArvidson.Infect. Immun. 2006,74:2767).Microarrayexperimentsarebeingperformed withRocheNimbleGen60-merarrays.Nitrogenaseactivity,withC2H2
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Final Program and Abstracts assubstrate,and H2 productionwerequantitatedbyacombinationof gaschromatographywith,respectively,aflameionizationanda thermalconductivitydetector,. Results:Comparedtophototrophicgrowth,photoheterotrophic growthonfructoseincreasedA. variabilis‘specificH2 productionrate 30-fold,andnitrogenaseactivitytwo-fold.Culturesformicroarray studieswereharvestedfourdaysafterinoculation.Cellpurification andcell-specificRNAextractionwereconfirmedbymicroscopic examinationandRT-qPCRwithcell-specificgenes,respectively.We areusingcDNAmicroarrayexperimentstostudycell-specificgene regulationinresponsetochangesingrowthconditions,from phototrophictophotoheterotrophicandtofullyheterotrophic.To groupgenesaccuratelythatshowsimilarexpressionpatternsineach growthconditionorineachcelltype,expressiondatawillbeclustered usingaself-organizingtreealgorithmandvisualizedasheatmaps.We expecttobeabletocategorizetypesofresponsesasconnected directlyorindirectlytoH2 production. Conclusions: Wewillreportmicroarraystudiesofcelltype-specific geneexpressioninafilamentouscyanobacterium.Weexpectthatour resultswillanswerpreviouslyunansweredquestionsoncell-specific generegulationandH2 metabolism.
physiologicalstress. Duringtheadaptationphasetonitrogenlimitationwhenformationof additionalheterocystsisinducedforassimilationofatmospheric nitrogen,theintracellularCYNcontentofAphanizomenon strains increasesuptotwicetheamountfoundincellsadaptedtonitrogen saturation.Incontrastinbatchcultureswithlimitingfactorsotherthan nitrogen,intracellularCYNdecreasescontinuouslyandCYNreleaseis substantiallyhigherthaninnitrogenlimitedcultures. Conclusions: ThevariationofthetotalCYNconcentrationinstrains fromtemperatelakesrespondstoenvironmentalvariablesinarange similartotherangedescribedpreviouslyforthewellinvestigated cyanobacterialpeptidetoxins(microcystins). WeconfirmedthatCYNreleasecouldnotonlyberelatedtocelllysis andproposeafurtheractiveprocesswhichseemstoberegulatedby variousenvironmentalfactors.Incipientnitrogenlimitationreduced CYNreleasefromcells,whileincontrastotherphysiologicalstress conditions,particularlyphotoinhibitionenhancedrelease. Riskassessmentthusneedstotakeintoaccountthatlargeamountsof dissolvedCYNmayoccurinwaterbodiesevenwhilecyanobacterial bloomsareintact.
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P.125
FACTORS INFLUENCING PRODUCTION AND RELEASE OF THE CYANOBACTERIAL TOXIN CYLINDROSPERMOPSIN.
VITAMIN-E DEFICIENCY RESULTS IN OVER-ACCUMULATION OF GLYCOGEN IN THE CYANOBACTERIUM SYNCHOCYSTIS SP. PCC6803.
KarinaPreußel,JuttaFastner,IngridChorus. Umweltbundesamt(FederalEnvironmentalAgency),Berlin,Germany. Introduction: Thecyanobacterialsecondarymetabolite cylindrospermopsin(CYN)isaninhibitorofeukaryoticprotein synthesis.Unlike thewellexaminedcyanobacterialpeptidetoxins (microcystins),CYNoftenoccurswithahighproportionofextracellular dissolvedtoxininwaterbodiesaffected.ThedissolvedCYNfraction hasimportantimplicationsbothforaquaticecosystems andforhuman health:asCYNinhibitsanubiquitousprocessingrowthand maintenancemetabolismoforganisms,thedissolvedtoxinmay impactawiderangeofaquaticorganisms(includingplants) independentlyoftheirtrophicrelationshipstocyanobacteria. ItissofarunknownifthehighamountsofdissolvedCYNinpersisting cyanobacterialbloomsandinstationaryphasesofbatchculture experimentsarisefrompassiveleakageandsimultaneousenrichment duetoitspoorbiodegradability,orifspecificenvironmentalfactors mayenhanceanactiveCYNreleasefromcells. Ourinvestigationsaimatimprovingtheunderstandingofthefactors whichinfluenceCYNproductionandreleaseespeciallytowards humanhealthriskassessment. Methods: Semicontinousaswellasbatchcultureexperimentswith differentCYNproducingcyanobacterialstrainswereperformedto measureCYNproductionandreleaseundervariouscultivation conditions. Ninedifferentcombinedlight-temperatureconditionsaswellasthe influenceofnitrogenandphosphoruslimitation weretested. IntracellularandextracellularCYNcontentswereanalysedbyLCMS/MSandHPLC-PDA. Results: BiovolumerelatedCYNcontentsvariedatmaximumbya factorof2.7inAphanizomenon strainsattheconditionstested,with temperatureasthefactorcausingthestrongesteffects.Extracellular CYNconcentrationsamountto11–26%oftotalCYNbutmay increaseupto58%underconditionswhichwerepostulatedtoactas 140
LisaRosgaard, YumikoSakuragi. DepartmentofPlantBiologyandBiotechnology, FacultyofLife Sciences,UniversityofCopenhagen;VKRResearchCentre“Proactive Plant”;StrategicResearchConsortium“Fuel-for-Life”,FacultyofLife Sciences,UniversityofCopenhagen;Denmark. Introduction: Photoautrotrophiccyanobacteriaareoneofthemost importantprimaryproducersonEarth.Theyareresponsiblefornearly 70%ofCO2 assimilationintheaquaticenvironmentand understandingoftheircarbonmetabolismisthereforeveryimportant (1). ThisprojectisfocussedoninvestigatingtheroleofvitaminEandits roleincarbonmetabolisminthemodelcyanobacteriumSynechocystis sp. PCC 6803.Thepreviousworkhasdemonstratedthatthe Synechocystis mutantslr1736- whichisdeficientinvitaminE,exhibit ca.20%increasedphotosyntheticcapacitiesandincreasedtotalsugar content (2).WehypothesizethatvitaminEfunctionsasregulatorof photosynthesisandcarbonmetabolism,inadditiontoitsantioxidant activity (2). WewanttounderstandthemechanismbywhichvitaminE regulatesthecarbonmetabolismandinthisworkweexaminedthe carbohydratecontentoftheslr1736- mutant. Methods:Thetotalsugarcontentwasdeterminedbythephenolsulfuricacidmethodasdescribedin(3)andthetotalhexosecontent wasdeterminedusinganthroneinsulfuricacidaccordingto(4). Glycogenwasextractedbyethanolprecipitationfromliquidcultures ofwildtypePCC6803andtheslr1736- mutant.Theglycogenextract washydrolyzedbyamyloglucosidaseandtheamountofglucose releasedwasquantifiedbycolorimetricdeterminationusingaglucose oxidaseandperoxidasecoupledassay. Results: Analysisoftheslr1736- mutantshowedthatamountoftotal sugarswas40%highercomparedtothewildtype.Thecontentoftotal hexoses andglycogen wasalsodetermined;theslr1736- contained approximately30%morehexosesandca.70%moreglycogenas comparedtothewildtype.Theincreaseintotalhexosecontentwas largelyattributedtotheincreaseinglycogen. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes Conclusion: Theresultsconfirmthepreviousresultsshowing increasedtotalsugarcontentintheslr1736- strain.Theincreasein hexosecontentfoundintheslr1736- mutantcouldbeattributedtoan increaseinglycogencomparedtothewildtype.Thissuggestsa connectionbetweenvitaminEandglycogenmetabolismandfurther worktoelucidatethemechanismofglycogenaccumulationinslr1736isinprogressandwillbepresented. BryantDA(2003).Proc.Natl.Acad.Sci.USA17:9647-9640. SakuragiY,MaedaH,DellaPennaD,BryantDA(2006).PlantPhysiol. 141:508-52. Masuko,T.,Minami,A.,Iwasaki,N.,Majima,T.,Nishimura,S-I.,lee,Y. (2005).AnalyticBiochem.39:69-72. Fry,S.C.(1988).In:TheGrowingPlantCellWall.Wilkins,M(Ed).pp. 64-101.LongmanScientific&Technical,Harlow,UK.
P.126 OVEREXPRESSION OF PKNE (ALR3732) INHIBITS HETEROCYST DEVELOPMENT IN ANABAENA SP. STRAIN PCC 7120. SushantaKumarSaha,JamesW.Golden. DivisionofBiologicalSciences,MolecularBiologySection,University ofCalifornia,SanDiego,CA,USA. Introduction: Proteinkinasesplayakeyroleinsignalingpathwaysby modifyingtheactivityoftheirtargetproteins.Thenitrogen-fixing filamentouscyanobacterium Anabaena PCC7120containsseveral eukaryotic-typeserine/threonineproteinkinases.Reportedincreases ofintracellularCa2+ concentrationduringheterocystdifferentiation promptedustodeterminethespatiotemporalexpressionoffour putativeCa2+ dependentserine/threoninekinases,andthenfurther characterizetheroleofpknE inheterocystdevelopment. Methods: Anabaena strainswereconstructedtocontainGFP reporters,andtoknockoutandoverexpresspknE.Strainswere characterizedbystandardandfluorescencemicroscopy,determination ofgrowthcharacteristics,andwesternimmunoassayofproteins. Results: Weconstructedreporterstrainscontaininggfp reporter fusionswiththeupstreamintergenicregionsofthefourkinasegenes all2334,alr3732 (pknE),all4668,andall4838.Allofthesekinasegenes hadlowlevelsofGFPreporterexpressionwhengrownonBG-11 medium.Uponnitrogenstep-down,theGFPfluorescencewas increasedindifferentiatingcellsfromthepromotersPpknE andPall4668, notchangedfromPall2334,andverylowthroughoutthefilamentfrom Pall4838.WefurthercharacterizedpknE becauseitshowedstrongupregulationofGFPreporterexpression indifferentiatingcellsafter nitrogenstep-down.ThepknE genewasinactivatedbysingle recombination.Theresultingstrainwasnearlynormal,buthadshorter filamentswithslightlyhigherheterocystfrequencycomparedtothe wild typeafternitrogendepletion.OverexpressionofpknE fromits nativepromoteronamulticopyshuttleplasmidcompletelyinhibited heterocystformation,andthestrainwasunabletogrowonnitrogendepletedmedium.However, overexpressionfromthe copper-induciblepetE promoter,whichisexpressedinvegetative cells, didnotinhibitheterocystdevelopmentbutcaused a24hdelay inheterocystdifferentiation.ThePpetE-pknE strainalsoformedgranular pleomorphicvegetativecellsafter4daysofgrowthandhaddefective celldivisionresulting4to10cell-lengthslongcells onBG-11medium. WeexaminedexpressionofthehetR geneinpknE mutantand overexpressionbackgrounds.InthepknE mutantbackground,normal patternedexpressionofthePhetR-gfp reporterwasobservedduring heterocystdevelopment.Inthe strainoverexpressingpknE fromits ownpromoter,PhetR-gfp reporterexpressionwasinhibited;andwestern August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts blotimmunoassayshowed nodetectableHetRproteininthis strain. BecauseofhetR positiveautoregulation,thepknE overexpression phenotypecouldbecausedbyablockeitherupstreamor downstreamofHetR.Ingeneticepistasisexperiments,wefoundthat theintroductionofpknE onamulticopyplasmidintothreestrainsthat produce ahigherfrequencyofheterocysts,AMC451(∆patS), AMC1289(PpetE-hetRR223W), andAMC1537(∆hetR containinghetRHis6), causedcompleteinhibitionofnewheterocystformationafter nitrogenstep-down.Therefore,itappearsthatoverexpressionofpknE blocksheterocystdifferentiationdownstreamofHetR. Conclusions: ThepknE geneisdevelopmentallyregulated,andits properexpressionisrequiredforbothnormalvegetativecellgrowth andcelldivision,andfornormalheterocystdevelopment.
P.127 BIOCHEMICAL STUDIES OF THE PROTEINS ENCODED BY SOX GENES INVOLVED IN THIOSULFATE AND SULFIDE OXIDATION FROM THE GREEN SULFUR BACTERIUM CHLOROBACULUM TEPIDUM. HidehiroSakurai,TakuroOgawa,ToshinariFurusawa,MichikoShiga, RyoheiNomura,DaisukeSeo,NaomiHosoya-Matsuda,Kazuhito Inoue. Res.Inst.Photobiol.H2Production,KanagawaUniv.,Hiratsuka, Kanagawa,Japan. Introduction:ThegreensulfurbacteriumChlorobaculum tepidum, utilizesvarioussulphur compoundssuchassulfide,thiosulfateand elementalsulphur aselectrondonorsforphototrophicgrowth.Thesox (sulfur oxidizingsystem)geneclusterofthisbacteriumconsistsof soxF2XYZAKBW genes.Wehavepurifiedandcharacterizedthree proteins(thecoreTOMES,thiosulfateoxidizingmulti-enzymesystem) indispensableforinvitrothiosulfateoxidation.Wehavealsopurified SoxF2,andstudieditseffectsonthiosulfateoxidizingactivityofthe coreTOMESaswellasitssulfide oxidationactivities. Methods: C. tepidum cellswerephotoautotrophicallygrownwith sulfide andthiosulfateaselectrondonors,andproteinswerepurified toapparenthomogeneityasdescribedelsewhere(Ogawaetal.,2008, Ogawaetal.,inpreparation). Results:FromC. tepidum cells,wehavepurifiedthreefactors indispensableforthiosulfate-dependentreductionofsmall,monohemecytochrome(cyt)c-554:SoxYZ, SoxBandSoxAXK(thecore TOMES).Thelastcomponentistrimericprotein(alsocalledcytc-551) composedofmono-hemecytSoxA,mono-hemecytSoxX,andthe colour-lessproductofthehypotheticalORFCT1020 (soxK).TheSoxA, XandKwereseparatelyexpressedinEscherichia coli.Inthepresence ofothertwocoreTOMESfactors,therecombinantSoxAandSoxX showedalowbutdiscernibleactivityofthiosulfate-dependentcyt c554reduction.FurtheradditionoftherecombinantSoxKgreatly increasedtheactivity,andthetotalactivitywasashighasthatofthe nativeSoxAXKcomplex.TherecombinantSoxKparticipatedin formationofatightcomplexwithSoxAandSoxX,andisreferredtoas SAXB(SoxAX bindingprotein).ThehomologuesofSAXB gene(soxK) arefoundinroughlyaboutonethirdofthiosulfateoxidizingbacteria whosesox geneclustersequenceshavebeendepositedsofar, rangingoverChlorobiciae, Chromatiaceae, Hydrogenophilaceae, etc. EachofthededucedSoxAandSoxXproteinsofvariousbacteria constitutegroupsthataredistinctfromthosefoundinbacteriathat apparentlylacksoxK genehomologues. WehavepurifiedSoxF2asamonomericflavoprotein.Thecore TOMEScatalysedoxidationofboththiosulfateandsulfitewithvarious
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Final Program and Abstracts cytochromesaselectronacceptors,andSoxF2stimulatedtheformer andinhibitedthelatterreaction.SoxF2alonecatalysedoxidationof sulfidewithaKm valueofabout2μMwithvariouscytochromesas electronacceptors. Conclusions: ThecomponentsofthecoreTOMESofC. tepidum are basicallysimilartothosefoundinvariousthiosulfateoxidizingbacteria suchasthepurplebacteriumAllochromation vinosum andthe facultativeorganotrophicbacteriumParacoccus pantotrophus. However,wehavefoundthatSoxAXproteinadditionallybindsanew subunitSoxKwhichisabsentinsomeofthem. AlthoughsoxFgeneisfoundinthesox geneclusterofvarious bacteria,itsbiochemicalfunctioninthiosulfateoxidationhasnotbeen demonstratedbyinvitroexperiments.Wehaveshownforthefirst timethatitstimulatesthiosulfateoxidizingactivityofthecoreTOMES byreconstitutedsystem. References: Ogawa,T.etal.,SoxAXbindingprotein,anovelcomponentofthe thiosulfate-oxidizingmultienzymesysteminthegreensulphur bacteriumChlorobaculum tepidum.J.Bacteriol.190:6097-6110 (2008). Ogawaetal.,(inpreparation).
sequencesofAnabaena sp.PCC7120SPPSPS, andSuS-A, respectively.TheresultingHis-taggedfusionproteinsshowedSPP, SPS,andSuSactivity.Inaddition,cellfreeextractsfromM. aeruginosa werechormatographedthroughanionexchangecolumnandthe enzymeactivitieswereassayedintheelutedfractions.Whenwe studiedthebiochemicalpropertiesoftheenzymes,wefoundthatSPS specificitywassimilartothatofothercyanobacterial SPSs,and interestingly,SuSactivitycouldonlybemeasuredintheSuccleavage direction.ExpressionanalysesbyRT-PCRandNorthernblotting indicatedthatthethreegenesaretranscribedduringstandardculture conditionsofthecyanobacterium. Conclusions: ThisisthefirstreportshowingthatbothSucsynthesis andcleavagetakeplaceinapotentialtoxiccyanobacterium. Remarkably,weshowedthatSuSisalsopresentinaunicellular,nonN2-fixingstrain.ThefactthatthisenzymeactsonlyintheSuccleavage directionsuggeststhatitmightcorrespondtoanovelclassofSuS. Furtherstudiesonthisenzymemayshedsomelightintoitsreaction mechanismandintotheroleofSucinMicrocystis cells. References: CuminoAC,MarcozziC,BarreiroR,SalernoGL.PlantPhysiol.143, 1385-1397(2007) CurattiL,GiarroccoLE,CuminoAC,SalernoGL.Planta228,617-625 (2008)
P.128 SUCROSE METABOLISM IN MICROCYSTIS AERUGINOSA PCC 7806. MaríaA.Kolman,LauraE.Giarrocco,CorinaM.Berón,GracielaL. Salerno. CEBB-MDP,CIB,FIBA,MardelPlata,Prov.BuenosAires,Argentina. Introduction: Sucroseisoneofthemostcommonnon-reducing disaccharidesinNature.Ithasbeenextensivelystudiedinplants, whereitisthemaintransportphotosyntheticproduct,asourceof carbonandenergy,andamoleculeassociatedwithenvironmental stressandsignaltransduction.Duringthelastdecade,Sucmetabolism wasdemonstratedincyanobacteria.SucissynthesizedthroughatwosteppathwayinvolvingSPS (U/ADP-glucose:D-fructose-6-phosphate 2-α-D-glucosyltransferase)and SPP(sucrose-6F-phosphate-phosphohydrolase),eitherinunicellularorfilamentouscyanobacteriumstrains. However,SuccleavagebySuS(A/UDP-glucose:D-fructose2-α-Dglucosyltransferase),hasonlybeenreportedinfilamentous heterocyst-formingcyanobacteria,whereSucwasdemonstratedto playacrucialroleinN2 fixationandinpolysaccharideproduction. Objective: TheaimofthisstudywastoinvestigatetheroleofSucin Mycrocystis aeruginosa,a unicellularnon-N2 fixingstrain, wellknown asoneofthemostcommonbloom-formingcyanobacteriainfresh waterenvironments. Methods: M. aeruginosa PCC7806wasculturedinBG11medium. DNAfragmentscorrespondingtoSucmetabolismcandidategenes werePCRamplified.Theopenreadingframes(orfs)wereligatedinto thepRSETvector.TherecombinantHis–taggedproteinswere expressedinEscherichia coli BL21(DE3)pLysS.Proteinswerepurified byNi2+ affinitychromatography,whileproteinextractsandpurification, enzymeactivitiesandexpressionanalyseswereperformedasreported previouslyforAnabaena [Cuminoet al.2007;Curattiet al.2008]. Results: WesearchedintheM. aeruginosa genomeforsequences homologoustothoseoffunctionallycharacterizedgenescodingfor SPS,SPPandSuS.Weretrievedthreecontiguousorfs inthecontig 328: IPF_1566, IPF_1564,andIPF_1565whosededucedamino-acid sequencesareabout 55%,53%,and72%identicaltotheprotein 142
P.129 REGULATION OF AUTOTROPHIC AND HETEROTROPHIC METABOLISM IN SYNECHOCYSTIS SP. STRAIN PCC 6803 BY TWOCOMPONENT SYSTEM CLUSTERS ON BOTH THE CHROMOSOME AND A PLASMID. Louis.A.Sherman,SowmyaNagarajan. DeptartmentofBiologicalSciences,PurdueUniversity,WestLafayette, IN,USA. Introduction: Synechocystis sp.strainPCC6803(Synechocystis)isa freshwatercyanobacteriumwitha3.57Mbchromosomeand7 plasmids,including3over100kb.Thechromosomecontains80 ORFs(>2.5%)thatcodeforTwoComponentSystem(TCS)proteins thathelpthecelladapttoenvironmentalchanges.Weareinterested in theTCSinathree-geneclusterthatcontainsahistidinekinase (Hik31),aDNA-bindingresponseregulator(Rre34),andanupstream hypotheticalprotein(Uhp).Thesegeneswerestronglyupordownregulatedunderseveralgrowthandstressconditionsinthewildtype (WT)andvariousmutantsinmicroarraystudies.Importantly,thereare duplicate(>95%identical)operonsofthisTCSonthechromosome (sll0788-sll0790)andonthe plasmidpSYSX(slr6039-slr6041).No detailedstudyofplasmid-encodedgeneshasbeenperformedin Synechocystisandthepresenceofthesetwoclusters(withdifferent promoters)raisesimportantquestionsastothefunctionand differentialregulationoftheparalogs.ThisistheonlyduplicatedTCS inSynechocystis, andtheonlyclusterpresentonboththe chromosomeandaplasmid,among16sequencedcyanobacteria. Methods: Wehaveconstructeddeletionmutantsthatlackallthree genesintheputativeoperonorhik31 alone,oneitherthe chromosome ortheplasmid,andonboththechromosomeandthe plasmid.Methodologyincludedgrowthunderdefinedphysiological conditions,carefulmeasurementsofcelldoublingandmorphology, spectralanalysisandmicroarrays. Results:Wereportdifferencesinmorphology,pigmentcontentand growthbetweenthecultures.Initialphenotypeanalysissuggeststhat thechromosomaloperonisinvolvedinnegativecontrolofautotrophic August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes events,whereastheplasmidoperonisinvolvedinpositivecontrolof heterotrophicevents.First,thedeletionoftheentireplasmidoperon andthedeletionofbothoperonsresultedinstrainsthatwerenotable togrowinthepresenceof5mMglucoseunder12hL-12hD conditions.Secondly,theplasmidoperonmutantgrowsbetteron continuouslightthanunder12hLDconditions.Thirdly,thedouble hik31 mutantdemonstratedsignificantalterationsinpigment compositionwhengrowninthepresenceofglucoseunder moderatelyhighlight(150-200µmolesphotonsm-2s-1).Fourth,the doubleoperonmutantdemonstratedthepoorestgrowthunderall conditionsstudiedtodate.Finally,thechromosomeoperonmutant growsbestundermanyconditions,includingphotoautotrophicallyin highlightandmixotrophicallyinlowlight. Conclusions:Theseresultssuggesttheimportanceofgenesonboth thechromosomeandtheplasmidontheregulationofmajor metabolicprocesses.Inaddition,allthreegenesintheoperon appeartobefunctionallyimportant.Theuniquenatureofthese operonsandtheintriguingphenotypesofthemutantsindicatethat thissystemisavaluablemodelforthestudyofregulation.Supported byagrantfromtheDOE.
P.131 PROTEOME DYNAMICS IN NOSTOC SP. PCC 7120 AND NOSTOC PUNCTIFORME ATCC 29133. KarinStensjö1, SawYenOw2,TanaiCardona1,ArnaudTaton3,Ann Magnuson1,PeterLindblad1,PhillipC.Wright2. 1 DepartmentofPhotochemistryandMolecularScience,TheÅngström Laboratories,UppsalaUniversity,Uppsala,Sweden; 2Biological& EnvironmentalSystemsGroup,DepartmentofChemicalandProcess Engineering,TheUniversityofSheffield,Sheffield,UnitedKingdom; 3 CenterfortheStudyofBiologicalComplexity,Virginia CommonwealthUniversity,Richmond,VA,USA.
Introduction Nostoc sp.PCC7120andNostoc punctiforme ATCC 29133aremulticellularcyanobacteria capableoffixingatmospheric nitrogen.Thenitrogenfixingprocesstakesplaceinheterocysts; specializedcellswithalteredmetabolismtomediatetheN2-fixing process.Oneofouraimsistofurthertheunderstandingofthe metabolicbalancebetweentheheterocystsandthephotosynthetic CO2 fixingvegetativecellsthroughtheuseofquantitativeshotgun proteomics. Methods Relativecomparisonsbetweentheproteomesofenriched heterocysts,vegetativecellsandcompletefilamentswereachieved usingaquantitativeproteomicapproach.A4-plexor8-plexisobaric peptidetaggingtechnology(iTRAQ) workflowcoupledwith chromatographicpre-fractionationandtandemmassspectrometry wasused.Reliabilityinourdatasets isexemplifiedbybiological replicatesforallsamples. Results Morethan500proteinsfromeachstrainwereidentifiedwith confidentquantifications.Observationsprovidedbypurified heterocystanalysisenabledtheelucidationofthedominantmetabolic processesbetweentherespectivecelltypes.Approximately20%of theidentifiedproteomeshowedsignificantdifferentialregulation. Key indicatorslikethenitrogenaseenzymecomplexand proteinsofthe oxidativepentosephosphatecyclewereasexpected moreabundant inheterocystsofbothstrains.
Final Program and Abstracts Conclusions Byusingpurifiedheterocystsweareabletopresenta quantitativeinvestigationwithmuchhigherdetailrelatingNostoc sp. PCC7120andN. punctiforme heterocystspeciescomparedtoinour earlierinvestigation,providingresolutionsunachievablebywhole filamentproteomics.Complementarydatafromthetwo cyanobacterialstrainsexhibitsimilarmetabolictrends.Findings obtainedfromthisstudycontributetotheunderstandingofheterocyst specificmetabolismimportantfordownstreamresearchforbio-fuel productionfromN2 fixingheterocystouscyanbacteria. P.132 USING 23NA AND 31P NMR SPECTROSCOPY TO STUDY PH AND NA+ HOMEOSTASIS IN SYNECHOCYSTIS SP. STRAIN PCC 6803. JessicaLiTse,HaticeGizemYayla,MaryMAllen,NancyHKolodny. WellesleyCollege,Wellesley,MA,USA. Cyanobacterialiveinmanyextremeconditions,includinghypersaline, hyperosmotic,andofrecentinterest,increasinglyacidicenvironments causedbypollutionandindustrialwaste.Cellshavebeenshownto survivein,andadaptto,acidicconditionsbyraisingthepHoftheir environment[1],butthemechanismsthattheyusetoregulatetheir internal(pHin)andexternal(pHex)pHarespeculative.Wehypothesize thatthecyanobacteriauseNa+/H+ antiporterstoregulatetheirinternal pH.ThemodelorganismSynechocystis sp.strainPCC6803contains fiveputativeantiportergenes,whichcodefortransmembraneproteins thatactivelytransportNa+ intothecellcytoplasmandH+ outofthe cell,orvice-versa,dependingonthepHoftheenvironment [2].A BrukerAvance400MHzNMRspectrometerwasusedtodetect phosphorussignalsinsolution,suchasinternalandexternalinorganic phosphatetodeterminepHin andpHex,respectively,sugarphosphate, and -, -,and -ATP.Sodiumsignalsinsolution,bothinside(Na+in) andoutside(Na+ex)thecyanobacteria,weredetectedusing23NaNMR spectroscopy.SincetheNa+in andNa+ex peaksresonateatthesame frequency,ashiftreagent,Na4HTmDOTP,wasusedtoresolvethe Na+ex fromtheNa+in peakbyshiftingtheNa+ex peakdownfield.23Na and31PNMRspectrawereacquiredforcontrolcellsbufferedin minimalBG-11+Na2CO3 medium(pH≈10)andacid-stressedcells bufferedin60mMPIPESmedium(pH≈6.5).Aninitial23NaNMR spectrumwasacquiredafteradditionofNa4HTmDOTP,followedbya timedseriesof31PNMRspectratodeterminethepHin ofthecellsand afinal23NaNMRspectrum.23NaNMRspectrawereanalyzedto determinetheratiooftheintegralofNa+ex totheintegralofNa+in.The chemicalshiftsoftheinternalinorganicphosphatepeakshowthat althoughthecellsamplesweresuspendedindifferentpH environments,thecellswereabletomaintainapHin ofabout7.2.The pHex ofcontrolcellsdecreasedovertime,whilethepHex oftheacidstressedcellsincreased,suggestingthatpHregulationdoesoccur. Additionofexternalinorganicphosphateincontrolcellsproduced spectrainwhichtheinternalandexternalinorganicphosphatepeaks werecompletelyresolved,whichwasnotseeninspectraofacidstressedcells.Inboththecontrolandacid-stressedcellsamples,using 2.2mMNa4HTmDOTP,theintegralofNa+ex totheintegralofNa+in decreasedovertimebetweeninitialandfinal23NaNMRspectra acquisition,suggestingthattheamountofNa+in increasedovertime. [1]HuangJ.J. et al.(2002)Arch.Microbiol. 177,486–493. [2]WangH-L et al.(2002)Mol.Microbiol. 44,1493–1506.
Moreinterestingarethefindingsofproteins,suchasthioredoxins involvedinredoxregulationandsuperoxidedismutasesinvolvedin cellprotectiveprocesseswithadifferentabundanceinheterocystsas comparedtovegetativecells.
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P.133 WHICH IS THE INTRINSIC FORM OF PHOTOSYSTEM II, A MONOMER OR A DIMER? MaiWatanabe1,MasakoIwai2,ReiNarikawa1,MasahikoIkeuchi1. 1
UniversityofTokyo,DepartmentofLifeSciencesBiology;2Tokyo UniversityScience,FacultyScienceandTechnology,Departmentof AppliedBiologicalScience;Japan. Introduction: PhotosystemII(PSII)isamembraneproteincomplex consisting ofatleast20proteinsubunits,manypigmentsandlipids. Todate,bothdimericandmonomericPSIIcomplexeshavebeen preparedfromawiderangeofoxygenicphototrophs.However,the oxygenevolutionactivityofPSIIwasoftenhigherinthedimerthanin themonomer.Thus,itiswidelybelievedthatthePSIIcomplexmay functionasadimerinthethylakoidmembrane.Thecrystalstructures ofthedimericPSIIfromthermophiliccyanobacteriahavebeen determineduptotheresolutionof2.9Å.Here,wereportconversion betweenthedimericandmonomericcomplexesofPSIIbytreatment ofadetergentandgenedisruption. Methods: ThethylakoidmembranesfromThermosynechococcus elongatus weresolubilizedwithvariousconcentrationsofn-dodecyl-bD-maltoside(DM)andseparatedbyblue-nativePAGE(BN-PAGE)and subsequentSDS-urea-PAGE(16-22%acrylamide).Smallmembrane polypeptidegenepsbTc wasinactivatedbyreplacementwitha chloramphenicolcassetteandcompletesegregationwasconfirmed. Results: Whenthethylakoidmembranesweresolubilizedwithlow concentrationsofDM,therecoveryofthePSIImonomerwashigher thanthedimerinBN-PAGE,whilesolubilizationwithhigh concentrationsofDMgaverisetolowerrecoveryofthePSIImonomer thanthedimer.ThechlorophyllcontentinBN-PAGEestimatedby monochromaticscanningat670nmconfirmedtheinversecorrelation betweenthedimericandmonomericbandsdependingontheDM concentrations,despitethatmaximalrecoveryofthetotalPSIIwas achievedabove0.6%DM.Thiscorrelationwasverifiedasspot intensityinthetwo-dimensionalPAGE.Namely,spotsforthePSII integralsubunitsreflectedthechlorophylldistributioninBN-PAGE, beingexclusivelyfoundinthemonomerandthedimerbands.These resultssuggestthatsolubilizationwithhighconcentrationofDM artificiallyfacilitatesthedimerizationofthemonomer.Wealsostudied sequentialtreatmentofPSIIwith0.5%and4.5%DM.After solubilizationofthethylakoidswith0.5%DM,thesolubilizedPSIIwas recoveredbycentrifugationandfurthertreatedwithorwithout additionof4.5%DM(final5.0%).Resultsthusobtainedwerevery similartothoseintheone-stepsolubilization.Thisindicatesthata majorpartofthedimerisgeneratedfromthemonomerafter solubilizationbyactionoftheadditionalDMtreatment.Inthecrystal structure,therearefourDMmoleculesatthemonomer-monomer interfaceofPSII.ThismaysuggestthatthedimericPSIIinthecrystalis artificiallystabilizedbyinsertionoftheDMmolecules.Therearealso PsbTcpolypeptidesatthemonomer-monomerinterfaceinthePSII crystal.WestudiedthePsbTc-depletedPSIIbyBN-PAGEafter solubilizationwithDM.AtlowconcentrationsofDM,verylittleamount ofthePSIIdimerwasrecovered,whiletherecoveryofthedimerwas increasedathighconcentrations.ThisresultsuggeststhatPsbTcis essentialforthestabilityofthedimerinlowconcentrationsofDM,but isnotessentialforthedimerizationdrivenbyhighconcentrationsof DM.
144
Conclusion: WeconcludethatthePSIIcomplexexistsinthethylakoid asamonomeroralooselyassociateddimerwhichissurroundedby lipids.PsbTcisessentialforstabilizationofthedimerbutnotessential forthedimerizationdrivenbyhighconcentrationofDM.
P.134 THREE INDIVIDUAL POINT MUTATIONS IN SYNECHOCOCCUS PCC6301 RUBISCO CONFER INCREASED OXYGEN TOLERANCE IN VIVO UNDER AEROBIC CHEMOAUTOTROPHIC GROWTH CONDITIONS IN RHODOBACTER CAPSULATUS SBI/II-. BrianWitte,StephanieScott,SriramSatagopan,F.RobertTabita. OhioStateUniversity,Columbus,OH,USA. Introduction: Ribulosebisphosphatecarboxylase/oxygenase (RubisCO)isthekeyenzymeoftheCalvin-Benson-Bassham(CBB) pathway,andisdirectlyorindirectlyresponsibleformostofthe organiccarbononEarth.Despiteitsimportance,theenzyme possessesconfusedsubstratespecificityandoftenwastescellular carboninincorporatingO2 insteadofCO2.Thisstudyusesrandom mutagenesisoftheRubisCOgenefromSynechococcus PCC6301and apowerfulbiologicalselectionsystemthatwaspreviouslydeveloped inRhodobacter capsulatus toselectformutantenzymesthatconfer increasedoxygentolerancein vivo.Further,themutantRubisCOs havebeenpurifiedandcharacterizedin vitro.Wehypothesizethata specificregionoftheRubisCOlargesubunitisespeciallyimportantfor interactionswithO2. Methods: Thegenesencodingthelarge(rbcL)andsmall(rbcS) subunitsofRubisCOfromSynechococcus PCC6301wereclonedin tandemintopUC19.Thevectorwithinsertwasusedasthetemplate inrepeatedroundsofmutagenicpcr.ThepoolofmutagenizedrbcLS wassubclonedintothebroadhostrangeplasmidpRPS-MCS3and subsequentlytransferredintotheRubisCO-deletionstrainofR. capsulatus SBI/II-.Individualcolonieswereusedforcreatingreplica platesforincubationunderphotoheterotrophic(minimalsaltsplus malate,CO2/H2 atmosphere),photoautotrophic(minimalsalts,CO2/H2 atmosphere)andchemoautotrophic(minimalsaltsplates,CO2/H2/O2 atmosphere).LevelsofintracellularRubisCOweremonitoredusing Westernblotstoascertaintheapproximatefractionoftotalprotein comprisedofRubisCO.Mutanted6301genesthatcomplemented growthunderallautotrophicconditionsweresubclonedinto Escherichia coli expressionvectorpSKB3andtheproductspurified andcharacterizedin vitro. Results: Usingthe6301numberingscheme,mutationsatresidues 259,269and375enabled6301RubisCOtocomplementgrowthin SBI/II- underaerobicchemoautotrophicconditions.Growthcurvesof SBI/II- complementedwitheachmutant,aswellasWT6301,are provided.Thekineticdatawithrecombinantproteinsindicatedthat eachmutationconfersasubtlydifferenteffectonRubisCO mechanism,althoughKm(CO2), Km(O2) andkcat wereaffectedto differentdegrees. Conclusions: ItisdifficulttosaythatanyoneofthemutantRubisCOs istruly“better”althoughtheydodefinitelyconferanincreased toleranceforoxygenunderchemoautotrophicgrowthconditionsand enablegrowthunderconditionswherethewild–typeenzymedoes not.Interestingly,noneoftheresiduesidentifiedinthisstudyare locateddirectlywithintheactivesiteoftheenzyme.Previousstudies haveshownthatalterationstoactivesiteresiduesalwaysresultsina lossofenzymefunction.Rather,thisstudyseemstoindicatethe importanceofresiduesneighboringtheactivesitethatcouldaffect theaccessofgaseoussubstratestotheenediolofribulose bisphosphateinthereactioncenter.Itisalsopossiblethatoneor August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes moreoftheseresiduesalterinteractionswithR. capsulatus intracellular chaperonessuchthat theobservedgrowthunderaerobicconditions maybedueinparttogreaterstabilityofthemutantenzymes. Nevertheless,thecombinationofinvivocomplementationandgrowth underselectiveconditionscombinedwithin vitro enzymological studies,projectsanintriguingnewdirectionforresearchintothebasis forRubisCO’sinteractionwithoxygen.
P.135 THE REDOX-SENSING MECHANISM OF REGB IN RHODOBACTER CAPSULATUS. JiangWu,CarlBauer. IndianaUniversity,Bloomington,Indiana,USA. RegB/RegAisaglobalredox-respondingtwo-componentsystemthat regulatesmanyenergy-generatingandenergy-utilizingprocessesin Rhodobacter capsulatus. Previousstudieshaveshownthat thesensor kinaseRegBmodulatesitskinaseactivityandsenseredoxsignals throughtwodifferentsignal-inputsites:aubiquinone-bindingsite locatedinaperiplasmicloopwhichcanmonitorstheoxidationstates ofubiquinonepoolandaredox-activecysteineinthecytosolic domainthatrespondstocellularredoxstate.Theubiquinone-binding siteandredox-activecysteineappeartoworkindependentlyandboth controlapartofRegBactivity. TofurtherelucidatethemechanismbywhichRegBsensestheredox signal,therolesoftwohighlyconservedresidues inthe transmembranedomain,R31andQ38,havebeenanalyzedby mutagenesisstudy.Mutationsatbothoftheseresiduescompromised theinhibitoryeffectofoxidizingconditiononRegBactivity,indicating thatR31andQ38areinvolvedinredoxsensing.Toinvestigatehow exactlyRegBinteractswithitsredoxsignalubiquinone,thebinding affinitiesofoxidizedubiquinoneanalogQ0andreducedQ0 toRegB havebeendeterminedbyIsothermalTitrationCalorimetry(ITC).ITC determinedtheKaofoxidizedQ0 toRegBwhilethereisno detectablebindingaffinityforreducedQ0.ConsistentwithITCdata,in vitro phosphorylationassayshowedthattwomutationsinthe ubiquinone-bindingsite,N110QandF112Y,willgiveRegBhigher activityinthepresenceofoxidizedQ0.In vivo, bothmutantshad elevatedphotosystemexpressionlevelsunderaerobicconditions. Thus,weconcludethatoxidizedubiquinoneistheformthatbindto RegBandinhibitRegBactivity,whenubiquinoneisconvertedto reducedform,itwillbedissociatedfromRegBandRegBactivitywill bereleased.
P.136 DISCOVERY OF NOVEL O2-TOLERANT NIFE-HYDROGENASES FROM ENVIRONMENTAL MICROBES TO CONSTRUCT A CYANOBACTERIAL RECOMBINANT FOR SOLAR H2 PRODUCTION. QingXu,GergelyMaroti,WalterVargas,YingkaiTong,ShibuYooseph. J.CraigVenterInstitute,Rockville,MD,USA. Hydrogenisacleanalternativetogasolineandotherfossilfuels. Photosyntheticcyanobacteriahaveattractedconsiderableattentionin recentyearssincetheycanphotolyticallysplitwatertosupportH2 productionthroughhydrogenasecatalysis.However,onemajor drawbackofthebiophotolysisprocessisthattheirH2-evolving hydrogenasesareextremelysensitivetoO2.TodevelopanO2tolerantbiophotolyticsystem,wearesearchingforO2-tolerant NiFe-hydrogenasesthatcanbetransferredintocyanobacteria.The oceansharboranabundanceofmicroorganismswithH2-production capabilityandtheyaregoodresourcesforidentifyingnovelO2August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts toleranthydrogenases.TheVenterInstituterecentlyconducteda metagenomicstudyofthemarineplanktonicmicrobesintheglobal ocean.Wesearchedthemetagenomicdataofthisglobalocean samplingprojectforputativenovelNiFe-hydrogenasesbyusing probabilisticmodelingmethods.Oneofthenovelhydrogenases identifiedfromtheglobaloceanwasclonedfromenvironmentalDNA samples,whichshowsstronghomologytoaknownO2-tolerant hydrogenasefromaphototropicbacteriumThiocapsa roseopersicina. TheclonedenvironmentalDNAcontainsthestructuralgenes(hynS andhynL)ofthisnovelhydrogenase.Locatedimmediatelyupstream ofhynS/hynL,twoaccessorygenes(hupH andhynD)werealso identified.ThroughheterologousexpressioninT. roseopersicina,we convertedtheenvironmentalDNAintoafunctionalNiFehydrogenase.TransferringthenovelNiFe-hydrogenaseinto cyanobacteriumSynechococcus elongates PCC7942isinprogress.
P.137 RHODOBACTER CAPSULATUS HBRL AND IRR: TWO MORE TRANSCRIPTION FACTORS INVOLVED IN THE REGULATION OF THE HEME BIOSYNTHESIS. SébastienZappa,CarlE.Bauer. DepartmentofBiology,IndianaUniversity,Bloomington,IN,USA. ThepurplephotosyntheticbacteriumRhodobacter capsulatus presentsaversatilemetabolism,capableofgrowingfromdarkaerobic tolightanaerobicconditions.Inordertoachievesuchmetabolic adaptations,thesynthesisofpigmentshastobetightlyregulatedand thebalancebetweenthemajortetrapyrrolesheme, bacteriochlorophyllandcobalaminmaintainedatadequatelevels.It hasbeenshowninthelabthatseveraltranscriptionfactorsare involvedintheregulationoftetrapyrrolesynthesis, i.e. RegA,CrtJ, AerR.Morerecently,studieswereundertakenabouttwonew transcriptionfactors,namelyHbrLandIrr.Whilethefirstoneisa memberoftheLysRfamily,thesecondisaFurrepresentative.Both werestudiedwithregardtotheireffectonhemebiosynthesis,asa functionoftheexogenoushemecontentortheavailabilityofironin thegrowthmedium.Theirrespectiveroleswereinvestigatedby geneticandbiochemicalapproaches.Indeed,genedisruptedstrains wereproducedandtheirphenotypes,regardingtheexpressionof hemesynthesisgenes,wereanalyzed.Inaddition,bothproteinwere heterologouslyexpressedinEscherichia coli TUNER(DE3),usingthe pSUMOvector.Purificationoftherecombinantproteinbyaffinity chromatographyenabledtoundertakestudiesregardingDNAand/or co-factorbinding.ItappearedthattheHbrLdeletedstrainisvery sensitivetoiron-chelatedmedia,whiletheIrrmutantshowsacellular hemecontentapproximately25%higherthanthewild-typeandthe HbrL-deletedstrains.Regardinggeneexpressionoftheheme biosynthesispathway,whileHbrLshowsacontrolonhemA,hemBand hemZ,itlookslikeIrrhasaneffectonhemCandhemE.Finally,the biochemicalcharacterizationgavesomeinsightsoftheDNAbinding mechanism,suchtheasthebindingofHbrLtopromoterssuchas hemAandpuf,asshownbygelmobilityshiftanalysis,andpreliminary DNAseIfootprintingassay.Asaconclusion,thisworkconfirmsthat HbrLandIrrareinvolvedintheregulationofthecomplextetrapyrrole synthesispathway,inadditiontopreviouslycharacterizedtranscription factors.
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P.138 METABOLIC ENGINEERING OF SYNECHOCYSTIS SP. PCC 6803 A SYSTEMS BIOLOGY APPROACH. YvonneZilliges,Jan-ChristophKehr,WolfgangLockau,Thomas Börner. Humboldt-UniversitätzuBerlin,InstitutfürBiologie, Berlin,Germany. Introduction: Cyanobacteria,alsoknownasblue-greenalgae, arethe onlyknownoxygenicphotoautotrophicprokaryotes.Theyplayamajor roleinglobalecologyandtheassimilationofinorganiccarbonfrom theatmosphere.Thegenomeoftheunicellularcyanobacterium Synechocystis sp.StrainPCC6803wasthefirsttobefullysequenced ofanyphototrophicorganism.Thisstrainthusrepresentsanideal candidateforasystems-leveldescriptionofprimarymetabolismand itsregulation–ultimatelyaimingataniterativeconstructionofa computationalmodelofthecoremetabolicprocesses.The combinationofametabolicnetworkmodelwith biochemicaland physiologicaldataallowforadeepunderstandingofphotosynthetic andmetabolicprocessesandtheirregulationinasystemsbiology approach. Methods and Results: Inourstudies,weanalyzedseveralmutants whereenzymesofglycolysisandespeciallyofpyruvatemetabolism,of metabolismofstoragecompoundsandoftheCalvincyclewere manipulated.Thesemutantswerecharacterizedwithrespectto growthparameters,pigmentationandintra-aswellasextracellular metabolitesbyionchromatographyandopticenzymatictestsfor metabolicfingerprinting.Thesedataareusedforasystems-level descriptionofprimarymetabolismanditsregulation.Thisapproachto amodelisalsoassistedbytranscriptomicdataandwillbefurther refinedbytheincorporationofexperimentallydeterminedkinetic parametersofselectedenzymes. Conclusion: Theestablishmentofanaccuratemodelcombines differentdatafromtranscriptomics,metabolomicsandkinetics.Using theestablishedmethodologyofflux-balanceanalysis,weevaluatethe mainmodesofmetabolism,identifymetabolicbottlenecksand correlatedreactionsets. Basedonthecomputationalmodel,mutationscouldbeintroduced andtheresultingphenotypescomparedtothetheoreticalpredictions. Thefinalmodelisexpectedtopermittheselectionofcandidate genesfortheoptimizationofbiotechnologicalprocesses.
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Exhibitors
ENVIRONMENTAL PROTEOMICS
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Notes
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Index of Presenters Abed,Raeid
P.090
Dragnea,Vladimira
P.009
Iwamori,Shunsuke
Adams,David
OC-11.6,P.035
Dragomani,Tierna
P.098
Jackson,Owen
Ahn,Chi-Yong
P.066
Druga,Bogdan
P.042
Jacobsen,Jacob
P.046
Akinwole,Philips
P.057
Duggan,Paula
P.099
Johansen,HenningKristian
P.064
P.036
Eaton-Rye,JulianJ
Al-Shehri,Abdulrahman Alexova,Ralitza
OC-12.5
Allison,L.F.
P.037
Eddie,BrianJ. Eisenhut,Marion
OC-6.1 P.100 OC-6.6
Elhai,Jeff
OC-18.3
Espie,George
P.101
Karp,Peter
Ferreira,Daniela
P.043
Katayama,Mitsunori
Kaplan,Aaron
Attia,Magdy
P.038
Autenrieth,Caroline
P.001
Fewer,David
OC-13.2
Kawabata,Tadashi
Azai,Chihiro
P.002
Florencio,FranciscoJ.
OC-10.4
Keren,Nir
Bagheri,Homayoun
P.078
Flores,Enrique
OC-1.6 P.003
Bauer,Carl Beatty,J.Thomas
PL-6.1 PL-7.2,P.004
Bennette,Nicholas
P.091
Bergman,Brigitta
PL-2.1
Bibby,ThomasS.
OC-1.2
Bird,David Biswas,Avijit Blankenship,Robert
Forchhammer,Karl
PL-3.3
KhalfaouiHassani,Bahia
Fortin,Nathalie
OC-15.5
Kirilovsky,Diana
Frigaard,Niels-Ulrik
OC-13.1
Kitashima,Masaharu Knoop,Henning
P.114
Galmozzi,CarlaV.
P.102
Kobayashi,Masayuki
P.015
P.103
Komada,Jun
P.070
GarciaCostas,Amaya
P.061
Korn,Anja
P.016
P.092
Garcia-Pichel,Ferran Ghosh,Robin
OC-16.5,P.104 P.105
Gomolla,Dorothea
P.011
Bryan,Samantha
P.094
Gorlenko,Vladimir
PL-1.3,P.062
Grammel,Hartmut
OC-15.4
OC-9.2 P.006,P.007
Grossman,Arthur
Burroughs,Nigel
P.039
Gugger,Muriel
Campbell,Douglas
P.095
Haande,Sigrid
Cardona,Tanai
P.032
Hagemann,Martin
Kurmayer,Rainer Kushige,Hiroko
OC-11.1,P.017
PL-4.1
Laguna,Rick
P.047
Le,ThiAnhTuyet
P.048
Lechno-Yossef,Sigal
P.018
PL-2.2
Lee,Young-Ki Lindblad,Peter Liu,Jinjie
OC-5.4
P.063
Liu,Zhenfeng
OC-4.3
PL-3.2
Ludwig,Frank
P.081 P.019
Ludwig,Marcus
Christman,Harry
OC-5.1
Haimovich,Maya
OC-11.5
Luther,Amanda
Cox,RaymondP.
P.060
Hanson,ThomasE.
OC-4.1
Csotonyi,JuliusT.
P.040
Harwood,Caroline
PL-9.1
PL-5.1
Hervás,Manuel
P.012
OC-7.1
Hess,Wolfgang
OC-11.3
OC-14.2 P.079
P.071 OC-18.1
P.080
P.106
P.097
P.115
Kyndt,John
Hahn,Alexander
OC-17.3,P.041
OC-15.6
OC-3.3,P.044
P.008,P.096
August 9 to 14, 2009 • Montréal, QC, Canada
OC-8.1
Gao,Qunjie
Golden,Susan
Dorador,Cristina
P.113
Knaff,David
P.059
Doná,Clelia
P.014 PL-6.2
P.010
P.005
Dey,Swati
OC-9.4
P.077
Brown,IgorI.
DePhilippis,Roberto
P.013 OC-16.6
Gaertner,Katrin
Boulay,Clémence
Dangel,Andrew
P.112
P.058 PL-1.1
Daldal,Fevzi
OC-2.4
Fuhrmann,Eva
Giani,Alessandra
Chen,Min
OC-6.5,OC-10.1
Kerfeld,Cheryl
P.093
Burnap,Robert
OC-1.5
PL-3.1
Bonsu,ErnestO.
Bryant,DonaldA.
P.111
Jungblut,AnneD.
OC-10.5
Batyrova,Horcheska
OC-3.2
Joshi,Gauri
Appel,Jens
Ballot,Andreas
OC-5.2
Johnson,Zackary
Alvey,Richard
OC-14.1
P.110
Maeda,Isamu
P.116 OC-17.1,P.117
Magnuson,Ann
OC-12.3
Maness,Pin-Ching
OC-18.2
Masepohl,Bernd
PL-6.3
Hirose,Yuu
P.107
Masukawa,Hajime
Hosokawa,Norimune
P.108
Mazard,Sophie
Inoue-Sakamoto,Kaori
P.045
McNeely,Kelsey
OC-12.2
Ishizuka,Takami
P.109
Meeks,Jack
OC-13.3
P.049 P.065
149
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Index of Presenters and Chairs Mella-Herrera,Rodrigo Meyer,Terry Midorikawa,Takafumi Miller,Mette Mulkidjanian,ArmenY. Mullineaux,Conrad Mundt,Sabine Narikawa,Rei
P.118 P.020,P.021
Shastik,Evgeny
P.073
Shen,Gaozhong
OC-9.6
Wu,Jiang
P.135
Xu,Qing
P.136
P.119
Sherman,Louis
PL-5.2,P.129
Xu,Yu
P.056
OC-14.3
Shimura,Yohei
P.074
Yamamoto,Haruki
P.030
PL-1.2,P.022
Sicora,Cosmin
P.033
Yeager,Chris
OC-15.2
OC-5.3
Sivonen,Kaarina
PL-9.3
P.050
Song,Hong-Gyu
P.053
Yurkov,Vladimir
OC-2.2
Zannoni,Davide
OC-17.2
Yin,Liang
OC-7.2,P.031
OC-16.3
Spence,Edward
P.067
Nenninger,Anja
P.082
Stadnichuk,Igor
OC-11.2
Zappa,Sébastien
P.137
Nicolaisen,Kerstin
P.120
Stal,Lucas
OC-10.2
Zilliges,Yvonne
P.138
Steglich,Claudia
OC-11.4
Núñez-Cardona,M.T. Oh-oka,Hirozo Ohki,Kaori Ohta,Hisataka
P.072,P.083 P.121 OC-3.1 P.122
Stensjö,Karin
P.131
Summerfield,Tina
OC-12.1
Summers,Michael
OC-5.5
Otaki,Hiroyo
OC-2.6
Suzuki,Eiji
Ouchane,Soufian
OC-7.3
Suzuki,Iwane
Overmann,Jörg
OC-2.1
Tabita,F.Robert
KN-2.1
P.023
Takaichi,Shinichi
OC-8.2,P.054
Owttrim,GeorgeW. Palinska,KatarzynaA. Park,Jeong-Jin
OC-1.4 P.123
Partensky,Frederic
PL-5.3
Pedersen,MarieØ.
OC-9.1
Pereira,Sara
P.051
Pitt,FrancesD.
OC-10.3
Preuflel,Karina Razi,Saiqa Redding,Kevin
OC-12.4 PL-7.1
Tamagnini,PaulaM.
PL-9.2
Tang,Kuo-Hsiang
OC-4.2,P.026
Tank,Marcus
OC-1.1
Tekucheva,Darya
P.055
Thiel,Teresa
OC-5.6
Thiel,Vera
P.086
P.124
Tomitani,Akiko
P.087
P.052
Tse,JessicaLi
P.132
Tsukatani,Yusuke
P.027
OC-6.4
Rosgaard,Lisa
P.125
Tsygankov,Anatoly
Saha,SushantaKumar
P.126
Vasquez,Yasmin
P.034
Sakamoto,Toshio
OC-10.6
Ventura,Stefano
OC-1.3,P.068
Sakurai,Hidehiro
OC-15.1,P.127
Salerno,GracielaL.
P.128
Sandmann,Gerhard
OC-15.3
Vermaas,Wim
OC-6.2,OC-9.3
Vioque,Agustin
P.075
OC-8.3
Vogl,Kajetan
P.076
Schaefer,Steffani
OC-6.3
Walsby,A.E.
Schluchter,Wendy
OC-16.2
Watanabe,Mai
Schmetterer,Georg
OC-16.1
Watson,SusanB.
Schumann,Susanne Schwarz,Christoph Schwarz,Rakefet Setterdahl,Aaron Severin,Ina Shabeb,Mohamed
150
P.084 OC-2.5 OC-16.4 P.025 OC-2.3 P.085
KN-1.1 P.133 OC-12.6
Wegener,KimberlyM. Wilmotte,Annick Wilson,Adjélé Witte,Brian Wolk,PeterC. Woronowicz,Kamil
P.028
P.069,P.088,P.089 P.029 P.134 PL-4.2 OC-9.5
August 9 to 14, 2009 • Montréal, QC, Canada
FINAL PROGRAM and ABSTRACTS
www.ispp2009.com
August 9 to 14, 2009
WEEK-AT-A-GLANCE SUNDAY August 9
TUESDAY August 11
WEDNESDAY August 12
THURSDAY August 13
FRIDAY August 14
09:30
Plenary 1 R. Blankenship A. Mulkidjanian V. Gorlenko
Plenary 3 E. FLores M. Hagemann K. Forchhammer
Plenary 5 F. Daldal L. Sherman F. Partensky
Plenary 6 C. Bauer D. Kirilovsky B. Masepohl
Plenary 8 C. Harwood P. Tamagnini K. Sivonen
10:00
Coffee Break
Coffee Break
Coffee Break
Coffee Break
Coffee Break
Oral Communications 1&2
Oral Communications 5&6
Oral Communications 9 & 10
Oral Communications 11 & 12
Oral Communications 15 & 16
Lunch
Lunch
Lunch Régence C & Foyer
Régence C or Tour de Ville / Check your Ticket in your badge holder
Régence C or Tour de Ville / Check your Ticket in your badge holder
Régence C & Foyer
Oral Communications 3&4
Oral Communications 7&8
Oral Communications 13 & 14
Oral Communications 17 & 18
Plenary 7 I. Suzuki J.T. Beatty
Closing Keynote F.R. Tabita
07:30
MONDAY August 10
Hotel Delta Centre-Ville Montréal, QC, Canada
Poster Mounting 07:30 to 10:00
08:00 08:30 09:00
10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00 14:30 15:00 15:30 16:00
Registration
Poster Session
16:30 17:00 Welcome & Opening Keynote 18:00 A.E. Walsby 17:30
18:30 19:00 19:30
Opening Reception
Plenary 2 B. Bergman A. Grossman
Free Afternoon
Poster Session
Plenary 4 S. Golden P.C. Wolk
Poster Session
ISPP 2009 Party Sucrerie de la Montagne
20:00 20:30 21:00 21:30 22:00
Buses leave at 17:30 from Hotel Delta Centre-ville
Lunch
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Table of Contents General Information Floor Plans
2
Welcome / Previous ISPP Symposia
3
Committees
4
Symposium Information
5
About Montréal & Useful Information
6
Social Program
7
Scientific Program
9
How This Program Is Organized
10
Instructions to Oral and Poster Presenters
10
Invited Faculty
11
Detailed Program Sunday, August 9
13
Monday, August 10
15
Tuesday, August 11
19
Wednesday, August 12
23
Thursday, August 13
25
Friday, August 14
29
Exhibitors
147
Presenter Index
149
Abstracts Keynotes and Plenaries
33
Oral Communications
45
Posters
85
Symposium Secretariat
Printed in Canada, August 2009 Recycled paper
August 9 to 14, 2009 • Montréal, QC, Canada
c/o IS Event Solutions 1334 Notre-Dame Street West, 2nd Floor Montréal, Québec, Canada H3C 1K7 Tel.: (514) 392-7703 Fax: (514) 227-5083 E-mail: [email protected]
1
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Floor Plans
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Site Plan Floor C
VITRÉ
ROYER
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FOYER
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Welcome to Montréal Welcome to Montréal to ISPP 2009, the 13th International Symposium on Phototrophic Prokaryotes. This is only the second time that the ISPP series finds itself in North America, and we hope that you find Montréal, a major cultural and scientific hub of North America, a pleasant and exciting blend of Old World charm and New World vitality. This is one of my favourite conference series to attend because not only does it inform my specific research passions, it broadly educates me in ways that improve my teaching and increase my breadth of understanding in a variety of outside areas. Indeed, the ISPP series brings together a broad spectrum of interests, techniques, and disciplines. The planned plenary and parallel symposia sessions will permit oral communications by both established researchers and newcomers to this field, as well as promoting active audience participation and lively discussions. Poster sessions will complement the oral program. In addition to the planned social events, we hope that the venue, and the surrounding city filled with small cafés and bistros, will be conducive to productive and insightful informal discussions. One of the highlights of past conferences in this series is the high level of active participation by attendees. We count on you to make ISPP 2009 a great success.
Enjoy!
Patrick Hallenbeck ISPP 2009 Conference Chair Université de Montréal Montréal, Quebec, Canada
Previous ISPP
August 9 to 14, 2009 • Montréal, QC, Canada
1st
1973
Freiburg, Germany
2nd
1976
Dundee, United Kingdom
3rd
1979
Oxford, United Kingdom
4th
1982
Bombannes, France
5th
1985
Grindelwald, Switzerland
6th
1988
Noordwijkerhout, The Netherlands
7th
1991
Amherst, USA
8th
1994
Urbino, Italy
9th
1997
Vienna, Austria
10th
2000
Barcelona, Spain
11th
2003
Tokyo, Japan
12th
2006
Pau, France
3
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Committees
Local Organizing Committee Patrick Hallenbeck, Université de Montréal; Chair Tom Beatty, University of British Columbia David Bird, Université de Québec à Montréal Doug Campbell, Mount Allison University George Espie, University of Toronto George Owttrim, University of Alberta Charles Trick, University of Western Ontario Vladimir Yurkov, University of Manitoba
International Scientific Committee Chair: Jörg Overmann, Germany
4
Group I. Reaction Centres, Antennae, Bioenergetics
Group III. Cell Structure and Biology, Development
Robert Blankenship, USA
Dave G. Adams, United Kingdom
Richard J. Cogdell, United Kingdom
Judith Armitage, United Kingdom
David B. Knaff, USA
Cheryl Kerfeld, USA
Mette Miller, Denmark
Katsumi Matsuura, Japan
Conrad Mullineaux, United Kingdom
Frederic Partensky, France
Wim Vermaas, USA
Satoshi Tabata, Japan
Group II. Metabolism, Biosynthesis, Physiology
Group IV. Taxonomy, Ecology and Evolution
Christiane Dahl, Germany
Patrizia Albertano, Italy
Antonia Herrero, Spain
Ferran Garcia Pichel, USA
Neil Hunter, United Kingdom
Akira Hiraishi, Japan
Masahiko Ikeuchi, Japan
Jörg Overmann, Germany
Oded Beja, Israel
Annick Wilmotte, Belgium
Paula M. Tamagnini, Portugal
Vladimir Yurkov, Canada
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Symposium Information Book of Abstracts
Lost and Found
The abstracts have been included in this publication, immediately following the detailed program section. Abstracts are also available online.
Any lost and found items will be held at the information desk in room Royer for the duration of the event. For any unclaimed items after the conference, please contact IS Event Solutions.
Business Centre
Luggage
The business centre can take care of photocopies, sending faxes etc. It is located one level below the lobby in the administrative offices of the hotel.
During the ISPP conference, whether you are staying at the Hotel Delta Centre-Ville or at a different hotel, you can leave your suitcases with the concierge.
Eco-friendly Event
Meals and Refreshments
We incorporated the following considerations into the planning of this conference: • Online abstract review requiring no printing • Onine registration system requiring no printing • E-mail marketing requiring no printing • Pitchers instead of bottled drinks • Milk and sugar containers instead of individual packaging • Recyclable and environmentally-friendly delegate bags
Coffee breaks will be served daily in the morning and afternoon in the foyer area. For times of service, please refer to the detailed program section.
The hotel is a 4 green key’s property which means that it has shown national industry leadership and commitment to protecting the environment through wide ranging policies and practices. The hotel has mature programs in place that involve management, employees, guests, and the public, and which have shown substantial and measurable results.
Lunch will be served in in different locations: Monday and Friday – Buffet lunch in Régence C and foyer Tuesday and Thursday – Régence C and Tour de Ville (check your lunch ticket!)
Poster Area Posters are installed in room Cartier AB. While delegates can visit posters at their convenience, we have scheduled specific poster sessions on Monday, Tuesday and Thursday.
Emergency/First Aid
Public Notice
For any emergency or first aid services inside the Hotel Delta Centre-Ville, please dial “0” for the operator from any available house phone.
The Hotel Delta Centre-Ville specifically and all Quebec public spaces including terraces in general are smoke-free environments. Smoking is permitted only outside of buildings.
Information
Registration
Should you require any assistance during the Conference, please call 514-991-3851. The Conference Services Desk will be located in the foyer on Sunday and Monday. As of Tuesday, the information desk is located in room Royer (across from Plenary room).
Registration is located in the foyer area of Régence. As of Tuesday, the information desk will be available in the room Royer.
Internet Access Wireless Internet is available for delegates carrying a laptop and can be accessed in the lobby area. Laptops must be wi-fi enabled. Some Internet stations are available inside room Royer. This room will be open during conference hours.
Registration Hours Sunday, August 9
15:00-20:00
foyer
Monday, August 10
07:30-17:00
foyer
Tuesday, August 11
07:30-17:00
Royer
Wednesday, August 12
08:00-12:00
Royer
Thursday, August 13
08:00-17:00
Royer
Friday, August 14
08:00-15:00
Royer
Language Although French is the official language of Quebec, visitors will have no problem communicating in English throughout Montréal. English is the official language of the congress.
August 9 to 14, 2009 • Montréal, QC, Canada
Name Badges For identification and security purposes, delegates must wear their name badges when onsite at the Hotel Delta Centre-Ville. 5
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
About Montreal – Activities You will love Montréal in August. This European-style, multicultural city invites you to enjoy the warm balmy days of summer when Canadians love to be outside enjoying parks and the lively cityscapes. We invite you to enjoy a contagious joie de vivre with a vibrant social life in cafés and on outdoor terraces! The Hotel Delta Centre-Ville is located downtown, close to many attractions, restaurants and shopping.
Rogers Cup
Les Tam-tams du Mont-Royal
August 8-16 • Jarry Park Stadium The best tennis players in the world meet in one of the most prestigious tournaments on the men's professional tour.
Ongoing • Mont-Royal Every Sunday, this free, unofficial festival takes place in the vast green spaces of Mount Royal Park on Avenue du Parc, exactly half-way between Avenue des Pins and rue Mont-Royal.
International Vestibules of Montréal August 12-16 • Downtown 10th edition of Relève en Blues contest
Aqua Ongoing • Montréal Science Centre The global vision that reflects the interdependence between nature and human beings will unfold as the story of water is shared. AQUA is a voyage into the world of water; a non-stop adventure through three distinct spaces where the spectator is completely immersed in the magic of water without a single word being spoken. A signature experience by Guy Laliberté’s ONE DROP foundation.
Piknic Électronik Ongoing • Park Jean-Drapeau Every Sunday, discover the best of the electronic music scene in an environment created to stimulate the senses.
Pirates, Privateers and Freebooters Ongoing • Pointe-à-Callière Discover the amazing stories of these terrors of the high seas as you delve into the history of piracy.
Aires Libres Ongoing • Downtown Sainte-Catherine Street turns into a pedestrian zone for artists and visitors between Berri and Papineau Streets.
About Montreal – Useful Information Credit Cards
Important Numbers
In order to report lost or stolen credit cards, these numbers will provide assistance.
Information Desk Delta Centre-Ville Taxi Service Tourism Montréal Montréal Trudeau Airport
American Express Mastercard Visa
1-800-268-9805 1-800-263-2263 1-800-847-2911
514-991-3851 514-879-1370 514-725-9885 514-873-2015 514-394-7377
Shopping Getting Around Montreal Clean, safe and comfortable, the underground subway system, called the METRO, is an ideal and economical way to reach many of Montreal’s attractions. You can also connect to the city bus system with the same fare. A 1-day pass costs $9 CAD and a 3-day pass costs $17 CAD.
Dress Code The dress code during the conference is casual.
6
Montreal’s reputation as an international fashion and shopping centre is well established. With major department stores and countless boutiques lining the city’s downtown streets and the unique underground city, Montreal offers creations by the bestknown names on the local, national and international fashion scene. Find upscale boutiques, art galleries and jewellers a few blocks north, near the Museum of Fine Arts on Sherbrooke Street. A 29-kilometre network of underground passageways provides access to 1,700 boutiques, department stores, restaurants, movie houses, theatres and exhibition halls as well as 7 major hotels, 1,615 housing units and thousands of offices.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Social Program and Excursions
Sunday, August 9, 2009
Wednesday, August 12, 2009
Welcome to Montréal Opening Reception
Conference Party at Sucrerie de la Montagne, Rigaud, Québec
18:30-20:00
19:00 to 23:00
Hotel Delta Centre-Ville, Foyer of Régence, 777 University Street (Included in full conference registration fee for delegates and registered accompanying persons)
Buses leave at 17:30 from Hotel Delta Centre-ville – approx. 45 minute bus ride
Dress Code: Casual
Buses return around 22:00 (Reservation fee of $25 CDN applies.) Dress Code: Comfortable wear!
An exciting evening awaits as ISPP 2009 officially opens with a keynote address followed by a reception with light hors d’oeuvres and beverages. Come and network with your colleagues to set the tone for the meeting.
REMEMBER TO BRING YOUR TICKET!
What’s a Sucrerie? It is a traditional Sugar Shack. In Quebec, sugaring-off is a hearty, typically French Canadian celebration. A traditional event that dates back to pioneer days, it celebrates the end of a hard winter. This activity is held in a sugar bush. The hosts have recreated the rural setting of the last century, with buildings made of authentic barn timbers and fieldstone ovens. This establishment was singled out by the New-York Times Food Editors for its typical decor and the quality of its sugartime feast. Quebecois folklore at its best! All Quebec food specialties are yours to eat as much as you like. A traditional bakery is in operation and a horse drawn caleche ride is part of the fun. Musicians will set the mood.
The traditional Sugar Shack meal includes: • Green Salad • Farm-Style Crusty Bread • Canadian Salted Back Bacon • Québécois Maple-Smoked Ham • Wood-Fired Baked Beans • Farm-Style Omelet • Traditional Meatball Stew • Country-Style Sausages • Old-style Mashed Potatoes • Meat Pie from Quebec's Beauce Region • Homemade Fruit Ketchup & Pickles • Dessert: Pancakes with Maple Syrup, Sugar pie, tea and coffee Vegetarian options are available.
August 9 to 14, 2009 • Montréal, QC, Canada
7
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Social Program and Excursions Departure from hotel at 1:30 pm. Buses leave from St. Jacques Street.
TOUR 1:
TOUR 3:
OLD MONTRÉAL WALK AND VISIT TO THE MUSEUM OF ARCHEOLOGY
BOTANICAL GARDEN and BIODÔME
Duration: 3 hours Departing from Hotel Delta Centre-Ville, you will first walk through the historic quarter into the life of the first settlers of Montréal. You will stroll through a maze of narrow cobblestone lanes and old buildings, providing a perfect opportunity to discover the history and charm of Old Montréal. En route you will see Place d'Armes, Jacques Cartier Square and Montréal City Hall. One of the highlight of this tour is the museum of Archeology, where you will discover the history of Montréal, starting with the multimedia show, and watch thousands of years of history spin by in 18 fascinating minutes. You will then explore the remains in the Museum at your own pace, through a vibrant and colourful exhibition. You will walk back to the hotel.
Duration: 4 hours Travel along the St. Lawrence River to the second largest Botanical Garden in the world displaying 20,000 species of flowers and plants in 30 outdoor gardens and 10 greenhouses. Of special interest: a remarkable collection of bonsai and orchids. You can choose to visit the Exhibition Greenhouses, the Japanese Pavilion, the Chinese Garden, the Arboretum or the Insectarium. The next visit will bring you to the the Biodôme, a unique concept of a "living" museum. Wander through four full scale natural ecosystems of the Americas: the Tropical Forest, the St. Lawrence Marine, the Laurentian Forest and the Polar World, complete with landscapes, waterfalls, rivers, flora and birds, animals and fish.
TOUR 2: CITY TOUR: MONTRÉAL C'EST MAGNIFIQUE! Duration: 3 hours This exciting tour will introduce you to various parts of the city that make Montréal what it is today. You will have the chance to see how culturally diverse Montréal is, as well as visit landmarks and new developments that are so very important in making Montréal such a unique and wonderful city. See Montréal's elegant upper class communities, our universities and part of our Underground City. Also, the Plateau Mont-Royal, one of the top four "hippest" neighbourhoods in North America, known for its distinctive Montréal architecture, with its spiral staircase and finely wrought cornices. Along the way, the tour includes a stop at the summit of Mt. Royal Park for a panoramic view of the city and a guided visit of Notre-Dame Basilica, an outstanding example of neo-gothic architecture.
8
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
SCIENTIFIC PROGRAM August 9 to 14, 2009 • Montréal, QC, Canada
9
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
How This Programme Is Organized ALL ORAL PRESENTATIONS
LEGEND OF POSTER SESSIONS
(INVITED SPEAKERS AND ABSTRACT PRESENTERS) All sessions have been coded to facilitate indexing and reference to presentations in the book of abstracts. The codes consist of two letters (representing the session type) followed by a number from 1 to 18 (2 Keynote, 9 Plenary and 18 Oral Communication sessions in total), followed by a number designating the sequence of presentations.
P = Poster Session
Poster sessions are coded as follows: P.
18
Poster
Poster Number
LEGEND OF ORAL SESSIONS Posters have been organized by Topic, followed by Alphabetic Order.
Keynote Speakers= KN Plenary Speakers = PL OC = Oral Communications OC-
1.
Oral Communications
Session Number
1
First Presentation in Session
Instructions for Oral and Poster Presenters Invited Speakers and Oral Abstract Presenters who Uploaded Their Presentation Pre-Symposium
Invited Speakers and Oral Abstract Presenters who Did NOT Upload Their Presentation Pre-Conference
Even if you have successfully uploaded your presentation, we want to make sure that your presentation functions smoothly onsite. Therefore, we have assigned a review time period for you where you should present yourself at the technician’s table in the room Le Royer. The technician will open your presentation for you to review the functionality of all slides. There won’t be time for you to make major changes. As backup, we request that you bring your presentation on a USB key or CD-rom.
Speakers MUST check in and submit their PowerPoint presentation at least three hours prior to their presentation, we have assigned two computers for you where you could briefly upload and preview your slides. Please see the registration staff for instructions.
Please proceed to the session room 15 minutes before the start of your session in case that the program needs to be adjusted due to last-minute cancellations.
You will be required to provide your presentation on USB key or CD-Rom. PLEASE NOTE THAT ALL PRESENTATIONS WILL BE DELETED FROM THE SERVER AT THE END OF THE DAY. INDIVIDUAL LAPTOP COMPUTERS ARE NOT PERMITTED FOR PRESENTATIONS!
Poster Presenters Poster presenters should mount their posters in the designated poster area (room Cartier) between 07:30-10:00 on Monday, August 10 and remove their posters no later than 18:00 on Thursday, August 13. Conference staff is available to assist. Velcro will already be attached to your board when you arrive.
10
Any posters that are not taken down by 18:00 on Thursday will be taken down by conference staff and stored at the registration desk. Posters unclaimed by 18:00 on Friday, August 14 will be discarded.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Invited Faculty Carl Bauer Professor of Biology Indiana University Bloomington, IN, USA
Caroline Harwood Professor of Microbiology University of Washington Seattle, WA, USA
J. Thomas Beatty Professor, Microbiology & Immunology University of British Columbia Vancouver, BC, Canada
Diana Kirilovsky Institut de Biologie et Technologies de Saclay CEA and CNRS Gif sur Yvette, France
Brigitta Bergman Professor, Department of Botany Stockholm University Stockholm, Sweden
Bernd Masepohl Chair, Microbiology Ruhr-Universität Bochum Bochum, Germany
Robert Blankenship Professor of Biology and Chemistry Washington University St. Louis, MO, USA
Armen Y. Mulkidjanian University of Osnabrueck Osnabrueck, Germany
Fevzi Daldal Professor of Biology University of Pennsylvania Philadelphia, PA, USA Enrique Flores Professor of Research Instituto de Bioquímica Vegetal y Fotosíntesis CSIC-Universidad de Sevilla Sevilla, Spain Karl Forchhammer Professor and Head Department of Microbiology / Organismic Interactions University Tübingen Tübingen, Germany Susan Golden Professor Division of Biological Sciences Department of Biology University of California-San Diego La Jolla, CA, USA Vladimir Gorlenko Professor and Head of Laboratory Ecology and Geochemical Activity of Microorganisms Winogradsky Institute of Microbiology Russian Academy of Sciences Moscow, Russia Arthur Grossman Professor, Carnegie Institution of Washington and Department of Biological Sciences Stanford University Stanford, CA, USA Martin Hagemann Bioscience Institute, Plant Physiology Department Universität Rostock Rostock, Germany
August 9 to 14, 2009 • Montréal, QC, Canada
Frederic Partensky Marine Photosynthetic Prokaryotes Team Station Biologique, CNRS and Université Paris06 (UMR 7144) Roscoff, France Louis Sherman Professor, Biological Sciences Purdue University West Lafayette, IN, USA Kaarina Sivonen Professor, Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland Iwane Suzuki Associate Professor University of Tsukuba Tsukuba, Ibaraki, Japan F. Robert Tabita Professor; Ohio Eminent Scholar of Microbiology Ohio State University Columbus, OH, USA Paula M. Tamagnini Departamento de Botânica, Fac. Ciências IBMC – Instituto de Biologia Molecular e Celular Universidade do Porto Porto, Portugal Anthony E. Walsby Emeritus Professor of Microbiology School of Biological Sciences University of Bristol Bristol, United Kingdom Peter C. Wolk Professor Michigan State University E. Lansing, MI, USA
11
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
12
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Sunday, August 9, 2009
17:30–18:30
OPENING KEYNOTE
RÉGENCE AB
WELCOME TO ISPP 2009 AND INTRODUCTION OF KEYNOTE SPEAKER. Patrick Hallenbeck, Symposium Chair and Université de Montréal, Montréal, QC. WELCOME FROM THE CHAIR OF THE INTERNATIONAL SCIENTIFIC COMMITTEE. Jörg Overmann, Planegg-Martinsried, Germany. KN-1.1 A SCIENTIST OF THE FLOATING WORLD. Anthony E. Walsby, University of Bristol, United Kingdom. Biography I trained as a botanist at the University of Birmingham and moved to Westfield College London for research with G. E. Fogg on cyanobacteria, which at that time masqueraded as blue-green algae. My first paper, with Peter Fay in 1966, described the serendipitous isolation of heterocysts. Bill Stewart saw the opportunity to test N2-fixation by isolated heterocysts; my contribution was to measure 15N labelling by mass spectrometry. This technique led to my analysing the gas in gas vesicles of cyanobacteria and a life-time of research on these fascinating structures. In the early 70s I spent two years on sabbatical leave in Berkeley, where I met and married Fausta Segrè. Returning to the UK, I spent another seven years with Fogg in marine science at the University College of North Wales and then, despite never having worked on a real plant, I moved to the Chair of Botany at the University of Bristol. Throughout my career chasing gas vesicles I sought collaborations with experts in other fields: on crystallography with Allen Blaurock in London; microscopy with Daniel Branton at Berkeley; protein sequencing with John Walker at Cambridge; and mathematical physics with John Simpson at Menai Bridge. My machines for measuring gas vesicles suggest inspiration by Heath Robinson (or Rube Goldberg). The results were the mechanics of gas vesicles, measurements of cell turgor pressure and estimates of gas diffusion rates into heterocysts. At Bristol I enjoyed a long collaboration with Paul Hayes and some excellent students. We took the physiology and molecular biology of cyanobacteria from the laboratory to analyse populations in lakes, seas and salterns. We had many nice collaborations with local ecologists, including those with Ferdinand Schanz on Lake Zürich, Lucas Stal on the Baltic Sea, and Colin Reynolds on the English Lakes. Sometimes I returned with unexpected trophies: Dactylococcopsis from Solar Lake, the square archeon Haloquadratum from the Sinai, and hundreds of Planktothrix strains from Lake Zürich.
18:30–20:00
WELCOME RECEPTION
FOYER – RÉGENCE
See page 7 for details.
August 9 to 14, 2009 • Montréal, QC, Canada
13
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
14
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Monday, August 10, 2009
08:30–10:00
PLENARY 1: PHYLOGENY, TAXONOMY AND DIVERSITY
RÉGENCE AB
Co-Chairs: Johannes Imhoff, Kiel, Germany and Min Chen, Sydney, Australia 08:30
PL-1.1
EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHIC BACTERIA DEDUCTED FROM WHOLE GENOME COMPARISONS. Robert Blankenship, Washington University, St. Louis, MO, USA.
09:00
PL-1.2
ORIGIN AND EVOLUTION OF PHOTOSYNTHETIC SYSTEMS. Armen Y. Mulkidjanian, University of Osnabrueck, Osnabrueck, Germany.
09:30
PL-1.3
ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA: DIVERSITY, NEW TAXONS. Vladimir Gorlenko, Russian Academy of Sciences, Moscow, Russia.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 1: PHYLOGENY TAXONOMY AND DIVERSITY
FOYER
RÉGENCE C
Chair: Robert Blankenship, St. Louis, MO, USA OC-1.1 SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETIC MARKERS FOR PURPLE SULFUR BACTERIA. Marcus Tank, Vera Thiel, Johannes F. Imhoff. IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University), Kiel, Germany. OC-1.2 BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTING GENES IN MARINE PHYTOPLANKTON. Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2. 1 School of Ocean and Earth Sciences, National Oceanography Centre, Southampton, United Kingdom; 2School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia. OC-1.3 PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THE CYANOBACTERIAL GENERA ANABAENOPSIS AND CYANOSPIRA. Stefano Ventura, Cristina Mascalchi, Claudio Sili. Institute of Ecosystem Study, National Research Council, Sesto Fiorentino, Italy. OC-1.4 CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATS OF A TROPICAL LAGOON, TIKEHAU ATOLL, TUAMOTU ARCHIPELAGO. Katarzyna A. Palinska1, Raeid M. M. Abed2†, Katja Wendt1, Maria Łotocka3, Loic Charpy4 & Stejpko Golubic5. 1 Institute of Chemistry and Biology of the Marine Environment, Geomicrobiology Dep. and CvO University of Oldenburg, Oldenburg, Germany; 2College of Science-Biology Department, Sultan Qaboos University, Al-Khod, Muscat, Sultanate of Oman; 3Institute of Oceanology, Polish Academy of Sciences, Powsta ców Warszawy, Sopot, Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167 (CYROCO), Traverse de la Batterie des Lions, Marseille, France; 5Biological Science Center, Boston University, Boston, MA, USA. OC-1.5 METAGENOMIC AND PHYLOGENETIC ANALYSES OF CYANOBACTERIAL MATS IN EXTREME HIGH ARCTIC ENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICAL FUNCTION. Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3, Warwick F. Vincent1. 1 Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté de Médecine, Université Laval; 3Québec-Océan, Dép. de Biologie, and Institut de biologie intégrative et des systèmes (IBIS); Université Laval; Québec, QC, Canada. OC-1.6 GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENON SPP. AND ANABAENA SPP. IN NORTHEAST GERMAN LAKES. Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1. 1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes, Neuglobsow; 2Federal Environmental Agency, Berlin; 3Brandenburg Technical University, Department of Freshwater Conservation, Bad Saarow; Germany. August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Monday, August 10, 2009
10:30–12:30
ORAL COMMUNICATIONS 2: ECOLOGY I
RÉGENCE AB
Chair: Mary Allen, Wellesley, MA, USA OC-2.1 MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS IN PHOTOTROPHIC CONSORTIA. Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller. Section Microbiology, Department Biology I, University of Munich, Planegg-Martinsried, Germany. OC-2.2 LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBIC ANOXYGENIC PHOTOTROPHS IN A SEMIARID REALM. J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. OC-2.3 ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MAT- NITROGEN FIXATION. Ina Severin, Lucas J. Stal. Department of Marine Microbiology, NIOO-Center for Marine and Estuarine Ecology, Yerseke, The Netherlands. OC-2.4 BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCED PHOTOSYNTHETIC MICROBES. Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer. SRI International, Menlo Park, CA, USA. OC-2.5 DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACE WATERS OF MARINE AND FRESHWATER ENVIRONMENTS WITH AN EMPHASIS ON CYANOBACTERIA. Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, Torsten Staller2, Rüdiger Schulz2, Jens Appel1. 1 School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Botanisches Institut, Universität Kiel, Kiel, Germany. OC-2.6 HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRING MICROBIAL MATS DOMINATED BY THE FILAMENTOUS ANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUS AGGREGANS. H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1. 1 Department of Biology, Tokyo Metropolitan University, Tokyo; 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki; Japan.
12:30–14:00
16
LUNCH
RÉGENCE C AND FOYER
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Monday, August 10, 2009
14:00–15:00
ORAL COMMUNICATIONS 3: ECOLOGY II
RÉGENCE AB
Chair: Vladimir Gorlenko, Moscow, Russia OC-3.1 CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROM THE HALOCLINE OF THE SALINE MEROMICTIC LAKE, LAKE SUIGETSU, JAPAN. Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya. Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan. OC-3.2 THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIA PROCHLOROCOCCUS. Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA. OC-3.3 THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTAL CYANOBACTERIA COMMUNITY AND ITS CONTRIBUTION TO TOXICITY IN A TROPICAL BRAZILIAN RESERVOIR. Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos. Department of Botany, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
14:00–15:00
ORAL COMMUNICATIONS 4: PHYSIOLOGY I
RÉGENCE C
Chair: David Adams, Leeds, United Kingdom OC-4.1 ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUM TEPIDUM: GENES, METABOLITES AND LINKAGES TO LIGHT HARVESTING. Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan, Rachael Morgan-Kiss. College of Marine and Earth Studies and DBI, Newark, DE, USA. OC-4.2 CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS. Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E. Blankenship1. 1 Departments of Biology and Chemistry; 2Department of Energy, Environment and Chemical Engineering; Washington University, St. Louis, MO, USA. OC-4.3 STRUCTURAL AND FUNCTIONAL STUDIES OF A PHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGH COMPARATIVE METATRANSCRIPTOME ANALYSIS. Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt, Lynn Tomsho, Stephan C. Schuster, David M. Ward, Donald A. Bryant. The Pennsylvania State University, University Park, PA, USA.
15:00–17:00
POSTER SESSION
17:00–18:00
PLENARY 2: ECOLOGY
CARTIER AB
RÉGENCE AB
Co-Chairs: Jörg Overmann, Planegg-Martinsried, Germany and Vladimir Yurkov, Winnipeg, MB, Canada 17:00
PL-2.1
MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMIC AND PROTEOMIC ANALYSES Brigitta Bergman, Department of Botany, Stockholm University, Stockholm, Sweden.
17:30
PL-2.2
PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATION AND POTENTIAL INTERACTIONS Arthur Grossman, Department of Plant Biology, The Carnegie Institution, Stanford, CA, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
17
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
18
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
08:30–10:00
PLENARY 3: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES
RÉGENCE AB
Chairs: Jack Meeks, Davis, CA, USA and James Golden, La Jolla, CA, USA 08:30
PL-3.1
MULTICELLULARITY IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA. Enrique Flores, Professor of Research, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain.
09:00
PL-3.2
EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY GLYCOLATE METABOLISM IN CYANOBACTERIA. Martin Hagemann, Plant Physiology, University Rostock, Inst. Biosciences, Rostock, Germany.
09:30
PL-3.3
THE NETWORK OF INTERACTIONS OF PII SIGNALING IN CYANOBACTERIA. Karl Forchhammer, Department of Microbiology, University Tübingen, Tübingen, Germany.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 5: PHYSIOLOGY II
FOYER
RÉGENCE AB
Chairs: David Knaff, Lubbock, TX, USA and Ann Magnuson, Uppsala, Sweden OC-5.1 HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOC PUNCTIFORME DESTINED TO BECOME HORMOGONIA. H. Christman, E.L. Campbell, J.C. Meeks. Section of Microbiology, University of California, Davis, CA, USA. OC-5.2 PLANT CELL WALL EPITOPES ARE EXPRESSED BY CYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS. Owen Jackson, J. Paul Knox, David G. Adams. University of Leeds, West Yorkshire, United Kingdom. OC-5.3 REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE IN HETEROCYST-FORMING CYANOBACTERIA. Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, Enrique Flores2, David G. Adams3. 1 School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty of Biological Sciences, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom. OC-5.4 SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS IN ANABAENA SP. STRAIN PCC 7120. Jinjie Liu, C. Peter Wolk. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA. OC-5.5 THE INVOLVEMENT OF TWO SIGMA FACTORS IN CYANOBACTERIAL AKINETE FORMATION. Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers. Department of Biology, California State University, Northridge, CA, USA. OC-5.6 SEQUENCES REGULATING NITROGENASE GENE EXPRESSION IN THE CYANOBACTERIUM ANABAENA VARIABILIS. Teresa Thiel, Justin L. Ungerer. Department of Biology, University of Missouri St. Louis, St. Louis, MO, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
19
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
10:30–12:30
ORAL COMMUNICATIONS 6: BIOENERGETICS I
RÉGENCE C
Chairs: Michael Summers, Northridge, CA, USA OC-6.1 THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES AT THE MONOMER-MONOMER INTERFACE OF PHOTOSYSTEM II IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley. Department of Biochemistry, University of Otago, Dunedin, New Zealand. OC-6.2 PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO IN SYNECHOCYSTIS. Danny Yao, Dan Brune, Wim Vermaas. School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. OC-6.3 IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FOR THE OXYGEN DEPENDANCE OF THE PHOTOSYNTHETIC ACTIVITY OF ROSEOBACTER DENITRIFICANS. Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and Andreas Labahn1. 1 Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany; 2Department of Cell Biology, Harvard Medical School, Boston MA, USA. OC-6.4 VARIABLE FLUORESCENCE IN HELIOBACTERIUM MODESTICALDUM CELLS: OBSERVATION AND EXPLANATION. Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, Stefano Santabarbara1,2, Robert Blankenship3. 1 Arizona State University, Dept. of Chemistry and Biochemistry, Tempe, AZ, USA; 2Institut de Biologie Physico-Chimique, Paris, France; 3Washington University, Departments of Biology and Chemistry, St Louis, MO, USA. OC-6.5 NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM II PROTECTS THE CYANOBACTERIUM MICROCOLEUS SP. AGAINST EXCESS LIGHT STRESS. Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2. Departments of 1Biological Chemistry, 2Plant and Environmental Sciences and 3The Interuniversity Marine Institute, Eilat, The Hebrew University of Jerusalem, Israel. OC-6.6 THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-Mari Aro. Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland.
12:30–14:00
20
LUNCH
RÉGENCE C AND TOUR DE VILLE Please check for a ticket inside your badge holder. Only those with a ticket in their badge holder are dining in the Tour de Ville.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Tuesday, August 11, 2009
14:00–15:00
ORAL COMMUNICATIONS 7: PHYSIOLOGY III
RÉGENCE AB
Chairs: Enrique Flores, Sevilla, Spain OC-7.1 PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA (PRRA), TRANSCRIPTIONAL REGULATORS OF THE CBB OPERONS (CO2 FIXATION) IN RHODOBACTER SPHAEROIDES. Andrew W. Dangel, F. Robert Tabita . Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, OH, USA. OC-7.2 PpsR AS A NEW TYPE OF HEMIN SENSOR. Liang Yin, Vladimira Dragnea, Carl Bauer. Indiana University, Bloomington, IN, USA. OC-7.3 ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN: AN EXPERIMENTAL SCENARIO FOR A STRICT ANAEROBIC PHOTOTROPH. Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg, Françoise Reiss-Husson, Chantal Astier, Soufian Ouchane. CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; Université Paris-Sud, Orsay; Université Pierre et Marie Curie, Paris; France.
14:00–15:00
ORAL COMMUNICATIONS 8: BIOENERGETICS II, PROTEINS, GENOMICS II
RÉGENCE C
Chair: Susan Golden, La Jolla, CA, USA and Willem Vermaas, Tempe, AZ, USA OC-8.1 CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BY FERREDOXIN:THIOREDOXIN REDUCTASE, FERREDOXIN AND THIOREDOXIN M. David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, Masakazu Hirasawa, Marcellus Ubbink. Texas Tech University, Lubbock, TX, USA. OC-8.2 CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA. Shinichi Takaichi1, Mari Mochimaru2. 1 Department of Biology, Nippon Medical School, Kawasaki; 2Department of Natural Sciences, Komazawa University, Setagaya, Tokyo; Japan. OC-8.3 EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROM PURPLE BACTERIA AND CYANOBACTERIA. Gerhard Sandmann, Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Frankfurt, Germany.
15:00–16:30
POSTER SESSION
16:30–17:30
PLENARY 4: PHYSIOLOGY, METABOLISM, AND GLOBAL RESPONSES
CARTIER AB
RÉGENCE AB
Chair: Fevzi Daldal, Philadelphia, PA, USA 16:30
PL-4.1
A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME. Susan Golden, Department of Biology, University of California-San Diego, La Jolla, CA, USA.
17:00
PL-4.2
EFFECTS OF INSERTION SEQUENCES ON THE GENOME OF ANABAENA SP. STRAIN PCC 7120. Peter C. Wolk, Michigan State University, E. Lansing, MI, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
21
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
22
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Wednesday, August 12, 2009
08:30–10:00
PLENARY 5: BIOENERGETICS, PROTEINS AND GENOMICS
RÉGENCE AB
Chairs: F. Robert Tabita, Columbus, OH, USA 10:00
PL-5.1
CYTOCHROMES STRUCTURE FUNCTION AND BIOGENESIS. Fevzi Daldal, University of Pennsylvania, Philadelphia, PA, USA.
10:30
PL-5.2
BETTER LIVING THROUGH CYANOTHECE-UNICELLULAR DIAZOTROPHIC CYANOBACTERIA WITH HIGHLY VERSATILE METABOLIC SYSTEMS. Louis Sherman, Professor, Biological Sciences, Purdue University, West Lafayette, IN, USA.
11:00
PL-5.3
PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENT EVOLUTION SCHEMES FROM A COMMON ANCESTRAL GENOME. Frederic Partensky, Marine Photosynthetic Prokaryotes Team, Station Biologique, CNRS and Université Paris, Roscoff, France.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES
FOYER
RÉGENCE AB
Chairs: Bernd Masepohl, Bochum, Germany OC-9.1 THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM – A STRUCTURAL MODEL BASED ON SOLID-STATE NMR DATA COMBINED WITH MOLECULAR SIMULATION STUDIES OF CD AND ABSORPTION SPECTRA. Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1, Peter Højrup2, Anders Giessing2, Juha Linnanto3, Niels-Ulrik Frigaard4, Mette Miller2 and Niels Chr. Nielsen1. 1 Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus, Århus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 3Department of Chemistry, University of Jyväskylä, Finland; 4Department of Biology, University of Copenhagen, Copenhagen, Denmark. OC-9.2 THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLS IN CHLOROSOMES OF CHLOROBACULUM TEPIDUM. Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, Michael Reus, Aline Gomez Gomez Maqueo Chew, Alfred R. Holzwarth, Egbert J. Boekema, Huub J. M. de Groot. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. OC-9.3 MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANES IN CYANOBACTERIA. Sawsan Hamad, Wim Vermaas. School of Life Sciences and the Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. OC-9.4 PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OF FUNCTION IN THE CARBOXYSOME. Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C. Bagby3, Sabine Heinhorst4, Fei Cai4, Gordon Cannon4, Sallie W. Chisholm3. 1 US Department of Energy, Joint Genome Institute, Walnut Creek, CA; 2Department of Plant and Microbial Biology, University of California, Berkeley, CA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; 4Department of Biochemistry, University of Southern Mississippi, Hattiesburg, MS; USA.
August 9 to 14, 2009 • Montréal, QC, Canada
23
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Wednesday, August 12, 2009
10:30–12:30
ORAL COMMUNICATIONS 9: BIOENERGETICS III, SUPRAMOLECULAR STRUCTURES
RÉGENCE AB
Chairs: Bernd Masepohl, Bochum, Germany OC-9.5 PROTEOMIC ANALYSIS OF THE DEVELOPING INTRACYTOPLASMIC MEMBRANE DURING CHROMATIC ADAPTATION IN RHODOBACTER SPHAEROIDES. Kamil Woronowicz, Robert A. Niederman. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA. OC-9.6 BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODING THE MEMBRANE-ASSOCIATED THIOREDOXIN PROTEIN PLAYS AN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OF PS I. Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1. 1 Department of Biochemistry and Molecular Biology; 2Department of Chemistry; The Pennsylvania State University, University Park, PA, USA.
10:30–12:30
ORAL COMMUNICATIONS 10: PHYSIOLOGY IV
RÉGENCE C
Chair: J. Thomas Beatty, Vancouver, BC, Canada OC-10.1
A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THE APPARENT PHOTOSYNTHETIC AFFINITY FOR CO2 BY MODULATING LOW-CO2-INDUCED GENE EXPRESSION. Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2, Yukako Hihara2, Ariel Gaathon3, Mario Lebendiker4, Aaron Kaplan1. 1 Dept. of Plant and Environmental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; 2 Dept. of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan; 3Bletterman Laboratory, Interdepartmental Equipment Unit, Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre, Hebrew University of Jerusalem, Jerusalem, Israel.
OC-10.2
THE EFFECTS OF TEMPERATURE AND OXYGEN ON NITROGENASE ACTIVITY IN THE THERMOPHILIC CYANOBACTERIUM FISCHERELLA SP. Lucas J. Stal, Department of Marine Microbiology, Netherlands Institute of Ecology, Yerseke, The Netherlands.
OC-10.3
PHOSPHATE SCAVENGING IN AN UNPREDICTABLE ENVIRONMENT, HOW DO CYANOBACTERIA MEET THE CHALLENGE? Frances D. Pitt, David J. Scanlan. Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom.
OC-10.4
REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY IN CYANOBACTERIA. IDENTIFICATION OF THE REGIONS INVOLVED IN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS. Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J. Florencio. Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla-CSIC, Sevilla, Spain.
OC-10.5
BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS. Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A. Bryant1. 1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2 Department of Biological Sciences, University of New Orleans, New Orleans, LA; USA.
OC-10.6
ACCUMULATION OF TREHALOSE IN RESPONSE TO DESICCATION AND CONTROL OF TREHALASE IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE. Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima. Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan.
FREE AFTERNOON AND EXCURSIONS
17:30 24
ISPP 2009 PARTY (SEE PAGE 7) August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Thursday, August 13, 2009
08:30–10:00
PLENARY 6: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION
RÉGENCE AB
Chairs: Donald A. Bryant, University Park, PA, USA and Kaori Inoue-Sakamoto, Ishikawa, Japan 08:30
PL-6.1
OXYGEN AND LIGHT CONTROL OF TETRAPYRROLE BIOSYNTHESIS IN RHODOBACTER SPECIES. Carl Bauer, Indiana University, Bloomington, IN, USA.
09:00
PL-6.2
A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHT INTENSITY SENSOR IN CYANOBACTERIA PHOTOPROTECTION. Diana Kirilovsky, Institut de Biologie et Technologies de Saclay, CEA and CNRS, Gif sur Yvette, France.
09:30
PL-6.3
NITROGEN AND MOLYBDENUM CONTROL OF NITROGEN FIXATION. Bernd Masepohl, Chair, Microbiology, Ruhr-Universität Bochum, Bochum, Germany.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 11: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I
FOYER
RÉGENCE AB
Chairs: Iwane Suzuki, Tsukuba, Ibaraki, Japan OC-11.1
FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAIN IN RHODOSPIRILLUM CENTENUM PPR. J.A. Kyndt, T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
OC-11.2
SHORT-TERM LIGHT ADAPTATION STRATEGIES OF PHYCOBILISOME-CONTAINING PHOTOSYNTHETICS, CYANOBACTERIA AND RED ALGAE. Igor Stadnichuk, PA.N.Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
OC-11.3
REGULATORY RNAS IN CYANOBACTERIA AND BEYOND. W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1, J. Vogel2, A. Wilde3. 1 Freiburg Initiative in Systems Biology and Faculty of Biology, University Freiburg; 2Max-Planck-Institute for Infection Biology Berlin; 3Justus-Liebig University Giessen, Institute of Microbiology and Molecular Biology; Germany.
OC-11.4
FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUM PROCHLOROCOCCUS: WHAT CAN WE LEARN? C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, Joerg Vogel3, Wolfgang Hess1. 1 University of Freiburg, Freiburg, Germany; 2University of Algarve, Faro, Portugal; 3Max Planck Institute for Infection Biology, Berlin, Germany.
OC-11.5
REGULATION BETWEEN PHOTOAUTOTROPHIC AND PHOTOMIXOTROPHIC GROWTH AND ITS DEPENDENCE ON THE CO2 LEVEL IN SYNECHOCYSTIS PCC 6803. Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy Lieman-Hurwitz1, Aaron Kaplan1. 1 Plant and Environmental Sciences, The Hebrew University of Jerusalem; Israel; 2Department of Biochemistry and Molecular Biology, Saitama University, Japan.
OC-11.6
CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY IN OSCILLATORIA. Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson, Dave Adams. University of Leeds, Leeds, West Yorkshire, United Kingdom.
August 9 to 14, 2009 • Montréal, QC, Canada
25
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Thursday, August 13, 2009
10:30–12:30
ORAL COMMUNICATIONS 12: ENVIRONMENTAL SENSING AND SIGNAL TRANSDUCTION I
RÉGENCE C
Chairs: Karl Forchhammer, Tübingen, Germany and Soufian Ouchane, Gif-sur-Yvette, France OC-12.1
GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGEN CONDITIONS AND THE ROLE OF THE HISTIDINE KINASE, HIK31, IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Tina C. Summerfield1, Louis A. Sherman2. 1 Botany Department, University of Otago, PO Box 56, Dunedin, New Zealand; 2Purdue University, Department of Biological Sciences, West Lafayette, IN, USA.
OC-12.2
INCREASING HYDROGEN PRODUCTION BY PERTURBING FERMENTATIVE METABOLISM IN THE MARINE, UNICELLULAR CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002. Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G. Charles Dismukes. Princeton University, Princeton, NJ, USA.
OC-12.3
PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATION TRANSFER TO PHOTOSYSTEM I IN THE FILAMENTOUS CYANOBACTERIUM NOSTOC PUNCTIFORME. Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring, Ann Magnuson. Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden.
OC-12.4
GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC 7942. Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, Yasunori Nakamura. Akita Prefectural University, Akita, Japan.
OC-12.5
IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTIS AERUGINOSA UNDER IRON LIMITATION. Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1. 1 School of Biotechnology and Biomolecular Sciences; 2School of Civil and Environmental Engineering; University of New South Wales, Sydney, Australia.
OC-12.6
THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITE WITH ECOPHYSIOLOGICAL ROLES. S.B. Watson1, F. Juttner2. 1 Environment Canada, Canada Centre for Inland Waters, Burlington, ON, Canada; 2Limnological Station, University of Zurich, Switzerland.
12:30–14:00
26
LUNCH
RÉGENCE C AND TOUR DE VILLE Please check for a ticket inside your badge holder. Only those with a ticket in their badge holder are dining in the Tour de Ville.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Thursday, August 13, 2009
14:00–15:00
ORAL COMMUNICATIONS 13: PHYSIOLOGY VI
RÉGENCE C
Chair: Peter Lindblad, Uppsala, Sweden OC-13.1
COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND PURPLE SULFUR BACTERIA. N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3. 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Plant Biology Department, University of Geneva, Geneva, Switzerland; 3Department of Biochemistry and Molecular Biology, Pennsylvania State University, State College, Pennsylvania, USA.
OC-13.2
THE GENOME OF THE TOXIC BLOOM-FORMING CYANOBACTERIUM ANABAENA SP. 90. David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2, Kaarina Sivonen1. 1 Department of Microbiology and Applied Chemistry, University of Helsinki, FIN-00014, Helsinki, Finland; 2 Beijing Institute of Genomics of the Chinese Academy of Sciences, Beijing Genomics Institute, Beijing, China.
OC-13.3
CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPMENT AND BEHAVIOR OF HORMOGONIA OF NOSTOC PUNCTIFORME. Jack Meeks, Elsie Campbell, Rui Chen. Department of Microbiology, University of California, Davis, CA, USA.
14:00–15:00
ORAL COMMUNICATIONS 14: BIOENERGETICS IV
RÉGENCE AB
Chair: Peter C. Wolk, East Lansing, MI, USA and Teresa Thiel, St. Louis, MO, USA OC-14.1
COMPARISON OF NONCODING FEATURES OF CYANOBACTERIAL GENOMES. Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, José Costa5. 1 Virginia Commonwealth University, VA, USA; 2University of California at Davis, CA, USA; 3University of Pennsylvania, PA, USA; 4Uppsala University, Uppsala, Sweden; 5University of Porto, Porto, Portugal.
OC-14.2
HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) AS ELECTRON DONOR? SEARCHING FOR THE ANSWER IN THE MEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM 263. Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, Ulrich Fischer1. 1 Zentrum für Umweltforschung und nachhaltige Technologien (UFT) and Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie, Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie; 3Fachbereich Biologie/Chemie, Laboratorium für Allgemeine Mikrobiologie, Universität Bremen; Bremen, Germany.
OC-14.3
PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS: INSIGHTS INTO THE SULFUR METABOLISM OF A PHOTOTROPHIC GREEN BACTERIUM. Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, Anders Mellerup2, Kirsten S. Habicht3, Raymond P. Cox1, Jens S. Andersen1 & Niels-Ulrik Frigaard2. 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK5230 Odense; 2 Department of Biology, University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center for Earth Evolution and Institute of Biology, University of Southern Denmark, Odense; Denmark.
August 9 to 14, 2009 • Montréal, QC, Canada
27
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Thursday, August 13, 2009
15:00–16:00
PLENARY 7: ENVIRONMENT AND BIOENERGETICS
RÉGENCE AB
Chairs: Davide Zannoni, Bologna, Italy and Igor Stadnichuk, Moscow, Russia
28
15:00
PL-7.1
HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVES THE ENVIRONMENTAL STIMULI? Iwane Suzuki, University of Tsukuba, Tsukuba, Ibaraki, Japan.
15:30
PL-7.2
GENOMICS AND METAGENOMICS APPROACHES TO THE EVOLUTION AND REGULATION OF GENE TRANSFER AGENTS (GTAS). J. Thomas Beatty, University of British Columbia, Vancouver, BC, Canada.
16:00–18:00
POSTER SESSION
CARTIER AB
18:00
POSTER REMOVAL
CARTIER AB
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Friday, August 14, 2009
08:30–10:00
PLENARY 8: BIOREMEDIATION, SECONDARY METABOLITES, AND APPLIED ASPECTS (BIOFUELS AND CLIMATE CHANGE)
RÉGENCE AB
Chairs: Jens Appel, Tempe, AZ, USA and Anatoly Tsygankov, Pushchino, Russia 08:30
PL-8.1
PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGEN PRODUCTION. Caroline Harwood, University of Washington, Seattle, WA, USA.
09:00
PL-8.2
THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICAL APPLICATIONS. Paula M. Tamagnini, Departamento de Botânica, Fac. Ciências , IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
09:30
PL-8.3
CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES, ACTIVITIES AND BIOSYNTHESIS. Kaarina Sivonen, University of Helsinki, Helsinki, Finland.
10:00–10:30
HEALTH BREAK
10:30–12:30
ORAL COMMUNICATIONS 15: APPLIED ASPECTS I
FOYER
RÉGENCE AB
Chairs: Caroline Harwood, Seattle, WA, USA and Hidehiro Sakurai, Hiratsuka, Japan OC-15.1
A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALE PHOTOBIOLOGICAL HYDROGEN PRODUCTION UTILIZING CYANOBACTERIA. Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue. Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka, Kanagawa, Japan.
OC-15.2
SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTION AMONG DIVERSE HETEROCYSTOUS CYANOBACTERIA. Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples, Polly Berseth, Tommy Sessions. Savannah River National Laboratory, Aiken, SC, USA.
OC-15.3
THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLE BACTERIA WITH DARK FERMENTATIVE HYDROGEN PRODUCTION AND HYDROGEN ELECTRODE. A. Tsygankov, Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
OC-15.4
HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUM RUBRUM UNDER RESPIRATORY DARK CONDITIONS. Lisa Zeiger, Christiane Rudolf, Hartmut Grammel. Max-Planck-Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
OC-15.5
DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA IN MISSISQUOI BAY, QUEBEC, USING Q-PCR. Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, Frances Pick3, David Bird4, Charles W. Greer1. 1 National Research Council, Biotechnology Research Institute, QC; 2Environmental Health Center, Health Canada, ON; 3Biology Department, University of Ottawa, ON; 4Biological Sciences, University of Quebec at Montreal, QC, Canada.
OC-15.6
EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENES AND THE GENETIC POPULATION STRUCTURE OF TOXIC CYANOBACTERIA. Rainer Kurmayer, Guntram Christiansen. Austrian Academy of Sciences, Institute for Limnology, Mondsee, Austria.
August 9 to 14, 2009 • Montréal, QC, Canada
29
Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Friday, August 14, 2009
10:30–12:30
ORAL COMMUNICATIONS 16: PHYSIOLOGY VII
RÉGENCE C
Chairs: Louis Sherman, West Lafayette, IN, USA and Kaarina Sivonen, Helsinki, Finland OC-16.1
THE RESPIRATORY TERMINAL OXIDASES OF THE CYANOBACTERIUM SYNECHOCOCCUS SP. STRAIN PCC7942. Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder, Dominik Aschenbrenner. Institute of Physical Chemistry, Vienna, Austria.
OC-16.2
PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA: STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN POST-TRANSLATIONAL MODIFICATION. Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1, Gaozhong Shen2, Donald A. Bryant2, Wendy M. Schluchter1. 1 Department of Biological Sciences, University of New Orleans, New Orleans, LA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; USA.
OC-16.3
SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVEL CYANOBACTERIOCHROME-TYPE PHOTORECEPTOR AnPixJ. Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose, Tomoo Shiba, Shigeru Itoh, Genji Kurisu, Masahiko Ikeuchi. University of Tokyo, Meguro, Tokyo, Japan.
OC-16.4
CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OF SYNECHOCOCCUS ELONGATUS REVEALS A NEW ANTIDOTE MOLECULE. Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz. Bar-Ilan University, Ramat-Gan, Israel.
OC-16.5
HOW CYANOBACTERIA BORE (AND WHY LATERAL HETEROCYSTS EXIST). F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao. School of Life Sciences, Arizona State University, Tempe, AZ, USA.
OC-16.6
FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEEN IRON HOMEOSTASIS AND OXIDATIVE STRESS. Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2, Nir Keren1. 1 The Alexander Silberman Institute of Life Sciences, Department of Plant and Environmental Sciences, Hebrew University, Givat Ram, Jerusalem, Israel; 2Department of Biological Sciences Purdue University, West Lafayette, IN, USA; 3 Department of Botany, University of Otago, Dunedin, New Zealand.
12:30–14:00
30
LUNCH
RÉGENCE C AND FOYER
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Friday, August 14, 2009
14:00–15:00
ORAL COMMUNICATIONS 17: APPLIED ASPECTS II
RÉGENCE C
Chair: Paula Tamagnini, Porto, Portugal OC-17.1
RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS, HARBORING THE CRTI REPORTER GENE TO MONITOR ENVIRONMENTAL TOXIC METALS. Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.
OC-17.2
THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTER CAPSULATUS CELLS VIA ACETATE PERMEASE. Roberto Borghese, Davide Zannoni. Department of Biology, University of Bologna, Bologna, Italy.
OC-17.3
CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CR-PLATING INDUSTRY WITH EXOPOLYSACCHARIDE-PRODUCING CYANOBACTERIA. Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis. Deparment of Agricultural Biotechnology, University of Florence, Firenze, Italy.
14:00–15:00
ORAL COMMUNICATIONS 18: BIOENERGETICS V AND APPLIED ASPECTS III
RÉGENCE AB
Chair: Peter Lindblad, Uppsala, Sweden OC-18.1
TRANSCRIPTIONAL REGULATION AND MATURATION OF CYANOBACTERIAL HYDROGENASES. Peter Lindblad, Department of Photochemistry & Molecular Science, Uppsala University, Uppsala, Sweden.
OC-18.2
PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803. Carrie Eckert, Jianping Yu, and Pin-Ching Maness. National Renewable Energy Laboratory, Golden, CO, USA.
OC-18.3
ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATION OF THE BIDIRECTIONAL [NIFE]HYDROGENASE FROM SYNECHOCYSTIS SP. PCC 6803. Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2, Rüdiger Schulz3 and Frauke Germer3. 1 School of Life Sciences, Arizona State University, Tempe, AZ, USA; 2Max-Volmer-Laboratorium, Technische Universität, Berlin, Germany; 3Botanisches Institut, Universität Kiel, Kiel, Germany.
15:00–16:00
CLOSING KEYNOTE
RÉGENCE AB
Chair: Patrick Hallenbeck, Montréal, QC, Canada KN-2.1
INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN, AND SULFUR METABOLISM: THE CENTRAL ROLE OF THE CALVIN-BENSON-BASSHAM CYCLE. F. Robert Tabita, Ohio State University, Columbus, OH, USA.
August 9 to 14, 2009 • Montréal, QC, Canada
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Notes
32
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
KN-1.1
KN-2.1
A SCIENTIST OF THE FLOATING WORLD.
INTEGRATIVE CONTROL OF CARBON, NITROGEN, HYDROGEN, AND SULFUR METABOLISM: THE CENTRAL ROLE OF THE CALVINBENSON-BASSHAM CYCLE.
Anthony E. Walsby, School of Biological Sciences, University of Bristol, United Kingdom. My invitation to give the Keynote Address proposed a lecture with reflections and a history of research on phototrophs. Before accepting, I ought to have reflected on Kazuo Ishiguro’s novel An Artist of the Floating World, in which Mr Ono, an aging painter, looks back on his life noticing how his reputation has faltered and attitudes towards him and his work have changed. The Floating World in which I wandered for over forty years is that of planktonic prokaryotes that derive buoyancy from gas vesicles. I was launched into this by the discovery of Bowen & Jensen (1965) that gas vacuoles of cyanobacteria were made up of cylindrical gas vesicles, packed like the cells of a honeycomb. Their report ended a hiatus of over twenty years since the scholarly review on gas vacuoles by G.E. Fogg, then a 21 year-old graduate student of F. E. Fritsch. Hans Klebahn had been the dominant figure but his fêted ‘hammer, cork and bottle’ experiment, demonstrating the loss of gas vauoles under pressure, belonged to Ahlborn (1894). Years later, the report of Houwink (1956) of gas vesicles in halobacteria was also eclipsed, by the later investigations on cyanobacteria. Discoveries made in isolation can become consigned to obscurity. I was using a mass spectrometer for 15N2-fixation studies in 1965 and I thought I would use the technique to identify the gas-vacuole gas. Two years later, using a modified “blood-gas manometer” of Barcroft and Haldane (1902), I showed that gas vesicles were freely permeable to gases. This led to investigations of the gas vesicles’ collapse under pressure, which affected many aspects of these structures. For example, cell turgor presure which presses on gas-vesicles, could be determined from the modified collapse pressure. The rigid, protein wall of the gas vesicle, little more than one molecule thick, obeyed the principles of engineering used in making pipes and submarines, principles also relevant to the provision of buoyancy of cyanobacteria in lakes, where gas vesicles must survive the pressure increasing with depth. In deeper lakes are cyanobacteria that evolved stronger gas vesicles, which are necessarily narrower and more expensive to make; in shallow salty pools, where cells have no turgor pressure, the weakest, widest and cheapest gas vesicles suffice.
F. Robert Tabita, Ohio State University, Columbus, OH, USA For many years our laboratory has studied the biochemistry and regulation of CO2 assimilation in phototrophic prokaryotes. In this talk, the central role that RubisCO and the Calvin-Benson-Bassham (CBB) pathway play in controlling many different aspects of metabolism will be stressed. Some 15 years ago, we discovered that the molecular control of key processes such as CO2 fixation, nitrogen fixation, hydrogen metabolism, and sulfur oxidation/reduction are linked in photosynthetic bacteria. Indeed, when the primary means by which these organisms balance their intracellular redox state is blocked (via inactivating the CBB reductive pentose pathway such that the preferred metabolic electron acceptor, CO2 , is no longer used) interesting adaptive mutant strains may be selected. One adaptive “trick” that we discovered in four separate organisms is that the nitrogenase complex may be derepressed so that this enzyme system acts exclusively as a hydrogenase and reduces protons to balance excess redox equivalents that are produced via the oxidation of organic carbon during photosynthetic growth. Such strains eructate massive quantities of molecular hydrogen, even in the presence of fixed nitrogen; some strains more than others. However, it is clear that the carbon assimilatory process influences this capability. It will also be shown that RubisCO and the control of the CBB cycle plays a central role in mediating other aspects of normal metabolism of these organisms.
In my researches on gas vesicles I depended on collaborations with specialists in diverse disciplines – the mathematical physicist John Simpson, the freeze-etching pioneer Daniel Branton, the crystallographer Allen Blaurock, and the molecular biologist John Walker; Paul Hayes, who started as my apprentice in things molecular, soon became my mentor, the co-superviser of some clever students and collaborator with visitors to our lab. Other valued contributers were the biologists drawn into my obsession with gas vesicles and buoyancy when I visited their institutes – Colin Reynolds in the English Lake District; Lucas Stal, who organised our Baltic cruises; and Ferdinand Schanz who sampled Planktothrix for a decade from Lake Zürich. I suspect that the fruitless attempts of Fogg (1941) to induce gasvacuole formation in Anabaena, led to his discovery that fixed nitrogen inhibits heterocyst formation. My career began as his graduate student with papers on the first isolation not of gas vesicles, but of heterocysts (with Peter Fay), and then with Bill Stewart, Fay and Fogg, the theory of nitrogen fixation by heterocysts, now one of microbiology’s forgotten discoveries. As Mr Ono discovered, attitudes changed. August 9 to 14, 2009 • Montréal, QC, Canada
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-1.1
PL-1.2
EVOLUTIONARY RELATIONSHIPS AMONG PHOTOTROPHIC BACTERIA DEDUCTED FROM WHOLE GENOME COMPARISONS.
ORIGIN AND EVOLUTION OF THE PHOTOSYNTHETIC FUNCTION.
Robert Blankenship, Departments of Biology and Chemistry, Washington University, St. Louis, USA. Photosynthesis is a central biological process that has a long and complex evolutionary history. The photosynthetic machineries found in the existing groups of phototrophs have both common and divergent characters, suggesting an evolutionary process that combines de novo gene appearance, gene duplication, gene and pathway recruitment and loss, with both vertical and horizontal genetic transfer. The earliest phototrophs were almost certainly anoxygenic and were in existence on Earth by at least 3.4 billion years ago and possibly somewhat earlier. Oxygenic phototrophs were undoubtedly in existence by 2.4 billion years ago when free molecular oxygen, the waste product of oxygenic photosynthesis, began to accumulate in the atmosphere. These organisms may have been present up to several hundred million years before that time. Several lines of evidence, including molecular evolution analysis, structural comparisons, and biochemical and biophysical data, suggest that all modern photosynthetic reaction centers are derived from a single ancient common ancestor and that the anoxygenic phototrophs preceded oxygenic ones. The transition from anoxygenic to oxygenic photosynthesis was accompanied by a number of evolutionary innovations, including multiple gene duplication and divergence events, modification of the pigment biosynthesis pathways from anaerobic to oxygen-requiring, invention of the oxygen evolution center and a dramatic increase in the reaction center protein subunit complexity. The precise evolutionary pathway that led to the current diversity of different types of phototrophs was not linear and involved significant amounts of horizontal gene transfer.
Armen Y. Mulkidjanian1,2, Michael Y. Galperin3, Eugene V. Koonin3. 1
School of Physics, University of Osnabrueck, Osnabrueck, Germany; A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; 3NCBI, NLM, National Institutes of Health, Bethesda, MD, USA. 2
Introduction: Based on a comparative analysis of 15 cyanobacterial genome sequences as well as complete genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus and Heliobacillus mobilis, we have suggested that the (bacterio)chlorophyll-based photosynthesis had originated in the anoxygenic ancestors of cyanobacteria (‘procyanobacteria’) and then spread to other phyla via lateral gene transfer [1]. Results: This scheme will be considered in a broader context of the evolution of complex cellular systems and, in particular, of the energyconverting machinery [2-4]. It will be argued that whether or not an increased complexity of a cellular system would be preserved in the course of evolution is determined by a trade-off between the gains that are brought by the system and its maintenance costs. Conclusions: The evolution of the primeval pro-cyanobacterial photosynthetic machinery can be considered as going in two directions, namely (1) towards the sophisticated photosynthetic apparata of the modern cyanobacteria that, besides being able to capture the energy of light, can exploit water as an unlimited source of electrons and 2) towards the “spare” photosynthetic appliances of other bacteria that have tighter energy budgets. References: 1. Mulkidjanian, A.Y., E. V. Koonin, K. S. Makarova, S. L. Mekhedov, A. Sorokin, Y. I. Wolf, A. Dufresne, F. Partensky, H. Burd, D. Kaznadzey, R. Haselkorn, M. Y. Galperin (2006) The cyanobacterial genome core and the origin of photosynthesis, Proc. Natl. Acad. Sci. U.S.A., 103: 13126-13131. 2. Mulkidjanian, A.Y., M.Y. Galperin, K.S. Makarova, Y.I. Wolf, E.V. Koonin (2008) Evolutionary primacy of sodium bioenergetics, Biol Direct 3 13. 3. Mulkidjanian, A.Y., P. Dibrov, M.Y. Galperin (2008) The past and present of the sodium energetics: May the sodium-motive force be with you, Biochim. Biophys. Acta, 582, 238-242. 4. Mulkidjanian, A.Y., M.Y. Galperin, E.V. Koonin (2009) Co-evolution of membranes and membrane bioenergetics, Trends Biochem. Sci., in press, DOI: 10.1016/j.tibs.2009.01.005.
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-1.3
PL-2.1
ALKALIPHILIC ANOXYGENIC PHOTOTROPHIC BACTERIA: DIVERSITY AND NEW TAXONS.
MARINE NITROGEN-FIXING CYANOBACTERIA METAGENOMIC AND PROTEOMIC ANALYSES. Birgitta Bergman, Department of Botany, Stockholm University, Stockholm, Sweden.
Vladimir Gorlenko, Irina Bryantseva, Ekaterina Boldareva. Vinogradsky Institute of Microbiology RAS, Moscow, Russia. The first isolates of alkaliphilic purple sulfur bacteria (PSB) obtained from the Vadi Natrun soda lakes included moderately halophilic members of the genus Ectothiorhodospira (Ect. haloalkalophila, Ect. vacuolata, and Ect. shaposhnicovii) and extremely halophilic Halorhodospira species (Hlr. halophila, Hlr. abdelmalekii, and Hlr. halochloris). We have described two new alkaliphilic genera of the family Ectothiorhodospiraceae, Thiorhodospira and Ectothiorhodosinus. The members of these new genera PSB differed from the known species in the topography of their photosynthetic membrane structures (PS). Recent research revealed big purple spirilla, “Ect. magna”, sp. nov., closely related to Ect. shaposhnicovii. Simialr to Thiorhodospira sibirica, their PS contained irregular intracellular lamellae and precipitated sulfur in the periplasm, like to members of the family Chromatiaceae. Microorganisms with gas vacuoles, described as Ect. variabilis, are widespread in the soda lakes where the mineralization is higher than ion seawater; this species is phylogenetically different from Ect. vacuolata. Thus, our findings confirm the low taxonomic value of the presence of intracellular gas vacuoles. Bacteria morphologically and phylogenetically related to Ect. shaposhnicovii were recently isolated from the Mono Lake (California); they are capable of a new type of photosynthesis with arsenite as an electron donor. The first alkaliphilic member of Chromatiaceae, Thioalkalicoccus limnaeus (containing BChl b), was isolated from Transbaikalian soda lakes. We investigated new strains of this species of alkaliphilic phototrophic bacteria. The second member of Chromatiaceae, Thiocapsa imhoffii (containing BChl a), was recently described. In the lakes with low salinity, alkalitolerant members of the genera Allochromatium and Thiocapsa are also present; the systematic position of these species has not yet been determined. Among nonsulfur purple bacteria (NPB), two Rhodobaca species are alkaliphilic. The new NPB Rubribacter polymorphus, was isolated from a soda lake in the Barguzin Valley. Some species of the genus Rhodovulum were also shown to be alkaliphilic. Close phylogenetic similarity was revealed between NPB and anaerobic, bacteriochlorophyll a-containing bacteria (ABC) of the genus Roseinatronobacter; this finding indicates their evolutionary relatedness. Similarity exists between NPB and ABC in their reaction centers and the electron transfer system. The new members of the genus Roseococcus, Rsc. suduntuensis and Rsc. vulcaneus were demonstrated to be alkaliphilic (natronophilic) but not halophilic. The alkaliphilic genus Heliorestis contains three natronophilic species. It may be concluded that obligate alkaliphily and natrononophily developed as the result of evolutionary selection caused by prolonged existence of the microorganisms under the alkaline conditions of soda environments. The taxonomic rank of obligate alkaliphiles is usually that of a genus. Such secondary adaptations as active motility by means of flagella, formation of gas vesicles and of cyst-like microcolonies are important from the ecological point of view. The intracellular membranes are diverse and correlate with the cell size and bacteriochlorophyll type.
August 9 to 14, 2009 • Montréal, QC, Canada
It has become increasingly clear that diazotrophic cyanobacteria play a crucial role in maintaining life in oceans, major players being members of the genus Trichodesmium. We are interested in understanding the structural and molecular background related to the maintenance of diazotrophy in Trichodesmium IMS101, a non-heterocystous cyanobacterium fixing nitrogen in light/day. We have approached this by examining events at the structural, transcriptomic and proteomic level, with focus on cellular dynamics during the development of the nitrogen-fixing cells, the diazocytes. The data obtained demonstrated a diurnal separation of basic physiological processes, with nitrogenfixation and photosynthesis confined to the light and cell division and diazocyte development to the dark period. When non-diazotrophic cultures were subject to nitrogen depletion a subset of cells differentiated into nitrogenase containing diazocytes, while addition of combined nitrogen abolished development. Comparative proteomic analysis revealed that ninty-four proteins were differentially expressed in diazotrophic (with diazocytes), as opposed to in the nondiazotrophic cultures, such as the nitrogenase enzyme and proteins related to supporting reducing equivalents and lowering oxygen tensions. Some recent metagenomic analyses of size fractionated microbes from a transect spanning the Indian Ocean will also presented.
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-2.2
PL-3.1
PHYSIOLOGY OF MICROBES IN THE HOT SPRINGS: REGULATION AND POTENTIAL INTERACTIONS.
MULTICELLULARITY IN THE HETEROCYST-FORMING CYANOBACTERIUM ANABAENA.
Arthur R. Grossman1, Anne-Soisig Steunou2, Rosario Gomez-Garcia1, Melissa Adams1, Michael Kühl3, John Heidelberg4, Devaki Bhaya1.
Enrique Flores, Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain.
1 The Carnegie Institute for Science, Department of Plant Biology, Stanford, CA, USA; 2CNRS, Centre de Génétique Moléculaire, Gif-surYvette, France; 3University of Copenhagen, Marine Biology Laboratory, Department of Biology, Helsingør, Denmark; 4University of Southern California, Department of Biological Sciences, Avalon, CA, USA.
Filamentous, heterocyst-forming cyanobacteria have been described to reproduce by random trichome breakage implying that their unit of growth is the filament. Under combined nitrogen deprivation, growth of filaments depends on the activity of two different types of cells, nitrogen-fixing heterocysts and photosynthetic vegetative cells, which exchange nutrients and regulatory compounds making the filaments true multicellular organisms. They carry a Gram-negative type of cell envelope in which, as shown by electron microscopy, the outer membrane is continuous along the filament without entering the septa between cells. The periplasmic space, which lies between the cytoplasmic and outer membranes, is therefore continuous along the filament, and this is so not only between vegetative cells but also between vegetative cells and heterocysts. In the model organism Anabaena sp. PCC 7120, a periplasmic form of the green fluorescent protein produced in (pro)heterocysts can be detected at a distance in the periplasm of vegetative cells showing that the periplasm is also functionally continuous and suggesting that it could constitute a communication conduit between the cells in the filament. Recent studies have shown that the Anabaena outer membrane is a permeability barrier for some sugars and amino acids that are important in the diazotrophic physiology of these organisms. On the other hand, electron microscopy also shows the presence in the intercellular septa of thin structures perpendicular to the cytoplasmic membranes of adjacent cells. In Anabaena sp. PCC 7120, a protein located at the cell poles in the intercellular septa, which consists of a predicted N-terminal extracytoplasmic domain and a C-terminal permease domain, has been identified and named SepJ, and two proteins, FraC and FraD, required for focused localization of SepJ have also been identified. Mutants of the genes encoding these proteins show a filament fragmentation phenotype indicating that they contribute to keep cells together in the filament. A method for testing intercellular molecular exchange in filamentous cyanobacteria has been developed that consists in loading of a fluorescent dye, calcein, in the cytoplasm of the cells and measuring calcein fluorescence in Fluorescence Recovery After Photobleaching (FRAP) experiments. Calcein exchange takes place rapidly between the cells in Anabaena filaments but is impaired in sepJ, fraC and fraD mutants suggesting that these proteins are involved in intercellular molecular exchange. Our current view of the Anabaena filament is that of a string of cells which are encapsulated by a continuous outer membrane and share the periplasm, and that are connected by proteinaceous structures that could also be involved in intercellular communication. Identifying which compounds move in the filament through the periplasm or the cell-to-cell joining structures is a major topic for future research.
Introduction: Hot spring microbial mats are natural biofilms composed of oxygenic photosynthetic cyanobacteria as well as heterotrophic microbes. These organisms are distributed along horizontal thermal and vertical light and oxygen gradients. The mats that we have been studying are from Octopus and Mushroom Springs of Yellowstone National Park. They are generally 1-2 cm thick, with densely packed cyanobacteria of the genus Synechococcus in the upper mat layer. The Synechococcus of the hot spring mats can harvest light energy for photosynthetic CO2 fixation at temperatures exceeding 70oC. Methods: Our work involves sequencing genomes from specific Synechococcus ecotypes that reside at different temperatures in the mat and also generating metagenome sequence information. Moreover, we have analyzed the abundances of specific transcripts using reverse transcriptase quantitative PCR and measured levels (immunologically) and activities of specific proteins over the diel cycle in situ. Results and Conclusions: The genomes of two Synechococcus variants from the hot springs of Yellowstone National Park were sequenced, and while they have similar gene content, they are remarkably different with respect to gene arrangement. Furthermore, the genomes of both of the cyanobacterial isolates harbor a full complement of nearly identical nitrogen-fixation (nif) genes. The nif genes are expressed in the mat toward evening, when the mat becomes anoxic, although the highest in situ N2 fixation rates are measured in early morning, when the mat is still anoxic but light starts to drive photosynthesis and the production of ATP. The mat community also switches from oxidative metabolism in the day, when cells are photosynthesizing, to fermentation metabolism in the evening. This metabolic switching is reflected by rapid changes in gene expression in response to anaerobic/aerobic conditions. We have also cultured Synechococcus variants from the hot spring mats and show that they can use various forms of nutrients, including phosphonates as a sole source of phosphate. I will discuss the physiology, metabolism and energetic constraints within the mat environment over the diel cycle.
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-3.2
PL-3.3
EVOLUTION AND IMPORTANCE OF PHOTORESPIRATORY 2-PHOSPHOGLYCOLATE METABOLISM IN CYANOBACTERIA.
THE NETWORK OF PII SIGNALING PROTEIN INTERACTIONS IN CYANOBACTERIA .
Martin Hagemann, University of Rostock, Institute of Biological Sciences, Plant Physiology, Rostock, Germany.
Karl Forchhammer, Institut für Mikrobiologie der Eberhard-KarlsUniversität Tübingen, Tübingen, Germany.
Introduction: Cyanobacteria fix most of the carbon using the CalvinBenson cycle with RubisCO as the carboxylating enzyme. However, Rubisco has a rather low affinity to CO2 and exhibits in the presence of oxygen also an oxygenase activity resulting in the toxic byproduct 2phosphoglycolate (2PG). This intermediate is detoxified by the photorespiratory 2PG cycle in plants. Cyanobacteria evolved an inorganic carbon concentrating mechanism (CCM) allowing them to cope with low carbon concentrations in the presence of oxygen. Active uptake systems for inorganic C accumulate high internal bicarbonate amounts. Moreover, RubisCO is concentrated in carboxysomes, where bicarbonate is transferred to CO2 leading to high CO2 concentration in the vicinity of RubisCO making oxygenase activity in cyanobacteria unlikely. Accordingly, it was widely accepted that cyanobacteria do not perform photorespiratory 2PG metabolism. During the last years we analyzed the mode of 2PG metabolism and its possible diverse function with the cyanobacterial model strain Synechocystis sp. PCC 6803.
Introduction: The family of PII signal transduction proteins is represented in all domains of life, and is especially abundant in organisms capable of inorganic nitrogen assimilation. The elementary mode of PII action is sensing the state of central metabolism/nitrogen assimilation and in response, orchestrating cellular processes related to nitrogen assimilation by binding to key factors involved in nitrogen control. Sensing the state of central metabolism is achieved by interdependent binding of effector molecules to PII, in particular, 2oxoglutarate and adenyl-nucleotides (ATP and ADP), and eventually by additional covalent modification of PII by modifying enzymes.
Methods:Defined mutants of Synechocystis defective in candidate genes for glycolate metabolism were generated by interposon mutagenesis (1, 3). Moreover, microarray analyses were used to search for genes coding for alternative routes in the glycolate metabolism (2). Results:Single-mutants in candidate genes for 2PG metabolism of Synechocystis showed only small changes, while the generation and characterization of double- and triple-mutants showed that an active 2PG metabolism employs three different routes: A) Glycolate is degraded by a plant-like C2 cycle. B) A route exists corresponding to the bacterial glycerate pathway. C) Additionally, the complete decarboxylation of glycolate via oxalate and formate occurs. A complete block in all three postulated routes resulted in a high-CO2requiring (HCR) phenotype as it is known from photorespiratory mutants in higher plants. Bioinformatic analyses were used to search for corresponding genes in the completed genomes of other cyanobacterial strains, which indicated that generally all cyanobacteria are able to metabolize 2PG despite the existing strain-specific differences in the occurrence of single pathways. The photorespiratory metabolism contributes also to high light acclimation in Synechocystis, since a combined mutation of 2PG metabolism and the so-called Mehler reaction resulted in higher light sensitivity. Conclusions: We could show that cyanobacteria perform an active photorespiratory metabolism despite the activity of the CCM. The 2PG metabolism uses three different routes in Synechocystis. A complete block of 2PG metabolism resulted in a HCR phenotype, which shows that this metabolism is essential for organisms performing oxygenic photosynthesis in the present day atmosphere. These findings gave rise to the hypothesis that an early photorespiratory has evolved already in ancient cyanobacteria and was conveyed endosymbiontically into plants. The main function of the glycolate metabolism is directed to the detoxification of critical intermediates. However, it is also involved in the acclimation to high light conditions. 1 – Eisenhut et al. (2006) Plant Physiol 142:333-342 2 – Eisenhut et al. (2007) Plant Physiol 144:1946-1959 3 – Eisenhut et al. (2008) PNAS 105:17199-17204
August 9 to 14, 2009 • Montréal, QC, Canada
In most cyanobacteria, one highly conserved gene, termed glnB, encoding a PII homologue has been identified in the genomic sequences. Some strains, such as Gloeobacter violaceus, Synechococcus sp. WH5701 or Acharyochloris marina harbor one or two further potential glnB paralogues. Functions and properties of cyanobacterial PII signalling were mostly worked out with the proteins from strains Synechococcus elongatus and Synechocystis sp. PCC 6803. There, PII is covalently modified by S49 phosphorylation, but this apparently does not apply to all cyanobacteria. Here, I will present interaction studies of PII with different target molecules, PII-phosphatase PphA, N-acetylL-glutamate kinase (NAGK) and PipX. PII - phosphatase interaction: The structure of the phospho-PII phosphatase PphA from Thermosynechococcus elongatus was resoved recently (1). Based on this structure, mutants were generated to elucidate the basis of substrate recognition of PphA. Further, an assay was established to stabilize PII-PphA interaction intermediates. Metal 1 (M1) of the trinuclear metal centre of PphA, a PPM family phosphatase, is absolutely required for binding PII-P. Furthermore, several residues at the periphery of the enzyme could be identified to be required for productive PII-P binding, but without affecting the activity of the catalytic centre towards artificial substrates. PphA binding of PII responds specifically to the presence of PII effector molecules ATP/ADP and 2-oxoglutarate, indicating that the entry of the phosphorylated T-loop of PII into the catalytic cavity is controlled by the effector molecules. PII-NAGK interaction: NAGK, the controlling enzyme of arginine biosythesis, by binding PII, is catalytically activated and relieved from arginine inhibition. The structure of the PII-NAGK complex from Synechococcus elongatus was resolved (2) and the catalytic activation of NAGK by PII was investigated in detail. 2-oxoglutarate antagonizes the PII mediated activation of NAGK. Compared to PII/NAGK from Arabidopsis, the principal mode of PII activation - in particular its response to 2-oxoglutarate - is conserved in these oxygenic phototrophs (3). By random mutagenesis combined with bacterial-two hybrid screening, we could identify several PII mutants with novel phenotypes regarding NAGK and effector metabolite binding, leading to novel insights in PII function. This involves in particular residues at the B-loop of PII, which appears to play a key role in the intra-molecular transmission of metabolite signals. PII-PipX interaction: A further PII target, PipX, appears to be a transcriptional co-activator of NtcA. PII competes with NtcA for binding to PipX, thereby antagonizing the activation of NtcA (Espinosa et al., 2007). Whereas binding of PipX to NtcA requires 2-oxoglutarate, 2-OG in concert with ATP im37
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts pairs PipX binding to PII. When PII and NtcA compete for PipX in presence of constant 2-oxoglutarate levels, PipX swaps form PII to NtcA when ATP is added. The NtcA-PipX complex binds to DNA, with PipX not affecting the DNA binding properties of NtcA. When PipX and NAGK compete for PII binding in absence of 2-OG, PII preferentially binds to NAGK.
PL-4.2 EFFECTS OF INSERTION SEQUENCES ON THE GENOME OF ANABAENA SP. STRAIN PCC 7120. C. Peter Wolk, Sigal Lechno-Yossef.
Other PII interactions:
Michigan State University, East Lansing, MI, USA.
Further to the above-mentioned targets, PII was shown to bind to a MscS-like protein from Synechocystis PCC 6803, PamA (5). In contrast to PipX or NAGK, PamA is not highly conserved in cyanobacteria, and it remains to be elucidated what the physiological function of this protein is. Mutants in the PII signalling system show impaired ability to regulate nitrate utilization (6) implying PII interactions with components of the nitrate utilization pathway.
Introduction: Many cyanobacteria closely related to Anabaena sp. strain PCC 7120 make akinetes and gas vacuoles, but PCC 7120 lacks those properties despite having homologs of the akinete-marker gene avaK (1) and gas vacuole-related orfs all2247 through asl2254. It bears ca. 145 orfs that are annotated as encoding transposases (http://bacteria.kazusa.or.jp/cyanobase/), which are diagnostic of insertion sequences (ISs). Some of the ISs surely transpose (2-4, and compare 5 and 6), and can thereby mutagenize. Our laboratory earlier identified three evidently IS891-interrupted regulatory genes in Anabaena sp. (7). Using the newly available sequence data for various cyanobacteria, we have returned to an in-silico attempt to examine features of the genome of PCC 7120 as it may have appeared prior to entry of those ISs.
The overall organization of the PII signal transduction network will be discussed. 1. Schlicker et al., 2008 J. Mol. Biol. 376: 570-581 2. Llacer et al, 2007 Proc. Natl. Acad. Sci. U.S.A. 104: 17644-17649 3. Beez et al., 2009 J Mol Biol. 389: 748-758 4. Espinosa et al., 2007 Microbiology 153: 711-718 5. Osanai et al., J. Biol. Chem. 280: 34684–34690 6. Kloft & Forchhammer, 2005 J. Bacteriol. 187: 6683-6690
PL-4.1 A CYANOBACTERIAL MODEL FOR HOW CELLS TELL TIME. Susan Golden, Department of Biology, Professor Division of Biological Sciences University of California-San Diego, La Jolla,CA, USA. Cyanobacteria, like diverse eukaryotes, possess circadian clocks that allow cells to coordinate physiological processes with the predictable daily patterns of environmental fluctuations on Earth. An autonomous oscillator comprised of three distinctive proteins, KaiA, KaiB, and KaiC, underpins the circadian clock of the cyanobacterial model organism Synechococcus elongatus PCC 7942. Our work applies genetic, genomic, biochemical, and structural approaches, integrated with cell biology, to achieve a comprehensive understanding of how the S. elongatus cell harnesses this oscillator to control cellular activities. The emerging picture is one of a clock that is set daily by sampling the cellular redox environment as a proxy for light and which uses protein-protein interactions and subcellular localization to coordinate gene expression, cell division, and chromosome dynamics. A functional genomics project has produced inactivation alleles of nearly all S. elongatus loci. Gene expression assays of the 700 mutants tested so far suggest that about 10% of loci affect circadian period or phasing.
Methods: To “remove” an IS, one must know how far it extends outwards from its transposase orf(s) and how many base pairs it replicates upon insertion. Such information is provided by: transposition of ISs to known genetic backgrounds; alignments of sequences containing related ISs to see where their commonality ceases, with the ISs often terminating in inverted repeats and frequently bracketed by direct repeats; and the finding that in other organisms, sequences that are nearly identical to those that flank ISs are joined. Results: Depending on whether neighboring transposase orfs are part of the same IS, PCC 7120 may have ca. 100-150 ISs. We have excised most, in silico, with convincing accuracy; and have identified further elements that, although lacking an annotated transposase, appear to be mobile. Once ISs are removed, we find: nearby orfs extended by replacement of their initiation or termination codons; flanking orfs fused in-frame; orfs previously unrecognized, including those of underlying ISs, found; orfs lost that consisted principally of ends of ISs; and extended intergenic regions that appear empty of orfs. Anabaena variabilis orthologs of two of the kinase genes intercepted in PCC 7120 may also have been intercepted by ISs. Conclusions: We have not identified a phenotype that is surely attributable to the presence of an IS. However, our “archeological genomics“ permits a view of at least part of the genome of PCC 7120 in a more ancestral form. We have identified presumptive orfs, operons, and regulatory genes whose restoration might enlarge PCC 7120‘s phenotype, including its ability to protect itself against entering DNA. Finally, we have added to knowledge of the ISs of PCC 7120 (and other bacteria): have they inverted terminal repeats, do they generate direct repeats upon insertion, and how widespread is their taxonomic dispersion? The excavation continues. References: 1. R Zhou and CP Wolk, J Bacteriol 184: 2529 (2002). 2. Y Cai and CP Wolk, J Bacteriol 172: 3138 (1990). 3. J Alam et al., J Bacteriol 173: 5778 (1991). 4. Y Cai, J Bacteriol 173: 5771 (1991). 5. I Bancroft and CP Wolk, J Bacteriol 171: 5949 (1989). 6. T Kaneko et al., DNA Res 8: 205 (2001). 7. M Ohmori et al., DNA Res 8: 271 (2001).
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-5.1
PL-5.2
CYTOCHROME C MATURATION, DISULFIDE BOND FORMATION AND PERIPLASMIC PROTEIN DEGRADATION IN RHODOBACTER CAPSULATUS.
BETTER LIVING THROUGH CYANOTHECE--UNICELLULAR DIAZOTROPHIC CYANOBACTERIA WITH HIGHLY VERSATILE METABOLIC SYSTEMS.
Ozlem Onder, Serdar Turkarslan, Carsten Sanders, Fevzi Daldal.
Louis A. Sherman1, Hongtao Min1, Jorg Toepel1, Michelle Liberton2, Hanayo Sato2, Jana Stöckel2, Himadri B. Pakrasi2.
University of Pennsylvania, Department of Biology, Philadelphia, PA, USA. Current knowledge on cytochrome c maturation process in the facultative phototrophic bacterium Rhodobacter capsulatus will be reviewed, and the crucial role of periplasmic thio-redox homeostasis (DsbA-DsbB versus CcdA-CcmG) involved in forming and breaking of apocytochrome c disulfide bonds during this process will be described. In addition, we will report the unusual observation that DsbA-null mutants are proficient in photosynthesis but are defective in respiration, especially in enriched growth medium at 35 ºC. Using combined proteomic and molecular genetic approaches we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originate from the overproduction of the periplasmic protease DegP, which render them temperature sensitive for growth. The DsbAnull mutants revert frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. We have shown that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for the proteasechaperone activity. Remarkably, although R. capsulatus DsbA-null or DegP-null mutants are viable, DegP-null DsbA-null double mutants are lethal at all growth temperatures. We found that this is unlike E. coli, indicating that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations amongst the DegP homologues, including a critical disulfide bond-bearing region, correlate well with the differences seen between various species like R. capsulatus and E. coli, and illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.
1 2
Dept. of Biological Sciences, Purdue University, West Lafayette, IN; Dept. of Biology, Washington University, St. Louis, MO; USA.
Cyanothece sp. ATCC 51142 is a unicellular, diazotrophic cyanobacterium with a versatile metabolism and very pronounced diurnal rhythms. Since nitrogen fixation is exquisitely sensitive to oxygen, Cyanothece utilizes temporal regulation to accommodate these incompatible processes in a single cell. When grown under 12h light-dark (LD) periods, it performs photosynthesis during the day and N2 fixation and respiration at night. During this process, carbohydrates and amino acids are compartmentalized in granules in the light and dark, respectively. In essence, Cyanothece creates an O2limited intracellular environment to perform oxygen-sensitive processes such as N2-fixation and H2 production during the night. This strain also grows exceedingly well heterotrophically (on glycerol). The excellent synchrony of a culture under LD diazotrophic conditions permits analysis of cellular morphology, mRNA levels, proteomics and metabolomics as a function of time. The genome sequence of Cyanothece sp. ATCC 51142 (Welsh et al (2008) PNAS 105: 15094-99) and that of 5 other Cyanothece sp. are complete and that of a seventh is in progress. Cyanothece sp. ATCC 51142 has 2 chromosomes—a 4.9 Mb circular chromosome and a 0.43 Mb chromosome with a total of 5,300 genes. This strain has the capability of producing high-energy compounds, such as ethanol and hydrogen and Cyanothece sp. PCC 7822 can produce these compounds as well as rather large quantities of parahydroxyalkanotes (PHAs). We are particularly interested in the regulation of these metabolic processes and the way in which these organisms respond to environmental cues such as light, the lack of combined nitrogen and changing O2 levels. Analysis at both the transcriptomics (with microarrays, Toepel et al (2008) J Bact 190:3904 and Stockel et al (2008) PNAS 105:6156) and proteomics levels in Cyanothece sp. ATCC 51142 has demonstrated the relationship of the metabolic synchrony with gene expression and has given us insights into diurnal and circadian regulation throughout a daily cycle. The Cyanothece strains (51142 and 7822 have been the most studied) produce copious amounts of H2 under different types of physiological conditions. Both the bidirectional hydrogenase (encoded by the hox genes) and the nitrogenase (nif genes) are capable of producing measurable hydrogen under appropriate conditions. However, nitrogenase produces far more H2 than the hydrogenase, in part because the nitrogenase levels are extremely high under N2-fixing conditions. With Cyanothece 51142 cultures grown in NO3-free media, either photoautotrophically or mixotrophically with glycerol, we have obtained rates of H2 produced of 150 mmoles/mg Chl/hr and 300 mmoles/mg Chl/hr, respectively. We anticipate even higher values once we are able to construct specific mutants and develop optimal physiological conditions. This work was sponsored by grants from the DOE and from the EMSL at the Pacific Northwest National Labs.
August 9 to 14, 2009 • Montréal, QC, Canada
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13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
PL-5.3
PL-6.1
PROCHLOROCOCCUS AND SYNECHOCOCCUS: DIVERGENT EVOLUTION SCHEMES FROM A COMMON ANCESTRAL GENOME.
OXYGEN AND LIGHT CONTROL OF TETRAPYRROLE BIOSYNTHESIS IN RHODOBACTER SPECIES.
Frédéric Partensky1, Alexis Dufresne1, 2, Martin Ostrowski3, David J. Scanlan3 and Laurence Garczarek1 . 1
UMR 7144 CNRS & Université Paris 6, Station Biologique, Roscoff, France; 2UMR 6553 EcoBio, Université Rennes, Rennes, France; 3 Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. Prochlorococcus and Synechococcus are the two most abundant oxyphototrophs in the ocean. These two marine picocyanobacteria are phylogenetically closely related and together make a branch well separated from all other cyanobacteria, including freshwater Synechococcus but also marine cyanobacteria of larger cell size, including Trichodesmium thiebautii and Crocosphaera watsonii. The large number of genomes of Prochlorococcus (13) and marine Synechococcus (11) that have recently been sequenced, covers most of the diversity known within these groups. This makes it possible to draw insights about the mechanisms that have presided genome evolution in these ecologically important groups. It is now clear that Prochlorococcus is a fairly recent group, which is highly specialized to life in warm, oligotrophic waters. It appeared quasi concomitantly with the highly diversified Synechococcus subcluster 5.1, whose members are seemingly the best represented in the ocean nowadays. Light has been a key environmental factor in the evolution of both genera. Prochlorococcus has first colonized the bottom of the euphotic zone then, more recently, the upper layer of oligotrophic zones. As a result, Prochlorococcus has differentiated itself into low- and high light ecotypes, a phenomenon that has allowed this genus to colonize the whole sunlit layer. For Synechococcus, however, light quality has seemingly been the key factor, since the diversification of its pigment content has allowed this genus to colonize the whole range of environments over the coastal to offshore gradient. But other environmental factors, including salinity, temperature or nutrient availability are also important in shaping the genomes. In this talk, current scenarios for genome evolution in these two key organisms that originate from a common ancestor will be presented. In particular, the impact of environmental factors (mainly light) on the genome content and structure of the different ecotypes within each genus will be discussed.
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Carl Bauer, Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. The purple photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides synthesizes three tetrapyrrole endproducts, bacteriochlorophyll, heme and cobalamine (vitamin B12). All three tetrapyrroles are the product of a complex pathway that is responsible for controlling synthesis of these endproducts in response to light intensity and oxygen tension. Over the past decade our laboratory has identified numerous transcription factors that are responsible for the control of tetrapyrrole gene expression. Recent biochemical analysis of several of these transcription factors has demonstrated that many also utilize heme or cobalamine as cofactors. Our current understanding of the control of tetrapyrrole biosynthesis in these species will be presented as well as what is known about the biochemical properties of key regulators.
PL-6.2 A PHOTOACTIVE CAROTENOID PROTEIN ACTING AS LIGHT INTENSITY SENSOR IN CYANOBACTERIA PHOTOPROTECTION. Adjélé Wilson, Clémence Boulay, Claire Punginelli, Diana Kirilovsky. Commissariat à l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS) and Centre National de la Recherche Scientifique (CNRS), 91191 Gif sur Yvette, France. Because too much light can be lethal for photosynthetic organisms, photoprotection against excess absorbed light energy is an essential and universal attribute of oxygenic photosynthetic organisms. In plants and algae, the membrane embedded chlorophyll antenna, LHCII, reversibly switchs from a very efficient energy collection state into a photoprotective state. This state by converting the excess energy into heat decreases the energy arriving at the reaction centers under high light conditions. The existence of an equivalent process in cyanobacteria, which use the extramembranal phycobilisomes to harvest energy instead of LHCII, was only recently discovered. The study of the photoprotective thermal energy dissipation mechanism in phycobilisome- and Photosystem II-mutants of the cyanobacterium Synechocystis PCC6803 by fluorescence measurements allowed to demonstrate that this mechanism involves a specific decrease of the fluorescence emission of the phycobilisomes and a decrease of the energy transfer from the phycobilisomes to the reaction centers. The process is induced by the absorbance of blue-green light by a soluble orange carotenoid protein, the Orange-Carotenoid-Protein (OCP). In Synechocystis , the OCP is encoded by the slr1963 gene which is constitutively expressed. OCP genes appear to be highly conserved among phycobilisome-containing cyanobacteria with few exceptions, indicating that the OCP related photoprotective mechanism is widespread. The strains containing a whole OCP gene can perform the blue-light induced photoprotective mechanism. In contrast, strains containing only N-terminal and/or C-terminal OCP like genes or no OCP like genes at all lack this mechanism and they were more sensitive to high light illumination. These strains to longer survive under stress conditions degrade the phycobiliproteins very fast to avoid the appearance of a population of dangerous, functionally disconnected phycobilisomes. The OCP containing strains increase the expression of the OCP and as a consequence increase the conversion to heat of the excess energy absorbed by the phycobilisomes. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes The OCP is a photoactive protein. The absorbance of blue-green light by the OCP induces structural changes in the carotenoid and the protein, converting its dark stable orange form into a relatively unstable active red form. The presence of the red OCP form is essential for the induction of the photoprotective mechanism. In the absence of the formation of the red form due to point mutations in the OCP, no fluorescence quenching is induced by strong white or blue-green light. OCP is fully active when binds hydroxyechinenone or echinenone. When these carotenoids are absent, OCP binds zeaxanthin but light is unable to photoconvert the dark form into a light active form. In the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. These results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity. Moreover, Trp288 and Tyr 201 forming hydrogen bonds to the carbonyl are essential for the photoconvertion and cannot be replaced by other amino-acid without loosing activity. The OCP is the first photoactive protein containing a carotenoid as the photoactive chromophore and its photocycle is completely different from those of other photoactive proteins.
Final Program and Abstracts The Mo-responsive regulators MopA and MopB are modular in structure consisting of an N-terminal DNA-binding and a C-terminal Mo-binding domain. They bind to conserved palindromic promoter sequences, so-called Mo-boxes. Affinity of the regulators to their target promoters is clearly enhanced by molybdenum. MopA and MopB can substitute for each other in repressing transcription of several target genes including the mopA-modABC operon and the anfA gene. In addition to its role as a repressor, MopA acts as an activator of the mop gene coding for the molbindin-like Mop protein. The genes coding for the Mo-responsive regulators, MopA and MopB, belong to two divergently transcribed operons, mopA-modABC and mopB. While transcription of the mopA operon is repressed by molybdenum, mopB is constitutively expressed, suggesting that the MopA-MopB ratio changes in response to the cellular Mo status. MopA and MopB form homodimers and heteromers. Formation of heteromers is thought to deplete the pool of homodimers, and thus might represent a fine-tuning mechanism controlling expression of Mo-regulated genes.
PL-7.1
PL-6.3
HOW THE CYANOBACTERIUM SYNECHOCYSTIS PERCEIVES ENVIRONMENTAL STIMULI?
NITROGEN AND MOLYBDENUM CONTROL OF NITROGEN FIXATION.
Iwane Suzuki.
Bernd Masepohl, Alexandra Müller, Jessica Wiethaus. Ruhr University Bochum, Bochum, Germany. The phototrophic purple bacterium Rhodobacter capsulatus has the capacity to grow with atmospheric dinitrogen (N2) as sole source of nitrogen. Reduction of N2 to ammonia is catalyzed by two nitrogenases, molybdenum-nitrogenase (Mo-nitrogenase) and an alternative iron-only nitrogenase (Fe-nitrogenase). Fe-nitrogenase exhibits lower specific activity than Mo-nitrogenase, and thus, Monitrogenase is the preferred enzyme as long as molybdenum is available. Upon Mo-depletion, R. capsulatus synthesizes the highaffinity Mo-uptake transporter ModABC. Since nitrogen fixation is a highly energy-demanding process, synthesis and activity of both nitrogenases is tighly controlled. Only in the absence of ammonium the sensor kinase NtrB autophosphorylates, and subsequently, phosphorylates its cognate response regulator NtrC. In turn, NtrC activates transcription of nifA and anfA coding for the transcriptional activators of all the other nitrogen fixation genes. Transcription of anfA is repressed by the Mo-responsive regulators MopA and MopB preventing expression of Fe-nitrogenase in the presence of molybdenum. Finally, activity of both nitrogenases is controlled by a switch-off/switch-on mechanism involving reversible covalent modification, thus enabling R. capsulatus to respond very fast to subtle changes in ammonium availability. Like many other bacteria, R. capsulatus contains two genes, glnB and glnK, coding for PII-like signal transduction proteins, which play central roles in regulation of nitrogen fixation and assimilation. GlnB and GlnK control activity of many target proteins by transient interaction in response to changes in the cellular nitrogen status. Among these target proteins are the sensor kinase NtrB, the transcription activator NifA, and the switch-off protein DraT. Affinity of GlnB and GlnK to their target proteins is modulated by binding of oxoglutarate and ATP, and uridylylation at a conserved tyrosine residue. The uridylylation state of GlnB and GlnK reflects the cellular glutamine concentration, with both proteins being uridylylated when glutamine concentrations are low. Upon ammonium addition both PII proteins are sequestered to the membrane by binding to the ammonium transporter AmtB. August 9 to 14, 2009 • Montréal, QC, Canada
University of Tsukuba, Tsukuba, Ibaraki, Japan. Introduction: Two-component systems, which generally consist of a sensory histidine kinase and a response regulator, are major signaling pathways in most of bacteria. In general, histidine kinases possess a conserved transmitter domain associating kinase activity at the Cterminus and a unique signal input domain (SID) at the N-terminus. The SIDs in the histidine kinases are thought to play important roles in perception of the specific stimuli and regulation of the kinase activity of the transmitter domain, however, the actual molecular mechanisms of signal perception are not clarified yet. I developed a system to express chimeric histidine kinases containing a transmitter domain of SphS, a phosphate-deficient sensor, and SID from other histidine kinase in the cyanobacterium Synechocystis sp. PCC 6803. It might be a useful strategy to investigate the functions of the histidine kinases. Methods: I constructed genes for chimeric sensors containing transmitter domain of SphS, which include either the N-terminal region of a Ni2+-sensor, NrsS, or SID of Hik33. The chimeric histidine kinases were driven by the native promoter of the sphS gene. Effects of the chimera proteins on the activity of SphS transmitter domain were examined by measuring expression levels of the phoA gene for an alkaline phosphatase as an internal reporter. Results: The N-terminal region of SphS contains both a hydrophobic region and a PAS domain. In order to evaluate the function of the Nterminal region of SphS, deletion mutants under the control of the native promoter were analyzed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from NrsS resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi-availability. These results indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and the PAS domain is involved in perception of Pi-availability. This strategy was also applied to characterize the SID in Hik33, which regulate gene expression under cold, high light, oxidative, high salt, and hyperosmotic stress conditions. The SID includes two transmembrane helices, a periplasmic loop, a HAMP and a PAS 41
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Final Program and Abstracts domain. When the SID was fused with the transmitter domain of SphS, it activated the phoA expression under the standard growth condition and repressed under the stress conditions. The repression of expression of phoA was dependent on the PAS domain in SID. The detail results will be discussed in the presentation. Conclusions: The chimeric sensor system may be a useful approach to investigate the function of SID of the histidine kinases.
PL-7.2 EVOLUTION AND REGULATION OF THE RHODOBACTER CAPSULATUS GENE TRANSFER AGENT. J. Thomas Beatty1, Molly Leung1, Sarah Florizone1, Jeanette A. Johnson1, Ryan Mercer2, and Andrew S. Lang2. Department of Microbiology & Immunology, the University of British Columbia1, BC; Department of Biology, Memorial University of Newfoundland2, NF; Canada. Introduction: The gene transfer agent (GTA) produced by the purple non-sulfur bacterium Rhodobacter capsulatus is a model for several virus-like elements that seem to function solely for mediating gene exchange. Some GTA structural genes in R. capsulatus are related to bacteriophage genes, but the cellular regulatory mechanisms that control GTA production indicate that GTA is more than just a defective prophage. Genome sequencing projects have shown that most prokaryotes contain prophage-like gene clusters, but possible relationships to the GTA gene cluster were unclear. The R. capsulatus GTA is produced maximally in stationary phase cultures, but it was not known what aspect of stationary phase induces the expression of GTA structural genes. Our research was done to investigate the presence of GTA genes in a variety of prokaryotes, and to study factors that affect the stationary phase production of GTA in R. capsulatus. Methods: R. capsulatus GTA protein sequences were used as queries in BLAST searches of genome sequences, and the genome context of high-scoring hits was subsequently evaluated by eye. Sequences were aligned with Clustal X v1.81 and the alignment was used to construct neighbor-joining trees with PAUP* v4.0. Cultures of R. capsulatus were grown photoheterotrophically, and cells separated from GTA by centrifugation and filtration through a 0.2 µm filter. The production of GTA was evaluated in gene transduction experiments, or by probing western blots with antiserum raised against the GTA major capsid protein over-expressed in E. coli. Results: Complete genome sequences were searched for GTA gene homologues to identify candidate GTA-producing species. Although homologous GTA gene clusters are widespread, they are found exclusively within the α-proteobacteria. A phylogenetic tree comparing 16S rRNA and GTA gene homologues shows that these two types of gene have evolved in concert. Work on R. capsulatus GTA regulatory gene mutants indicates three independent pathways that affect the production of extracellular GTA particles. One pathway involves the response-regulator CtrA, which is essential for transcription of GTA structural genes. A second pathway transmits a quorum-sensing signal, produced by a homoserine lactone synthase, which is needed for maximal induction of transcription of GTA genes. A third pathway requires the sensor-kinase homologue CckA for release of GTA particles from cells. An evaluation of the effects of carbon, nitrogen and phosphorus deprivation on the synthesis and release of GTA particles indicates that different environmental signals affect the production of GTA, similarly to the effects of regulatory mutations. 42
Conclusions: It appears that GTA arose in the α-proteobacteria after divergence of this line from other prokaryotes. The similarity of 16S rRNA and GTA gene descent to present-day species indicates that the transmission of GTA was largely vertical, with little or no horizontal gene transfer. We propose a speculative model in which three environmental signals affect GTA production: 1) the availability of phosphate and ammonium affect the balance between phosphorylated and non-phosphorylated CtrA, which in turn determines whether GTA genes are transcribed; 2) cell population density-dependent accumulation of a homoserine lactone molecule stimulates transcription of GTA genes; 3) a decrease in the availability of carbon stimulates a pathway involving CckA, which is needed for release of GTA particles from cells.
PL-8.1 PURPLE NONSULFUR BACTERIA AS CATALYSTS FOR HYDROGEN PRODUCTION. Caroline S. Harwood, University of Washington, Seattle, WA USA. In the world today, large quantities of hydrogen gas are used in petroleum refining and ammonia production for fertilizer and in the future there is the potential for hydrogen to be used in huge amounts as a transportation fuel. Although still under development, there are several microbial routes for hydrogen production that depend on the environmentally benign use of biomass, solar energy and other readily available resources. The development of an efficient process for hydrogen production by purple nonsulfur bacteria will require that the biocatalyst for hydrogen production, the nitrogenase, be synthesized and active under all growth conditions and it will be important to develop strains that cannot recapture and dissipate the hydrogen that they produce. Beyond this, the metabolic modules of photophosphorylation, reductant generation and nitrogenase activity that feed the process of hydrogen production, must each operate with maximum efficiency for hydrogen generation as opposed to for ammonia production. Obviously, the integrated functioning of these modules must also be maximally efficient. With this framework in mind there are numerous strategies for strain development that can be expected to lead to improvements and stabilization of the hydrogen production process. In addition to their practical usefulness, the application of such strategies will lead to an improved understanding of the hydrogen production process as it operates in the context of whole cells. In this talk I will discuss three strategies to improve hydrogen production. These are 1) Removal of regulatory constraints on nitrogenase, 2) Optimization of electron flow to nitrogenase, and 3) Design of efficient bioreactors.
PL-8.2 THE POTENTIAL OF CYANOBACTERIA FOR BIOTECHNOLOGICAL APPLICATIONS Paula Tamagnini1,2, 1IBMC - Instituto de Biologia Molecular e Celular; 2 Faculdade de Ciências, Departamento de Botânica; Universidade do Porto, Porto, Portugal. Introduction: Cyanobacteria combine the ability to perform oxygenic photosynthesis with typical prokaryotic features like the ability to fix nitrogen. Many strains produce exopolymers, mainly of polysaccharidic nature, named exopolysaccharides (EPS). Due to their characteristics, cyanobacteria have high potential for biotechnological applications, such as the (i) production of biohydrogen and (ii) removal of heavy metals from contaminated waters. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes (i) BioModularH2 uses a synthetic biology approach to design reusable, standardized molecular building blocks that integrated into a chassis will produce a photosynthetic bacterium containing engineered chemical pathways for competitive, clean and sustainable hydrogen production [1]. (ii) The EPS project aims at optimizing the production of cyanobacterial EPS to establish an effective system to remove metallic ions from polluted waters. Methods: (i) BioModularH2 - Generation of mutants without redundant parts (e.g. genes) and identification of neutral sites using standardized vectors compatible with the BioBrick™; design and construction of synthetic parts and its assembly into modules/devices; characterization of the devices in the cyanobacterial chassis. (ii) EPS project - Metal removal assays, determination of EPS monosaccharidic composition by ion-exchange chromatography, identification of the functional groups responsible for metal-binding by potentiometric titration and Diffuse Reflectance Infrared Fourier Transform spectrometry, and sequence comparisons using bioinformatics tools. Results: (i) Preparation of the chassis starting with the (i) deletion of genes encoding the bidirectional hydrogenase in Synechocystis sp. PCC 6803 (redundant part) and (ii) construction of mutants on putative neutral sites (to integrate synthetic parts/devices such as an efficient heterologous hydrogenase). Concomitantly, several parts (e.g. promoters), and oxygen consuming devices (to provide a microaerobic environment required for an optimal heterologous hydrogenase activity) are being synthesized, characterized and tested. (ii) Copper removal assays performed with Gloeothece sp. PCC 6909 wild type and its sheathless mutant revealed that the mutant is more efficient in the process. Subsequent studies showed that although the mutant does not possess a sheath, it releases large amounts of polysaccharidic material (RPS) into the medium, and that its RPS possess higher amount and/or more accessible functional groups (e.g. carboxyl and amide groups) [2]. An in silico analysis of cyanobacterial genomes revealed the presence of genes encoding proteins that, in other organisms, are involved in the last steps of EPS production, although with a different physical organization. Based on these findings a putative mechanism for the biosynthesis and export of cyanobacterial EPS was proposed [3]. Conclusions: (i) BiomodularH2 - The standardized parts, modules and devices generated will be introduced to the chassis for photobiological H2 production, as well as will be available for other biotechnological applications. (ii) The future implementation of heavy metal removing systems based on cyanobacterial EPS depends on the knowledge about the biosynthetic pathways leading to their production. References: [1] http://www.biomodularh2.org [2] Micheletti E et al (2008) Appl Environ Microbiol 74: 2797-2804. 19: 139-185 [2] Pereira et al (2009) FEMS Microbiol Rev (accepted)
Final Program and Abstracts
PL-8.3 CYANOBACTERIAL BIOACTIVE COMPOUNDS: STRUCTURES, ACTIVITIES AND BIOSYNTHESIS. Kaarina Sivonen, Department of Applied Chemistry and Microbiology, Viikki Biocenter, University of Helsinki, Finland. Introduction: Cyanobacteria are a prolific source of bioactive compounds. These include potent toxins as well as biomedically interesting molecules, drug leads or useful probes in cell biology studies. Mass occurrences of neurotoxic and hepatotoxic cyanobacteria have caused number of animal poisonings and are a risk for human health. The cyclic hepatotoxic heptapeptides microcystins are most studied due to their worldwide occurrence. Methods: The structures of cyanobacterial compounds were determined by LC-MS and NMR. Bioactivities were investigated by enzyme inhibition tests and cell assays. Molecular methods (PCR, qPCR, cloning, sequencing, heterologous expression, microarrays) were used to identify the biosynthetic gene clusters and to detect hepatotoxic cyanobacteria. Shotgun sequencing and bioinformatic analyses were applied to genome of Anabaena strain 90. Results: Microcystins and nodularins (pentapeptide hepatotoxin occurring in brackish waters) are specific inhibitors of eukaryotic protein phosphatases and act as tumour promoters. Cyanobacterial bioactive cyclic and linear peptides may also display various bioactivities including serine protease inhibition and cytotoxicity. Novel bioactivities found recently include compounds acting as antidotes for microcystins and lipopeptides distroying the eukaryotic cell membrane. Microcystins and nodularin are products of a mixed non-ribosomal peptide and polyketide synthetase. The microcystins and nodularin gene clusters encode peptide synthetases, polyketide synthases and tailoring enzymes. This knowledge has been used to develop number of molecular techniques to detect hepatotoxin producing cyanobacteria. Studies on the evolution of microcystin/nodularin synthetase genes suggest that these genes are ancient and that present non-toxic strains have lost the genes and toxin production. The cyclic anabenopeptilides, and anabaenopeptins as well as linear spumigins are synthesised on non-ribosomal peptide synthetases. Interestingly, in anabaenopeptin biosynthesis a new way to create non-ribosomal peptide structural diversity was detected. The complete genome of Anabaena sp. strain 90 showed that 5% of the genome was dedicated to biosynthesis of bioactive compounds and that all these gene clusters are carried on the chromosome. It also revealed ribosomal peptide synthesis of a novel family of cyanobactins, anacycloamines. We demonstrated the widespread (48 out of 132 strains) but sporadic occurrence of the cyanobactin biosynthetic pathway among planktonic cyanobacteria. Conclusions: Cyanobacteria are a rich source of bioactive compounds for drug leads and the number of new compounds identified from cyanobacteria is increasing continuously. Cyanobacterial bioactive compounds may prove useful in combating various diseases in the future. In addition, their biosynthetic machineries (ribosomal and nonribosomal) provide enzymes to be used in combinatorial biosynthesis or chemoenzymatic synthesis to produce novel compounds. References: Sivonen, K. and T. Börner. 2008. Bioactive compounds produced by cyanobacteria. In: “The cyanobacteria: Molecular Biology, Genomics and Evolution”, Herraro, A. & E. Flores (Eds.). p. 159-197. Caister Academic Press, Norfolk, U. K. Sivonen, K. 2009. Cyanobacterial toxins. In: “Encyclopedia of Microbiology“, 3rd Edition, edited by M. Schaechter, pp. 290-307. Oxford:Elsevier.
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
OC-1.1 SUITABILITY OF PUFL AND PUFM GENES AS PHYLOGENETIC MARKERS FOR PURPLE SULFUR BACTERIA. Marcus Tank, Vera Thiel, Johannes F. Imhoff IFM-GEOMAR, Leibniz Institute of Marine Sciences (at Kiel University), Kiel, Germany. Introduction: Purple Sulfur Bacteria (PSB) are photoautotrophic bacteria phylogenetically grouped to the order Chromatiales within the Gammaproteobacteria. They perform anoxygenic photosynthesis under anaerobic conditions generally using reduced sulfur compounds (e.g. H2S, S2-, thiosulfate) as e--donator. PSB are ubiquitously distributed but mainly restricted to aquatic environments containing adequate light conditions, low/no oxygen tension and moderate sulfide concentrations. pufL and pufM are essential in photosynthesis of PSB and encode for polypeptides of the photosynthetic reaction centres which are located in intracytoplasmic membranes (ICM). The phylogenetic relationship of purple sulfur bacteria (PSB) of the Chromatiales (Gammaproteobacteria) was analysed based on photosynthetic gene sequences of the pufL and pufM genes and the results compared to phylogenetic trees and grouping of the 16S rRNA gene. Methods: Primers for pufL and pufM genes were constructed and used to successfully amplify the pufLM genes of members of 16 genera of Chromatiales. pufLM and 16S rRNA gene sequences of 66 PSB strains, including 29 type strains and 28 new isolates, were determined and phylogenetically analyzed. The phylogenetic relationships based on the functional and ribosomal genes were compared to each other as well as to current taxonomic classification. Results: The inferred phylogenetic trees of pufLM and 16S rRNA genes reflect a largely similar phylogenetic development suggesting coevolution of these essential genes within the PSB. The two functional genes displayed similar phylogenetic relationship of the PSB regardless of the used gene (pufL, pufM or pufLM) and of weather nucleotides or deduced amino acid sequences were used. pufLM phylogeny is in good agreement to current taxonomic classification of PSB. Conclusion: It is concluded that horizontal gene transfer of pufLM genes within the PSB is highly unlikely, which contrasts the situation in other groups of anoxygenic phototrophic bacteria belonging to Alphaproteobacteria and Betaproteobacteria. A phylogenetic classification of PSB to the genus level is possible based on their pufL or pufM sequences, in many cases even to the species level. In addition, our data support a correlation between Puf protein structure and the type of internal photosynthetic membranes (vesicular, lamellar or tubular).
OC-1.2 BIOGEOGRAPHY OF PHOTOSYNTHETIC LIGHT-HARVESTING GENES IN MARINE PHYTOPLANKTON. Thomas S. Bibby1,2, Yinan Zhang2, Min Chen2. 1
School of Ocean and Earth Sciences, National Oceanography Centre, Southampton, United Kingdom; 2School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia. Introduction: Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial genera Prochlorococcus and Synechococcus that are defined by two distinct August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts light-harvesting systems, chlorophyll-bound protein complexes or phycobilin-bound protein complexes, respectively. Here, we use the Global Ocean Sampling (GOS) Project as a unique and powerful tool to analyze the environmental diversity of photosynthetic lightharvesting genes in relation to available metadata including geographical location and physical and chemical environmental parameters. Methods: All light-harvesting gene fragments and their metadata were obtained from the GOS database, aligned using ClustalX and classified phylogenetically. Each sequence has a name indicative of its geographic location; subsequent biogeographical analysis was performed by correlating light-harvesting gene budgets for each GOS station with surface chlorophyll concentration. Results: Using the GOS data, we have mapped the biogeography of light-harvesting genes in marine cyanobacteria on ocean-basin scales and show that an environmental gradient exists in which chlorophyll concentration is correlated to diversity of light-harvesting systems. Three functionally distinct types of light-harvesting genes are defined: (1) the phycobilisome (PBS) genes of Synechococcus; (2) the pcb genes of Prochlorococcus; and (3) the iron-stress-induced (isiA) genes present in some marine Synechococcus. At low chlorophyll concentrations, where nutrients are limited, the Pcb-type lightharvesting system shows greater genetic diversity; whereas at high chlorophyll concentrations, where nutrients are abundant, the PBStype light-harvesting system shows higher genetic diversity Conclusions: We interpret this as an environmental selection of specific photosynthetic strategy. Importantly, the unique lightharvesting system isiA is found in the iron-limited, high-nutrient low-chlorophyll region of the equatorial Pacific. This observation demonstrates the ecological importance of isiA genes in enabling marine Synechococcus to acclimate to iron limitation and suggests that the presence of this gene can be a natural biomarker for iron limitation in oceanic environments.
OC-1.3 PHYLOGENETIC AND TAXONOMIC ANALYSIS OF THE CYANOBACTERIAL GENERA ANABAENOPSIS AND CYANOSPIRA. Stefano Ventura, Cristina Mascalchi, Claudio Sili Institute of Ecosystem Study, National Research Council, Sesto, Fiorentino, Italy. Introduction: Planktic heterocytous cyanobacteria from tropical alkaline environments have been studied in our group since long ago. We described the genus Cyanospira, with its two species C. rippkae and C. capsulata, that are among the few cyanobacterial taxa which hold a formally valid taxonomic description under the Bacteriological Code. The existence of the genus Cyanospira and of its two species and its separation from the genus Anabaenopsis has been repeatedly questioned. To contribute to clarify the relationships between the two genera, we present a phylogenetic analysis of Anabaenopsis and Cyanospira and a detailed study of the morphology of several undescribed strains of Cyanospira. Methods: Strains have been isolated from natron coming from Chad that contained large numbers of dormant akinetes. Phylogenetic analysis has been performed using the ARB and SILVA phylogenetic tools and databases, which supply a constantly updated and aligned database with quality evaluation of the sequences. Morphology has been studied in laboratory cultures inoculated with dormant desiccated akinetes. The entire life cycle has been studied and documented, from the akinete germination to the full development of vegetative forms. 45
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Final Program and Abstracts Results: Cultures of Cyanospira, obtained from germination of desiccated akinetes, were characterized by variable filament dimensions and coiling degrees. Akinete development in vegetative filaments was typically apoheterocytic; akinetes developed in chains that continuously expanded along the filament; in old cultures, all cells in the filament can be transformed into akinetes. Filaments of Cyanospira were often surrounded with a thick mucilaginous capsule. The development pattern of akinetes in Cyanospira was definitely different from that of Anabaenopsis. After a selection of sequences based on length and quality, that is inherent to the SILVA database, all publicly available sequences identified as belonging to the genera Anabaenopsis and Cyanospira, plus new sequences of our strains have been compared with selected entries of Nostoc, Thricormus, Nodularia, and Azolla cyanobionts. The phylogenetic analysis produced several well defined clusters. The assemblage of the two genera Anabaenopsis and Cyanospira formed a phylogenetically coherent and well defined unit, mostly related to the genus Nodularia. Inside this unit, sequences of Anabaenopsis were subdivided into three clusters, two housing freshwater strains, and one, more apart, housing representatives of the species A. abijatae which came from mineralized (alkaline) lakes in Ethiopia. Sequences of Cyanospira formed three distinct phylogenetic clusters that were more related to the A. abijatae cluster. Conclusions: As already demonstrated for hypersaline environments, alkaliphilic cyanobacteria stayed apart from their freshwater counterparts. Among the alkaliphiles, the genus Cyanospira was subdivided into three phylogenetic subunits. The relationships of two of these units with A. abijatae should be investigated in more details by means of a precise morphological study. Our data support the taxonomic robustness of the genus Cyanospira as the alkaliphilic counterpart of Anabaenopsis, and the existence of more than one species inside it.
OC-1.4 CRYPTIC DIVERSITY OF CYANOBACTERIA IN MICROBIAL MATS OF A TROPICAL LAGOON, TIKEHAU ATOLL, TUAMOTU ARCHIPELAGO. Katarzyna A. Pali ska1, Raeid M. M. Abed2†, Katja Wendt1, Maria Łotocka3, Loic Charpy4 & Stejpko Golubic5. 1
Institute of Chemistry and Biology of the Marine Environment, Geomicrobiology Dep.; CvO University of Oldenburg, Oldenburg, Germany; 2College of Science-Biology Department, Sultan Qaboos University, Al-Khod, Muscat, Sultanate of Oman; 3Institute of Oceanology, Polish Academy of Sciences, Powsta ców Warszawy, Sopot, Poland; 4Centre d’Oceanologie de Marseille, IRD, UR R167 (CYROCO), Traverse de la Batterie des Lions, Marseille, France; 5 Biological Science Center, Boston University, Boston, MA, USA. Introduction: Cultivation of microorganisms is known to underestimate the real diversity of bacterial communities thriving under natural conditions (Amann et al., 1995; Abed et al., 2008) and the isolates are often selected by the supplied culture conditions. However, these minor bacterial populations in the field may become dominant in response to environmental changes or in the case of certain perturbation events that favor their growth. While recent studies in microbial ecology focuses on identifying dominant field bacterial populations using culture-independent molecular tools, with the assumption that they are responsible for most bacterial activities, minor populations are often ignored. In this study, we investigated the diversity of opportunistic cyanobacteria that respond to the high load of nutrients provided by the cultivation medium. We postulate that 46
these autochthonous assemblages of taxa are of ecological importance. Methods: Twelve strains of filamentous and unicellular cyanobacteria were isolated from microbial mats of the lagoon of Tikahau atoll, Tuamotu Archipelago, the most oligotrophic Pacific waters; identified morphologically and using 16S rRNA-based phylogenetic analysis. Taxonomic identity of the studied strains was further supported by obtaining CpcBA-IGS sequences. Cultures were isolated from natural populations growing on the bottom of the lagoon formed of calcareous sand from the surface to a depth of ca. 20m. We investigated strains with respect to their pigmentation, as well as their potential to perform chromatic adaptation, and nitrogen fixation. Results: Using direct microscopy, 12 different cyanobacterial morphotypes were identified. They were classified in six morphogenera sensu Geitler (1932) and Anagnostidis & Komarek (1988): Cyanocystis, Leptolyngbya, Aphanothece, Phormidium, Chlorogloea and Pseudanabaena. The genera Cyanocystis and Chlorogloea were genotypically characterized for the first time Most of the strains posses’ phycoerythrin and have the ability to fix nitrogen.. Non-heterocystous cyanobacteria with narrow trichomes were classified under Leptolyngbya and Phormidium following botanical recommendation, but homogeneity of their grouping was not genotypically confirmed. Conclusions: The strains represent autochthonous cryptic diversity, which dominated cultures but not the natural populations. Taxa studied here clearly responded to higher nutrient supply provided by the standard artificial medium, which may simulate conditions in nature following the impact of tropical storms, thus recruiting opportunistic taxa, which are normally present at low frequencies. They may be considered important members of microbial successions during restoration of the ecosystem following catastrophic impacts. With increasing man-made disturbances, such as eutrophication, pollution or dredging, it is expected that these taxa will increase in abundance. While recent studies in microbial ecology focuses on identifying dominant field bacterial populations using culture-independent molecular tools, with the assumption that they are responsible for most bacterial activities, minor populations are often ignored. We presume that these less-dominant populations are equally important and can only be identified by enrichment cultivation simulating possible environmental perturbations and changes.
OC-1.5 METAGENOMIC AND PHYLOGENETIC ANALYSES OF CYANOBACTERIAL MATS IN EXTREME HIGH ARCTIC ENVIRONMENTS: BIOGEOGRAPHY AND BIOGEOCHEMICAL FUNCTION. Anne D. Jungblut1, Connie Lovejoy2, Thibault Varin3, Jacques Corbeil3, Warwick F. Vincent1. 1
Centre d’Études Nordiques (Centre for Northern Studies); 2Faculté de Médecine, Université Laval; 3Québec-Océan, Département de Biologie, and Institut de biologie intégrative et des systèmes (IBIS); Université Laval, Québec, QC, Canada. Introduction: Mat-forming cyanobacteria are among the oldest known prokaryotes and are widely distributed in more extreme environments, and in environments where grazing pressure is reduced or absent. The physical form of the cyanobacteria living within the mats and the three dimensional structure of mats themselves is much conserved with fossils dating from more than 2 billion years. However given the wide
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes variety of habitats where mats tend to be abundant, a major question is whether such morphologically similar structures harbour similar species and what particular genetic adaptations or metabolic pathways may be present in different environments. Polar aquatic ecosystems are dominated by microbes and abundant mat-forming cyanobacteria are widely distributed in shallow lakes, streams and meltwater ponds on ice shelves of the High Arctic. During summer, oxygenic photosynthesis by cyanobacteria is the primary energy source supporting a complex community within the mats. In contrast to physically similar mats from other habitats, such as hot springs, there is relatively little data on the taxonomic and functional diversity of these polar cyanobacteria-dominated mat communities. Without such information, questions on the evolutionary persistence of cyanobacteria mats during both warming and glacial epochs, and on their global biogeography cannot be addressed. Therefore, we characterised High Arctic microbial mats using metagenomics to uncover adaptive functional genes and targeted 16S rRNA gene libraries and phylogenetics to determine cyanobacterial diversity. Methods: Microbial mats were investigated from freshwater ecosystems of different nutrient and physical regimes in the Canadian High Arctic. Microbial community analysis was performed using pyrosequencing and bioinformatic analysis. The phototrophic diversity was determined using signature pigment analysis. Cyanobacterial diversity was targeted using strain isolation, and culture independent methods including microscopic examination, and phylogenetic analyses based on 16S rRNA gene clone-libraries. Results: High Arctic microbial mats had chlorophyll a values ranging from 3.9 to 46.2 µg cm-2 with high concentrations of cyanobacterial pigments such as scytonemin. Metagenomic analyses of two microbial mat communities from ice shelves identified Cyanobacteria and Proteobacteria as dominant bacterial groups. Other bacteria, Archaea, Eukaryota and viruses were also identified. A high proportion of genes were implicated in metabolic pathways of major biogeochemical cycles with evidence of specific molecular adaptations to the cryosphere. Cyanobacterial diversity consisted of phylotypes within Chroococcales, Oscillatoriales, Nostocales and Stigonematales based on morphological and 16S rRNA gene analyses. Phylotypes within the orders Chroococales and Nostocales had highest similarity to temperate environments, whereas the majority of oscillatorians had closest similarity (>99) to Antarctic and alpine sequences, including to taxa previously considered to be endemic to the Antarctica. Conclusions: High Arctic microbial mats contain diverse assemblages of microbes that perform an ensemble of biogeochemical cycling processes, similar to cyanobacteria-dominated mats from other climate zones. Our results also imply that low-temperature cyanobacterial ecotypes have a cosmopolitan distribution throughout the cold terrestrial biosphere.
OC-1.6 GENOTYPE AND CHEMOTYPE DIVERSITY OF APHANIZOMENON SPP. AND ANABAENA SPP. IN NORTHEAST GERMAN LAKES. Andreas Ballot1 Jutta Fastner2, Jaqueline Rücker3, Claudia Wiedner1. 1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Limnology of Stratified Lakes, Neuglobsow; 2Federal Environmental Agency, Berlin; 3Brandenburg Technical University, Department of Freshwater Conservation, Bad Saarow; Germany.
Introduction: In the last decades hepatotoxic cylindrospermopsin (CYN) and neurotoxic saxitoxins (STX) were detected in several lakes in August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts northeast Germany. CYN and STX are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, or Aphanizomenon. So far only Aphanizomenon sp. was confirmed as producer of CYN in two German lakes, while the source of STX is not yet identified in German water bodies. In this study we attempted to identify STX- and CYN producers in German water bodies and characterized them morphologically, genetically, and biochemically in comparison to non toxin producing strains. Methods: In 2007 and 2008, 200 strains of Aphanizomenon spp., Anabaena spp., and Anabaenopsis spp. from 8 selected lakes in Brandenburg, Germany were isolated and cultivated. The isolated strains were identified morphologically using light microscopy and genetically using different genetic markers (cpcBA-IGS and RBCL). Primers were designed for a fast detection of the sxtA part of the STX gene cluster. For the detection of the CYN gene cluster primers to amplify the peptide synthetase (PS) gene were used. To confirm potential STX and CYN producers ELISA as a biochemical method and liquid chromatographic tandem (LC-MS/MS) methods were applied. Results: The isolated strains were assigned morphologically to four different species of Aphanizomenon and four different species of Anabaena and one Anabaenopsis species. Our sequence data and phylogenetic analyses confirmed the formation of intermixed clusters by planktic Anabaena and Aphanizomenon strains. Aphanizomenon gracile and Aphanizomenon flos-aquae could not be divided as separate species. Using polymerase chain reaction (PCR), 14 strains of Aphanizomenon cf. gracile, three strains of Aphanizomenon issatschenkoi, and one strain of Anabaenopsis sp. were identified as potential STX producers due to the possession of fragments of the sxtA gene. The ability to produce STX was confirmed with ELISA and LC-MS for the 14 strains of Aphanizomenon cf. gracile. Each strain produced 6 STX variants in variable amounts The STX concentrations found were between 400 and 1300 µg/g cyanobacterial dry weight. No CYN producer was detected using genetical methods and ELISA. Conclusions: This is the first study to confirm Aphanizomenon cf. gracile as saxitoxin producer in German water bodies. As strains of Aphanizomenon issatschenkoi and Anabaenopsis sp. also possessed fragments of the STX gene cluster other nostocalean producers of STX are very likely in German water bodies.
OC-2.1 MOLECULAR BASIS OF THE BACTERIAL SYMBIOSIS IN PHOTOTROPHIC CONSORTIA. Jörg Overmann, Roland Wenter, Kajetan Vogl, Johannes Müller. Section Microbiology, Department Biology I, University of Munich, Planegg-Martinsried, Germany. Introduction: Phototrophic consortia represent the most highly developed bacterial symbiosis and consist of green sulfur bacterial epibionts surrounding a central chemotrophic Betaproteobacterium. A rapid and specific exchange of multiple signals occurs between both bacterial partners and allows tactic behaviour towards light and chemical stimuli. Laboratory cultures of the consortium “Chlorochromatium aggregatum“ as well as of the isolated epibiont have recently been established, providing the opportunity to elucidate the molecular mechanisms underlying the symbiotic interaction. Methods: Subcellular structures were studied by high resolution analytical scanning electron and transmission electron microscopy. Putative symbiosis genes of the epibiont were identified by suppression subtractive hybridization of genomic DNA against that of 16 relatives and bioinformatics. Transcription was studied by reverse
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Final Program and Abstracts transcriptase-PCR. Cross-linking experiments were conducted with bis(sulfosuccinimidyl)suberate or 3,3´dithiobis(sulfosuccinimidylpropionate). Results: In “C. aggregatum”, numerous periplasmic tubules extend from the outer membrane of the central bacterium and are in direct contact to the epibiont. Our search for specific recognition and signal transduction proteins identified a total of 189 genes of the epibiont of “C. aggregatum“ to be absent in all other green sulfur bacteria. Most noticable was the presence of four genes which were related to virulence factors of human or plant pathogenic bacteria. These genes were transcribed constitutively in the epibiont. The hemagglutinin-like putative gene products of ORFs Cag0614 and Cag0616 probably arose by gene duplication and represent the largest bacterial open reading frames so far documented for bacteria. Cag1920 codes for a putative hemolysin whereas the gene product of Cag1919 is a putative repeat in toxin (RTX)-like protein containing a Ca2+- binding beta roll motif. In fact, intact consortia disaggregated upon the addition of EGTA. The RTX-type C-terminus coded by Cag1919 exhibits a significant similarity to RTX modules of various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium. This conclusion is further supported by the presence of three genes encoding transposases which are located immediately upstream of ORF 1919. Most recently, cross-linking experiments of intact consortia revealed the presence of a corresponding filamentous hemagglutinin/adhesin in the central bacterium which appears to be involved in maintaining the multicellular structure of the phototrophic consortium. Conclusions: While the adaptation of green sulfur bacteria to a symbiotic lifestyle in phototrophic consortia requires only a comparatively low number of niche-specific genes, it involves several unique genes which most likely were acquired by lateral gene transfer from a proteobacterium. Our study of the molecular mechanisms of symbiosis indicates that typical bacterial virulence factors have been exploited by green sulfur bacteria to establish a mutualistic interaction and enabled these bacteria to occupy a novel ecological niche.
OC-2.2 LIVING FOSSILS FROM BIOLOGICAL SOIL CRUSTS: AEROBIC ANOXYGENIC PHOTOTROPHS IN A SEMIARID REALM. J.T Csotonyi, J Swiderski, E Stackebrandt, V. Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. The discovery of aerobic anoxygenic phototrophs from biological soil crusts (BSC) is reported. Living on an ecological knife-edge of habitability, BSC are drought-tolerant communities composed primarily of microorganisms that form a cohesive, erosion-resistant veneer on the surface of soil. These communities inhabit semiarid to arid soils and are composed of cyanobacteria, bacteria, fungi and mosses. They are receiving increased attention from a land management perspective because they reduce soil erosion and enhance moisture and nutrient status. From sandy soils (including sand dunes) in Manitoba and Alberta, Canada, 21 strains were isolated that possessed the diagnostic light harvesting pigment, bacteriochlorophyll. Anoxygenic phototrophs constituted 0.1 to 5.9% of the cultivable biological soil crust bacterial community. Sequencing the 16S rRNA gene of 16 isolates revealed that nine strains were closely related to the genus Methylobacterium, while seven fell within the alpha-1- and alpha-4-proteobacteria, related to typical aerobic anoxygenic phototrophs such as Paracraurococcus, and to soil crust 48
heterotrophs such as Belnapia. This study is the first demonstration of proteobacterial anoxygenic phototrophs from semiarid environments. The presence of anoxygenic phototrophs in BSC implies a higher efficiency of light harvesting by soil curst organisms than previously realized. Utility of near-IR radiation not used by oxygenic phototrophs facilitates light-accelerated turnover of soil organic carbon content.
OC-2.3 ON THE ROLE OF CYANOBACTERIA IN MICROBIAL MATNITROGEN FIXATION. Ina Severin, Lucas J. Stal. Department of Marine Microbiology, NIOO-Center for Marine and Estuarine Ecology, Yerseke, The Netherlands. Microbial mats on intertidal beaches and sand flats are often pioneer systems. Following their establishment, colonization by higher plants occurs and rigorously changes the morphodynamics of the ecosystem. One perquisite for this succession is the enrichment of the sediment with combined nitrogen as the result of N2 fixation. Cyanobacteria are usually the most conspicuous structural part of microbial mats. These organisms have long been the focus of research on N2 fixation in microbial mats. However, there is growing evidence that Cyanobacteria are sometimes minor players in terms of N2 fixation. We investigated a coastal microbial mat with regard to N2 fixation using the acetylene reduction assay as well as with regard to the diversity of the 16S rRNA and nifH genes and transcripts. Quantitative RT-PCR was used to access daily changes of nifH-gene expression of the dominant phylotypes and groups as well as the whole mat community. The daily patterns of nitrogenase activity differed with respect to the point of time when the maximum was reached. The mat higher up in the littoral zone (station I) showed several nitrogenase activity-maxima between sunset and sunrise and harboured a variety of heterocystous and non-heterocystous Cyanobacteria. The mat situated close to the low water mark (station II) was mainly composed of non-heterocystous Cyanobacteria with Lyngbya aestuarii being the most dominant diazotroph. Accordingly, a night time-maximum of nitrogenase activity was recorded. A clear light dependency of nitrogenase activity implied phototrophs as main diazotrophs. 16S rRNA gene clone libraries showed that both microbial mats were dominated by Cyanobacteria and Proteobacteria. Filamentous non-heterocystous Cyanobacteria and Gammaproteobacteria were predominant at both stations but the relative contribution of taxa differed. Analysis of the nifH gene clone libraries confirmed that Cyanobacteria were the dominant diazotrophs in both communities. In accordance with 16S rRNA gene clone libraries, Gammaproteobacteria-related nifH sequences were most frequently retrieved at station I whereas the delta-subdivision made up for almost all Proteobacteria-related nifH sequences at station II. The analysis of nifH transcripts of station I revealed a dynamic community with changing relative abundances of nifH transcripts of members of the community over time. While Cyanobacteria-related sequences were predominant from midday to midnight, Proteobacteria-related nifH transcripts were found to become more important from midnight on. A high proportion of these proteobacterial sequences were most closely related to known anoxygenic phototrophs. Relative contribution of sequences to cDNA clone libraries differed considerably from those observed for DNA clone libraries, highlighting active diazotrophs. Gene expression levels yielded further insight into diel nifH gene expression patterns of key diazotrophs in comparison to whole mat community nitrogenase activity. The results suggest that species composition affects the maximum attainable August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes nitrogenase activity and its diel pattern. Oxygenic as well as anoxygenic phototrophs were found to be the key players in N2 fixation in the investigated microbial mats.
Final Program and Abstracts
OC-2.5
OC-2.4
DISTRIBUTION ANALYSIS OF HYDROGENASES IN SURFACE WATERS OF MARINE AND FRESHWATER ENVIRONMENTS WITH AN EMPHASIS ON CYANOBACTERIA.
BIOCYC: PATHWAY/GENOME DATABASES FOR SEQUENCED PHOTOSYNTHETIC MICROBES.
Christoph Schwarz1, Martin Barz2, Christian Beimgraben2, Torsten Staller2, Rüdiger Schulz2, Jens Appel1.
Peter D. Karp, Pallavi Kaipa, Ron Caspi, Alexander Shearer.
1
SRI International, Menlo Park, CA, USA. The BioCyc [1] collection of 409 Pathway/Genome Databases (PGDBs) is available at URL BioCyc.org. BioCyc includes the genomes, and predicted metabolic networks and operons of eleven photosynthetic microbes. BioCyc also includes the EcoCyc [2] database (DB), which describes the metabolic and genetic regulatory network of Escherichia coli; and the MetaCyc DB, which describes more than 1200 metabolic pathways that were experimentally elucidated in more than 1,600 organisms. BioCyc contains PGDBs for most organisms with completely sequenced genomes. Each BioCyc PGDB combines the annotated genome sequenced with predicted metabolic pathways, predictions of pathway hole fillers, and operon predictions. BioCyc is generated on a regular ongoing basis using the MetaCyc reference database of experimentally elucidated pathways. BioCyc offers many capabilities to researchers for analysis and investigation of genomes, metabolic networks, and regulatory networks, and for analysis of high-throughput datasets. A genome browser allows exploration of a microbial genomes, and comparison of conserved genome regions. The predicted metabolic network of each organism can be queried and visualized using a variety of bioinformatics tools that display information about metabolites, enzymes, reactions, and individual pathways. Computational tools for analysis of metabolic networks include tools for finding dead-end metabolites, and for performing reachability analysis of metabolic networks. In addition, each PGDB provides a diagram of the complete metabolic map of the organism, and of the complete regulatory network when it has been curated for that organism. These tools support analysis of large-scale omics datasets [3], such as gene-expression and metabolomics data, by painting those data onto the full metabolic map diagram and regulatory network diagram to place omics data within a biological context. A toolkit of comparative analysis tools permits comparisons of the genome and biochemical networks of multiple Pathway/Genome Databases. BioCyc can be accessed at BioCyc.org, as a set of downloadable data files, and as an installable software distribution. The BioCyc.org Web site and data files are freely available to all; the downloadable software is freely available to research institutions. [1] R. Caspi et al, “The MetaCyc Database of Metabolic Pathways and Enzymes and the BioCyc Collection of Pathway/Genome Databases,” Nucleic Acids Research 36:D623-31 2008. [2] Karp, P.D. et al, “Multidimensional annotation of the Escherichia coli K-12 genome,” Nucleic Acids Research 35:7577:90 2007. [3] Paley, S.M. et al, “The Pathway Tools Cellular Overview Diagram and Omics Viewer,” Nucleic Acids Research 34:3771-8, 2006.
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2
School of Life Sciences, Arizona State University, Tempe, AZ, USA; Botanisches Institut, Universität Kiel, Kiel, Germany.
Introduction: After methane hydrogen is the second most abundant trace gas in the atmosphere, making up around 0.5 ppm to 0.6 ppm. Surface waters of different aquatic environments have been shown to evolve hydrogen. The ocean is estimated to be the major natural source of H2. Still its origin and the proportion of biological and nonbiological production and consumption processes are unknown. Hydrogen concentrations of surface waters above the expected equilibrium prompted us to investigate the presence and distribution of all known hydrogenases in marine and fresh water environments with a special emphasis on cyanobacteria. Methods: DNA was isolated from samples from the North Sea, the Baltic Sea, the North Atlantic, the Mediterranean Sea and two German lakes (Westensee and Selenter See). Degenerated primers were used to amplify part of the gene of the large subunit of the bidirectional NAD(P)-linked hydrogenase (hoxH) from these samples. In addition the available genomes of cyanobacteria, marine bacteria and the GOS database were searched for the presence of all the known hydrogenase genes. Results: The search of the cyanobacterial genomes confirms that their uptake hydrogenase (hupL) is strictly linked to the nitrogenase genes. This is not surprising since it is used to recycle the hydrogen inevitably produced by the nitrogenase in each catalytic cycle. But nevertheless there are some strains that do have the nitrogenase genes but no uptake hydrogenase indicating that the hydrogenase is not essential to compete and might not be necessary under all environmental conditions. The distribution of the hoxH sequences in the complete genomes as well as in the different samples analyzed indicates a strong bias to freshwater environments and coastal waters. It seems to be absent in the open ocean, which is confirmed by the database searches also in other marine bacterial genomes. Therefore, the presence of this hydrogenase is especially linked to the probability to encounter anaerobiosis. In approximately 20 % of all marine genomes hydrogenases were found. The presence of H2-sensing hydrogenases in photosynthetic bacteria such as Roseovarius strains and other marine bacteria suggests that H2 triggers the expression of the structural genes of their uptake hydrogenases. Conclusions: Our results show that in marine waters all the genetic complement is present for biological hydrogen production as well as uptake. Therefore, biological processes alone might be responsible for the measured H2 turnover in these habitats. The sequences found suggest that hydrogen might be used as an additional energy source by marine bacteria in nutrient-limited environments. In addition, our data supports the conclusion that the bidirectional NAD(P) linked hydrogenase is especially needed for adaptation to rapidly changing redox conditions and unnecessary in the open ocean.
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Final Program and Abstracts
OC-2.6
OC-3.1
HYDROGEN PRODUCTION AND CONSUMPTION IN HOT SPRING MICROBIAL MATS DOMINATED BY THE FILAMENTOUS ANOXYGENIC PHOTOSYNTHETIC BACTERIUM CHLOROFLEXUS AGGREGANS.
CHARACTERIZATION OF PICOCYANOBACTERIA ISOLATED FROM THE HALOCLINE OF THE SALINE MEROMICTIC LAKE, LAKE SUIGETSU, JAPAN.
H. Otaki1, R.C. Everroad1, S. Hanada1,2, S. Haruta1, K. Matsuura1.
Faculty Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan.
1
Department of Biology, Tokyo Metropolitan University, Tokyo; 2 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki; Japan. Introduction: The filamentous anoxygenic phototrophic bacteria Chloroflexus aggregans and C. aurantiacus are often present in hot springs dominated by cyanobacteria. In some hot springs however, there are distinctive microbial mats that lack cyanobacteria and are instead dominated by Chloroflexus spp. At Nakabusa hot spring (Nagano, Japan) at 65°C, C. aggregans develops mats by growing autotrophically using hydrogen sulfide as an electron donor for photosynthesis. These mats also contain a thermophilic sulfatereducing bacterium, Thermodesulfobacterium sp., which produces hydrogen sulfide. It is known that this sulfate-reducing bacterium can utilize hydrogen, but it is still unknown whether this bacterium consumes hydrogen in the mats or whether biological hydrogen production even occurs in this community. In this study, we investigated net hydrogen production in the 65°C microbial mats at Nakabusa. Methods: Samples of these mats, mainly composed of C. aggregans, were collected and hydrogen production was measured in artificial hot-spring water at 65°C under various treatments in anaerobic bottles. Results: In the absence of any treatment, hydrogen was not detected, but significant production was observed in the presence of molybdate, an inhibitor of sulfate reduction or with a homogenization treatment of the mat. This net hydrogen production largely decreased under illumination. These results suggest that the metabolism resulting in hydrogen production was not photosynthetic; we hypothesize that hydrogen was produced by fermentative bacteria breaking down organic compounds supplied by anoxygenic photosynthetic bacteria. This hydrogen was then consumed by sulfate-reducing bacteria as they reduced sulfate to sulfide. The decrease of net hydrogen production under illumination can be explained by the anoxygenic photosynthetic bacteria using hydrogen as an electron donor or competing with fermentative bacteria for organic compounds. Conclusions: Hydrogen was shown to be produced and consumed within the mats, and net hydrogen production could not be detected without the inhibition of sulfate reduction or physical blocking of hydrogen consumption. In the 65°C mats at Nakabusa, anoxygenic photosynthetic bacteria produce organic compounds using hydrogen sulfide as an electron donor. Fermentative bacteria use this carbon as an energy source and release hydrogen as a byproduct. This hydrogen is then used by sulfate-reducing bacteria that produce hydrogen sulfide. Thus, electrons cycle via organic compounds, hydrogen and hydrogen sulfide in the community.
Kaori Ohki, Shinya Yoshikawa, Mitsunobu Kamiya.
Introduction: The surface layer of Lake Suigetsu (35 35’N, 135 52’E, coast of the Japan Sea in Fukui Prefecture, Japan) has O2-containing fresh water, while the deeper layer is anoxic salt water where a permanent halocline (salinity is ca.0.4 to 1.4%) is created between these two water layers. The concentration of O2 decreases to zero at about the middle of the halocline; and H2S, a potent inhibitor of oxygenic photosynthesis, is detected deeper below the oxic-anoxic boundary layer (OABL). Only weak green light (less than 1% of surface intensity) penetrates to the OABL. We found small size cyanobacteria (<2 μm, PCy) were distributed through the year within the halocline as the dominant phytoplankton (0.5 to 5×105 cells/ml) during four years 2005 - 2008. The attempt to determine the adaptation mechanism(s) of PCy to their habitat, phylogenetic and physiologic properties of PCy were studied using isolated strains from the halocline of Lake Suigetsu. Methods: Water samples were collected from the different depths within the halocline of Lake Suigetsu during the summer of 2005-6. Unicyanobacterial isolates were obtained by isolating colonies on agar plates using various salinities in artificial media (0.4 to 1.6 %). Morphology observations were performed using epifluorescence and electron microscope. Phycobiliprotein (PB) compositions were determined spectrophotometrically using intact cells and/or isolated phycobilisomes. The most part of the 16S rRNA and the internal transcribed spacer (ITS) between 16S and 23S rRNA genes were sequenced from the genomic DNA of the isolated PCy; and molecular phylogenetic trees were constructed using the neighbor-joining method. Results: C-phycoerythrin (R-type) or C-phycocyanin (G-type) was the major PBs. The isolated clones (about 100) were grouped into six subgroups using the PB composition and sequence homology of the ITS between the 16S and 23S rRNA genes. Further analyses were performed using six clones from the different sub-groups (three Rtypes and three G-types). The six clones were included within the freshwater Synechococcus-Cyanobium clade, but they were not monophyletic in the 16S rRNA-based tree. Cells were spherical or ellipsoidal at 0.5 to 1.5 μm in diameter or width and reproduced using transverse binary fission in a single plane. The thylakoids were peripheral and were oriented parallel to the cytoplasmic membrane. They were able to grow in a wide range of salinities (0.2 to 2 % or more). Significant growth was observed under weak green light (2 μmol·m-2·sec-1, ca. 460 to 600 nm). Whereas, the cells were bleached irreversibly under the white light at relatively high intensities (≥15 μmol·m-2·sec-1 for the R-type; ≥25 μmol·m-2·sec-1 for the G-type). At least four clones survived in medium containing sulfide (0.3 to 1.2 mM as S2-, pH7.6) for more than a week. Conclusion: The physiological properties found in this study show PCy survive under unique physicochemical environments in the halocline of the saline meromictic lake, Lake Suigetsu. The ability to use weak green light for photosynthesis and sulfide tolerance may provide an advantage over other phytoplankton under sulfide-rich environments. The present phylogenetic analyses suggested this ability was acquired by PCy in Lake Suigetsu more than once.
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Final Program and Abstracts analyses of sequences obtained from part of the 16S rRNA gene.
OC-3.2 THE GLOBAL IMPORTANCE OF THE MARINE CYANOBACTERIA PROCHLOROCOCCUS. Z.I. Johnson, Duke University, 135 Marine Lab Rd., Beaufort, NC, USA. The marine cyanobacteria Prochlorococcus is thought to be the most numerically abundant phototropic prokaryote in the global oceans and as such it plays a critical role in the ecology and biogeochemistry of marine ecosystems. Recently we have completed several global ocean surveys covering vast oceanic regions where we measured the abundance and photosynthesis (primary production) of this important phototrophic organism. We find that over these regions, the integrated water column abundance of Prochlorococcus is remarkably conserved, in spite of significant variability in the vertical distributions. Further, although these surveys covered many oceanographic biogeochemical provinces, each with unique nutrient, temperature and other environmental variables, the contribution of Prochlorococcus to primary production remains highly conserved and ranges between 25 – 50% of the total production rates. Quantitative phylogenetic analyses using clade specific qPCR shows that while the taxonomic composition of Prochlorococcus can change dramatically in response to these environmental variables (and in particular temperature), the integrated abundance and production of the genus remains relatively consistent. Together, these global abundance and primary production estimates suggest that the Prochlorococcus niche is more consistent and larger than previously thought.
OC-3.3 THE IMPORTANCE OF PICOCYANOBACTERIA IN THE TOTAL CYANOBACTERIA COMMUNITY AND ITS CONTRIBUTION TO TOXICITY IN A TROPICAL BRAZILIAN RESERVOIR. Alessandra Giani, Juliana S.M. Pimentel, Camila A. Campos. Department of Botany, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. Introduction: Autotrophic picoplankton (APP) represents an important part of the planktonic communities in aquatic systems and may be responsible for a large amount of carbon fixation. Even though these organisms are widespread and abundant, in the past their populations were often not recognized because of their diminutive size (0.2-2 µm). A large part of the picoplanktonic community is represented by cyanobacteria. The importance of picocyanobacteria for global primary production and biomass has received less attention in freshwater than in marine systems. The development of molecular techniques opened new possibilities for the study of the diversity of these organisms and even their quantification. In the present work we present results obtained from a large mostly oligotrophic reservoir located in Southeastern Brazil, Furnas Reservoir. Methods: Samples were taken regularly over a two years period, concentrated on glass fibre filters and frozen until extraction. Samples for phytoplankton counting were preserved unfiltered with a Lugol’s iodine solution. Extraction for molecular analyses were performed by the phenol-chloroform technique. Extracts were analyzed by conventional PCR and real-time quantitative PCR (Step One Applied Biosystem). Primers were designed aimed at the detection of the cpcB region (total cyanobacteria) and mcyD (toxic cyanobacteria) genes in natural populations. New primers were designed for the q-PCR. Some samples were selected for the investigation of cyanobacterial diversity by the technique of “Amplified Ribosomal DNA Restriction Analysis” (ARDRA) using primers for amplification of part of the 16S rRNA gene and the entire internal transcribed spacer (ITS 16S-23S), and the August 9 to 14, 2009 • Montréal, QC, Canada
Results: Results from q-PCR revelead the importance and the numerical dominance of picocyanobacteria in the overall cyanobacteria community in the reservoir but not a sgnificant contribution in its toxicity. The percent of mcyD genes copies relative to cpc-B varied between 0.1 to 1.6 % but increased up to 55% when picoplanktonic cells were eliminated. The analyses of ARDRA patterns showed that although eighteen patterns were found, fifteen of them were related with the sequences of just two picoplanktonic genera (Cyanobium and Synechococcus). Conclusions: Our results suggest that the cyanobacterial APP community can be extremely important in tropical freshwater systems and may represent a significant contribution in the plankton diversity. Further development of the genomic approach to photosynthetic picoplankton will lead to new understanding of their roles in warm oligotrophic systems.
OC-4.1 ANAEROBIC SULFUR OXIDATION IN CHLOROBACULUM TEPIDUM: GENES, METABOLITES AND LINKAGES TO LIGHT HARVESTING. Thomas E. Hanson, Jennifer L. Hiras, Leong-Keat Chan, Rachael Morgan-Kiss. College of Marine and Earth Studies and DBI, Newark, DE, USA. Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is a model green sulfur bacterium for studying light harvesting in phototrophs that utilize chlorosomes as the major antenna complex and pathways of anaerobic sulfur oxidation. Current studies are focused on delineating pathways of anaerobic sulfur oxidation, including required gene products and metabolites, and how these pathways interact with global physiology in this organism. Methods: C. tepidum is capable of phototrophic growth with sulfide or thiosulfate as the sole electron donor or with both substrates in combination. This flexibility enabled the genetic dissection of sulfur oxidation pathways by targeted transposon mutagenesis, random transposon mutagenesis, and the addition of His-tags to specific genes in the C. tepidum chromosome. The resulting mutant strains and wild type grown under various sulfur, light and temperature regimes were analyzed by techniques of photobiology and a method for detecting and characterizing thiol metabolites. Results: C. tepidum mutant strains were recovered that implicated previously unsuspected gene products in thiosulfate oxidation and clearly showed that two of three sulfide:quinone oxidoreductase (SQR) homologs display in vitro SQR activity and are required for C. tepidum to grow on high concentrations of sulfide. Furthermore, mutant strains and the wild type grown under different conditions displayed altered chlorosome properties and altered energy transfer between the chlorosome and reaction center. Analysis of thiol metabolites revealed that C. tepidum contains structurally novel thiols and that thiol pool sizes are significantly altered in mutant strains defective for sulfur oxidation and in the wild type grown under a variety of conditions. Conclusions: Taken together, the results suggest that C. tepidum is capable of a range of responses to altered environmental and physiological states. These responses presumably allow C. tepidum to coordinately regulate and balance fluxes of light energy and reducing power in order to grow under a wider variety of conditions than was appreciated when it was first isolated. Furthermore, it appears that even a small genome can encode metabolic surprises as evidenced by novel thiol metabolites that we hypothesize are integral to both regulatory and anaerobic sulfur oxidation pathways. 51
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Final Program and Abstracts
OC-4.2
OC-4.3
CARBON ASSIMILATION IN ROSEOBACTER DENITRIFICANS.
STRUCTURAL AND FUNCTIONAL STUDIES OF A PHOTOSYNTHETIC MICROBIAL COMMUNITY THROUGH COMPARATIVE METATRANSCRIPTOME ANALYSIS.
Kuo-Hsiang Tang1, Xueyang Fang2, Yinjie Tang2, Robert E. Blankenship1. Departments of Biology and Chemistry; 2Department of Energy, Environment and Chemical Engineering; Washington University, St. Louis, MO, USA.
Zhenfeng Liu, Christian G. Klatt, Jason Wood, Nicola E. Wittekindt, Lynn Tomsho, Stephan C. Schuster, David M. Ward, Donald A. Bryant.
Introduction: Aerobic anoxygenic phototrophs (AAPs) are the only known organisms performing photosynthesis requiring oxygen but not producing oxygen. The AAPs are potentially major contributors to global carbon metabolism as they make up at least 10% of the microbial community in some euphotic upper ocean. Previous genetic analysis indicated the lack of the key genes required for the Calvin cycle, and physiological characterization suggested the requirement of organic carbon for Roseobacter denitrificans (R. denitrificans) growth. It is not clear how the R. denitirificans (and other AAPs) can assimilate carbon for the formation of the cellular components without autotrophic growth.
Introduction: The microbial mats in alkaline siliceous hot springs in Yellowstone National Park are model systems for microbial community studies, especially communities constructed by photosynthetic prokaryotes. Recently developed metatranscriptome analyses using high-throughput sequencing technology have revealed valuable information about the composition, in situ gene expression activities and gene regulation behavior of members of this microbial community.
1
Methods: We optimized the growth conditions of R. denitrificans with different defined carbon sources, and used metabolomic approaches, biochemical studies, and gene expression profiles to investigate the organic carbon metabolism, organic carbon and CO2 incorporation rate, and the carbon assimilation pathway(s). Results: We have investigated the R. denitrificans growth with various defined carbon sources in different growth conditions. The optimized growth conditions without non-specific carbon sources (such as yeast extract) are critical to minimize the background for the following metabolomic studies. The spectra of the LHII antenna complex and LHI-RC complex for the cultures with either pyruvate or glucose as the sole carbon source are very similar to those of the cultures grown in the rich growth medium. Metabolomic studies to probe the proposed carbon assimilation pathway in R. denitrificans with [1-13C]-labeled pyruvate, [1-13C]-labeled glucose, or 13C-labeled sodium bicarbonate were carried out. The N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide was used to derivatize amino acids for gas chromatography/mass spectrometry analysis. We have detected very low 13C-incorporation level of leucine and isoleucine and identified the CO2 assimilation to glutamate and proline. About 70% of alanine and valine are 13C-labeled. The incorporation rate of pyruvate, glucose, or CO2 was estimated with various biochemical approaches. Moreover, we used gene expression studies to identify the transcriptional level of genes encoding four anaplerotic carbon fixation enzymes in R. denitrificans under various growth conditions. In all of the conditions we tested, the transcript level is higher for malic enzyme (RD1_0421) and PEP carboxykinase (RD1_1376) than for pyruvate carboxylase (RD1_3376) and PEP carboxylase (RD1_4248), and different gene expression profiles for the anaplerotic carbon fixation enzymes are observed. Conclusions: We have successfully grown the R. denitrificans with either pyruvate or glucose as the sole carbon source, without the addition of yeast extract as previous literature reported. Our metabolomic studies confirm the biosynthesis of leucine and isoleucine, in which the citramalate pathway is favorable compare to derive from theronine for isoleucine biosynthesis, and incorporation of CO2 to oxaloacetate is suggested. The incorporation of pyruvate is more efficient than that of CO2. With the studies of the metabolomic (relatively low 13C-incorporation on several amino acids), biochemical and gene expression profiling, a working hypothesis on the carbon assimilation pathway in R. denitrificans is presented herein. 52
The Pennsylvania State University, University Park, PA, USA.
Methods: Samples of the photosynthetic microbial mat at Mushroom Spring in Yellowstone National Park at 61-64 °C were collected on 1011 July 2008 at four different time points in one 24-h period: at sunset (2100 h), prior to sunrise (0515 h), in dim light after sunrise (0640 h) and shortly after full illumination of the mat (0840 h). Total RNAs were extracted from these samples and were reverse transcribed into cDNAs. Reads generated from 454-pyrosequencing of the cDNAs were searched by blastN against reference genomes and databases. Taxonomic composition information was obtained by analysis of rRNA reads while analyses of mRNA reads revealed functional characteristics and regulatory patterns. Results: Pyrosequencing generated 409,567 reads, totaling 94,692,205 basepairs for the four samples. For each sample, from 90 to 95 percent of the reads were derived from rRNA, most of which could be assigned to different taxa. Ribosomal RNAs related to six chlorophototrophic microbial sources, Roseiflexus sp. RS-1, Synechococcus sp. JA-2-3B’a, Synechococcus sp. JA-3-3Ab, one or more Chlorobi species, Chloroacidobacterium thermophilum and a Chloroflexus species, accounted for nearly 90% of the total rRNA reads. The fact that the composition of this microbial community is so well defined, and that reference genomes of the major community members are available, allowed an in-depth survey of the 3.5 to 8 percent of reads corresponding to mRNA reads. Comparative analyses uncovered not only which genes of the major species are most actively transcribed, but also how they are regulated at light transition periods. This information sheds new light on the metabolism of the chlorophototrophic microbes in the community and their interactions. Insights on other components of the mat will also be discussed. Conclusions: This study vastly improves our understanding of a model photosynthetic microbial community because it provides a comprehensive view of the physiology of major mat phototrophic community members. It should also serve as a model for future studies of this and other microbial communities.
OC-5.1 HETEROCYST SPECIFIC GENES ARE EXPRESSED IN NOSTOC PUNCTIFORME DESTINED TO BECOME HORMOGONIA. H. Christman, E.L. Campbell, J.C. Meeks. Section of Microbiology, University of California, Davis, CA, USA. Introduction: Nostoc punctiforme is a developmentally diverse filamentous cyanobacterium capable of differentiating three distinct cellular forms- heterocysts, hormogonia and akinetes. Different signals give rise to these different states, however there is some August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes overlap. Cultures growing with ammonium can be induced to make hormogonia or heterocysts upon removal of combined nitrogen. Methods: Our DNA microarray of 6,893 genes predicted from the 9.05 Mb annotated genome sequence has been used to generate a global transcription data set consisting of seven time points over a 24 hour period of nitrogen deprivation that results in heterocyst formation and a similar profile that results in hormogonia formation. Results: 1 hour after nitrogen deprivation leading to heterocyst differentiation, there is enhanced expression of nitrate utilization and urea transport genes, and the nitrogen response regulator nrrA, which is required for timely development of heterocysts. Within 3 hours, the positive acting heterocyst master regulator hetR shows enhanced expression. From 6 to 12 h, enhanced levels of heterocyst structural genes, such as polysaccharide and glycolipid synthesis genes, and functional genes, such as those encoding two additional sets of cytochrome oxidase specific to heterocysts were observed. At 18 to 24 h the nif genes encoding nitrogenase are transcribed. In the nitrogen starvation induced (NSI) hormogonia time course, the nitrate utilization genes are enhanced at 1 h and nrrA is up regulated from 3 to 18 hours. hetR is maintained at a repressed level throughout the time course. The urea transport and cytochrome oxidase genes are enhanced transiently at the 12 hour time point and there is no change in expression of the heterocyst structural genes. At 24 h, the nif genes show up to 8-fold enhanced expression. Conclusion: The heterocyst time course is consistent with previous data, demonstrating a sequence of events required to form a fully functional heterocyst. NSI hormogonia show an early nitrogen response, but lack enhanced expression of heterocyst regulatory genes that would be expected to elicit transcription of nif genes. This is in contrast to previous work that has shown nif gene transcription to be under heterocyst developmental control. These data indicate a link in the regulatory decision to become either hormogonia or heterocysts. In order to sort out this regulation, examination of expression profiles on heterocyst deficient mutants is underway.
OC-5.2 PLANT CELL WALL EPITOPES ARE EXPRESSED BY CYANOBACTERIA IN THE GUNNERA-NOSTOC SYMBIOSIS. Owen Jackson, J. Paul Knox, David G. Adams. University of Leeds, West Yorkshire, United Kingdom. Introduction: Gunnera is the only angiosperm to host a cyanobacterial symbiont. Uniquely for plant-cyanobacteria symbioses the cyanobiont, Nostoc punctiforme, is intracellular. The plant benefits from the nitrogen-fixing properties of the bacterium, and in return Nostoc receives physical protection, an uncompetitive ecological niche and nutrients. The Gunnera-Nostoc system has potential uses in developing new, beneficial symbioses between nitrogen-fixing cyanobacteria and crop plants. However, when compared to the highly characterised relationship between leguminous plants and bacteria of the genus Rhizobium, the NostocGunnera relationship is not well understood. We present results showing the presence of arabinogalactan-protein (AGP) and pectin epitopes to be associated with both partners in the symbiosis. AGPs and pectic polysaccharides are important plant cell wall components implicated in cell growth, cell development and cell interactions. Methods: We have used a variety of immunochemical techniques (including ELISAs and epifluorescence microscopy) with anti-AGP and anti-pectin monoclonal antibodies, along with β-glycosyl Yariv reagent (known to specifically interact with AGPs), to investigate the presence and localisation of these polymers. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Results: Use of anti-AGP antibodies indicated the presence of AGPs associated with cyanobacterial strains grown in liquid media. The same set of antibodies also indicated the presence of AGPs in the gland mucilage of uninfected Gunnera glands, and associated with the walls of plant cells surrounding symbiotic Nostoc colonies in infected Gunnera glands. Anti-pectin antibodies revealed the presence of pectins on the surfaces of symbiotic cyanobacterial colonies. Conclusions: Cyanobacterial strains are capable of producing and expressing AGPs on their surfaces independent of their plant symbiotic partner, and are associated with pectins when in a symbiotic relationship with Gunnera. Both AGPs and pectin are present at the symbiotic surface at different times during the symbiotic relationship, and hence we discuss their potential roles in the initiation and continuation of a stable symbiosis
OC-5.3 REGULATION OF INTERCELLULAR MOLECULAR EXCHANGE IN HETEROCYST-FORMING CYANOBACTERIA. Conrad W. Mullineaux1, Anja Nenninger1, Vicente Mariscal2, Enrique Flores2, David G. Adams3. 1 School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Sevilla, Spain; 3Faculty of Biological Sciences, Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.
Introduction: When heterocyst-forming filamentous cyanobacteria such as Anabaena sp PCC7120 are growing diazotrophically, they must exchange metabolites between cells in the filament. Amino acids synthesised in the heterocysts must be transferred to the vegetative cells, and sugars synthesised in the vegetative cells must be transferred to the heterocysts. Recently we showed that intercellular molecular diffusion in Anabaena could be visualised by loading the cytoplasm with calcein, a fluorescent tracer dye. Fluorescence Recovery after Photobleaching (FRAP) was used to quantify rates of exchange of the dye among vegetative cells and heterocysts. We further showed that FraG, a protein with a DME permease domain located at the cell-cell interface, is essential for the diffusion of molecules from cell to cell (Mullineaux et al, 2008, EMBO J. 27, 12991308). FraG is probably a central component of pore structures (“microplasmodesmata”) that link the cells in the filament. Here we test the possibility that the activity of the microplasmodesmata is regulated according to the requirement for exchange of metabolites between cells. Methods: We used confocal FRAP to quantify rates of exchange of calcein at vegetative-vegetative and heterocyst-vegetative cell junctions, and GFP-tagging and confocal microscopy to quantify levels of FraG at cell junctions. Changes in both parameters were measured following nitrate step-down and heterocyst formation, and then after adding nitrate back to the cultures. Results: FraG is constitutively present at cell junctions, even when filaments are grown on nitrate. This is consistent with EM studies showing that the microplasmodesmata are always present at cell junctions. However, intercellular molecular exchange is slow in nitrategrown filaments. The rate of exchange between vegetative cells increases by more than a factor of 10 following nitrate step-down. When nitrate is added back to diazotrophically-growing cultures, molecular exchange becomes slower again. Conclusions: 1. Rates of intercellular diffusion are controlled according to the requirement for metabolite exchange. When nitrate is added 53
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Final Program and Abstracts back to differentiated, diazotrophic filaments, exchange becomes much slower. We suggest that the reduced supply of sugars to the heterocysts is a major factor leading to their subsequent death and disappearance. Under these conditions the vegetative cells may benefit by minimising the loss of sugars to the heterocysts. 2. The intercellular channel structures are constitutively present, even when molecular exchange is slow. Therefore there must be posttranslational mechanisms that regulate the opening and closing of the channels. We argue that the channels are constitutively present because they can only be formed during cytokinesis. Channels are synthesised in undifferentiated filaments so they are ready and waiting to be activated when the supply of combined nitrogen runs out.
but not of alr3442 and all0192, form heterocysts closer together than does the wild type strain, and that over-expression of patA resulted in closer heterocysts than in the wild-type strain. Conclusions: Yeast two-hybrid analysis provided evidence of proteins that interact with PatA. Six genes that consistently showed interaction with PatA were tested by mutation in Anabaena sp., looking for a possible alteration in pattern. Because the phenotype of an all3305 mutant has a phenotype like that of patA, there is a substantial likelihood that they interact in vivo. We are trying to test whether they interact in vitro. Over-expression of patA by use of PpetE elicited closer spacing of heterocysts.
OC-5.4
1. Liang J et al. PNAS 89: 5655 (1992); 2. Risser DD, Callahan SM. J Bacteriol 190: 7645 (2008); 3. Makarova KS et al. Bioinformatics 22: 1297 (2006); 4. Prentki P et al. Gene 103: 17 (1991).
SEARCH FOR PROTEIN(S) WITH WHICH PATA INTERACTS IN ANABAENA SP. STRAIN PCC 7120.
OC-5.5
Jinjie Liu, C. Peter Wolk.
THE INVOLVEMENT OF TWO SIGMA FACTORS IN CYANOBACTERIAL AKINETE FORMATION.
MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA. Introduction: In wild type Anabaena sp., heterocysts form with an average interval of ca. 10 intervening vegetative cells. patA (all0521) affects the regulation of the pattern of heterocyst differentiation: heterocysts of a patA mutant form nearly exclusively at the ends of filaments (1). PatA has also been reported to control the level of HetR (2). We have sought to identify the protein(s) with which PatA interacts and what their roles may be in the process of pattern formation. Methods: We used PatA as bait to identify interacting proteins in a yeast two-hybrid (Y2H) library. The library was constructed by partially digesting Anabaena sp. DNA separately with seven 4-bp restriction endonucleases whose products match 3 different cloning sites in the prey vectors. Different size ranges of the digested genomic DNA were recovered separately and ligated into appropriately digested prey vectors. Products of ligation were electro-transformed into Stratagene competent cells of E. coli XL10 Gold, isolated, and re-transferred to Saccharomyces cerevisiae strain AH109. Bait vector was transferred to S. cerevisiae strain Y187. The two yeast strains were mated on plates of SD/-Ade-His-Leu-Trp with X-alpha-galactosidase. Blue colonies were selected, purified, their inserts amplified by colony PCR, and the PCR products of several hundred clones were sequenced and analyzed. Because PatA appears to be a two-component response regulator that lacks an intact DNA-binding domain, we focused our attention on 11 prey genes that presumptively encode kinases and on genes for which multiple, independent clones showed interaction with PatA. Prey genes that survived those tests were mutated in Anabaena sp. by double reciprocal recombination with insertion of the Omega interposon (4). Mutations in six of those genes have been fully segregated. Results: The 11 candidate prey clones, all of which when re-tested by Y2H showed interaction with PatA, were tested for interaction with the REC, the PATAN, or both domains of PatA. According to Y2H analysis, All3305 interacted with the PATAN, but not with the REC, domain. Double-recombinant mutants of the prey genes alr3304, all3305, all5210, all2699, alr3442 and all0192, respectively, have been fully segregated. The all3305 mutant showed a phenotype very similar to that of a patA mutant. Preliminary observations and statistical analyses with the Wilcoxon rank-sum test suggest that mutants of alr3304, all5210, and all2699, 54
Karen LeGrand, Svetlana Rose, Peter Holmquist, Michael Summers. Department of Biology, California State University, Northridge, CA, USA. Introduction: Akinetes are desiccation and cold-resistant spore-like resting cells formed from vegetative cells in certain heterocyst forming cyanobacteria. In Nostoc punctiforme, akinetes first appear midway between heterocysts, or randomly along a filament lacking heterocysts, following phosphate or potassium limitation and low light. A previous DNA microarray study comparing vegetative cells to akinetes differentiated in a zwf metabolic mutant indicated that two putative sigma factors (NpR4091 and NpF4153) were up-regulated during this process. Almost nothing is known about the genetic regulation involved in the morphogenesis of vegetative cells into akinetes. To elucidate if these sigma factors were involved in akinete differentiation, each was over-expressed to test the hypothesis that over-expression of the sigma factor would lead to increased akinete formation. Methods: Random amplification of cDNA ends (RACE) was used to map the transcriptional start sites for each gene. DNA fragments bearing promoter regions were cloned into the green fluorescent protein (GFP) vector pSUN119, electroporated into N. punctiforme and reporter strains visualzed by epifluorescence microscopy following induction of akinetes by phosphate starvation. PCR fragments containing the entire reading frame of both sigma factors were amplified from the N. punctiforme genome and cloned into the BamHI site of pRL490 in the correct orientation to drive constitutive expression from the strong tac promoter. The expression plasmids were electroporated into the wild-type strain and neomycin selection used to obtain over-expression strains. DNA microarray analysis of the over-expression strain expression relative to wild-type is in progress to determine the regulon of each sigma factor. Results: NpR4091 encodes for an alternate group 2 sigma factor similar to SigB2 in Anabaena sp. strain 7120. Two promoters were identified, and a GFP reporter containing both was more highly expressed in akinetes relative to neighboring vegetative cells. Electroporants overexpressing NpR4091 exhibited large numbers of akinetes in filaments growing on non-inducing plates containing A&A medium containing Mops and ammonium. Over-expression strains in A&A/4 Mops and ammonium liquid culture grew slowly relative to wild-type cultures. Microscopic examination revealed large numbers of abnormally large cells that had the characteristics of akinetes when growing in non-inducing conditions. NpF4153 encodes for an Extracytoplasmic Function (ECF) sigma factor similar to SigG in August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes Anabaena sp. strain 7120. Two promoters were identified; the upstream promoter conferred akinete-specific expression of a GFP reporter, and the downstream promoter did not, although it was required for the higher-level gene expression in akinetes when both were present. The over-expression strain exhibited large numbers of akinetes under non-inducing conditions, and slow growth in liquid culture, similar to that of NpR4091. Conclusions: Cell-type specific gene expression or transcriptional reporters and altered cell morphology of over-expression strains support the hypothesis that the sigma factors encoded by NpR4091 and NpF4153 are involved in akinete formation.
Final Program and Abstracts similar stem-loop structure exists in the nifU-nifH intergenic region. We hypothesize that the required sequences upstream the transcription start site of nifH act to prevent transcriptional termination of nifBSU or function in transcript processing. NtcA activates expression of various genes under conditions of nitrogen deprivation. We show that NtcA does not directly regulate nifHDK, however it is likely that NtcA regulates nifBSU because this promoter contains an NtcA-binding site. This supports the hypothesis that nifHDK expression results from nifBSU promoter activity. 1
Wilson, J., Pierrard, J., Hubner, P (1993) Gene, 133: 39-46
OC-6.1 OC-5.6 SEQUENCES REGULATING NITROGENASE GENE EXPRESSION IN THE CYANOBACTERIUM ANABAENA VARIABILIS.
THE ROLE OF THE LOW-MOLECULAR-WEIGHT POLYPEPTIDES AT THE MONOMER-MONOMER INTERFACE OF PHOTOSYSTEM II IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Teresa Thiel, Justin L. Ungerer.
Julian J. Eaton-Rye, Hao Luo, Roger Young and Fiona K. Bentley.
Department of Biology, University of Missouri St. Louis, St. Louis, MO, USA.
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Introduction: The goal of this research is to identify and describe the function of key genetic elements that govern expression of the nitrogenase structural genes in heterocysts of cyanobacteria. Once we understand how specific sequences affect high-level, heterocystspecific expression of nitrogenase, we can use these regulatory elements to provide heterocyst specific expression of other oxygensensitive genes such as hydrogenases or alternative nitrogenases. The nifHDK regulatory elements would also be useful for expressing genes for bio-diesel production in heterocysts; an environment rich in lipids that may serve as the precursors to bio-diesel.
Introduction: X-ray diffraction data of Photosystem II (PS II) crystals from Thermosynechococcus elongatus have revealed a dimeric structure of PS II at 2.9 – 3.5 Å resolution [Ferreira et al. (2004) Science 303: 1831-1838; Guskov et al. (2009) Nat. Struct. Mol. Biol. 16: 334342]. Three low-molecule-weight polypeptides, PsbL, PsbM and PsbT have been identified at the monomer-monomer interface. We have used the model organism Synechocystis sp. PCC 6803 to investigate the role of these polypeptides in PS II dimer formation, assembly, turnover and electron transport.
Methods: Transcriptional lacZ fusions of various sized nifH1 promoter fragments, either wild type or containing specific mutations, were used to identify important nifH1 promoter sequences. Activity of the promoter fragments was determined using a standard β-galactosidase assay. A fluorescent substrate for β-galactosidase was used to visualize localization of expression of the reporter. The results from the lacZ expression study were confirmed by reconstructing the most interesting mutations or deletions directly in the upstream nifHDK region on the chromosome. Results: We have determined that the first 100 bp upstream of the nifH transcription start site are necessary but insufficient to provide normal regulated expression of nifH. Deletions in this region severely limit expression of nifHDK, whereas upstream sequences up to the start codon of nifB can be deleted from the chromosome without affecting expression. Conversely, the same required region and/or upstream sequences are not capable of driving high-level expression of nifH or the lacZ reporter in trans, though the limited expression that is detected is localized to heterocysts. This nifH1 upstream region contains a non-canonical NtcA binding site, however mutations of the NtcA binding site do not affect expression of nifHDK, therefore NtcA does not directly regulate nifHDK. Conclusions: The 100 bp upstream of the nifH transcription start site are sufficient to determine heterocyst specific expression of the nifHDK transcript; however, it is insufficient to provide high-level expression. Our data suggest that transcriptional activation of nifH may be under control of the nifB promoter. No region between the nifH start codon and the nifB promoter can drive high-level expression of lacZ; therefore, expression must result from the nifB promoter. It is possible that the known nifH1 transcription start site is a result of processing of a larger nifBSUHDK transcript, as there is evidence of nif transcript processing at stem-loop structures in other species 1. A August 9 to 14, 2009 • Montréal, QC, Canada
Methods: Mutagenesis, biochemical characterization and physiological measurements were carried out as described in [Hao et al. (2008) Photosynth. Res. 98: 337-347]. Chlorophyll a fluorescence induction and decay kinetics were measured with a fluorometer supplied by PSI Instruments, Brno, Czech Republic. Polysomes from thylakoid and cytosolic fractions were isolated according to [Tyystjärvi et al. (2001) Mol. Microbiol. 40: 476-484]. Results: Mutations in the cytosolic N-terminal extension of PsbL destabilized PS II dimers, although the monomeric complexes retained their ability to split water. Mutations in this region of PsbL also activated diuron-insensitive cyclic electron transfer around PS II. Additionally, truncation of 4 residues at the C-terminus was sufficient to prevent association of PsbL with a subcomplex of the photosystem thereby blocking assembly of active PS II complexes and creating an obligate photoheterotrophic strain. The absence of PsbM decreased the number of assembled PS II centers by one third and the remaining centers exhibited an increased susceptibility to high-light-induced photodamage suggesting turnover and assembly were slowed when compared with PS II centers in wild type. Similarly, absence of PsbT resulted in a strain that was highly susceptible to high-light-induced stress. ∆PsbT cells were able to recover from photodamage when transferred from high- to low-light conditions. Recovery was observed to be dependent on protein synthesis and light, and was prevented in the absence of Psb27. Moreover, evidence for a specific role for PsbT in PS II turnover following photodamage was found since accumulation of psbA transcripts with thylakoid-associated polysomes was observed in wild type but not seen in ∆PsbT cells. Conversely, following exposure to high light, elevated levels of psbA transcripts with polysomes isolated from the cytosolic fraction were only observed in ∆PsbT cells. Electron transfer between the PS II primary and secondary plastoquinone acceptors, QA and QB, was also impaired in the ∆PsbT strain. Unexpectedly, loss of oxygen-evolving activity under high light was reversed by addition of bicarbonate in 55
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Final Program and Abstracts ∆PsbT cells and oxygen evolution was abolished by addition of formate under ambient CO2 levels in the presence of the artificial electron acceptors 2,5-dimethyl-p-benzoquinone (DMBQ) and K3Fe(CN)6. Our results suggest that DMBQ may bind and inhibit at the QA binding site in ∆PsbT cells and in some PsbL mutants. Conclusions: C-terminal mutations of PsbL prevent association with PS II blocking assembly at the CP43-less inactive monomer. N-terminal mutations within PsbL alter the acceptor side and stability of PS II, as does the absence of PsbM and PsbT. The absence of PsbT may also destabilize the association of polysomes with thylakoid membranes following high light stress and therefore PsbT could play a key role in efficient repair of PS II following photodamage.
OC-6.2 PHOTOSYSTEM II PROTEIN LIFETIMES IN VIVO IN SYNECHOCYSTIS. Danny Yao, Dan Brune, Wim Vermaas. School of Life Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. Introduction: The lifetime of at least one of the photosystem II protein components, PsbA (D1), is short (less than an hour at high light intensity) but little information is available on the lifetimes of most other photosystem II subunits. As the subunits all form a complex in the membrane, different lifetimes of components raise interesting questions regarding mechanisms of repair. The lifetime of chlorophylls (determined by in vivo labeling with 13C or 15N) in Synechocystis is more than a week, suggesting that chlorophylls are recycled efficiently if photosystem II breaks down. Small Cab-like proteins (SCPs) have been shown to play a role in the stability of the chlorophyll, with the chlorophyll lifetime decreasing in the absence of SCPs. The questions addressed in this research are what the range of lifetimes of photosystem II proteins is, and whether SCPs also affect the stability of proteins in the photosystems. Methods: Stable isotope labeling and mass spectrometry have proven to be a powerful combination to determine lifetimes. Using 15 N-ammonium nitrate labeling that is added to growing cultures at a specific time, both chlorophyll and protein are labeled, and the disappearance of unlabeled (old) compounds can be followed over time after isolation of photosystem II that carries a His-tagged CP47. For these experiments we used photosystem I-less strains that were grown at a light intensity of 4 mol photons m-2 s-1. Results: The lifetimes of specific photosystem II proteins (PsbA (D1), PsbB (CP47), PsbC (CP43), PsbD (D2), PsbE and PsbF (cytochrome b559), PsbH, PsbO and Psb27) in assembled photosystem II complexes were followed in living Synechocystis cells under standard growth conditions by monitoring the amount of unlabeled protein that could be extracted from membranes using the His tag on CP47. The halflifetime of D1 was short as expected (about 2 hours at low light intensity), but particularly PsbE and PsbO first showed a ~25% increase in unlabeled protein integrated into photosystem II complexes in the first 9 hours after the start of labeling before unlabeled protein amounts decreased and labeled protein became more prevalent. This suggests that a significant amount of PsbE (and PsbO) is present in cells that is not incorporated into photosystem II complexes, and that may serve as an anchor for photosystem II assembly. The role of SCPs in photosystem protein stability will be presented as well.
and –in contrast to radioactive labeling- can detect both “old” (unlabeled) and newly synthesized protein. The lifetime of polypeptides in photosystem II complexes varies greatly, implying that the polypeptides are replaced independent of each other. Also, the presence of a pool of photosystem II proteins that are not yet incorporated into a mature photosystem II complex but that may form the nucleus of a new photosystem II complex is demonstrated.
OC-6.3 IMPLICATIONS OF THE RC-LH1 CORE COMPLEX STRUCTURE FOR THE OXYGEN DEPENDANCE OF THE PHOTOSYNTHETIC ACTIVITY OF ROSEOBACTER DENITRIFICANS. Steffani Schäfer1, Richard K. Hite2, Thomas Walz2 and Andreas Labahn1. 1 Institut für Physikalische Chemie, Universität Freiburg, Freiburg, Germany; 2Department of Cell Biology, Harvard Medical School, Boston MA, USA.
Introduction: Roseobacter denitrificans belongs to the group of obligate aerobic photosynthetic bacteria. The typical bacterial photosynthesis is an anaerobic process where the formation of the photosynthetic apparatus is down-regulated in the presence of oxygen. Roseobacter produces bacteriochlorophyll a as well but is incapable of performing photosynthetic energy transduction without oxygen. Only under aerobic conditions, the protein components of the photosynthetic apparatus are synthesized and photoinduced electron transport is operative. Methods: Core complexes, consisting of reaction center and light harvesting complex 1 (RC-LH1) were solubilized from the photosynthetic membranes of Roseobacter denitrificans with decylmaltoside and purified by anion exchange chromatography and sucrose density gradient centrifugation. Single particle electron microscopy was used to perform a first structural characterization of the core complex . 30,000 particles were selected from micrographs of negatively stained samples, aligned to each other and classified to produce class averages. Results: The class averages showed different views of the core complex which could thus be combined to calculate a threedimensional reconstruction. The density map at 30 Å resolution shows that the LH1 complex surrounding the RC forms a closed circle. Although the resolution of the reconstruction is not yet sufficient to resolve possible gaps in the LH1 ring, this tight ring around the reaction center may prevent the exchange of dihydroquinone and thus light-induced cyclic electron transfer. Conclusion: With the RC-LH1 complex from R. denitrificans we have isolated and structurally characterized a core complex from an obligate aerobic photosynthetic bacterium for the first time. A tight LH1 ring surrounding the reaction center was found which may inhibit the exchange of dihydroubiquinone with the quinone pool. Thus, we conclude that light-driven energy production is insufficient for bacterial growth and that oxygen is not only required for enabling photosynthetic electron transport but mainly for alternative metabolic pathways. We will now 2D-crystallize the core complex for cryo-electron microscopy to obtain improved resolution and a detailed reconstruction of its 3D structure.
Conclusion: Stable-isotope labeling in combination with mass spectrometry is a powerful method for determining protein lifetimes,
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Final Program and Abstracts
OC-6.4
OC-6.5
VARIABLE FLUORESCENCE IN HELIOBACTERIUM MODESTICALDUM CELLS: OBSERVATION AND EXPLANATION.
NON-RADIATIVE CHARGE RECOMBINATION IN PHOTOSYSTEM II PROTECTS THE CYANOBACTERIUM MICROCOLEUS SP. AGAINST EXCESS LIGHT STRESS.
Kevin Redding1,2, Fabrice Rappaport1, Aaron Collins3, Stefano Santabarbara1,2, Robert Blankenship3. 1
Arizona State University, Dept. of Chemistry and Biochemistry, Tempe, AZ, USA; 2Institut de Biologie Physico-Chimique, Paris, France; 3 Washington University, Departments of Biology and Chemistry, St Louis, MO, USA. Introduction: Heliobacteria have the simplest photosynthetic apparatus of all known phototrophs. They possess a single type I homodimeric reaction centre (RC), which harvests light using a pool of 22-34 bacteriochloropyll g (Bch g) as part of each RC’s intrinsic antenna. There is no extrinsic antennae. The immediate electron donor is a membrane-attached cytochrome c, which is re-reduced by a diheme-containing cytochrome bc complex, and the immediate electron acceptor is a di-cluster ferredoxin. At this point, it is not clear how the cycle is closed to allow light-driven proton pumping. Also unclear is the role of the pool quinone (menaquinone-9) – each RC contains 2 of these molecules, but there is conflicting evidence as to their role in electron transfer. Although heliobacteria are photoheterotrophs and do not fix CO2, all species examined so far fix N2, and nitrogenase is expected to be a large consumer of lowpotential electrons under N2-fixing conditions. Methods: We have examined in vivo kinetics of electron transfer within living cells of Heliobacterium modesticaldum using a combination of transient absorption and fluorescence spectroscopy. Results: We found that H. modesticaldum cells exhibit the phenomenon of variable fluorescence, although the characteristics are quite different from those seen in oxygenic phototrophs. It usually took ~100 ms of illumination with actinic light before fluorescence began to rise. Variable fluorescence could be abolished by blocking re-reduction of P800 + (oxidized primary electron donor), either by addition of ferricyanide (oxidant) or stigmatellin (Qo inhibitor). Contrariwise, reduction of intracellular electron acceptors by addition of dithionite and PMS (as mediator) increased variable fluorescence, as did inhibition of nitrogenase by addition of ammonia. Under conditions that led to high variable fluorescence, it was observed that the yield of photo-oxidizable P800 (in the µs timescale) was lower. We can explain all of these data by a model in which saturation of the electron acceptor pool leads to reduction of the FX Fe4S4 cluster in the RC, provoking a back-reaction from the P800 + A0 state, which produces delayed fluorescence. We tested this model by using pumpprobe spectroscopy in the ns timescale and found that a ~20-ns back-reaction from the P800 + A0 state was indeed observed under conditions that produce high variable fluorescence. Moreover, we can quantitatively account for almost all of the variable fluorescence as being due to back-reaction. The effects of QB-type inhibitors upon variable fluorescence provide evidence in favor of the participation of menaquinone in forward electron transfer within the RC. Conclusions: These results demonstrate a new phenomenon in heliobacterial physiology – variable fluorescence – and explain it as a consequence of delayed fluorescence stemming from a fast backreaction within the heliobacterial RC. This demonstrates that the intracellular electron acceptor pool can easily become saturated under strong illumination conditions, and indicates the importance of electron donor/acceptor dynamics. These facts likely explain why this organism does not require a large antenna (as it is not required) August 9 to 14, 2009 • Montréal, QC, Canada
Itzhak Ohad1, Nir Keren2, Dan Tchernov3, Aaron Kaplan2. Departments of 1Biological Chemistry, 2Plant and Environmental Sciences and 3The Interuniversity Marine Institute, Eilat, The Hebrew University of Jerusalem, Israel. Photosynthetic organisms are exposed to light-induced oxidative stress due to photoinactivation of the oxygen evolving photosystem II (PSII). We investigated the persistence of PSII activity of the desiccation-tolerant Microcoleus vaginatus, a cyanobacterium inhabiting biological desert crusts where it is exposed to high light intensities. Surprisingly, CO2-dependent oxygen evolution persisted at light intensities 2-3 times higher than saturation. In contrast, light intensities close to or higher than required to saturate oxygen evolution triggered extensive loss (85-90%) of radiative PSII charge recombination measured as variable fluorescence or thermoluminescence (TL) emission. Radiative charge recombination, which involves the generation of singlet oxygen, recovered slowly following exposure to low light intensity but did not recover in darkness. Light induced loss of fluorescence and its recovery are not inhibited by herbicides that bind to the PSII-QB site, indicating that reduction of plastoquinone or O2 is not involved. The temperature required for maximum TL emissions resulting from QA-/S2 charge recombination (Q band, 22oC) indicated a significant upshift of the PSII-QA redox potential as compared to “model” photosynthetic organisms (Q band, 10-15 oC). This property of Microcoleus may reduce harmful radiative charge recombination as a function of light intensity, thereby lowering the generation of 1O2 and related oxidative stress. On the basis of our findings we present a novel model whereby a non-radiative charge recombination within PSII lowers photoinactivation damage under excess illumination and a possible reason why this effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacterium to the higher plant chloroplast.
OC-6.6 THE PROTECTIVE ROLE OF FLAVODIIRON PROTEINS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Marion Eisenhut, Pengpeng Zhang, Yagut Allahverdiyeva, Eva-Mari Aro. Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, Finland. Introduction: The prokaryotic photoautotrophic cyanobacteria (bluegreen algae) are favored model organisms to investigate photosynthetic mechanisms. Stress conditions like high light or limitation in inorganic carbon often lead to restriction in the availability of the terminal electron acceptors of the photosynthetic electron transfer chain. To ensure the delicate balance of energy input and consumption, thus preventing the generation of dangerous reactive oxygen species, photosynthetic organisms developed different strategies. It is suggested that flavodiiron proteins (Flv) are involved in those adaptation strategies. Though quiet well studied in (facultative) anaerobic Bacteria and Archaea, knowledge on Flvs in cyanobacteria is very poor. However, the modular character (a β-lactamase-like domain containing a diiron center, a flavodoxin domain with FMN binding site and an additional C-terminal flavin reductase domain in 57
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Final Program and Abstracts cyanobacterial Flvs) of those proteins strongly suggests their participation in electron transfer processes [1]. Methods: Detailed material and methods are described in [2]. Results: The genome of the cyanobacterial model strain Synechocystis sp. PCC6803 comprises four genes, encoding the hypothetical flavodiiron proteins Flv1, Flv2, Flv3 and Flv4. While Flv1 and Flv3 were shown to function in the Mehler reaction as NAD(P)H:oxygen oxidoreductases [3], the physiological function of Flv2 and Flv4 remains open. RT-PCR and Western Blot analyses show that the expression of flv2 and flv4 is high under LC (air level of CO2) and negligible at HC (3% CO2). Moreover, the rate of accumulation of flv2 and flv4 transcripts upon shift of cells from HC to LC is strongly dependent on light intensity. Characterization of Flv-mutants in Synechocystis revealed a specific decline in PSII centers and impaired translation of the D1 protein in the mutants ∆flv2 and ∆flv4 when grown at LC, whereas at HC the Flvs were dispensable. ∆flv2 and ∆flv4 were also more susceptible to high light induced inhibition of PSII than WT or ∆flv1 and ∆flv3. In fact, ∆flv1 and ∆flv3 seemed to have more functional PSII centers, and in both mutants the photoinhibition of PSII was slightly less severe than in the WT. Analysis of published amino acid sequences revealed that cyanobacterial genomes always contain two up to six genes encoding distinct Flvs within the same organism. Furthermore we could find cyanobacteria-like Flvs also in some oxygenic photosynthetic eukaryotes like green algae, mosses and lycophytes. The occurrence of Flvs in pairs seems to be a special feature for oxygenic photosynthetic organisms. Conclusions: We demonstrate a novel and crucial function of flavodiiron proteins, particularly Flv2 and Flv4, for oxygenic photosynthesis in Synechocystis cells at LC. There seems to be an evolutionary trend in the diversity and function of the flavodiiron proteins: In anaerobic microbes the activity of Flvs is directed against O2/NO toxicity. In oxygenic photosynthetic organisms Flv1 and Flv3 reduce oxygen to water while Flv2 and Flv4 protect the oxygen evolving PSII complex against photoinhibition. Higher plants lack Flvs and distinctly different mechanisms have evolved for photoprotection of PSII. [1] Vicente JB, Justino MC, Gonçalves VL, Saraiva LM, Teixeira M (2008) Methods Enzymol 437: 21-45 [2] Zhang P, Allahverdiyeva Y, Eisenhut M, Aro EM (2009) PLoS ONE [3] Helman Y, Tchernov D, Reinhold L, Shibata M, Ogawa T et al. (2003) Curr Biol 13: 230-235
OC-7.1 PROTEIN-PROTEIN INTERACTION BETWEEN CBBR AND REGA (PRRA), TRANSCRIPTIONAL REGULATORS OF THE CBB OPERONS (CO2 FIXATION) IN RHODOBACTER SPHAEROIDES.
and RegA are DNA binding proteins with helix-turn-helix motifs for DNA interaction. DNA binding sites for both proteins are found in the regulatory regions of the cbbI and cbbII operons in Rhodobacter sphaeroides. RegA has four DNA binding sites in the cbbI promoter. The CbbR binding site and RegA binding site 1 overlap each other upstream of the cbbI operon in R. sphaeroides, as demonstrated by footprint analysis. Results: CbbR and RegA interact and CbbR must be bound to DNA for this protein-protein interaction to occur. Conversely, cbbI promoter binding by RegA is not required for interaction with CbbR. The presence of both CbbR and RegA in cbbI promoter/protein complexes was confirmed. In addition, the presence of RegA enhances the ability of CbbR to bind the cbbI promoter. Gel mobility shift analysis demonstrates that RegA binds to cbbI promoter DNA and forms incrementally larger multimeric complexes with DNA as the concentration of RegA increases, referred to as oligomerization. When RegA binding sites 1, 2 and 3 are present in the cbbI promoter, RegA forms a complex with the DNA at a significantly lower concentration and a greater mobility compared to complex formation with RegA binding sites 1/2, 3 or 3/4, separately. Specific site-directed mutants of RegA have been generated that cannot bind CbbR and also have reduced oligomerization function. These mutations of the RegA protein are located in the N-terminus receiver domain as well as in the DNA binding domain. Methods: Methods include gel mobility shift experiments using 32Plabeled DNA probes and chemical cross-linking analysis using dimethylpimelimidate. Isolation of the protein/DNA complexes from native acrylamide gels and immunoblot analysis using antibodies specific to CbbR and RegA was employed to determine the composition of promoter/protein complexes. Conclusions: There is strong interaction between RegA and CbbR, and the presence of RegA increases the quantity of CbbR that binds the cbbI promoter. Possibly, RegA lowers the activation energy required for CbbR to bind DNA, thereby increasing the DNA binding affinity of CbbR. CbbR must be bound to DNA to interact with RegA, possibly ensuring that RegA only binds transcriptional regulators such as CbbR at the appropriate promoter site. RegA sites 1, 2 and 3 are necessary for optimal binding of RegA to the cbbI promoter. There is communication or cooperation between RegA binding site 1/2 and site 3 that allows RegA/DNA complex formation at a lower concentration of RegA. An intermediate loop structure could be formed to facilitate the communication between RegA site 1/2 and site 3. In addition, several specific regions of the RegA protein are important for interaction with CbbR.
OC-7.2 PpsR AS A NEW TYPE OF HEMIN SENSOR. Liang Yin, Vladimira Dragnea, Carl Bauer.
Andrew W. Dangel, F. Robert Tabita.
Indiana University, Bloomington, IN, USA.
Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, OH, USA.
My research focuses on PpsR-AppA system. To date, this regulatory system is the only cascade identified that coordinates light- and redox- signaling in transcription control. PpsR was identified as a DNA-binding transcription factor in R. sphaeroides. Under aerobic conditions, PpsR blocks the transcription of the tetrapyrrole biosynthetic and the photosynthetic genes, which are responsible for biosynthesis of the pigments and the light harvesting II complex (1). AppA, an antirepressor of PpsR, is able to inactivate PpsR by forming a PpsR2-AppA complex under anaerobic and dark conditions (1). In the past year, our results indicate that hemin provides another level of control in this system, showing the direct link between free hemin pool
Introduction: CbbR and RegA (PrrA) are transcriptional regulators of the cbbI and cbbII (Calvin-Benson-Bassham CO2 fixation pathway) operons in Rhodobacter sphaeroides. CbbR is a LysR type transcriptional regulator (LTTR) which is the most commonly utilized protein family for gene regulation in prokaryotes. RegA is part of a two-component signal transduction system also involving the membrane-bound histidine kinase, RegB (PrrB). RegA is phosphorylated by RegB to activate its regulatory function. Both CbbR 58
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes and photosynthesis for the first time. We demonstrated that PpsR is a hemin-binding protein with stoichoimetry of 1:1 ([PpsR]/[hemin]). The presence of hemin inhibited the DNA-binding ability of PpsR significantly, while other tetrapyrrole products did not show any inhibition. Experiments with truncated PpsR suggested that it bound to hemin via its C-terminal region, possibly through the Cys located in its DNA-binding domain. In summary, our results suggest that PpsR is likely to be a hemin sensor. Most of well characterized hemin-binding proteins have tightlycoordinated hemin to perform many different tasks, such as electron transfer, oxygen storage and gas molecule sensing (2). Only in recent years people started to appreciate the hemin sensors, which are able to sense the free hemin in vivo, from bacteria (3) to mammal (4). Yet all the identified hemin sensors to date contain the conserved -Cys-Promotif, which PpsR does not have. Our research will provide us new insights about hemin sensors, which seem to be more diverse than we used to think. 1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623 2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L., Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073 3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A 96, 13056-13061 4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc Natl Acad Sci U S A 105, 76-81
OC-7.3 ADAPTATION TO THE APPEARANCE OF ATMOSPHERIC OXYGEN: AN EXPERIMENTAL SCENARIO FOR A STRICT ANAEROBIC PHOTOTROPH. Bahia Khalfaoui Hassani, Anne-Soisig Steunou, Sylviane Liotenberg, Françoise Reiss-Husson, Chantal Astier, Soufian Ouchane. CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; Université Paris-Sud, Orsay; Université Pierre et Marie Curie, Paris; France. Introduction: The appearance of oxygen in Earth’s atmosphere as a result of oxygenic photosynthesis required strict anaerobes and obligate-phototrophs to cope with the presence of this potentially toxic molecule. Nitrogen-fixing organisms, for example, have evolved a number of strategies to protect nitrogenase from environmental oxygen. Among these strategies, oxygen depletion by specific oxidases was shown to play an important role. We have previously detailed the AcsF/BchE enzyme replacement and suggested a similar HemF to HemN replacement during the shift from aerobic to anaerobic environment to emphasize the O2 dependent and independent metabolic networks. Here, we further investigate the problem of O2 and photosynthesis (PS) in the anoxygenic phototroph bacterium Rubrivivax (R) gelatinosus and show how the terminal oxidases (cbb3, bd and caa3) expand the physiological range of O2 environments under which this bacterium and probably many other “aerobic” phototrophs can carry out photosynthesis. Methods: PCR-based cloning allowed the identification of four oxidase encoding operons in R. gelatinosus. Mutations were generated to disrupt each operon to study the respiratory chain of this bacterium. Mutants were further analyzed to check how photosynthesis and respiration or O2 detoxification are functionally related in this microorganism. Results: We have shown that: August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts 1. Only two terminal oxidases are functional in wild type R. gelatinosus: the cbb3 cytochrome c oxidase, and the bd type quinol oxidase. 2. Unlike the wild type and the single mutants, the cbb3--bd- deficient double mutant can carry out photosynthesis only under strict anaerobicity. 3. In oxygenated environments, this mutant survives nonphotosynthetically until the O2 tension drops. Co-inoculation with aerobes (E. coli) and PS deficient strains restores PS growth of this mutant even in oxygenated medium. 4. Spontaneous suppressor mutations especially in caa3 cytochrome c oxidase that restore respiration simultaneously restore photosynthesis in oxygenated environment. Conclusions: The cbb3, bd and caa3 oxidases are therefore not only required for respiratory growth but also play an essential role in the reduction of the environmental O2 pressure during anaerobic photosynthesis. These results demonstrate that photosynthetic and other anaerobic metabolisms in environments which are (at least transitorily) exposed to oxygen are critically dependent on the O2detoxifying action of terminal oxidases.
OC-8.1 CHARACTERIZATION OF THE TERNARY COMPLEX FORMED BY FERREDOXIN:THIOREDOXIN REDUCTASE, FERREDOXIN AND THIOREDOXIN M. David Knaff, Xingfu Xu, Peter Schürmann, Sung-Kun Kim, Masakazu Hirasawa, Marcellus Ubbink. Texas Tech University, Lubbock, TX, USA. Introduction: Ferredoxin:thioredoxin reductase (FTR), catalyzes the two-electron reduction of thioredoxins in chloroplasts and in cyanobacteria (thioredoxins f and m and perhaps other thioredoxins are substrates), using reduced ferredoxin as the electron donor. Reduced thioredoxins then play important roles in redox regulation. FTR, a heterodimer with a unique [4Fe-4S] cluster as its sole prosthetic group, has a single binding site for ferredoxin and a separate single binding site for thioredoxin. The binding sites for FTR from the cyanobacterium Synechocystis sp. PCC 6803 on ferredoxin and on thioredoxin in the 1:1 complexes of the enzyme with its two protein substrates have been mapped and the ternary complex between all three proteins has been characterized. Methods: Perturbations of the two-dimensional NMR spectra of both ferredoxin and thioredoxin arising from complex formation with FTR have been used to identify specific amino acids on the two proteins involved in complex formation with FTR. Results: A mono-gallium analog of the [2Fe-2S] Synechocystis sp. PCC 6803 ferredoxin was obtained by reconstituting apo-ferredoxin in a gallium-containing refolding buffer. The use of this diamagnetic Ga structural analog eliminates the paramagnetic broadening of NMR resonances of amino acids in the vicinity of the [2Fe-2S] cluster in native ferredoxin. This has allowed the first complete mapping of the interaction interface of a [2Fe-2S] ferredoxin for a target enzyme. NMR has also been used to contain a three-dimensional structure of the Gaferredoxin in solution, confirming that its structure is almost identical to that of the native, iron-containing protein. NMR spectroscopy was also used to map the interaction domain for FTR on thioredoxin m in a 1:1 complex of the two proteins. Both similarities and differences are seen in the thioredoxin m interaction domain for FTR in the noncovalent complex examined by NMR and in a disulfide-linked covalent complex of FTR and thioredoxin m for which an X-ray crystal structure 59
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Final Program and Abstracts has been obtained. NMR has also been used to characterize a ternary complex between ferredoxin, FTR and thioredoxin m in solution. Conclusions: The interaction domains on both ferredoxin and thioredoxin for FTR appear to consist largely of hydrophobic amino acids. The fact that FTR can form a ternary complex with ferredoxin and thioredoxin simultaneously confirms the presence of separate binding sites on FTR for its two substrates. Furthermore, Gasubstituted ferredoxin has been established as a valuable, non-paramagnetic analog for ferredoxin for NMR investigations of the interaction of ferredoxin with its target enzymes.
OC-8.2 CAROTENOIDS AND CAROTENOGENESIS IN CYANOBACTERIA. Shinichi Takaichi1, Mari Mochimaru2. 1
Department of Biology, Nippon Medical School, Kawasaki; Department of Natural Sciences, Komazawa University, Setagaya, Tokyo; Japan. 2
Introduction: Cyanobacteria grow by photosynthesis, and essentially contain chlorophyll and various carotenoids, whose main functions are light-harvesting and photoprotection. We have summarized carotenoids, characteristics of carotenogenesis enzymes and genes, and carotenogenesis pathways in some cyanobacteria, whose both carotenoids and genome DNA sequences have been determined (Takaichi & Mochimaru (2007) Cell. Mol. Life Sci. 64: 2607-2619). Methods: Carotenoids were extracted from cyanobacterial cells and purified. They were identified based on absorption spectra, retention times on C18-HPLC, MS, 1H-NMR and CD analyses (Iwai et al. (2008) Plant Cell Physiol. 49: 1678-1687). Knockout mutants of carotenogenesis genes were constructed for characterization of enzymes (Mochimaru et al. (2008) J. Bacteriol. 190: 6726-6733).
Results & Discussion: Cyanobacteria contain various carotenoids; βcarotene, its hydroxyl derivatives, such as zeaxanthin and nostoxanthin, or keto derivatives, such as echinenone and canthaxanthin, and carotenoid glycosides, such as myxol glycosides and oscillol diglycoside. Both ketocarotenoids, such as echinenone and 4-ketomyxol, and the carotenoid glycosides are the unique carotenoids among phototrophic organisms. Some cyanobacteria, e.g. Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, contain both unique carotenoids, while others, e.g. Synechococcus elongatus PCC 7942 and Prochlorococcus marinua, do not contain such carotenoids. From these findings and characteristics of carotenogenesis enzymes, certain carotenogenesis pathways can be proposed. The different compositions of carotenoids might be due to the presence or absence of certain gene(s), or to different enzyme characteristics. For instance, two distinct β-carotene hydroxylases, CrtR and CrtG, are bifunctional enzymes; CrtR catalyzes β-carotene to zeaxanthin and deoxymyxol to myxol, CrtG catalyzes zeaxanthin to nostoxanthin and myxol to 2-hydroxymyxol, and substrate specificities of CrtR vary across species. Two distinct b-carotene ketolases, CrtO and CrtW, are found only in the first group, and properly used in two pathways, β-carotene to echinenone and myxol to 4-ketomyxol, depending on the species. Lycopene cyclases of CrtL from Synechococcus elongatus PCC 7942, CruA and CruP from Synechococcus sp. PCC 7002, and CrtL-b and CrtL-e from Prochlorococcus marinus have been functionally confirmed, while those in other species are not found yet. In Synechococcus sp. PCC 7002, 1’,2’-hydratase (CruF) for myxol synthesis is functionally confirmed, and novel unique carotenoid of synechoxanthin is found. At present, the number of functionally confirmed genes is limited, and
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only a few species are examined. Therefore, further studies of carotenoids, characteristics of carotenogenesis enzymes and genes, and carotenogenesis pathways are needed.
OC-8.3 EVOLUTION OF CAROTENE DESATURATION: INSIGHTS FROM PURPLE BACTERIA AND CYANOBACTERIA. Gerhard Sandmann. Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, Frankfurt, Germany. Introduction: Carotenoids are essential pigments for photosynthetic organisms. Their predominant function is protection of photosynthesis from photooxidation. This antioxidative property depends on the conjugatd double bond system of the carotenoid molecule which is formed in a series of desaturation steps. During evolution of carotenogenesis, two basically different desaturation pathways were acquired. The “ancient” pathway exists in archeae, bacteria and fungi involving a single CrtI type enzyme which can catalyze up to six desaturations and isomerizes cis to trans double bonds. Among the purple bacteria, CrtI desaturases exist either as 4-step desaturases starting the spirilloxanthin pathway as in Rhodospirillum rubrum or as 3-step desaturases as in Rhodobacter species synthesizing spheroidene derivatives. In contrast, double bonds are formed by structurally and mechanistically unrelated CrtP type desaturases in cyanobacteria, Chlorobaculum species and photosynthetic eukaryotes. The most likely evolutionary root of CrtP is a CrtU desaturase which is responsible for aromatization of b-ionone rings in several groups of bacteria including Chlorobiaceae. Methods: Carotenogenic genes were cloned from different species. They were heterologously expressed and the resulting enzymes used to carry out enzyme kinetic studies including determinations of substrate and product specificities Results: Within cyanobacteria, one finds among the existing species a relic of the CrtI desaturation pathway, Gloeobacter violaceous, which is the only known cyanobacterium with a CrtI-type desaturase. In contrast to the CrtI-catalyzed desaturations, the desaturation products of CrtP-related reactions are poly-cis carotenes. Consequently, the resulting tetra-cis lycopene has to be isomerized to all-trans by an additional isomerase CrtH which developed from CrtI by loosing its entire desaturation function but retaining its isomerization function. There is an enzyme resembling an evolutionary link between CrtI and CrtH in the cyanobacterium Nostoc PCC7120. It resembles an additional CrtI-related enzyme named CrtQa which is a z-carotene desaturase producing all-trans products. Its participation in the polycis desaturation pathway of Nostoc PCC7120 could be specified. The evolutionary modification of the product specificity of the CrtI desaturases was studied in Rubrivivax gelatinosous in which CrtI resembles a transitional state yielding 3-fold and 4-fold desaturated products, simultaneously. The in vitro and in vivo results were compared to a 3-step desaturase from Rhodobacter. We could find that the kinetic property of this phytoene desaturase is one factor to determine the type of reaction product. However, structural properties can prevent that the last desaturation step is catalyzed. The reaction stops at the level of neurosporene which is no longer a substrate for the desaturase. Conclusion: Among the prokaryotes, two basically different carotenoid desaturation pathways were acquired. Our enzyme kinetic investigations were focussed on two desaturases, a missing link between both pathways and one desaturase comprising all
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes characteristics of a 3- and of a 4-step desaturase. The results will help to understand the evolutionary origin and the divergence of carotene desaturation.
OC-9.1 THE CHLOROSOME BASEPLATE OF CHLOROBACULUM TEPIDUM – A STRUCTURAL MODEL BASED ON SOLID-STATE NMR DATA COMBINED WITH MOLECULAR SIMULATION STUDIES OF CD AND ABSORPTION SPECTRA. Marie Ø. Pedersen1, Morten Bjerring1, Jarl Underhaug1, Jens Dittmer1, Peter Højrup2, Anders Giessing2, Juha Linnanto3, Niels-Ulrik Frigaard4, Mette Miller2 and Niels Chr. Nielsen1. 1
Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus, Århus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 3Department of Chemistry, University of Jyväskylä, Finland; 4 Department of Biology, University of Copenhagen, Copenhagen, Denmark. Introduction: Green sulfur bacteria possess two external lightharvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. A small 6.1 kDa chlorosome protein, CsmA, plays a major role by connecting these two antennae physically and functionally. CsmA is located in the chlorosome baseplate - a paracrystalline structure observed by freeze fracture electron microscopy. Each CsmA coordinates one molecule of bacteriochlorophyll (BChl) a, and reconstitution experiments have shown that the CsmA-BChl a complex forms dimers and higher aggregates (1). The CsmA baseplate is one of the simplest known antenna systems. Thus, a structural characterization might provide new insights into how photosynthesis evolved, as well as serve as inspiration to the development of new photosynthetic devices. Methods: We have deduced a solution NMR structure of monomeric CsmA in a mixed solvent system, which shows CsmA to be an αhelical protein (2). To find the native organization of the CsmA-BChl a complex in the baseplate, we have studied isolated BChl c-less chlorosomes (so-called carotenosomes (3)) by solid-state NMR. In this sample, CsmA is the dominating protein so it can be investigated directly without further purification. The native state of the pigmentprotein complex was investigated by absorption and CD spectroscopy and used for molecular simulation studies. Results and Conclusions. Based on NMR and CD data, we present a model of the chlorosome baseplate in which CsmA is arranged symmetrically in long rows of dimers with BChl a molecules in a hydrophobic pocket between the dimers. Literature: (1) Pedersen, M.Ø.; Pham, L.; Steensgaard, D.B.; Miller, M. (2008). “A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a.” Biochemistry 47(5): 1435-1441. (2) Pedersen, M. Ø.; Underhaug, J.; Dittmer, J.; Miller, M.; Nielsen, N.C. (2008). “The three-dimensional structure of CsmA: A small antenna protein from the green sulfur bacterium Chlorobium tepidum.” FEBS Letters 582(19): 2869-2874. (3) Frigaard, N. U., Li, H.; Martinsson; P., Das, S.K.; Frank, H.A.; Aartsma, T.J.; Bryant, D.A. (2005). “Isolation and characterization of carotenosomes from a bacteriochlorophyll c-less mutant of Chlorobium tepidum.” Photosynthesis Research 86(1-2): 101-111. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts
OC-9.2 THE STRUCTURAL ORGANIZATION OF BACTERIOCHLOROPHYLLS IN CHLOROSOMES OF CHLOROBACULUM TEPIDUM. Donald A. Bryant, Swapna Ganapathy, Gert T. Oostergetel, Michael Reus, Aline Gomez Gomez Maqueo Chew, Alfred R. Holzwarth, Egbert J. Boekema, Huub J. M. de Groot. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. Introduction: Chlorosomes are remarkably efficient light-harvesting structures that allow green sulfur bacteria to live at light intensities at which no other phototrophs can survive. Each chlorosome can contain up to 250,000 bacteriochlorophyll (BChl) c/d/e molecules, and cells can contain up to 50 million or more BChl c/d/e molecules. Because these BChls are not bound to proteins and are self-organized, the structural basis for BChl organization has been of intense interest as models for biomimetic systems for solar energy applications. Methods: Chlorosomes were isolated from wild type or triple-mutant strain bchQ bchR bchU of Chlorobaculum tepidum (1) by standard procedures using 2M sodium isothiocyanate. Cryo-electron microscopy and negative stain microscopy were performed as previously described (2). Magic-angle spinning NMR studies were performed with uniformly 13C labeled chlorosomes as described (3). Results: The NMR assignments of the 13C and 1H signals of BChl d molecules in chlorosomes from the bchQRU mutant were obtained from two-dimensional 13C-13C and 1H-13C magic-angle spinning (MAS), solid-state NMR dipolar correlation spectra collected from 13C enriched chlorosome preparations. The observed interstack correlations provided convincing evidence that syn-anti monomer stacking is the basic building block in the bchQRU chlorosome structure. Additional structural constraints were obtained from Fourier transforms of cryoEM images of chlorosomes. Supramolecular models were constructed for different orientations of the stacks relative to the tube axis. With stacks running perpendicular to the tube axis along the circumference of a cylinder in rings, the simulated image and its Fourier transform reproduced the strong periodicity of 0.83 nm and the distinct striped appearance with a spacing of 2.1 nm that is observed in the cryoEM images of the chlorosomes of the bchQRU mutant. Wild-type chlorosomes also had BChl c nanotubes with the same 2.1-nm lamellar spacing. However, indicating that the stacks are differently oriented relative to the long-axis of the chlorosome, they also exhibited a weak layer line at 1.25 nm, which corresponds to the distance between repeating syn-anti pair units in the direction of the stacks. Conclusions: Computational integration of two different bio-imaging techniques, solid-state NMR and cryoEM, revealed a previously undescribed syn-anti stacking mode and showed how ligated BChl c and d self-assemble into coaxial cylinders to form concentric tubularshaped suprastructures. The close packing of BChls via π-π-stacking and the helical H-bonding networks present in both the mutant and wild-type chlorosomes forms the basis for ultrafast, long-distance transmission of excitation energy. The structural framework is robust and can accommodate extensive chemical heterogeneity in the BChl side chains for adaptive optimization of the light-harvesting functionality in low-light environments. In addition, syn-anti BChl stacks form sheets that allow for strong exciton overlap in two dimensions, which enables triplet exciton formation for efficient photoprotection.
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Final Program and Abstracts References Gomez Maqueo Chew, A, Frigaard, NU & Bryant, DA (2007) J Bacteriol 189: 6176-6184. Oostergetel, GT, et al. (2007) FEBS Lett 581: 5435-5439. Ganapathy, S, et al. (2009) Proc. Natl. Acad. Sci. USA, in review.
OC-9.3 MODIFICATION OF THE AMOUNT OF THYLAKOID MEMBRANES IN CYANOBACTERIA. Sawsan Hamad, Wim Vermaas. School of Life Sciences and the Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, AZ, USA. Introduction: The process of biosynthesis of photosynthetic membranes in oxygenic phototrophs remains elusive. The first protein that was linked directly to thylakoid membrane biosynthesis was Vipp1, a membrane-associated polypeptide. The vipp1 gene (sll0617) could not be fully deleted from the cyanobacterial genome. In order to determine the mechanism of Vipp1 function we have now overexpressed this protein in Synechocystis. Methods: A second vipp1 gene, identical in sequence to the native one, was placed under the control of the strong psbA3 promoter. In addition, a YFP or His tag sequence was fused to the second copy of the vipp1 gene as appropriate for localization or for identification of co-isolating proteins. Results: Overexpression of vipp1 resulted in a significantly enhanced content of thylakoid membranes and of vesicles inside cyanobacterial cells, and in an increased amount of photosystem II. Moreover, large inclusions with membranous content were frequently observed in Vipp1 overexpresser cells, often near the constriction site for the next cell division; these inclusions may be used to provide the building blocks for membrane biosynthesis. The Vipp1-YFP fusion protein in Vipp1-overexpressing Synechocystis was localized at specific locations near the periphery of the cell, again often near the constriction site for the next cell division, suggesting that Vipp1 is associated with the large membranous inclusions in the cell that may be used for thylakoid biogenesis. Slr1641, which is one of the ClpB chaperones in Synechocystis, was found to be the only protein that co-isolated with His-tagged, overexpressed Vipp1 from a cyanobacterial protein extract. Conclusion: Our results suggest Vipp1 to be critical for and to regulate thylakoid membrane biogenesis, and Vipp1 appears to be associated with inclusions at specific membrane biogenesis sites in the cell. This work also demonstrates the value of overexpression of native proteins if corresponding segregated deletion mutants cannot be obtained.
OC-9.4 PROGRESS IN ELUCIDATING THE STRUCTURAL BASIS OF FUNCTION IN THE CARBOXYSOME. Cheryl A. Kerfeld1,2, Michael Klein1, James N. Kinney1, Sarah C. Bagby3, Sabine Heinhorst4, Fei Cai4, Gordon Cannon4, Sallie W. Chisholm3. 1
US Department of Energy, Joint Genome Institute, Walnut Creek, CA; Department of Plant and Microbial Biology, University of California, Berkeley, CA; 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; 4Department of Biochemistry, University of Southern Mississippi, Hattiesburg, MS; USA. 2
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Introduction: Cyanobacteria and many chemoautotrophic microbes have evolved a special mechanism for dramatically enhancing CO2 fixation by encapsulating RuBisCO and carbonic anhydrase into subcellular microcompartments called carboxysomes. Methods: Bioinformatic and Structural Studies Results: Our recent structural studies revealed the basic architectural principles underlying construction of the carboxysome shell. Expansion of these studies into the Prochlorococcus model system has led to the identification and elucidation of the structures of a new type of building block for the carboxysome shell, one with the potential for gated transport across the shell. The carboxysome is the best studied bacterial microcompartment; bioinformatic surveys of genomic sequence reveal that the tendency to form bacterial microcompartments is widespread. For example, structurally related micrcompartments are found in Rhodopseudomas palustris and Rhodospirillum rubrum that appear to be involved in functions other than CO2 fixation. Conclusions: The carboxysome provides general insights into a surprisingly widespread strategy for biological compartmentalization; recent data provide an increasingly sophisticated view of its function and regulation.
OC-9.5 PROTEOMIC ANALYSIS OF THE DEVELOPING INTRACYTOPLASMIC MEMBRANE DURING CHROMATIC ADAPTATION IN RHODOBACTER SPHAEROIDES. Kamil Woronowicz, Robert A. Niederman. Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA. Introduction: Although the primary photochemical events and energy transduction processes in Rba. sphaeroides are well understood, much less is known about mechanisms involved in the formation of the participating light-harvesting (LH) and photochemical reaction center (RC) complexes, or the interactions of their apoproteins with numerous complex-specific and general assembly factors in the site-specific assembly of functional photosynthetic units in the growing intracytoplasmic membrane (ICM). Here, we report a detailed structural and functional proteomic analysis of the ICM assembly process during a downshift in a light intensity. Methods: Proteomic approaches have focused upon identifying proteins temporally expressed during ICM development and spatially localized in both membrane growth initiation sites, isolated as an upper pigmented band (UPB) after rate-zone sedimentation in sucrose density gradients, and in the mature ICM, isolated as the main band (chromatophores). The isolated membrane fractions have been further purified by a two-phase partitioning procedure and subjected to nondenaturing clear gel electrophoresis (CNE). Bands are excised from the gels and subjected to LC-MS/MS analysis. Results: CNE gives rise to four distinct bands: a top band containing the LH1 (B875)-RC core complex; a bottom band containing the LH2 (B800-850) peripheral antenna complex; and two bands of intermediate migration in which the LH2 and core complexes remain associated. Proteomic analysis yielded a large array of proteins associated with the gel bands and the resulting profiles can be correlated with the local organization of ICM as revealed by atomic force microscopy (AFM). The PuhA RC-H subunit was the most abundant protein, while surprisingly, the Puc2A LH2-α polypeptide, encoded by puc2BA operon, was the second most abundant protein with PucB LH2-β and Puc2B LH2-β comprising the other detectable August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes LH2 subunits. The spectral counts of several of the F1FO– ATP synthase subunits were unexpectedly high, given the low abundance of the ATP synthase in chromatophores and the inability to detect this coupling factor, as well as the more abundant cytochrome bc1 complex in AFM. Moreover, the high levels of ATP synthase and subunit IV of the bc1 complex found associated with the LH2-enriched fractions is consistent with their localization at ICM vesicle edges, thought to contain LH2-only domains, which are outside the flat ICM vesicle regions imaged by AFM. Significant levels of a preprotein translocase YidC homolog were found, along with lesser amounts of other general membrane assembly factors such as TatA, the twin-arg translocation system subunit, the SecY preprotein translocase subunit and the bacterial type 1 signal peptidase. In contrast, no complex-specific assembly factors were found in any of the bands. Forty-five proteins of unknown function were associated with the gel bands. One hypothetical protein, RSP6124, was observed in high abundance and is associated with the LH2 band, with its appearance preceding the increase of LH2 levels during chromatic adaptation. Conclusions: Proteomic analysis of bands containing the LH1-RC and LH2 complexes obtained by CNE of chromatophore from low light adapting cells, yielded a variety of proteins that could be correlated with their localization determined in AFM topographs and the possible roles of general membrane assembly factors that may be involved in the insertion of their nascent polypeptides into the ICM. (Supported by DOE Grant No. DE-FG02-08ER15957).
Final Program and Abstracts organisms. Inactivation of the Syn7002_A0564 gene in Synechococcus sp. PCC 7002 leads to about 40% reduction of chlorophyll content, which is due to impairment of PS I accumulation as demonstrated by measurements of the low temperature fluorescence emission spectroscopy and PS I analysis. However, no obvious difference could be observed in the composition of PS I, as shown in the results of SDS-PAGE analysis of isolated PS I complexes. Under photoautotrophic growth conditions the mutant exhibited a slower growth rate and higher sensitivity to high light. The stress response of the mutant is possibly related to an increase of ROS in cells. As measured by using a cell-permeant ROS probe and compared to the wild type, the ROS content is two-times higher in the mutant cells even when grown in reduced light. A 30-min treatment in high light led to a 6-fold increase in ROS content. As shown by RT-PCR analysis, expression of the sodB gene is significantly up-regulated in the mutant cells grown under the reduced light conditions. An enhanced photoautotrophic growth rate was observed for the mutant when cells were grown under semi-anaerobic growth conditions by bubbling with 99% N2 and 1% CO2. Conclusion: Our results demonstrate that the PDI-like protein Syn7002_A0564 is involved in biogenesis of PS I reaction center. It may function as a membrane-associated dithiol/disulfide oxidoreductase or may play an important role in maintaining the balance of oxidizing/reducing equivalents of thiol redox in PS I assembly and regulation, especially in facilitating cofactor insertion and maturation.
OC-9.6 BIOGENESIS OF PHOTOSYSTEM I: A NOVEL GENE ENCODING THE MEMBRANE-ASSOCIATED THIOREDOXIN PROTEIN PLAYS AN ESSENTIAL ROLE IN REGULATION AND ACCUMULATION OF PS I.
OC-10.1
Gaozhong Shen1, Fei Gan1, John H. Golbeck1,2, Donald A. Bryant1.
Judy Lieman-Hurwitz1, Maya Haimovich1, Gali Shalev-Malul1, Ai Ishii2, Yukako Hihara2, Ariel Gaathon3, Mario Lebendiker4, Aaron Kaplan1.
1
2
Department of Biochemistry and Molecular Biology; Department of Chemistry; The Pennsylvania State University, University Park, PA, USA. Introduction: The biogenesis of PS is a complex, multi-step process in which many factors are involved, mostly in assembly of the protein component and insertion of cofactors. In recent years, advances in genome sequencing of several cyanobacteria (including Synechococccus sp. PCC 7002) and functional genomics of photosynthetic genes have provided much new information for exploring assembly and regulation of the PS I reaction center. In this study, we describe a novel gene (Syn7002_A0564) encoding a protein disulfide isomerase (PDI)-like protein that is found be involved in assembly and/or regulation of PS I Methods: DNA and protein sequence analyses were performed with MacVector software. Homologous sequences of the DPI-like Syn7002_A0564 were retrieved from the IMG and Cyanobase databases. A Syn7002_A0564 mutant was generated through insertion of a kanamycin-resistance cartridge at the unique EcoRV site within Syn7002_A0564 coding sequence. Growth rates of the wild type and mutant were measured at different conditions. The low-temperature fluorescence emission spectroscopy was applied to compare changes of PS I and PS II complexes in cells. PS I complexes were isolated for analysis of subunit composition. The cell-permeant indicator CMH2DCFDA was used for probing intracellular reactive oxygen species (ROS). Results: Sequence analyses indicate that Syn7002_A0564 encodes a PDI-like protein that is composed of two thioredoxin-like domains and that it is predicted to be membrane-associated. Homologs of Syn7002_A0564 can be found in all oxygenic photosynthetic
August 9 to 14, 2009 • Montréal, QC, Canada
A CYANOBACTERIAL ABRB-LIKE PROTEIN AFFECTS THE APPARENT PHOTOSYNTHETIC AFFINITY FOR CO2 BY MODULATING LOW-CO2-INDUCED GENE EXPRESSION.
1 Dept. of Plant and Environmental Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; 2 Dept. of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan; 3Bletterman Laboratory, Interdepartmental Equipment Unit, Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem Israel; 4The Wolfson Centre, Hebrew University of Jerusalem, Jerusalem, Israel.
In Synechocystis sp. strain PCC 6803, over 450 genes are up-regulated following transfer of the cells from a high (1-5% CO2 in air, HC) to a low level of CO2 (as in air or lower, LC). This includes sbtA, ndhF3 and cmpA involved in inorganic carbon (Ci) uptake. Earlier studies implicated NdhR in the regulation of LC-induced genes but there are indications that additional components are involved. Following extraction of proteins from cells grown under HC and (NH4)2SO4 fractionation, we have identified LexA and two AbrB-like proteins, Sll0359 and Sll0822 that bind to a fragment of the sbtA promoter. Using extracts prepared from LC-grown cells, Sll0822 did not bind to the sbtA promoter despite its presence in the cells, suggesting that it may serve as a repressor of LC-induced genes. This is supported by the fact that sbtA, ndhF3, and cmpA normally expressed only under LC in the wild type are transcribed under both HC and LC in a ∆sll0822 mutant. When grown under HC this mutant exhibits an elevated apparent photosynthetic affinity to Ci, typically observed in the wild type only under LC. Clearly, expression of genes essential for Ci uptake was sufficient to raise the apparent photosynthetic affinity for external Ci.
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OC-10.2
OC-10.3
THE EFFECTS OF TEMPERATURE AND OXYGEN ON NITROGENASE ACTIVITY IN THE THERMOPHILIC CYANOBACTERIUM FISCHERELLA SP.
PHOSPHATE SCAVENGING IN AN UNPREDICTABLE ENVIRONMENT, HOW DO CYANOBACTERIA MEET THE CHALLENGE?
Lucas J. Stal, Department of Marine Microbiology, Netherlands Institute of Ecology, Yerseke, The Netherlands.
Frances D. Pitt, David J. Scanlan.
Introduction: Nitrogenase – the enzyme complex responsible for the fixation of dinitrogen in diazotrophic prokaryotes - is irreversibly inactivated by oxygen. Cyanobacteria evolved strategies that allowed the coexistence of the two incompatible processes of dinitrogen fixation and oxygen-evolving photosynthesis. Heterocysts are differentiated cells that contain nitrogenase but have lost the oxygenic photosystem. The heterocyst possesses a thick glycolipid cell envelope that serves as a gas diffusion barrier. Any oxygen that enters the heterocyst is respired thereby guaranteeing an anoxic interior of the heterocyst. However, a too efficient glycolipid cell envelope would also limit dinitrogen from entering the heterocyst. Hence, the gas diffusion barrier must be a trade-off between the entrée of dinitrogen and a limitation of the entrée of oxygen. Ideally, the amount of gas that diffuses into the heterocyst should be as high as possible so that the respiratory system can still keep it to sufficient low intracellular concentrations. This would require a dynamic gas diffusion system of the heterocyst in order to adequately respond to changes in the environmental conditions. Methods: I have selected the thermophilic heterocystous cyanobacterium Fischerella sp. to investigate the question of the dynamic response of gas diffusion in the heterocyst by measuring nitrogenase activity by an on-line, near real-time acetylene reduction assay (ARA) using a laser photoacoustic ethylene detector. Fischerella sp. was grown under continuous light at 27oC. Results: I measured ARA at temperatures from 18-39oC at 3oC intervals. At each temperature light response curves of ARA were recorded. The light response curves were fitted to the rectangular hyperbola function yielding the following parameters. Nd and Nm were the dark- and maximum light-dependent acetylene reduction rates, respectively and Ntot was the sum of these and represented the actual nitrogenase activity measured at light saturation. The light saturation constant Ik equals Km in Michaelis-Menten kinetics and is the light intensity at which 0.5Nm. Ik equals Nm/α. The light affinity constant α is the initial slope of the light response curve at zero light. I found that nitrogenase activity increased with temperature with a Q10 of 6, three times higher than what one would expect from enzyme kinetics. The dimensionless factor Ntot/Nd equaled ~2 and was constant over the temperature range. Nd increased with temperature and this is only possible when the amount of oxygen that enters the heterocyst increased. This would indicate a possible dynamic regulation of the oxygen (gas) diffusion into the heterocyst. In order to investigate this further, I measured ARA at a range of oxygen concentrations from 090%. I found that Ntot remained constant in the range of 5-30% oxygen. This was the result of an increasing Nd and simultaneously decreasing Nm. Above 30% oxygen acetylene reduction decreased, obviously because an increasing part of the reducing equivalents were necessary for respiration and nitrogenase activity became electron limited. Conclusions: I conclude that the heterocyst did not dynamically regulate the entrée of oxygen at a constant temperature but that it happened as a function of temperature. The changes of the photosynthetic parameters Ik or α were fully attributed to changes in Nm and therefore photosynthesis itself did not affect dinitrogen fixation. 64
Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom. Introduction: Cyanobacteria are ubiquitous in aquatic and marine environments where they play a key role as primary producers. Their growth is limited by the availability of nutrients, particularly phosphorus (P), and many species persist and flourish in environments with an unpredictable and constantly fluctuating supply of P. Whilst genome-wide analyses have revealed many details of the P uptake and regulatory machinery there are still very few reports that demonstrate the functional significance of individual, and apparently redundant, components of the cyanobacterial Pho regulon. For example, Synechocystis sp. PCC6803 encodes duplicate ABC transporters (i.e. two PstCAB operons) with at least three associated periplasmic binding proteins (two of the PstS type and one of the SphX type). This contrasts with virtually all other known bacteria which encode a single ABC transport system. Current research into the specific functional attributes of the multiple phosphate transporter arrangement in Synechocystis sp. PCC6803 aims to elucidate their interplay with other proteins involved in phosphate acquisition and regulation. By answering the question of evolutionary adaptation by biochemical or regulatory means, we aim to reveal new strategies for the acquisition of phosphate during conditions of environmental stress. Methods: In order to assess the role of multiple ABC transporter elements in Synechocystis sp. PCC6803 we created gene deletions of the two pstCAB operons as well as single knock-outs of the associated pstS / sphX genes. Gene expression profiles were generated using real time quantitative PCR to compare mutants with WT under phosphate replete and deplete conditions. Phosphate uptake assays using radiolabelled 32P were then performed to compare the kinetics of phosphate uptake in the various mutants. Results: Disruption of one of the PBPs in Synechocystis sp. PCC6803, pstS1 (sll0680) led to an impairment in the expression of specific genes of the pho regulon during P-deplete growth. In addition, the mutant was unable to alter the expression of various genes normally induced in WT in response to changes in external P concentration. This phenotype was not observed when the remaining phosphate binding proteins were disrupted suggesting that only one of the two transporters is involved in sensing and regulation of the pho regulon. Preliminary P uptake assays suggest potential differences in the binding affinities of the two transporters. Further uptake work using specific gene knock out mutants should clarify their contribution to phosphate uptake under different growth conditions. Conclusion: Differential expression of P binding protein genes in WT Synechocystis sp. PCC6803 during P replete and deplete conditions suggests that each ABC transporter performs a distinct functional role. This conclusion is supported by the phenotype of the pstS1 deletion mutant which displays impaired ability to respond to changes in external P. Preliminary results from P uptake assays go further and suggest that there may be differences in the P binding protein’s affinity for P, thus leading to the development of a novel strategy for P uptake in a freshwater cyanobacterium.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
OC-10.4 REGULATION OF GLUTAMINE SYNTHETASE ACTIVITY IN CYANOBACTERIA. IDENTIFICATION OF THE REGIONS INVOLVED IN THE PROTEIN-PROTEIN INTERACTION BETWEEN GS AND IFS. Lorena Saelices, Carla V. Galmozzi, M. Isabel Muro-Pastor, Francisco J. Florencio. Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de SevillaCSIC, Sevilla, Spain. Glutamine synthetase plays a central role in the regulation of nitrogen assimilation in cyanobacteria via the control of the carbon/nitrogen metabolic flow. Thus, this enzyme is tuned up depending on the nitrogen source and the carbon disponibility. The intracellular concentration of 2-oxoglutarate defines the carbon/nitrogen status of the cell and it is the key metabolite in the control of the GS activity, being high level of GS activity when 2-oxoglutarate concentration is high and low GS activity at low 2-oxoglutarate level. The regulation of cyanobacterial GS activity occurs by a original system consisting in the protein-protein interaction between the GS protein and two inactivating factors (IFs) named IF7 and IF17. This interaction provokes the inactivation of the enzyme when the carbon/nitrogen ratio is low, for example using ammonium as nitrogen source, but if it is nitrate, the enzyme is active and the IFs are absents. The expression of the IFs are controlled at the level of transcription by the global nitrogen regulator NtcA. Here, we have investigated the molecular interaction between GS and the IFs, we try to know which are the regions in both GS and IFs implicated in the protein-protein interaction by using two different approaches. The first one was to construct different GS chimeras using Synechocystis and Anabaena GSs, it is worth to note that both cyanobacteria possess the same system to inactivate GS, but the IF are different in the way that IF7 from Anabaena is able to inactivate both GS in vitro, but IF7 or IF17 are unable to inactivate the Anabaena GS. Since IFs from Synechocystis are unable to inactivate Anabaena GS, we expect to find a region responsible for the interaction in Synechocystis GS. The second method was to construct different site-directed mutants of IFs from Synechocystis in order to study their capacity to inactivate or not GS. We will present results indicating that the region required for the interaction resides in the carboxy-terminal part of the GS. We also found that a change of single residue is this region is sufficient to disrupt the interaction between GS and IFs in Synechocystis. With respect to the IFs interaction studies, we have found that at least 3 basic amino acid residues are essential to maintain the interaction with the GS and that these residues are localized in the central part of these proteins. The relevance of these results will be discussed in relation to a possible model that explain how these GS-IFs interaction promotes the GS inactivation.
OC-10.5 BIOSYNTHESIS OF UN-NATURAL BILIPROTEINS. Richard M. Alvey1, Avijit Biswas2, Wendy M. Schluchter2, Donald A. Bryant1. 1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2 Department of Biological Sciences, University of New Orleans, New Orleans, LA; USA. Introduction: Cyanobacteria employ a number of different tetrapyrrole/biliprotein combinations both as accessory pigments to augment their ability to utilize wavelengths of light which are not efficiently captured by chlorophyll and as sensors to perceive and August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts respond to light conditions in their environment. Up to four different linear tetrapyrroles may be incorporated into various phycobiliproteins: phycocyanobilin, phycoerythrobilin, phycoviolobilin and phycourobilin. When bound to its cognate protein, each of these bilins confers unique spectral properties, which enhance the ability of the cyanobacteria to exploit the light available in its environment. On the other hand, plants are only known to utilize biliproteins known as the phytochromes as sensors. Plant phytochromes uniquely incorporate the bilin, phytochromobilin. In the last several years the components involved in the assembly of biliproteins have been characterized. This study was conducted to determine the factors involved in maintaining the specificity between phycobiliproteins and their cognate chromophores and to determine the effects on a cyanobacterium when chromophores are misincorporated into a noncognate apoprotein. Methods: A dual-plasmid E. coli expression system was used to study the attachment of chromophores to phycobiliproteins in isolation. Use of this heterologous system allowed the co-expression of the minimal components necessary to assemble a holophycobiliprotein and made it possible to assay the effects of substitutions of various components, specifically, bilin lyases and bilin biosynthetic enzymes, on the final holophycobiliprotein made in E. coli. In addition, we studied the effects of potential mis-pairings of chromophores and apoproteins inside Synechococcus sp. PCC 7002 using site-specific integrations into the genome. Results: With various lyase and chromophore synthesis combinations, CpcA from Synechococcus sp. PCC 6803 can bind six different linear tetrapyrrole chromophores, including the plant-specific chromophore phytochromobilin and an A-ring isomerized version of phytochromobilin not known to occur naturally. Additionally, the heterologous over-expression of the phycoerythrobilin or phytochromobilin biosynthetic enzymes in Synechococcus sp. PCC 7002, a cyanobacterium that utilizes only phycocyanobilin as a chromophore, results in strains with readily apparent altered absorbance profiles. Conclusions: Different chromophore/apoprotein/lyase combinations can be used to yield several different colored versions of a single chromophore binding protein. The specificity of attachment of a linear tetrapyrrole chromophore to its cognate apoprotein is more general than once believed. Although in nature the misincorporation of chromophores is believed to occur only rarely, if at all, it can be made to occur quite readily in genetically modified E. coli and cyanobacteria. These studies shed new light on the assembly and evolution of phycobiliproteins, and the results have implications for the production of recombinant proteins with properties not found in natural proteins.
OC-10.6 ACCUMULATION OF TREHALOSE IN RESPONSE TO DESICCATION AND CONTROL OF TREHALASE IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE. Toshio Sakamoto, Takayuki Yoshida, Hiromi Arima. Graduate School of Natural Science and Technology, Kanazawa University, Ishikawa, Japan. Introduction: Desiccated organisms have little to no metabolic activity and rapidly resume metabolism upon rehydration. This phenomenon is termed “anhydrobiosis”. The terrestrial cyanobacterium Nostoc commune sustains the capacity for cell growth for over 100 years in a
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Final Program and Abstracts desiccated state; thus, N. commune is considered an anhydrobiotic microorganism with oxygenic photosynthetic capabilities. Since N. commune does not differentiate into akinetes (spores), the mechanisms of its extreme desiccation tolerance most likely involve multiple processes. Anhydrobiotic organisms accumulate trehalose, which protects biological membranes and proteins against the destructive effects of water removal by replacing the primary waters of hydration, as well as through the formation of amorphous glasses (vitrification). In this study, we investigated changes in photosynthetic activity and trehalose levels in the terrestrial cyanobacterium Nostoc commune responding to desiccation and the biochemical properties of the enzymes involved in trehalose metabolism to elucidate the mechanisms of trehalose accumulation. Methods: Trehalose was determined by gas-liquid chromatography (GLC) and by high performance liquid chromatography (HPLC). Photosynthetic O2 evolution during desiccation was measured using a gas-phase Clark-type oxygen electrode. The three genes for trehalose metabolism, treZ (maltooligosyltrehalose trehalohydrolase, Mth), treY (maltooligosyltrehalose synthase, Mts), and treH (trehalase), were found as a gene cluster, and the mRNAs for these genes were detected by RT-PCR. The activity of trehalose synthesis, consisting of Mts and Mth, was measured by the production of trehalose from soluble starch. The treH gene was heterologously expressed in E. coli cells in an active form with a molecular mass of 52 kDa and the activity of trehalase was measured by the production of glucose from trehalose.
13th International Symposium on Phototrophic Prokaryotes chromophore that typically absorbs 446 nm light, although there is some variation amongst species. The Ppr from the purple photosynthetic bacterium, Rhodospirillum centenum is a chimera of PYP, bacteriophytochrome (Bph), and histidine kinase. The function of Ppr is to regulate a polyketide synthase. When both photoreceptors are activated, the PYP domain appears to hasten the recovery of Bph to the dark state. However, the cause of this phenomenon is unclear. We have now analyzed the Ppr recovery kinetics and kinase activity in greater detail under three different light conditions. Methods: Ppr was produced in E. coli using a two plasmid based system. Purification was performed on a Q-sepharose column followed by a TALON His-tag column. Absorption spectra and kinetics were obtained using a Cary 300 spectrophotometer. Recovery kinetics were measured for up to 300 minutes and the data were fit using Sigmaplot 8.0 (Systat Software). Autophosphorylation was measured by adding 1mM ATP (containing ~ 2.5 μCi P32 ATP) and keeping the sample illuminated for 30 min. Six time points were taken and analyzed on SDS-page gels. The resulting autoradiogram was digitized using a Typhoon Imager (GE Healthcare) and intensities were quantified by volume using ImageQuant TL.
Results: As the water content decreased in N. commune colonies during desiccation, photosynthetic O2-evolving activity decreased and no activity was detected in desiccated colonies. No detectable trehalose was found in fully hydrated colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air-dried colonies. Moreover, NaCl treatment also induced trehalose accumulation and the levels of trehalose induced were equivalent to that in the desiccated colonies. The transcripts for treZ, treY and treH genes were detectable at similar levels during desiccation. Trehalase activity was strongly inhibited in the presence of 10 mM NaCl while trehalose synthesis activity remained active in the presence of salt.
Results: Bph normally absorbs 700 nm light in the Pr state and when illuminated with red light, it is red-shifted in most species to the photoreversable Pfr state absorbing near 750 nm. However, the Bph domain within Ppr bleaches upon red-light illumination to a form which weakly absorbs near 650 nm and cannot be photoreversed by near red light. Dark reversion normally takes from 20 to 60 minutes. However, recovery of the Bph 701 nm absorbance is accelerated in part to about 1 minute following white light illumination. White and blue light bleaches PYP, which initially recovers with kinetics similar to that of the PYP construct, however, only about half the absorbance at 434 nm is recovered in ~ 4 minutes, the remainder takes several days to recover due to formation of an intramolecular complex with Bph whose absorption is red-shifted. The Bph within the metastable complex can no longer be bleached by red light. The PYP remains locked in its bleached state which absorbs near 355 nm, but it can be photoreversed by UV light, in which case the Bph also returns to its initial 701 nm dark state. Autokinase activity is turned on by both red and white light, but is inactivated when photoreversed by UV light.
Conclusions: Trehalose accumulation and cessation of photosynthesis occurs during desiccation. Since transcriptional regulation of the gene cluster for trehalose synthesis and trehalose hydrolysis cannot fully explain trehalose accumulation in response to desiccation, posttranscriptional regulation most likely controls the cellular level of trehalose. Under water-stress conditions characterized by increased cellular solute concentrations, the rate of trehalose production exceeds that of trehalose hydrolysis because of the inhibition of trehalase. Control of trehalase plays an important role in trehalose accumulation under conditions of extreme desiccation.
Conclusions: The kinase activity of Bph is normally turned off in the dark and activated in red light. It is switched off again by far-red light with simultaneous return of the 700 nm absorbance. Because the Bph within Ppr does not form the Pfr state but is instead bleached by red light, it cannot be photoreversed in the usual way. Thus, the role of PYP is threefold, to block activation of Bph in blue light through formation of the metastable complex, to accelerate recovery of Bph absorbance in white light, and to photoreverse the effects of blue and white light by subsequent illumination in the UV. We therefore postulate that Ppr functions as a UV-red light sensor.
OC-11.1
OC-11.2
FUNCTION OF THE PHOTOACTIVE YELLOW PROTEIN DOMAIN IN RHODOSPIRILLUM CENTENUM PPR. J.A. Kyndt, T.E. Meyer, M.A. Cusanovich.
SHORT-TERM LIGHT ADAPTATION STRATEGIES OF PHYCOBILISOME-CONTAINING PHOTOSYNTHETICS, CYANOBACTERIA AND RED ALGAE.
Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
Igor Stadnichuk.
Introduction: Ppr is unique in that it has a blue light photoreceptor (PYP) N-terminally fused to a bacteriophytochrome (Bph) domain. Photoactive Yellow Protein (PYP) is related to the PAS domain superfamily of signalling proteins. It generally occurs as a freestanding cytoplasmic protein and has been found in 18 species of bacteria of diverse lifestyles. It contains a p-hydroxy-cinnamic acid
A.N.Bakh Institute of Biochemistry, Russian academy of Sciences, Moscow, Russia.
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Phycobilisome (PBS)-containing photosynthetics, the cyanobacteria and red algae, have evolved several short-term reversible adaptation mechanisms to survive under different light quality and quantity conditions. Realized on a timescale of minutes, all mechanisms do not August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes involve alterations in gene expression or photoinhibition of the pigment apparatus and are revealed as non-photochemical fluorescence quenching (NPQ) that includes alterations of the fluorescence emission from PBS. The appropriate phenomena changing rapidly the cross-sections of the photosystems I (PSI) and II (PSII) are: state transitions realized in cyanobacteria and red algae when light is normal to growth (≤ 150 microE m-2s-1); orange carotenprotein (OCP)-induced PBS quenching realized in cyanobacteria for relatively high light intensities (≥ 500 microE m-2s-1); induced by weak (~10 microE m-2s-1) far-red light increase of PBS emission, in the red algae. State transitions are well-known phenomenon of the adaptation of pigment apparatus to unbalanced light absorption by PSI and PSII first described 40 years ago ([1] and ref. there). Contrary to elucidation of the effect in green plants, the cause of state transitions in cyanobacteria and red algae is still in debate and different models have been proposed. In the spillover model, the excitation energy is transferred directly from PSII to PSI core chlorophylls. The partial dissociation of PBS from PSII and its reassociation without docking to PSI is the main event of the detachment model. In the mobile model, the PBS could reversibly migrate in the plane of the thylakoid membrane from the surface of PSII to PSI. It could be possible that none of the existed models would stay correct after additional investigations [2]. In cyanobacteria, most probably, the monomerisation of the PSII dimers is the main event of transition to state 1 [3]. In 2004, due to the registered action spectrum of the process, we have described [4] an additional to state transitions short-term mechanism of energy dissipation in PBS of cyanobacteria involving a carotenoid identified as OCP [5]. Light-activated OCP was found to cause reversible NPQ via interception the energy transfer from PBS to chlorophyll a of PSII as well as of PSI cores [2, 5]. Recently [5], we have demonstrated quite new display of short-term light adaptation of the pigment apparatus in the extremophilic microalga, Galdieria sulphuraria. Comparing to the dark-adapted cells, weak far-red light primarily absorbed by photosystem I (PSI) causes an increase in the amplitude of the PBS low-temperature fluorescence emission. Contrary to emission, the PBS peak in fluorescence excitation spectrum of PSI increases simultaneously with PBS quenching while excitation spectrum of PSII stays invariable. Therefore, in G. sulphuraria, the registered changes of 77 K fluorescence emission correspond to the reversible detachment of the PBS from the core complexes of PSI. The data indicates the adaptive decrease of cross-section and the diminishing of the PSI activity when it is not accompanied by PSII function. This kind of NPQ is not known for cyanobacteria where PBS docking to PSI has to be faster than the docking to monomeric PSI in the red algae. References 1. Murata N. (2009) Photosynth. Res. 99, 155-160. 2. Stadnichuk I. et al. (2009) Photosynth. Res. 99, 227-241. 3. Stadnichuk I. et al. (2009) FEBS Lett. in press. 4. Rakhimberdieva M. et al. (2004) FEBS Lett. 574, 85-88. 5. Wilson A. et al. (2006) Plant Cell 18, 992-107.
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Final Program and Abstracts
OC-11.3 REGULATORY RNAS IN CYANOBACTERIA AND BEYOND. W.R. Hess1, J. Georg1, I. Scholz1, J. Mitschke1, B. Voss1, C. Steglich1, J. Vogel2, A. Wilde3. 1
Freiburg Initiative in Systems Biology and Faculty of Biology, University Freiburg; 2Max-Planck-Institute for Infection Biology Berlin; 3 Justus-Liebig University Giessen, Institute of Microbiology and Molecular Biology; Germany. Regulatory RNA has been discovered in all three domains of life. However, transcriptional units that give rise to non-coding RNAs (ncRNAs) or antisense RNAs (asRNAs) are not identified during normal genome annotation, and in phototrophic bacteria only a small number of ncRNAs has been described. We have used 5 different methods for the identification of novel non-coding and antisense RNAs in various cyanobacteria, with a focus on model cyanobacteria (1-4). The reliability of computational predictions for the detection of ncRNAs and asRNAs in Synechocystis sp. PCC 6803 and for marine Synechococcus species was scrutinized in microarrays, complemented by deep sequencing of the small RNA population and validated by an extended set of Northern hybridizations and 5’ RACE experiments. The total number of asRNAs in Synechocystis sp. PCC 6803 alone is more than 350 and of classical trans-acting ncRNAs about 100. Thus, regulatory RNAs play a much more important role in this model organism and probably also in most other bacteria. Although functions are still unknown for the vast majority of regulatory RNAs, we will provide functional evidence for cyanobacterial ncRNAs acting against invading phages, controlling the expression of key photosynthetic genes, or serving as signals for RNA maturation, processing and degradation. References (1) Axmann et al. (2005), Genome Biol. 6, R73: 1-16. (2) Voss et al. (2007), BMC Genomics 8:375. (3) Steglich et al. (2008), PLoS Genetics 4 (8) e10000173. (4) Voss et al. (2009), BMC Genomics 10, 123.
OC-11.4 FUNCTIONAL SMALL RNAS IN THE MARINE CYANOBACTERIUM PROCHLOROCOCCUS: WHAT CAN WE LEARN? C Steglich1, M Futschik2, Cynthia Sharma3, Jan Mitschke1, Joerg Vogel3, Wolfgang Hess1. 1
University of Freiburg, Freiburg, Germany; 2University of Algarve, Faro, Portugal; 3Max Planck Institute for Infection Biology, Berlin, Germany. Small non-coding RNAs (ncRNAs) are functional RNA molecules, mostly without a protein-coding function, that have been found in all domains of life. In bacteria these functional RNA molecules range in size between 50 – 400 nt and frequently play a crucial role in regulatory networks particularly in response to environmental stress. ncRNAs are also known to control plasmid and viral replication, bacterial virulence and quorum sensing, while the function of others has remained unknown. A detailed survey for ncRNA was performed in the ecologically important marine cyanobacterium Prochlorococcus, which it is the most abundant phototroph in the vast nutrient-poor areas of the ocean. Genome analyses of several isolates revealed compact streamlined genomes that contain a core set of about 1,200 genes augmented with a variable number of ‘flexible’ genes. All sequenced Prochlorococcus isolates possess only a small number of 67
Final Program and Abstracts genes coding for regulatory proteins. Applying different methods for the detection of ncRNAs in Prochlorococcus MED4, the total number of ncRNAs, which have been experimentally confirmed by two different methods, rises to 26 (including Yfr1-6, 6S RNA, SRP RNA, tmRNA, RNase P RNA). Thus, unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNA genes are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism. Of the 12 completely sequenced Prochlorococcus strains only 2 possess the RNA chaperone Hfq. A 454 deep sequencing approach was conducted to compare the number of regulatory RNAs between the high light-adapted hfq-lacking strain MED4 and MIT9313 who is adapted to low light and contains an hfq homolog.
OC-11.5 REGULATION BETWEEN PHOTOAUTOTROPHIC AND PHOTOMIXOTROPHIC GROWTH AND ITS DEPENDENCE ON THE CO2 LEVEL IN SYNECHOCYSTIS PCC 6803. Maya Haimovich1, Shira Kahlon1, Yukako Hihara2, Judy LiemanHurwitz1, Aaron Kaplan1. 1 Plant and Environmental Sciences, The Hebrew University of Jerusalem; Israel; 2Department of Biochemistry and Molecular Biology, Saitama University, Japan.
Cyanobacteria represents a unique case were carbohydrates formation in photosynthesis and their breakdown in respiration occurs in the same compartment at the same time, unlike eukaryotes where the catabolic and anabolic activities are spatially separated. The mechanisms whereby these activities are regulated in the context of the environment is poorly understood. Glucose sensitive mutants of the cyanobacterium Synechocystis sp. PCC 6803 were isolated. Many of them are far more resistant to the presence of glucose when exposed to low level of CO2. Analyses of the expression of certain CO2-dependent genes in the wild type and the mutants as affected by the ambient CO2 concentration and presence of glucose revealed an intriguing but complex regulation between these processes. Our results shows for the first time the interconnectivity between CO2 availability and organic carbon supply and provide some insight into the molecular mechanisms involved.
OC-11.6 CELL WALL ULTRASTRUCTURE AND GLIDING MOTILITY IN OSCILLATORIA. Toby Tatsuyama-Kurk, Dan Whalley, Simon Connell, Neil Thomson, Dave Adams. University of Leeds, Leeds, West Yorkshire, United Kingdom. Introduction: When attached to a surface many filamentous cyanobacteria move by a process known as gliding. The mechanism of gliding is unknown, although two main theories, slime extrusion and surface waves, have been proposed to explain the generation of thrust. In the first, filaments are pushed along by the extrusion of slime from rows of junctional pores that are known to encircle each cell septum. The second theory proposes that gliding results from the rhythmical progression of waves along the cell surface and their interaction with the substrate to which the filament is attached. It has been suggested that such waves are created by protein fibrils beneath the outer membrane. We have been using Atomic Force Microscopy 68
13th International Symposium on Phototrophic Prokaryotes (AFM) and Field Emission Gun Scanning Electron Microscopy (FEGSEM) to study the cell walls and cell surfaces of Oscillatoria strains to identify structures that may be associated with motility. Methods: For FEGSEM, samples were fixed in 2.5% (vol/vol) glutaraldehyde and post-fixed with 1.0% (wt/vol) osmium tetroxide. Samples were then dehydrated in a graded ethanol series, criticalpoint dried and mounted on aluminum pin stubs. Finally, samples were coated with either gold or platinum-palladium and examined with a LEO 1530 series FEGSEM instrument operating at 3 kV. For AFM imaging of cells under liquid, cyanobacterial filaments were first immobilized by partial embedding in dental wax which had been softened by warming in an oven. Results: AFM scanning under liquid of filaments of Oscillatoria sp. strain A2 immobilised in wax confirmed the presence of an array of parallel 40 nm wide corrugations on the surface. These surface corrugations are a consequence of the presence of protein fibrils beneath the outer membrane. By increasing pressure on the AFM tip it was possible to remove the fibrillar array from a defined area of the cell surface. By contrast, FEGSEM images of the very large filaments of O. princeps revealed an irregular array of corrugations on the surface. These proved difficult to visualise by AFM under liquid, possibly because of a slime layer obscuring the finer surface details. However, the O. princeps surface is covered by raised structures which correspond in spacing to large pores, approximately 150 nm in diameter, seen in FEGSEM images to penetrate the extremely thick peptidoglycan layer. The function of these pores seems to be to bring the cytoplasmic and outer membranes into closer proximity. However, they may also have a function in slime production. The significance of these cell wall and surface features, in terms of the mechanism of gliding motility, will be discussed. Conclusions: It is possible to immobilise cyanobacterial filaments in dental wax in a way that preserves the filaments in a fully hydrated state and allows AFM scanning under liquid. The AFM tip can be used to remove surface layers, such as the slime layer, outer membrane and fibrillar array, and by repeat scans of the same area, it is possible to visualise surface changes. We hope in the near future to employ these techniques to visualise the putative surface waves that drive gliding motility.
OC-12.1 GLOBAL TRANSCRIPTIONAL RESPONSE TO LOW OXYGEN CONDITIONS AND THE ROLE OF THE HISTIDINE KINASE, HIK31, IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Tina C. Summerfield1, Louis A. Sherman2. 1 Botany Department, University of Otago, PO Box 56, Dunedin, New Zealand; 2Purdue University, Department of Biological Sciences, West Lafayette, IN, USA.
Introduction: In habitats such as hot springs, soils (e.g. rice paddies or estuarine mud), and eutrophic lakes, cyanobacteria experience low oxygen conditions. These conditions are associated with the ability of cyanobacteria to produce ethanol and H2 indicating decreased O2 may play an important role in this energy production. Methods: Using DNA microarrays, we examined gene expression in Synechocystis sp. PCC 6803 on transition from aerobic growth to low oxygen conditions. We observed up-regulation of the gene encoding the histidine kinase Hik31 under low oxygen conditions. To examine the role of this histidine kinase we compared gene expression in the wild type and a ∆Hik31 strain that lacked Hik31. Additionally, we measured growth, oxygen evolution and whole cell spectra during August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes incubation under low oxygen conditions. Results: In the wild type strain, expression of >13% of chromosomal genes were altered within 1 hour under low oxygen conditions. Similar numbers of genes showed increased and decreased transcript abundance. This included down-regulation of genes encoding photosynthesis proteins, regulatory proteins and chaperones. Upregulated genes included the hox genes encoding the bidirectional hydrogenase and the LexA-like transcription regulator (sll1626) that binds the hox promoter. Genes encoding proteins involved in transport, regulation and energy metabolism were up-regulated under low oxygen conditions, including an operon encoding Hik31 and response regulator (Rre34). The transition from aerobic to low oxygen conditions resulted in differential regulation of ~8% of chromosomal genes in the ∆Hik31 strain. The majority of these genes (~90%) showed increased transcript abundance under low oxygen conditions. Almost half these genes, including the hox operon, were up-regulated in the wild type under the same conditions. Genes up-regulated in the ∆Hik31 strain but not the wild type under low oxygen conditions encoded proteins involved in photosynthesis, such as phycobilisome components, and ribosomal proteins. Consistent with this, extended incubation under low oxygen conditions resulted in cells with increased phycobilisome content relative to chlorophyll content in the ∆Hik31 strain but not the wild type. Furthermore, unlike the wild type, the ∆Hik31 strain did not down-regulate genes encoding chaperones and ATP synthase. Conclusions. These data suggest that Synechocystis sp. PCC 6803 responds to low oxygen conditions by down-regulating genes involved in key processes such as photosynthesis and translation and that the histidine kinase, Hik31, plays a role in this negative regulation.
OC-12.2 INCREASING HYDROGEN PRODUCTION BY PERTURBING FERMENTATIVE METABOLISM IN THE MARINE, UNICELLULAR CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002. Kelsey McNeely, Yu Xu, Nicholas Bennette, Donald A. Bryant, G. Charles Dismukes. Princeton University, Princeton, NJ, USA. Introduction: Certain strains of cyanobacteria contain a bidirectional hydrogenase and make hydrogen as a byproduct of sugar catabolism during fermentation via the reaction: NADH + H+ NAD+ + H2. In the cyanobacterium Synechococcus 7002, nitrate reduction and lactate production are two pathways that utilize reductant during fermentation and therefore compete with hydrogenase. These pathways have been eliminated in this study. Lactate dehydrogenase utilizes one equivalent of NADH to reduce pyruvate to lactate during fermentation. Nitrate metabolism is largely unexplored during fermentative metabolism, but is expected to be analogous to photoautotrophic nitrogen metabolism in which nitrate is reduced to nitrate with 2 electrons from ferredoxin. Nitrite is further reduced to ammonium with 6 electrons. Methods: Cultures were grown photoautotrophically to stationary phase in medium A sparged with 2% CO2. Cultures were prepared for fermentation experiments by washing cells in media lacking nitrate and then resuspending cells media lacking nitrate or with noted concentration of nitrate added. Anoxic conditions were induced in 10mL vials with 3mL headspace by wrapping the vials in foil and purging with argon. The hydrogen concentration in the headspace was measured using gas chromatography. The metabolite concentrations in the extracellular media were quantified using proton NMR spectroscopy. Nitrate/nitrite concentrations in the media and August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts sugar concentrations in the cells were assayed chemically. The ldhA gene was insertionally inactivated to create the mutant ldhA. Results: In order to examine the effect of nitrate limitation under fermentative conditions, cells were resuspended in fresh medium A containing 0 µM, 200 µM, or 1 mM sodium nitrate. Eliminating nitrate from the media prior to fermentation gives a 10-fold increase in the rate of hydrogen production as compared to media containing 200 µM or 1 mM nitrate. The cells assimilate 100% of the extracellular nitrate added within one day of fermentation. Nitrite is detected in the extracellular media after four days of fermentation. The cells that assimilate 200 µM nitrate excrete 70 µM nitrite; the cells that assimilate 1 mM nitrate excrete 700 µM nitrite. The presence of nitrate in the fermentation media acts as an outlet for reductant during fermentation. Nitrate competes for reductant with hydrogenase as evidenced by the 10-fold increase in in vivo hydrogen when nitrate is absent. The primary product excreted by Synechococcus 7002 under fermentative conditions when nitrate is not present is lactate, at a constant rate of 21.6 mol*day-1 per 1017 cells over the 4 days experiment. To redistribute reductant flux away from lactate and towards hydrogen, a mutant lacking D-lactate dehydrogenase was examined under fermentative conditions. The ldhA mutant excretes no lactate, while all other metabolites (hydrogen, acetate, alanine, and succinate) are excreted in higher amounts compared to WT. Hydrogen production increases 5-fold in ldhA cultures, to a rate of 14.1 mol*day-1 per 1017 cells. Conclusions: We have gained insight into carbon and nitrogen metabolism of these photoautotrophs during fermentative metabolism, a branch of metabolism largely unexplored in cyanobacteria. We show increases of 10-fold, with removal of nitrate, and 5-fold, with removal of pyruvate reduction to lactate. Engineering photoautotrophs gives promise for obtaining substantial increases for producing biofuels.
OC-12.3 PHOTOSYSTEM II-INDEPENDENT CONTROL OF EXCITATION TRANSFER TO PHOTOSYSTEM I IN THE FILAMENTOUS CYANOBACTERIUM NOSTOC PUNCTIFORME. Tanai Cardona, Karin Stensjö, Peter Lindblad, Stenbjörn Styring, Ann Magnuson. Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden. Cyanobacteria as well as eukaryotic algae and plants have the capacity to adapt to varying light conditions by controlling the amount of excitation energy distributed to the photosystems. On the minute time scale this control leads to so-called state transitions, that in cyanobacteria involve the movement of the phycobilisomes (PBS) as well as non-photochemical quenching. On longer time scales, cyanobacteria regulate the size and protein composition of PBS as a response to light conditions (‘chromatic adaptation’) on the genetic level. State transitions have been suggested to be dependent on the redox state of the plastoquinone pool, which in part results from electron transfer between the photosystems, but in spite of decades of research, the molecular mechanisms behind the observed phenomena are still not fully understood. In the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 ca 5-10% of the vegetative cells differentiate into heterocysts. The process brings extensive structural reorganization of the thylakoid membranes, and due to oxygen sensistivity of the nitrogenase,
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Final Program and Abstracts Photosystem II is inactivated in the heterocyst. In the heterocyst thylakoids, cyclic electron transfer around Photosystem I (PSI) is essential for nitrogen fixation, but linear electron transfer between the photosystems does not take place. Nevertheless, isolated heterocysts display changes in the excitation transfer to PSI reminiscent of a state transition.
determined as DP 3 through 6 (maltotriose to maltohexaose).
Here we present a characterization of the composition and functionality of thylakoid membranes of the heterocysts. We have investigated the ability of the heterocyst to regulate excitation transfer to PSI, and present the outline of a mechanistic model for excitation re-distribution that does not depend on linear electron transfer.
OC-12.5
OC-12.4 GLYCOGEN CATABOLISM IN SYNECHOCOCCUS ELONGATUS PCC 7942. Eiji Suzuki, Natsuko Abe, Tsubasa Ashikaga, Satomi Ishikawa, Yasunori Nakamura. Akita Prefectural University, Akita, Japan. Introduction Our previous analysis of the mutants of Synechococcus elongatus PCC 7942 defective in glycogen biosynthesis indicated that they were highly susceptible to salt and oxidative stresses. It is therefore suggested that the storage of the polysaccharide and its efficient mobilization are very important for cyanobacteria to cope with various environmental stresses. It is postulated that the degradation of storage β-glucans in bacteria (glycogen or starch) is mediated by phosphorylase (PHO), debranching enzyme (DBE) and 1,4-glucanotransferase (disproportionating enzyme, DPE), encoded by glgP, glgX and malQ, respectively. However, their roles in carbohydrate metabolism of cyanobacteria have been poorly investigated. In the present study, mutants of glgP, glgX and malQ have been constructed and characterized for the catabolic activity of glycogen accumulated in the cell. S. elongatus PCC 7942 is suitable for the functional analysis of the genes involved in the glycogen catobolism, as the organism contains just one each of these genes. Methods The three genes of S. elongatus PCC 7942 were disrupted independently by insertional inactivation using antibiotic resistant markers. Inactivation of the targeted enzyme was confirmed by in vitro assays as well as non-denaturating electrophoresis using amylopectincontaining polyacrylamide gels, followed by iodine staining. Distribution of chain length (as expressed by degree of polymerization, or DP) in the debranched glycogen and maltooligosaccharides was determined by capillary electrophoresis. Results Comparison of the electrophoretic patterns of crude extracts between wild type (WT) and the mutants led us to the identification of the specific bands on the zymograms, corresponding to the activities of all three enzymes. When grown under continuous illumination, the phenotype of the mutants was not clearly distinguished from that of WT, except for the increased accumulation of glycogen and relative increase of short chains in the glycogen molecule (DP 2 through 6) in glgX mutant. The cellular content of glycogen was therefore examined in the continuous darkness, where the turnover of the polysaccharide was stimulated. After 72 hours of dark treatment, the glycogen content in WT cells was decreased to 27% of the initial level. The addition of 0.2 M NaCl to the culture further enhanced the glycogen breakdown (to 6% of the original level after 72 hours) in WT cells. Under the same conditions, the activity of glycogen degradation in the glgP, glgX and malQ mutants was markedly reduced as compared to WT. In addition, accumulation of malto-oligosaccharides was observed specifically in malQ mutant, irrespective of the growth conditions. The molecular species of the malto-oligosaccharides was 70
Conclusions These results demonstrated the pivotal roles of the three enzymes PHO, DBE and DPE in the glycogen catabolism of cyanobacteria.
IRON UPTAKE AND TOXIN SYNTHESIS IN MICROCYSTIS AERUGINOSA UNDER IRON LIMITATION. Ralitza Alexova1, Manabu Fujii2, T. David Waite2, Brett A. Neilan1. 1
School of Biotechnology and Biomolecular Sciences; 2School of Civil and Environmental Engineering; University of New South Wales, Sydney, Australia. Introduction: The production of toxins during cyanobacterial blooms poses a significant public health threat in water bodies globally and requires the urgent development of effective bloom management strategies. Microcystin is a hepatotoxin synthesised non-ribosomally by members of several cyanobacterial genera. Although the microcystin biosynthesis pathway is now characterised, the contribution of toxicity to the adaptation of cyanobacteria to environmental stresses, such as changing light intensity and nutrient limitation, is still unclear. Previously, microcystin synthesis has been proposed to be regulated by iron availability. The aim of this study was to compare the transcription of genes involved in iron uptake and redox control in toxic and non-toxic Microcystis aeruginosa subjected to moderate and severe iron stress.
Methods: Three strains of M. aeruginosa, the toxic PCC7806, the nontoxic PCC7005 and the mutant mcyH-, were grown in Fraquil media containing 10-1000 nM Fe. The transcription of a number of genes involved in iron uptake, oxidative stress response and toxin synthesis was accessed by quantitative real-time PCR (qRT-PCR). The possible involvement of the ferric uptake regulator homologues FurA, FurB and FurC in regulating toxicity and survival under redox stress was also assessed. Results: Both non-toxic strains (PCC7005 and mcyH-) used in this study showed pronounced bleaching by the end of the experiment, with the growth of mcyH- being significantly retarded under iron limitation. Toxin production in PCC7806 was increased in an irondependent manner and appeared to be regulated by FurA. The transcription of the genes studied here showed a high degree of variability between strains and the growth stage at which the cells were harvested. The inability to produce microcystin, either due to natural mutations in the mcy gene cluster or insertional inactivation of mcyH, affected the remodeling of the photosynthetic machinery in iron-stressed cells and the transport of Fe3+ in early exponential growth. Conclusion: This is the first comprehensive study on the effects of iron starvation in toxic and non-toxic M. aeruginosa. Our results show that the process of adaptation to iron stress is strain-specific and highly dynamic. Toxicity appears to protect microcystin-producing cyanobacteria in the early stages of exposure to severe iron stress and may protect the cell from reactive oxygen species-induced damage. We have initiated proteome studies to confirm this hypothesis.
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13th International Symposium on Phototrophic Prokaryotes
OC-12.6 THIOL PRODUCTION BY MICROCYSTIS: A POTENT METABOLITE WITH ECOPHYSIOLOGICAL ROLES. S.B. Watson1, F. Juttner2. 1
Environment Canada, Canada Centre for Inland Waters, Burlington, ON, Canada; 2Limnological Station, University of Zurich, Switzerland. Introduction: Microcystis species produce sulphides, a sometimes significant source of malodour. Although well documented in marine systems, little is known about freshwater photoautotrophic production of sulphides, which to date has been generally regarded as insignificant. However, the apparent increasing number of Microcystis blooms may in fact represent a significant source of these compounds in many inland waters. Methods: We investigated the physiology and cellular/ecological function of sulphide production by four axenic and non-axenic Microcystis aeruginosa strains. Using short-term incubations and an adapted Headspace-GC-MS technique and more focussed studies with the axenic strain using different light regimes, metabolic inhibitors (sodium azide, DCMU), antioxidant enzymes (superoxide dismutase, catalase) and isotopically labelled precursors (hydrogencarbonate, acetate and sulphate) Results: We found that isopropyl sulphides were of cyanobacterial in origin. Our work demonstrated that i) isopropyl thiol (ISH) is the initial cell product, with subsequent extracellular conversion to diisopropyl disulphide and trisulphide); iv) ISH is actively produced over the growth cycle, iii) is continuously excreted with no internal storage or post-lysis catalytic generation; iii) production occurs in both light and dark, increases up to an optimal light level and decreases significantly under very high irradiance; iv) labelling patterns indicate ISH is synthesized via the acetate pathway.
Final Program and Abstracts Methods: Genome sequence data of Thiodictyon sp. Cad16 was generated by pyrosequencing and annotated using software packages from Softberry Inc. (www.softberry.com). Results: The core genes of all analyzed GSB strains largely exhibit congruent phylogeny. Photosynthesis genes are largely in agreement with this core phylogeny. However, sulfur metabolism genes are often not congruent and are scattered across the GSB lineages suggesting extensive horizontal gene transfer events. The PSB Thiodictyon sp. Cad16 has an unexpectedly large genome (~8 Mbp), which reflects the large physiological diversity of this strain. CO2 fixation metabolism is supplemented by carboxysomes, previously not known to occur in PSB. Genes encoding sulfur metabolism exhibit unexpected combinations and similarities with both GSB and other PSB. Conclusions: GSB have relatively small genomes (2–3 Mbp) reflecting their limited physiological capabilities. Some PSB have very large genomes (~8 Mbp), whereas other PSB have much smaller genomes (3–4 Mbp), probably reflecting large variations in their environmental adaptation. Finally, whereas the cellular core and key metabolisms such as photosynthesis and carbon assimilation have rather different origins in GSB and PSB, their sulfur metabolism appears to share a common origin.
OC-13.2 THE GENOME OF THE TOXIC BLOOM-FORMING CYANOBACTERIUM ANABAENA SP. 90. David Fewer1, Hao Wang1, Leo Rouhiainen1, Zhijie Li2, Bin Liu2, Kaarina Sivonen1. 1
Department of Microbiology and Applied Chemistry, University of Helsinki, FIN-00014, Helsinki, Finland; 2Beijing Institute of Genomics of the Chinese Academy of Sciences, Beijing Genomics Institute, Beijing, China.
Conclusions: These experiments and related work using the two aquatic invertebrates Thamnocephalus and Daphnia indicate that isopropyl thiol plays several important physiological and ecological roles. It acts as an effective antioxidant against high levels of reactive oxygen species, particularly in surface blooms; it elicits avoidancerelated behavioural responses in grazer communities and at high levels, can be toxic to some invertebrates.
Introduction: Toxic cyanobacterial blooms are linked to the deaths of wild and domestic animals and are a health risk for human beings via recreational or drinking water. Strains of the genus Anabaena are important primary producers in freshwater bodies throughout the world and contribute to the formation of noxious cyanobacterial blooms through the production of a range of hepatotoxins and neurotoxins. Here we report the genome sequence of a hepatotoxic bloom-forming cyanobacterium Anabaena sp. 90.
OC-13.1
Methods: We constructed a whole-genome shotgun assembly from 119,316 sequencing reads from 2 kb, 6 kb and 40 kb libraries. These were organized in pairs by virtue of end-sequencing 6-kb and 50-kb inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 12.5 times.
COMPARATIVE GENOMICS OF PHOTOTROPHIC GREEN SULFUR BACTERIA AND PURPLE SULFUR BACTERIA. N.-U. Frigaard1, M. Tonolla2, D.A. Bryant3. 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Plant Biology Department, University of Geneva, Geneva, Switzerland; 3Department of Biochemistry and Molecular Biology, Pennsylvania State University, State College, Pennsylvania, USA.
Introduction: Green sulfur bacteria (GSB) and purple sulfur bacteria (PSB) are photoautotrophs that oxidize reduced sulfur compounds for growth. Whereas some aspects of their ecology are similar, other aspects of their physiology and evolution are rather different. We are interested in the evolution of these lineages and their photosynthetic, sulfur, and carbon metabolisms. Genome sequence data are available for >12 strains of GSB, but not for any PSB of the Chromatiaceae. We have therefore sequenced the genome of the PSB Thiodictyon sp. Cad16, which grows both photolithoautotrophically and chemolithoautotrophically.
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Results: The length of the genome amounts to 5,305,674 bp and is partitioned into two circular chromosomes and three plasmids. Essential house keeping genes were distributed across both chromosomes while the plasmids consisted largely of hypothetical open reading frames. A total of 5,750 putative ORFs were annotated from the genome with just over 20% of the predicted ORFs being unique to Anabaena sp. 90. This strain is known to produce toxins and enzyme inhibitors and over 5% of the genome is dedicated to the production of bioactive compounds. During the assembly we documented insertions, deletions and single nucleotide polymorphisms which in some cases has lead to the inactivation of biosynthetic pathways. Interestingly, approximately 7% of the genome is composed of repetitive autonomous and non-autonomous mobile DNA which have inactivated a number of genes. Together this suggests a mechanism by which Anabaena can tailor and customize its
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Final Program and Abstracts genomic repertoire through genomic rearrangements. Analysis of the genome revealed an investment in restriction modification systems explaining in part difficulties in genetic manipulation of this organism. Bioinformatic analysis revealed strong similarities to other heterocyst forming cyanobacteria and preliminary evidence that phage infection has shaped the genome of this bloom forming cyanobacterium. Conclusions: Further research will target a system biological investigation of this organism and is likely to reveal in-depth information of this ecologically relevant, bioactive compound producing cyanobacterium.
OC-13.3 CHEMOTAXIS-LIKE SIGNAL TRANSDUCTION SYSTEMS IN DEVELOPMENT AND BEHAVIOR OF HORMOGONIA OF NOSTOC PUNCTIFORME. Jack Meeks, Elsie Campbell, Rui Chen. Department of Microbiology, University of California, Davis, CA, USA. Introduction: The genome of the symbiotic nitrogen-fixing cyanobacterium Nostoc punctiforme contains five loci of genes that encode core complex proteins analogous to the chemotaxis proteins CheY, CheW, MCP and CheA of Escherichia coli. Genes in four of the loci appear to have evolved in the cyanobacterial lineage, while those in the fifth locus have roots in the proteobacteria. We have set out to characterize these genetic loci in terms of organization and physiological role. Methods: A DNA microarray of N. punctiforme was used to examine global transcription profiles during nitrogen starvation induction of heterocysts and hormogonia, and plant produced exudate containing a hormogonium inducing factor. The data were analyzed in the R statistical platform and normalized log2 of reference/experimental values were k-means-clustered using the program Genesis. Insertion mutants were constructed by gene replacement, and cellular differentiation and behavior phenotypes analyzed by standard techniques. Results: Global transcriptional analyses indicate that 13 out of 35 chemotaxis-like genes are expressed at a low level in ammoniumgrown cultures; these include 3 CheB methylesterase and 2 CheR methyltransferase-like adaptive proteins, all of which lack the response regulator receiver domain and their genes are not collocalized in the mentioned loci. Transcription of genes in the four cyanobacterial lineage loci are up-regulated only during hormogonium differentiation, except for three genes in different loci (a MCP in locus 1, and a cheA and cheW in locus 3) that are not transcribed under any condition examined. Insertion mutations in genes encoding MCP proteins in the four loci in the cyanobacterial lineage yielded two distinct phenotypes: a locus 2 MCP mutant fails to differentiate hormogonia and a locus 4 mutant is defective in phototaxis. Hormogonia of a locus 1 mutant have a slight morphological alternation, but are phototactic and infect a bryophyte symbiotic plant partner, while a mutant in the MCP of locus 3 has no detectable phenotypic defect. The chromophore bound to the MCP of locus 3 undergoes green-red light photoconversion. The genes in locus 4 (phototaxis) include duplicates encoding CheW and partial MCP and CheA proteins. Insertion mutations in the duplicate MCP and cheA genes yield phototaxis defective phenotypes. Only one MCP of locus 5 (NpR0247) is significantly up-regulated late in hormogonium differentiation, but the gene is difficult to manipulate in E. coli. Conclusions: The transcription and mutant phenotypes indicate a variable organization of the multiprotein sensory transduction
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complexes encoded in the four cyanobacterial lineage loci. We propose the proteins encoded by locus 4 assemble into a soluble complex as heterodimeric MCP, CheW and CheA proteins, with one or both CheY proteins communicating with the motility motor(s). Conversely, the proteins encoded in the membrane-associated locus 2 complex (development) assemble as homodimers. The homodimeric CheA and CheW of locus 1 may associate with the MCP encoded in a different locus. The MCP of locus 3 may assemble as a homodimer with homodimeric CheW and CheA proteins also encoded in a different locus, perhaps locus 1. The MCP of locus five may be a chemotactic sensor and interact with proteins encoded in other loci.
OC-14.1 COMPARISON OF NONCODING FEATURES OF CYANOBACTERIAL GENOMES. Jeff Elhai1, Michiko Kato2, Sarah Cousins3, Peter Lindblad4, José Costa5. 1
Virginia Commonwealth University, VA, USA; 2University of California at Davis, CA, USA; 3University of Pennsylvania, PA, USA; 4Uppsala University, Uppsala, Sweden; 5University of Porto, Porto, Portugal. Introduction: Insights from genomic sequences have been gained primarily analysis of the genes and protein functions inferred from them, but much can be learned beyond this analysis. Short repeated sequences that are not explained by inclusion within multicopy genes pose interesting biological questions: (1) By what mechanism are the repetitive units formed? (2) What selective forces act to preserve them? and (3) What effect do they have on cellular function? Methods: The sequences of 42 cyanobacterial genomes (36 complete) were analyzed using BioBIKE, an integrated knowledge base and programming interface. DNA methylation was inferred by the presence of orthologs of DNA methyltransferases with known methylation activity. Results: Sequences of at least 24 nucleotides that appear multiple times in the cyanobacterial genomes were dominated by three types: (1) long dispersed repeats often associated with transposes (hence probably insertion sequences), (2) repeated sequences interspersed with non-repeated elements, characteristic of CRISPRs (clustered regularly interspersed short palindromic repeats), and (3) short dispersed repeats (termed SDRs) not previously described. All of these are potentially mobile sequences. We have shown by intergenomic comparisons that SDR sequences, ranging in size from 21 to 28 nucleotides, have moved in recent evolutionary times. They may be the smallest mobile sequences known. Three families of SDR sequences (SDR4, SDR5, and SDR6). can assume the same conserved secondary structure, even though they do not share significant sequence similarity. This suggests that they act through an RNA intermediate. One family, SDR5, specifically targets HIP1 sites (GCGATCGC). For ease in discussion, we define Group F of cyanobacteria to consist of the filamentous cyanobacteria plus those unicellular cyanobacteria within the genera Synechocystis, Cyanothece, Crocosphaera, Microcystis, and Acaryochloris, as well as Synechococcus PCC 7002. Group P consists of all small marine Prochlorococcus and Synechococcus. The remaining cyanobacteria, Synechococcus PCC 7942/6301, Thermosynechococcus, hot spring Synechococcus, and Gloeobacter are ungrouped. All Group F cyanobacteria were found to possess a high frequency of HIP1 (GCGATCGC) sequences, as do Synechococcus PCC 7942/6301 and Thermosynechococcus but not other genomes. Insertion
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13th International Symposium on Phototrophic Prokaryotes sequences were found to be prominant in all Group F cyanobacteria but were seldom found in Group P cyanobacteria. CRISPR sequences were confined to Group F (though absent in many). Orthologs of dmtA (encoding a GATC-specific DNA methyltransferase) are found in all cyanobacterial genomes outside of Group P. GGCC- and CGATCGmethylation (inferred from the presence of orthologs of dmtB and dmtC, respectively is found exclusively within Group F cyanobacteria. SDR sequences were frequent in heterocystous (filamentous) cyanobacteria and very rare in other genomes. Conclusions: DNA sequence characteristics – the frequency of repeated sequences and methylation patterns – can be used to define cyanobacterial groups that make sense by comparison with other phylogenetic measures. The groupings may relate to underlying molecular properties, e.g. recombination (HIP1) or possibly DNA replication (GATC methylation). It is interesting that three conserved features have overlapping specificity: GATC-specific methylation, CGATCG- specific methylation, and HIP1 (GCGATCGC), suggesting overlapping function.
OC-14.2 HOW CAN GREEN SULFUR BACTERIA USE SOLID SULFUR (S0) AS ELECTRON DONOR? SEARCHING FOR THE ANSWER IN THE MEMBRANE PROTEOME OF CHLOROBACULUM PARVUM DSM 263. Clelia Doná1,2, Lena Hauberg3, Barbara Reinhold-Hurek3, Ulrich Fischer1. 1
Zentrum für Umweltforschung und nachhaltige Technologien (UFT) and Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie, Universität Bremen; 2Max Planck Institut für Marine Mikrobiologie; 3 Fachbereich Biologie/Chemie, Laboratorium für Allgemeine Mikrobiologie, Universität Bremen; Bremen, Germany. Intoduction: Green sulfur bacteria are anaerobic anoxygenic photolithoautotrophs provided with a photosystem of type I and with peculiar organelles, the chlorosomes, in which the light-harvesting pigments are organized. These bacteria couple the fixation of CO2 to the oxidation of inorganic sulfur compounds or, in only one known case, ferrous iron. When sulfide or thiosulfate are the electron donors, elemental sulfur (S0) is an intermediate product, which can be further oxidized to sulfate. S0 is thus a key substrate, whose insolubility in water constitutes a special task for the living cells. At present, it is unknown how green sulfur bacteria mobilize S0 and if the protein components involved are subjected to transcriptional control. Methods: The mesophilic strain Chlorobaculum parvum DSM 263, whose genomic sequence is now available, was chosen to investigate the mechanism of S0 mobilization in green sulfur bacteria. To preliminarily assess whether the cells need a direct contact with sulfur for its oxidation, previously prepared biogenic sulfur was embedded into agar and brought in contact with a living culture. The appearance of cleared zones at the border between cell culture and agar was then monitored. For the proteomics experiment, Clb. parvum DSM 263 was grown only on sulfide or on biogenic sulfur previously extracted from a culture of the same species. French press was used to disrupt the cells and ultracentrifugation to separate the soluble fraction from the membranes. 2-dimension gel electrophoresis with non-equilibriumpH-gradient-electrophoresis (NEPHGE) as first dimension and matrix-assisted-laser-desorption/time-of-flight (MALDI/TOF) mass spectrometry were used to identify which proteins are differentially August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts expressed in the membranes of the 2 populations. Results: Neither a clear zone could be observed in the agar with biogenic sulfur, nor a culture could grow if the embedded sulfur was the only electron donor present. These results allowed a focus on the membrane fraction, in which differences in the analysed protein patterns were found, a major one regarding a conserved hypothetical protein. Conclusions: We could show that cells of Clb. parvum DSM 263 need a direct contact with S0 for its mobilization and that at least some of the proteins involved in the utilization of this electron donor appear to be subjected to transcriptional control.
OC-14.3 PROTEOME ANALYSIS OF CHLOROBACULUM TEPIDUM TLS: INSIGHTS INTO THE SULFUR METABOLISM OF A PHOTOTROPHIC GREEN BACTERIUM. Mette Miller1, Lasse F. Nielsen1, Monika Szymanska1, Anders Mellerup2, Kirsten S. Habicht3, Raymond P. Cox1, Jens S. Andersen1 & Niels-Ulrik Frigaard2. 1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK5230 Odense; 2Department of Biology, University of Copenhagen, DK-2200 Copenhagen; 3Nordic Center for Earth Evolution and Institute of Biology, University of Southern Denmark, Odense; Denmark. Introduction: Green sulfur bacteria (GSB) use reduced sulfur compounds as electron donors for their photosynthetic carbon dioxide fixation. They are often found in stratified lakes at, or below, the level in the chemocline where sulfide is exhausted. The sulfur metabolism in GSB is complex; almost all green sulfur bacteria oxidize sulfide (HS-) and elemental sulfur (S0) but in addition some are able to utilize thiosulfate (S2O32-). GSB have a high affinity for sulfide and this substrate is taken up in preference for other sulfur substrates such as thiosulfate. Predictions from the genome sequence of Chlorobaculum (Cba.) tepidum TLS suggest that at least 60 gene products participate in sulfur oxidation pathways (1). Methods: To obtain information about gene expression during various stages of the sulfur metabolism of Cba. tepidum, we have grown the cells in batch culture in a medium containing both sulfide and thiosulfate. Proteome analyses were performed at two stages: once in the early exponential growth phase where the cells are consuming sulfide, and once at the end of the exponential growth phase where the cells have utilized almost all thiosulfate. Proteins were extracted, digested with trypsin and the peptides released were analyzed using an Agilent HPLC coupled to either a LTQ FT Ultra (Thermo) or a LTQ Orbitrap XL (Thermo) mass spectrometer. Proteins where identified using Mascot and quantified using MaxQuant. Samples were run in duplicate on each system and the relative expression of proteins was calculated on basis of the measured intensities. Results: In total 945 proteins of the 2,288 coding sequences on the Cba. tepidum genome were identified on the basis of at least two unique peptides. We detected 34 of the 60 proteins proposed to be involved in the sulfur metabolism of Cba. tepidum and most of these proteins were equally expressed in the two samples. However, in cells in the early exponential growth phase with active sulfide uptake we found a two-fold up-regulation of the dissimilatory sulfite reductase enzymes DsrE, DsrF, DsrH and the Rubisco-like enzyme RLP compared to cells growing on thiosulfate where the sulfur oxidizing enzymes SoxX, SoxY & SoxZ and quinone modifying oxidoreductase QmoA & QmoB and the soluble cytochrome c555, CycA were up-
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Final Program and Abstracts regulated. Results will also be presented on proteome analysis of a mutant of Cba. tepidum that lacks the DSR enzyme system and is deficient in elemental sulfur utilization. (1) Frigaard & Bryant, 2008. p 60-76. In C Dahl and CC Friedrich (eds.), Microbial sulfur metabolism. Springer, New York
OC-15.1 A SURVEY OF THE ECONOMICAL VIABILITY OF LARGE-SCALE PHOTOBIOLOGICAL HYDROGEN PRODUCTION UTILIZING CYANOBACTERIA. Hidehiro Sakurai, Hajime Masukawa, Kazuhito Inoue. Res. Inst. Photobiol. H2 Production, Kanagawa Univ., Hiratsuka, Kanagawa, Japan. Introduction: In order to meet for the demand for large-scale production of renewable energy, we have proposed nitrogenasebased photobiological production of H2 using maricultured cyanobacteria. Although most of the technologies are still in the beginning stages of development, a preliminary trial cost analysis was made in order to assess the future prospects for the economical viability of large-scale renewable energy production. Methods: Assumptions: 1) Total solar radiation on the surface of Earth: 2.7 x 106 EJ per year (or 5.3 x 109 J m-2 year-1, 1,680 kWh m-2 year-1 or 14.5 MJ m-2 day-1) on average. 2) Cyanobacteria photobiologically produce H2 at 1% efficiency vs. total solar radiation (5.3 x 107 J m-2 year-1). 3) Finally, available H2 energy: one and two thirds of biologically produced H2 energy, consuming two and one third needed for harvesting, purification, storage, and transportation to land (1.8 and 3.5 x 107 J m-2 year-1, respectively, in the final product). 4) Area required: 38.8 x 1018 divided by 1.8 and 3.5 x 107 = 2.2 and 1.1 x 1012 (m2) (=2.2 and 1.1 x 106 km2), respectively. Results: 1. A plausible general scheme 1) Cyanobacteria produce H2 with sunlight as the sole source of energy. The substrate is water and products are H2 and O2. The culture medium is simple in composition and contains mineral nutrients, and the gas phase in the bioreactor is Ar plus low concentrations of CO2 and N2. 2) The bioreactor consists of three layers plastic bag (inner: retains culture medium, middle: hydrogen gas barrier, outer: mechanically protects the inner twos) and is floated on a calm sea surface. 3) Repeated harvesting of gas. When H2 gas attains a certain concentration level (e.g. 25 - 30%, v/v), a factory ship harvests the gas mixture (main components: Ar, H2, O2) from bioreactors, and purifies H2 on board. 2. Cost analysis 1a) Nutrients. Potentially growth-limiting nutritional elements are added to the natural water (20 cm in depth) as ‘fertilizers’ akin to agricultural practices. [1.5 - 12 cents m-2 of the bioreactor surface] 1b) Water. Reference price of water for industrial use in Japan at about 7.5 - 60 cents per ton. [1.5 - 12 cents m-2] 1c) Bioreactor. The average plastic price of $2 - $4 per kg and the plastics are recycled at the half prices of the feed stocks [50 - 100 cents per m2 of bioreactor,] 1d) Gas (mostly Ar). FOB, bulk rate of Ar: $100 - 500 per ton. [9 cents 45 cents per m2] 74
13th International Symposium on Phototrophic Prokaryotes 1e) Other costs: the cost of growing cyanobacteria, the labor cost, the cost of ships, the interest on capital goods, and the cost of marine transportation of production materials. [Cost E]. 1f) Cost of gas harvesting, H2 separation, storage, and marine transportation to final destination. [Cost F] 2) Depending on the Cost E and F, the final cost of H2 at end-users: 1.8 - 13.4 (in cents MJ-1). Comparison; wind electricity: 2.0 - 5.2, solar photovoltanics: 10 - 50, crude oil: 0.8 - 2.4 (50 - 150 dollars barrel-1) or 2.4 - 7.2 (conversion efficiency of 1/3 into electricity). Conclusions: Our trial calculation indicates that it will be difficult for bio-solar H2 production to be competitive with fossil fuels at current prices, but as specific policies come into play such as carbon taxes, there will likely be an acceleration of its development. In spite of the technical as well as economical difficulties, we propose that largescale H2 production by mariculture is the most promising strategy to be pursued for “mitigating dangerous anthropogenic interference with the climate system” (the purpose of UNFCCC).
OC-15.2 SYSTEMATIC EVALUATION OF HYDROGEN PRODUCTION AMONG DIVERSE HETEROCYSTOUS CYANOBACTERIA. Chris Yeager, Charlie Milliken, Christopher Bagwell, Lauren Staples, Polly Berseth, Tommy Sessions. Savannah River National Laboratory, Aiken, SC, USA. Introduction: H2 generated by cyanobacteria is a highly attractive form of alternative energy. Much progress has been made to this end, yet an important element of the research continuum has been largely overlooked in recent years - the physiology and diversity of naturally occurring, H2-producing cyanobacteria. The overall goal of the research described here was to develop and apply a systematic approach for evaluating the H2 production capacity among diverse heterocystous cyanobacteria. Specifically we wished to: 1) compare several environmental parameters (O2 concentration, N2 concentration, light intensity, and carbon source) that control H2 production among diverse heterocystous cyanobacteria, and 2) establish benchmark activities/phenotypes against which to routinely evaluate newly discovered H2 producing cyanobacteria. Methods: Cyanobacteria (10 diverse heterocystous strains were tested) were cultured in Roux flasks containing BG11- media bubbled rapidly with air under a light (70-100 µmol m-2 s-1)/dark photocycle (16:8 h). To assay biological hydrogen production, cells were concentrated via centrifugation, washed, and placed in sealed vials where an argon atmosphere (≥ 98.5% argon) was generated. Headspace gas compositions of the vials were adjusted with N2, O2, or CO2 and gas samples were analyzed with MTI Model M200D micro gas chromatograph equipped with a TCD detector. Results: All strains exhibited an immediate inhibition of H2 production rates upon the addition of N2. Compared to the activity of cells incubated in vials containing <1% N2 headspace volume, H2 production rates measured for the individual strains decreased 38-61% in the presence of 5% N2, 71-92% in the presence of 20% N2, and 8997% in the presence of 80% N2. It was determined that although nitrogenase is sensitive to O2, low levels of O2 (typically 2.5-5% atm) are required for respiratory metabolism to generate sufficient reductant and/or ATP to drive H2 production. The addition of glucose (10 mM) accelerated H2 production in 7/10 strains examined, with rate increases ranging from 1.1-14.5 fold and yields increasing 1.4-88.1 fold. When cells were incubated in the absence of an exogenous carbon source, all of the strains exhibited dramatic increases in H2 yields (13.2-18.0 fold) when the light intensity was increased from 30 August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes to 200 µmol m-2 s-1. The stimulatory effect of increased light intensity on cyanobacterial H2 yield was generally not as pronounced when strains were incubated in the presence of glucose (1.4-10.5 fold increases). Of particular interest, cells of 4 strains exhibited near maximal H2 yields at light intensities <100 µmol m-2 s-1 when incubated with glucose Conclusions: The immediate, inhibitory effect of N2 on H2 production was quite similar across a phylogenetically diverse collection of heterocystous cyanobacteria, enabling us to establish a baseline from which to evaluate the “N2 sensitivity” of H2 production among these organisms. Our results also highlight that it is critical to optimize reductant availability for H2 production. Addition of sugars and low levels of O2 (2.5-5% atm) may be useful for many strains. The commonly held belief that the economic feasibility of cyanobacterial H2 production is dependent primarily on the light to H2 conversion efficiency may be fundamentally flawed. Mixotrophic conditions should be explored as a means to produce biohydrogen with cyanobacteria.
OC-15.3 THE INTEGRATION OF HYDROGEN PRODUCTION BY PURPLE BACTERIA WITH DARK FERMENTATIVE HYDROGEN PRODUCTION AND HYDROGEN ELECTRODE. A. Tsygankov. Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia. Introduction: Green and cost-effective H2 production and H2dependent electricity generation are of the key problems restricting the development H2-based economy. Purple bacteria produce H2 using light energy and simple organic compounds with high rates. The source of organic compounds is one of factors preventing development of biotechnological H2 producing systems based on purple bacteria. Last years in different laboratories the integration of purple bacteria with dark fermentation of organic wastes are under investigation. This presentation describes the integrated system consisting of dark fermentation of wastes with H2 and concomitant organic acids production, of purple bacterial H2 production under the light using organic acids, and of hydrogen enzyme electrode for the electricity generation using H2. Methods: Experiments used bioreactors for dark fermentation, photobioreactors for suspension and immobilized purple bacteria, and hydrogen enzyme electrodes with various hydrogenases separately and in combination. Results: The stability of dark fermentation and light-dependent H2 production by purple bacteria in continuous mode of operation was studied and it was shown that these processes are stable for months. Operational stability of hydrogen enzyme electrode in buffer system, as well as in medium from photobioreactor was studied also. Different combinations of reactors with photobioreactors, hydrogen electrodes with bioreactors were tested and results will be presented in detail. Conclusion: Results indicate that there are no principal factors preventing an integration of bioreactors and hydrogen enzyme electrode exist. However, the system needs in optimization for electricity output. The research was supported by Russian RAS Program for Basic Research “Chemical aspects of energetics” and by Russian Foundation of Basic Research.
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Final Program and Abstracts
OC-15.4 HIGH CELL DENSITY CULTIVATION OF RHODOSPIRILLUM RUBRUM UNDER RESPIRATORY DARK CONDITIONS. Lisa Zeiger, Christiane Rudolf, Hartmut Grammel. Max-Planck-Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany. Introduction: The aim of this study was to determine the maximal achievable cell concentration of the facultative photosynthetic bacterium Rhodospirillum rubrum in a bioreactor, thus allowing the estimation of potential volumetric yields in bioprocesses. Mass cultivation of purple non-sulfur bacteria, incl. R. rubrum under photosynthetic conditions is generally inefficient due to the inevitable limitation of light-supply when cell densities become very high. For R. rubrum however, a cultivation process has been described that allows the high-level expression of photosynthetic membranes (PM), equivalent to light-grown cultures, under completely dark conditions when grown semi-aerobically in a bioreactor. On the basis of this cultivation system, we developed a process for obtaining very high cell densities (HCD) in aerobic and semi-aerobic fed-batch cultivations. We also present a biochemical analysis of HCD cells and show that the physiology of R. rubrum is affected significantly by the applied conditions. Methods: For HCD fed-batch cultivations, an exponential feeding algorithm implemented in a computer process control system was applied. An unstructured computational model, based on mixedsubstrate kinetics was developed for optimization of the process in simulation studies. For aerobic fed-batch cultivation, the exponential feed algorithm was applied in combination with a pH-stat element to prevent substrate accumulation. Semi-aerobic fed-batch cultures were conducted with a different control strategy, using the culture redox potential as the manipulated variable. The obtained high density cells were characterized by biochemical analyses, absorption and fluorescence spectroscopy, and HPLC-MS. Results: Key growth parameters (saturation constants, inhibition constants, etc. ) were determined prior to the fed-batch experiment and used to parameterize the computational model and the controller algorithm. Two carbon substrates, fructose and succinate, were present in the feed because of the previously described elevated PM levels in cells grown semi-aerobically in this medium composition. The applied exponential fed-batch strategy resulted in a maximum cell density of 59 g L-1 cell dry weight in aerobically grown cultures. To our knowledge, comparable values have never been reached before with R. rubrum or related species. An interesting finding was that high density cells accumulated and excreted large amounts of tetrapyrrole intermediates which were identified as protoporphyrin IX and Mgprotoporphyrin IX monomethyl ester. Furthermore, attempts to induce PM expression by applying oxygen-limiting conditions surprisingly failed. Conclusion: In this study we demonstrate that it is possible to cultivate R. rubrum under chemoheterotrophic conditions to, so far inaccessible high cell densities. Biochemical analyses revealed regulatory imbalances in HCD cultures at the branchpoint of aerobic and anaerobic tetrapyrrole biosynthetic pathways. An additional finding was that the induction of PM expression is dependent on the cell density and PM formation is severely impaired in HCD cells. The results are important for the developments of bioprocesses using this organism, and provide an interesting experimental system for the investigation of regulatory mechanisms at the hemebacteriochlorophyll metabolic branch. 75
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13th International Symposium on Phototrophic Prokaryotes
OC-15.5
OC-15.6
DETECTION OF MICROCYSTIN-PRODUCING CYANOBACTERIA IN MISSISQUOI BAY, QUEBEC, USING Q-PCR.
EVOLUTIONARY LOSS OF MICROCYSTIN BIOSYNTHESIS GENES AND THE GENETIC POPULATION STRUCTURE OF TOXIC CYANOBACTERIA.
Nathalie Fortin1, Rocio Aranda-Rodriguez2, Hongmei Jing4, Frances Pick3, David Bird4, Charles W. Greer1. 1
National Research Council, Biotechnology Research Institute, QC; Environmental Health Center, Health Canada, ON; 3Biology Department, University of Ottawa, ON; 4Biological Sciences, University of Quebec at Montreal, QC, Canada. 2
Toxic cyanobacterial blooms, and their increasing global occurrence, pose a serious threat to human health, domestic animals and livestock. The frequency and severity of bloom events continue to rise, most probably as a direct result of increased nutrient loading of water systems worldwide. The number of lakes in Quebec, Canada, affected by blooms has been increasing steadily from 34 (2004), to 45 (2005), 84 (2006) and 197 (2007). Government agencies are under tremendous pressure to cope with escalating demands for water analysis, specifically for cyanotoxins. In Missisquoi Bay, Lake Champlain, public health advisories were issued from 2000 to 2008, and local microcystin concentrations found in lake water often exceeded the Canadian drinking water guideline of 1.5µg L-1. A quantitative-PCR (Q-PCR) approach was developed for the early detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the polyketide synthase (mcyD(KS)) and the first dehydratase domain (mcyD(DH)) of the mcyD gene, involved in microcystin synthesis. Q-PCR was used to monitor the blooms in the littoral and pelagic zones of Philipsburg, Missisquoi Bay, during the summers of 2006 and 2007. Two toxic bloom events were detected by Q-PCR during the summer of 2006: more than 60,000 copies of the mcyD(KS) gene mL-1 were detected in August and an average of 40,000 copies mL-1 were detected in September, when microcystin concentations were more than 4 µg L-1 and approximately 2 µg L-1, respectively. There was a good correlation between gene copy number and microcystin concentrations. The Microcystis aeruginosa population followed the same trend as the number of mcyD(KS) copies and microcystin concentration in the littoral zone. The HPLC profile revealed a change in the toxin composition of the two bloom events. Four microcystin analogues were observed during the bloom in August (MCYST-LA, LR, RR and YR). The MCYST-LA analogue was predominant in August and dominated the bloom in September. The other analogues identified in the second bloom were MCYST-LR and YR. Favorable conditions for a cyanobacterial bloom occurred only late in the summer of 2007. Approximately 300 copies of the mcyD(KS) gene mL-1 were identified in the pelagic zone at the end of August despite the low concentration of toxin. The Q-PCR method was more sensitive than standard chemical assays and allowed the detection of microcystin-producing cyanobacteria as early as June. This technique could be used for the efficient monitoring of the most at-risk water bodies.
Rainer Kurmayer, Guntram Christiansen. Austrian Academy of Sciences, Institute for Limnology, Mondsee, Austria. Introduction: Green- or red-pigmented cyanobacteria of the genus Planktothrix spp. frequently contain the toxic heptapeptide microcystin. We could previously show that non-toxic strains isolated from 28 water bodies in nine European countries have lost more than ninty percent of the large gene cluster (mcy) responsible for microcystin synthesis (Christiansen, G, Molitor, C, Philmus, B, Kurmayer, R. 2008, Mol. Biol. Evol. 25:1695-1704). This gene loss event probably happened million years ago. Due to its rather long history we hypothesized that the non-toxic strains meanwhile could have adapted to a variety of ecological factors not causally related to microcystin anymore. This would obscure a clear correlation of microcystin production and ecological fitness. Methods: In this study 139 red-/green-pigmented strains isolated from lakes of three continents (Europe: 101 strains, North America:29, Sub-Saharan East Africa:9), were analysed (i) for their pigmentation and the production of microcystin, (ii) for the genetic variation within five housekeeping gene loci (16S rDNA, 16S rDNA-ITS, PC-IGS, PSAIGS, RNase P), and (iii) for remnants of the mcy gene cluster (in non-toxic strains). Using established multi locus sequence typing techniques the strains were phylogenetically assigned to several lineages that may be considered as cryptic ecotypes (Cohan, FM 2002, Ann. Rev. Microbiol. 56:457-487). According to common theory clonal dependence can only be observed if ecological differentiation prevents genetic exchange and therefore favours genetic differentiation. Results: In total 58 non-toxic strains were found that were isolated from all three continents. These non-toxic strains were found to lack the mcy gene cluster but still contained at least one of the former flanking regions. The majority of them (42 strains) shared a common remnant of the mcy gene cluster, mcyT, a type II thioesterase. The sequencing of the five housekeeping gene loci revealed six major phylogenetic lineages. Those lineages consisted either of toxic strains (two lineages) or non-toxic strains (three lineages). Only one lineage contained both toxic and non-toxic strains. Taking all strains together the presence/absence of the mcy gene cluster was not related to pigmentation: All toxic lineages and one non-toxic lineage contained both green- and red-pigmented strains. Conclusions: The fact that the mcy gene cluster seems to be patchily distributed is due to a so far unrecognized clonal dependence. We conclude that the clonal lineages have been adapted to significantly different ecological niches forcing their genetic differentiation. The identification of mcy gene cluster remnants supports the gene loss hypothesis and provides evidence against the role of a recent horizontal gene transfer. Interestingly the reddish type of pigmentation evolved several times independently in one toxic and two non-toxic lineages (after the loss of the mcy gene cluster).
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August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
OC-16.1 THE RESPIRATORY TERMINAL OXIDASES OF THE CYANOBACTERIUM SYNECHOCOCCUS SP. STRAIN PCC7942. Georg Schmetterer, Otto Kuntner, Heinrich Burgstaller, Günter Walder, Dominik Aschenbrenner. Institute of Physical Chemistry, Vienna, Austria. Introduction: All cyanobacteria respire with dioxygen as the terminal electron acceptor. The key enzymes of respiration are the respiratory terminal oxidases (RTOs) that transfer electrons to oxygen with water as the reaction product. A search for genes encoding putative RTOs in those cyanobacteria whose genomic sequence has been determined, has revealed a surprising variability both in the types of RTOs (at least 7 different types) and in the number of different RTOs per cell (1 to 10). Many cyanobacterial RTOs have not yet been characterized beyond their sequence similarity to RTOs from other organisms. At least some cyanobacteria have respiratory chains both in their cytoplasmic membranes (CM) and in their intracellular menbranes (thylakoids or ICM). In the ICM the respiratory chain shares components with the photosynthetic electron transport chain. The best studied cyanobacterium in this respect is Synechocystis sp. strain PCC6803 that has three RTOs: one genuine aa3-type cytochrome c oxidase, one quinol oxidase of the cytochrome bd-type - both localized in the ICM - and one ARTO, (Alternate Respiratory Terminal Oxidase, a homolog of cytochrome c oxidase that apparently does not react directly with soluble cytochrome c) localized in the CM. The aim of this work was to study cyanobacterial genes that show sequence similarity to RTO of the cytochrome cbb3 type that is well known from several organisms (including purple bacteria). In cyanobacteria, the corresponding gene products have so far not been characterized at all to the point that at the beginning of our study it was even unclear whether they actually encode an RTO. Only a few cyanobacteria (Synechococcus sp. PCC7942, Synechococcus sp. PCC6301 and Trichodesmium erythraeum IMS101) seem to contain these genes and among these the strain of choice was Synechococcus sp. strain PCC7942, since it is well amenable to genetic manipulation. In other organisms cbb3-type RTO accepts electrons from soluble cytochrome c and transfers them to dioxygen. In addition to the putative RTO of the cbb3 type , the genomic sequence of strain PCC7942 shows the presence of one set of genes encoding an aa3-type (mitochondrialtype) cytochrome c oxidase and a set of genes encoding a bd-type qunol oxidase. Methods: Mutants in two putative cbb3 genes (ccoN and ccoO) and also a mutant with a deletion of the subunit I of cytochrome c oxidase (coxA) were kindly provided by Dr. Susan Golden. We have developed a method to separate and purify cyanobacterial CM and ICM from a large culture (10 liters) using gradient centrifugation in a zonal rotor. The membranes were tested for in vitro cytochrome c oxidase activity by dual wavelength spectroscopy in a Varian Cary5 spectrophotometer using horse heart cytochrome c reduced by ascorbic acid as the electron donor. Results: Wild type strain PCC7942 displayed in vitro cytochrome c oxidation both in the CM and in the ICM. Compared to other cyanobacteria, the cytochrome c oxidation activity of PCC7942 CM is high on a per mg protein basis. The mutant strain lacking subunit I of aa3 type cytochrome c oxidase also shows in vitro cytochrome c oxidation in both CM and ICM. In contrast, the mutant strain lacking the ccoN gene displayed no cytochrome c oxidase activity in the ICM , but some activity in the CM. Conclusions: We have shown for the first time that the ccoN gene of August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Synechoccoccus sp. strain PCC7942 encodes a protein that is part of an RTO enzyme able to oxidise reduced cytochrome c in vitro. Since no activity could be measured in the ICM of the ccoN deletion mutant, our results imply that the cbb3 enzyme of this strain is the sole cytochrome c oxidase in these membranes. The cytochrome c oxidase activity activity of the CM of the ccoN mutant of strain PCC7942 must be due to the aa3-type cytochrome c oxidase. This is in striking contrast to Synechocystis sp. strain PCC6803, where the aa3-type cytochrome c oxidase is the only such enzyme in the ICM and is not present in the CM.
OC-16.2 PHYCOBILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA: STRUCTURE AND FUNCTION OF ENZYMES INVOLVED IN POSTTRANSLATIONAL MODIFICATION. Avijit Biswas1, Nicolle Saunée1, Crystal Miller1, Shervonda Williams1, Gaozhong Shen2, Donald A. Bryant2, Wendy M. Schluchter1. 1
Department of Biological Sciences, University of New Orleans, New Orleans, LA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; USA. Introduction: Cyanobacterial phycobiliproteins can contain one or more of four different types of isomeric bilins: phycourobilin, phycoerythrobilin, phycoviolobilin (also called phycobiliviolin), and phycocyanobilin (PCB). These linear tetrapyrroles are derived from heme and are covalently attached via thioether linkages at cysteine residues within the α- and β-subunits. Phycobiliproteins also contain a unique, post-translational modification of a conserved asparagine (Asn) present at β-72, which occurs only on the β-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases responsible for attaching PCB to phycobiliproteins, and the Asn methyl transferase for β-subunits in Synechococcus sp. PCC 7002. Methods: Genes encoding putative enzymes involved in posttranslational modifications of phycobiliproteins in Synechococcus sp. PCC 7002 were identified and characterized using comparative genomics, reverse genetics, in vitro enzyme assays of the recombinant proteins, and in vivo heterologous expression in E. coli. Results: Two new families of bilin lyases that are involved in phycobiliprotein biosynthesis in Synechococcus sp. PCC 7002, first identified in Fremyella diplosiphon as cpeS and cpeT, were characterized. There are three cpeS-like genes (denoted cpcS, cpcU, cpcV) and one cpeT-like gene (denoted cpcT) within the genome of Synechococcus sp. PCC 7002. The CpcS and CpcU proteins form a heterodimer (1:1) and catalyze the addition of PCB to Cys-82 on CpcB (β-phycocyanin) and most allophycocyanin subunits (ApcA, ApcB, ApcD, and ApcF). CpcT attaches PCB to Cys-153 on CpcB. ApcE was shown to have intrinsic bilin lyase activity and has a region with similarity to CpcS/CpcU. Many cyanobacteria have one CpcS/CpeS protein that catalyzes bilin attachment. We hypothesized that these proteins may form homodimers. Consistent with this, a 3D structure of CpcS-III from Thermosynechococcus elongatus BP1 (tll1699) was recently solved, and it crystallized as a homodimer. This is the first structure solved for any bilin lyase, and we have verified it is the Cys82 bilin lyase for attaching PCB to CpcB, ApcA and ApcB by in vitro enzyme assays. The cpcM gene encodes a protein with sequence similarity to other Sadenosylmethionine-dependent methyltransferases. Recombinant CpcM methylated the β-subunits of phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding α-subunits (CpcA, ApcA, and ApcD). Based upon in vitro studies with various substrates, 77
Final Program and Abstracts we conclude that this methylation probably occurs after chromophorylation but before trimer assembly in vivo. Conclusions: All enzymes required for post-translational modification of all phycobiliproteins in Synechococcus sp. PCC 7002 have now been identified and characterized, and we are beginning to characterize the order in which these post-translational modifications occur during phycobiliprotein biosynthesis. Now that the first bilin lyase structure has been solved, we hope to gain more mechanistic insight into how these enzymes catalyze the specific addition of bilins to their phycobiliprotein substrates.
OC-16.3 SPECTRAL AND STRUCTURAL CHARACTERIZATION OF A NOVEL CYANOBACTERIOCHROME-TYPE PHOTORECEPTOR AnPixJ. Rei Narikawa, Norifumi Muraki, Yoshimasa Fukushima, Yuu Hirose, Tomoo Shiba, Shigeru Itoh, Genji Kurisu, Masahiko Ikeuchi. University of Tokyo, Meguro, Tokyo, Japan. Introduction: Phytochromes of plants, cyanobacteria and some bacteria are the photoreceptor superfamily that binds a linear tetrapyrrole and exhibits reversible photoconversion between the redabsorbing (Pr) and the far-red-absorbing forms. They exhibit photochemical isomerization between the C15-Z and C15-E isomers of the linear tetrapyrroles. GAF domain plays a crucial role in the function of the chromophore. Cyanobacteriochromes are the recently emerging photoreceptors in cyanobacteria whose GAF domains are related to but distinct from those of the phytochromes. Mutational studies in cyanobacteria have revealed that they are involved in various photoacclimation responses such as chromatic acclimation and phototaxis. GAF domains in the phototaxis regulator SyPixJ1 (from Synechocystis sp. PCC 6803) and TePixJ (from Thermosynechococcus elongatus BP-1) bind a phycoviolobilin and undergo the reversible photoconversion between blue-absorbing and green-absorbing (Pg) forms. Similar GAF domains identified in cyanobacterial genomes may function in novel photoreceptor proteins. Methods: The chromophore-binding GAF domains of the cyanobacteriochromes expressed in Synechocystis or phycocyanobilin (PCB) -producing Escherichia coli have been purified. The purified proteins were characterized by the spectral and crystallographic analyses Results: Putative phototaxis regulator AnPixJ (from Anabaena sp. PCC 7120) binds PCB and shows reversible photoconversion between Pr and Pg forms. Time-resolved spectral analysis suggest that the Pr form of AnPixJ is almost equivalent to that of the phytochromes and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the phytochromes, but is finally photoconverted to the unique Pg form. On the other hand, chromatic acclimation regulator SyCcaS (from Synechocystis) covalently binds PCB and shows similar reversible photoconversion between Pr and Pg forms. However, relationship of chromophore configuration and spectral properties is opposite between AnPixJ and SyCcaS. To understand the structural basis, we are now trying the X-ray crystallographic analyses. We determined the crystal structure of the GAF domain of AnPixJ in the Pr form. The backbone structure of the protein and the chromophore configuration are independently similar to those of the Pr form of bacterial phytochromes, although relative position of the chromophore bound to the protein is significantly deviated. Correspondingly, critical amino acid residues interacting with the chromophore are also diverged in position or orientation, and 78
13th International Symposium on Phototrophic Prokaryotes resultantly in the function. Together with the role of water molecules, a novel protonation mechanism of the chromophore is proposed. Unique features of a hydrophobic pocket to accommodate photoisomerization of the chromophore are discussed with regard to its unusual formation of the Pg form. We will discuss about structurefunction relationship of the tetrapyrrole-based photoreceptors. Conclusion: We found various spectral properties of the cyanobacteriochromes. Spectral analyses suggest that these properties are yielded by different chromophores, different conjugated systems or different photoconversion mechanisms. Structure of the Pr form of AnPixJ provides direct insights into the photoconversion mechanism. This new structural basis shed light on the universal aspects of the photosensory mechanism of linear tetrapyrrole-based photoreceptors.
OC-16.4 CHARACTERIZATION OF THE VAP TOXIN-ANTITOXIN SYSTEM OF SYNECHOCOCCUS ELONGATUS REVEALS A NEW ANTIDOTE MOLECULE. Eleonora Sendersky, Sagiv Shaar, Elizabeth Ginsberg, Rakefet Schwarz. Bar-Ilan University, Ramat-Gan, Israel. Introduction: Toxin-Antitoxin (TA) loci are ubiquitously present on chromosomal DNA of diverse bacteria. These modules were assigned a role in programmed cell death and it has been suggested that the population as a whole benefits from sacrificing individual cells. In other cases it has been shown that the un-neutralized toxin does not cause cell death, rather, it re-sets cell physiology and adjusts it to stress conditions. In the course of our global transcriptome analyses of the cyanobacterium Synechococcus elongatus, we noted that vapC, encoding for a toxin homolog, is highly induced upon nitrogen and sulfur starvation. Chromosomal TA systems of cyanobacteria have not been characterized thus far. Additionally, S. elongatus does not possess a homolog of VapB, the typical cognate antidote of known Vap-systems. Thus, it was highly interesting to examine if VapC acts as part of a TA system or functions as ‘solitary toxin’ and to address the cellular role of VapC in the context of a cyanobacterial cell. Methods: We employed genetic and molecular tools including gene inactivation, over-expression and protein tagging to characterize the nature and role of VapC and the new antidote we identified. Results: To test if VapC has a potential cytotoxic effect on S. elongatus cells, we expressed it using a strong promoter. VapC accumulation upon induction, as verified by Western analysis, was accompanied by rapid cell death. TA modules are typically encoded by operons, in which the gene for the antidote molecule precedes the toxin gene. Upstream of vapC, we noted an ORF and termed it VapX; this ORF is not defined as a potential coding region in the current annotation of S. elongatus genome. We bring evidence for the function of VapX, which is not homologous to proteins of known function, as an antitoxin. Over-expression of VapX together with VapC counteracts the killing effect observed when VapC is over-expressed by itself. Furthermore, Northern analysis and RT-PCR indicate that vapXC form a bicistronic operon, a typical feature of TA modules. Experiments involving expression of VapX and VapC in Escherichia coli further support the function of these components as a TA system. Additionally, these experiments indicate complex formation between VapC and VapX, in agreement with the suggested antidote function of the latter. Conclusions: VapC of S. elongatus has a cytotoxic effect when expressed by a strong promoter. Additionally, we present evidence
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
based on several observations supporting identification of VapX as a new antitoxin of the Vap-system. To the best of our knowledge, our study provides the first characterization of cyanobacterial chromosomal TA system. The role of the Vap-system of S. elongatus will be discussed in light of quantitative analyses of natively expressed VapX and VapC and inactivation of vapXC as well as additional TA systems of this cyanobacterium.
Calcium is then subject to trans-cellular transport along the filament and extruded at the lagging end to the outside aqueous medium. Proton antiport at the apical cell likely maintains charge and neutralizes alkalization by excess carbonate ions. Incidentally, an evolutionary pressure not to interfere with calcium trafficking, may be behind the unusual property of Mastigocoleus (and BC008) to develop lateral heterocysts.
OC-16.5
OC-16.6
HOW CYANOBACTERIA BORE (AND WHY LATERAL HETEROCYSTS EXIST).
FERRITIN FAMILY PROTEINS AT THE CROSS ROADS BETWEEN IRON HOMEOSTASIS AND OXIDATIVE STRESS.
F. Garcia-Pichel, E. Ramirez-Reinat, Q. Gao.
Sigal Shcolnick1, Tina Summerfield3, Lilia Reytman1, Louis Sherman2, Nir Keren1.
School of Life Sciences, Arizona State University, Tempe, AZ, USA. Introduction: Some cyanobacteria bore into carbonates. They are few, but ancient players in geologic phenomena such as the destruction of coastal limestones, the reworking of carbonate sands and the cementation of microbialites. They constitute important tools for paleoenvironmental reconstruction, and play a significant role as pests in marine aquaculture. In spite of their importance, the mechanism by which cyanobacteria excavate minerals remains unknown. In fact, this ability represents a geochemical paradox, in that autotrophic metabolism will push the carbonate system towards precipitation, not dissolution. We have advanced mechanistic models that may allow cyanobacterial boring to proceed and be still consistent with geochemistry. These are based on either temporal or spatial separation of photosynthesis and boring, or on the active extrusion of calcium through a cellular uptake and transport process. Until now, experimental approaches were hampered by the lack of cyanobacteria that could bore in the laboratory. We report here on investigations using a novel isolate of the filamentous heterocystous cyanobacterium Mastigocoleus/Fischerella strain BC008 that maintains boring in the laboratory. Methods: Growth and boring of axenic cultures of BC008 were investigated on different crystalline minerals, and under a variety of conditions. The microenvironments of cyanobacterial filaments actively boring on calcite chips were monitored quantitatively for free Ca2+ using in vivo fluorophore-enabled confocal fluorescence microscopy. The temporal dynamics of a variety of physical and chemical inhibitors on calcium transport dynamics in the cyanobacterium/mineral system were tracked using this system. PCR-based methods were used to detect and track the differential expression of genes putatively involved in boring. Results: We could easily reject hypotheses involving the action of accompanying heterotrophic bacteria or the temporal separation of photosynthesis and boring. The range of mineral carbonates bored (Ca-bearing, or those containing its biochemical analog Sr, but not those with Mg, Mg/Ca, Mn, Fe, K, or Na as metal) implicated Ca as necessary for boring. Extracellular Ca2+ levels in and around the boreholes of actively boring BC008 were consistent with an active uptake Ca2+ at the apical end cell (severe under-saturation) and extrusion at the surficial end of the filaments (supersaturation). This out-of-equilibrium state could be relaxed by i) ceasing illumination, ii) adding inhibitors of ATP generation, and iii) adding specific inhibitors of P-type calcium ATPases, indicating that cellular energy is required for this process, as are ATP-ase calcium transport systems. P-type ATPase genes could be detected in BC008’s genome.
1
The Alexander Silberman Institute of Life Sciences, Department of Plant and Environmental Sciences, Hebrew University, Givat Ram, Jerusalem, Israel; 2Department of Biological Sciences Purdue University, West Lafayette, IN, USA; 3 Department of Botany, University of Otago, Dunedin, New Zealand. Introduction: Iron is an essential nutrient for the survival of all organisms, including cyanobacteria. However, the same redox properties that makes iron a valuable cofactor also lead to oxidative interactions resulting in the formation of harmful radicals. Therefore, iron accumulation in the cells should be tightly regulated. Ferritin family proteins play an important role in iron homeostasis. Synechocystis sp. PCC 6803 contains two ferritin type storage complexes, bacterioferritin (BFR) and MrgA. Previous studies demonstrated the role of BFR and MrgA in iron storage. In addition, MrgA was found to play a key role in oxidative stress response. Methods: In the study presented here, we examined the dual role of the ferritin family proteins using physiological and transcriptomic approaches. These include measurements of growth of cyanobacterial cultures in media limited for iron by various chelators; analysis of internal transition metal quotas by ICP-MS; measurements of photosynthetic performance and transcriptional profiling by microarray analysis. Results: Microarray analysis of iron limited wild type and ∆mrgA cultures revealed a remarkable up-regulation of oxidative stress related genes in the mutant cells. The PerR regulator was found to play an important role in that process. Furthermore, we were able to demonstrate the connection between internal iron quota, the presence of the two storage proteins and the sensitivity to externally applied oxidative stress. These data suggests a pivotal role for the two ferritin type protein-complexes of Synechocystis 6803 cells in coordinating iron homeostasis and the oxidative stress response. Conclusions: The combined action of the two complexes allows for the safe accumulation and release of iron from storage by minimizing damage caused by the interaction reduced iron and oxygen radicals which are abundant in cyanobacterial cells due to the function of the photosynthetic apparatus.
Conclusions: Our experiments suggest that cyanobacteria bore by actively taking up Ca2+ ions from the leading end of a filament at the cost of energy, locally lowering extracellular calcium levels to the point that mineral equilibrium is displaced towards dissolution there. August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
OC-17.1 RECOMBINANT PURPLE BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS, HARBORING THE CRTI REPORTER GENE TO MONITOR ENVIRONMENTAL TOXIC METALS. Isamu Maeda, Kazuyuki Yoshida, Md. Harun-ur-Rashid. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan. Introduction: Genetically engineered bacterial biosensors utilize living cells as sensing elements that harbor a reporter gene linked to a transcriptional switch. The genetic engineering makes it possible for bacterial cells to elicit signal in response to a small amount of environmental chemicals as effectors. Reporter gene systems that do not require instruments and exogenous chromogenic reagents are advantageous in establishing simple monitoring protocols. By taking it into account, the usefulness of several genes involved in carotenoid biosynthesis pathways has been examined, and the crtI gene has been selected as the reporter gene system of the purple bacterium, Rps. palustris. In the crtI reporter gene system, the crtI gene placed downstream of a transcriptional switch is expressed, and complements the crtI deletion of host bacterium, only in the presence of effectors. In this study, Rps. palustris strains responding to arsenic and lead/cadmium were constructed, and their possibilities as biosensors for environmental monitoring were examined. Methods: The green mutant, no. 711, was constructed through deletion of crtI from the chromosome. The arsenic crtI biosensor (AsCrtIBS) was made of the no. 711 strain transformed with the crtI gene under regulation of the operator/promoter region of the ars operon and the arsR gene from Escherichia coli. The hmrR gene of Rps. palustris, encoding the heavy metal resistance transcriptional regulator HmrR, and its upstream region were chosen as the transcriptional switch of the lead/cadmium crtI biosensor (Pb/Cd-CrtIBS).
13th International Symposium on Phototrophic Prokaryotes approximately 1 mM. Further, its susceptibility to tellurite under respiratory conditions is affected by the carbon source. Under these conditions the MIC for tellurite varies from 8 μM, with malate and other dicarboxylic acids, to 300 μM with acetate. R. capsulatus is also characterized by a high tellurite uptake rate as compared to other bacterial species. Uptake activity measurements have shown that tellurite enters the cells at a much lower rate when acetate is present. This effect is not seen when dicarboxylic acids are used instead (1). These early data have led to propose that tellurite exploits an acetate transport system to enter R. capsulatus cells. In this work we show, for the first time in a bacterial system, that the main entry gate for tellurite is acetate permease (actP), and that an insertional mutation in this gene is sufficient to drastically reduce the oxyanion uptake. Methods: Resistant mutants were isolated on aerobic RCV standard medium in the presence of 100 μM tellurite. The actP insertional mutant was constructed using the pJP5603 suicide vector (2). Tellurite uptake was measured as described by Turner et al. (3). MIC determinations, conjugations, subcloning and PCR amplification were performed using standard molecular procedures. Results: The resistant mutant RTe36, used in this work, showed a MIC 50 fold higher than the wild type B100 and an uptake rate that was limited to 25% of the wild type control. Complementation of the mutant with a R. capsulatus cosmid library allowed the isolation of two cosmids which restored high level uptake and reduced the MIC well below that measured in the mutant, but still above the w.t. basal level. From the cosmids it was subcloned the acetate permease gene (actP), which was identified by DNA sequencing, and was shown to complement the mutant by restoring both the sensitivity to tellurite and the uptake level of the w.t.. Finally, to confirm the role of actP, an acetate permease insertion mutant was constructed. Initial experiments indicated that the actP – mutant has indeed a decreased uptake and a MIC for tellurite higher than wild type.
Results: As-CrtIBS and Pb/Cd-CrtIBS changed color in response to 10 μg/L or less As(III) and Pb(II)/Cd(II), respectively. This change could be distinguishable to the sense of sight after 24-hour culture without further manipulation. The hue angle of cultures in the CIE-L*a*b* color space shifted from greenish yellow toward red when concentration of the metals increased (Fig). As(III) supplemented to mineral waters on the market and environmental arsenic in Bangladesh well waters were monitored. Although the basal color in the absence of As(III) shifted to red with an increase in the hardness of mineral water, As-CrtIBS held responsibility to As(III). As-CrtIBS could screen the well waters indicating an arsenic concentration higher than 1 μg/L.
Conclusions: This work demonstrates, for the first time, that acetate permease is the main gate for tellurite entry into R. capsulatus cells. The residual uptake, still measurable in the mutant, indicates that tellurite can take advantage of other less efficient entry facilities. The failure to bring the MIC for tellurite in the complemented mutant, back to level of the wild type, as opposed to the complete restoration of the uptake rate, indicates that other mutations are present, determining the high level of resistance measured in the mutant. Work is already planned to address this question. Finally, it remains to be determined if acetate permease is involved in tellurite uptake in other bacterial species and, if so, how spread is this mechanism.
Conclusion: The crtI biosensors indicate practical detection limits and are applicable to monitoring of toxic metals in groundwater and freshwater by the naked eye.
Turner R.J., Weiner J.H. and Taylor D.E. (1992) Anal. Biochem., 204:292-295
OC-17.2
Borghese R., Marchetti D. and Zannoni D. (2008) Arch. Microbiol. 189:93-100 Penfold R.J. and Pemberton J.M. (1992) Gene, 118:145-146
OC-17.3
THE TOXIC OXYANION TELLURITE ENTERS RHODOBACTER CAPSULATUS CELLS VIA ACETATE PERMEASE.
CHROMIUM (VI) REMOVAL FROM WASTE WATERS OF A CRPLATING INDUSTRY WITH EXOPOLYSACCHARIDE-PRODUCING CYANOBACTERIA.
Roberto Borghese, Davide Zannoni.
Giovanni Colica, Pier Cesare Mecarozzi, Roberto De Philippis
Department of Biology, University of Bologna, Bologna, Italy.
Deparment of Agricultural Biotechnology, University of Florence, Firenze, Italy.
Introduction: Rhodobacter capsulatus is a purple non-sulfur phototrophic bacterium that shows a highly variable response to the toxic oxyanion tellurite. It is highly susceptible to the oxyanion while growing by respiration with a MIC (minimal inhibitory concentration) of 8 μM, and is very resistant when grown phototrophycally with a MIC of 80
Introduction: Chromium (VI) compounds are contained in the waste waters deriving from a large number of industrial processes. In the last twenty years, the possibility to use specific microorganisms for the removal of heavy metals from industrial waste waters has been widely August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes studied, but the possibility to use exopolysaccharide (EPS)-producing cyanobacteria for the removal of heavy metals in the anionic form was never tested. The aim of this study was the assessment of the capability of some EPS-producing cyanobacteria to remove chromate from waste waters of a plating industry in lab and in semi-pilot systems and to define the role of the different cell fractions (i.e. biomass and polysaccharidic layers) in the metal removal process. Methods: Exopolysaccharide-producing Cyanothece ET5, TI4, PE14, VI22, CE4, Cyanospira capsulata and Nostoc PCC7936, were tested for their ability to remove Cr (VI) from waste waters of a plating industry containing Cr (VI) 89.71% (w/w), Cr (III) 8.97%, other metals 1.32%. Lab experiments were carried out for 48h with 50 ml of acidpre-treated cyanobacterial cultures confined in dialysis tubing and dipped in 450 ml of the diluted industrial wastewater (pH 3; Cr (VI) concentrations in the range 10-200 mg L-1). Field experiments were carried out with acid-pre-treated cultures confined in three different semi-pilot systems (a filter press, a filter column and a dialysis membrane system) operating with 30 L of the same diluted waste water. Results: In lab experiments, Cr (VI) was removed by Cyanothece CE4 at a specific metal uptake of 18.0 ± 0.8 mg of Cr(VI) per g of cell dry weight, while three other strains, Cyanothece ET5 and PE14 and Nostoc PCC7936 showed a metal uptake in the range 10 to 12 mg (Cr) (g dry wt)-1. All the other strains showed a lower capability to remove the metal. The three semi-pilot systems operating with Nostoc PCC7936 biomass showed a very different behavior in the metal removal process: in the dialysis membrane system, a decrease of the concentration of Cr (VI) and of total Cr was observed, both concentrations reaching, after 72 hours, values corresponding to about 60% of the initial ones. In the filter press system, the metal removal was done by flushing 30L of the diluted waste water containing 112 mg L-1 of Cr (VI). In the experiment, a decrease in the concentration of Cr (VI) down to a value of 0.40 mg L-1 was observed after 48 h; at the same time, the total Cr concentration only decreased from 123 to 53 mg L-1, owing to a very significant increase in the amount of Cr (III) present in solution. In the column system, after 24 h almost all the Cr (VI) was reduced to Cr (III), without any significant decrease of the total Cr concentration. Thus, dried biomass of Cyanothece TI4, which is known to have a very high Cr3+ uptake, was added on the top of the column. In the following 120 h, the concentration of total Cr and of Cr (III) decreased to a value of 3.6 mg L-1, while the concentration of Cr (VI) reached the value of zero. Conclusions: The results obtained pointed out the complex role played by the cyanobacterial biomass in the removal of Cr (VI): the cells, when subjected to treatments removing the external polysaccharidic structures, carried out the reduction of Cr (VI) to Cr (III), while the polysaccharidic fraction, previously released by the cells and solubilized in the culture medium, was capable to remove the Cr (III) cations formed in the reduction process. The results obtained in the semi-pilot systems showed that EPS-producing cyanobacteria can be profitably used for the removal Cr (VI) from industrial waste waters.
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Final Program and Abstracts
OC-18.1 TRANSCRIPTIONAL REGULATION AND MATURATION OF CYANOBACTERIAL HYDROGENASES. Peter Lindblad. Department of Photochemistry & Molecular Science, Uppsala University, Uppsala, Sweden. Introduction: In cyanobacteria three enzymes are directly involved in the hydrogen metabolism: a nitrogenase that produces H2 as a byproduct of nitrogen fixation, an uptake hydrogenase (HupSL, encoded by hupSL) that recaptures H2 and oxidize it, and a bidirectional hydrogenase (encoded by hoxEFUYH, forming an enzyme with a hydrogenase part, HoxYH, and an electron transfer partner protein, HoxEFU) that can both oxidize and produce H2, see Tamagnini et al 2007. The transcription of cyanobacterial hup and hox operons are known to be regulated. The maturation of hydrogenases into active enzymes is a complex process and eg a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded by either hupW or hoxW. We have addressed trancriptional regulation and hydrogenase maturation in a few selected strains. Methods: The unicellular cyanobacterium Synechocystis PCC 6803 contains a single bidirectional hydrogenase. The filamentous cyanobacteria Nostoc punctiforme and Nostoc PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase, respectively. Molecular experiments were performed as detailed elsewhere. Results: Generally, little is known about the transcriptional regulation of cyanobacterial uptake hydrogenases. We showed that NtcA has a specific affinity to a region of the hupSL promoter of Nostoc punctiforme. Truncated versions of the promoter region of the hupSL operon were fused to gfp. All constructs showed heterocyst specific expression (Holmqvist et al 2009). Unexpectedly the shortest promoter fragment, covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. The cyanobacterial hox genes have been characterised in some strains. LexA interacts with the promoter region of the hox operon in Synechocystis PCC 6803. The hox genes of Nostoc PCC 7120 are transcribed as two different clusters with LexA interacting with both promoter regions. DNA affinity assays also identified an AbrB-type DNA-binding protein as interacting directly with the promoter region of the hox operon in Synechocystis PCC 6803. This protein binds to the its own promoter region and the promoter region of the hox operon, suggested to function as a positive regulator of hox gene expression. The presence and expression of hyp-genes were examined in the N2-fixing cyanobacterium Nostoc PCC 7120. RT-PCRs demonstrated that the six hyp-genes may be transcribed as a single operon. TSPs were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively (Agervald et al 2008). Five upstream ORFs located in between hupSL and the hyp-operon, and two downstream ORFs from the hyp-genes were shown to be part of the same transcript unit. Transcriptional analyses were performed of hupW in Nostoc punctiforme, and hupW and hoxW in Nostoc PCC 7120. We identified numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW) (Devine et al 2009). A phylogenetic tree of hupW & hoxW showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Bioinformatic studies revealed a so called “HOXBOX”; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.
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Final Program and Abstracts Conclusions: Significant advances have been made in the understanding of both the transcriptional regulations and the maturation of cyanobacterial hydrogenases. References: Agervald Å, Stensjö K, Holmqvist M, Lindblad P. 2008. BMC Microbiol 8: 69 Devine E, Holmqvist M, Stensjö K, Lindblad P. 2009. BMC Microbiol 9: 53
either an integration or expression plasmid. The Rubrivivax transgenes are cooL, U, and H encoding the small and large subunits of a typical NiFe-hydrogenase as well as hypA, putatively involved in nickel insertion for hydrogenase maturation. However, no hydrogenase activity was detected in the recombinant Synechocystis strain, suggesting the need to transfer additional Rubrivivax hydrogenase genes to yield activity.
Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris D, Heidorn T & Lindblad P. 2007. FEMS Microbiol Rev 31: 692-720
Conclusions: Our approach to generate knockout mutants lacking the various Hox subunits led to the conclusion that (1) HoxYH alone constitutes in vitro hydrogenase activity and (2) HoxEFU are required for complex stability and for NAD(P)H-supported H2 production, in vivo. Work is underway to express additional Rubrivivax coo hydrogenase and its hyp maturation genes into Synechocystis, presumably needed to confer activity for sustained photobiological H2 production.
OC-18.2
OC-18.3
PHOTOBIOLOGICAL HYDROGEN PRODUCTION IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC6803.
ISOLATION AND SPECTROBIOCHEMICAL CHARACTERIZATION OF THE BIDIRECTIONAL [NIFE]-HYDROGENASE FROM SYNECHOCYSTIS SP. PCC 6803.
Holmqvist M, Stensjö K, Oliveira P, Lindberg P, Lindblad P. 2009. BMC Microbiol 9: 54 Oliveira P & Lindblad P. 2008. J Bacteriol 190: 1011-1019
Carrie Eckert, Jianping Yu, and Pin-Ching Maness. National Renewable Energy Laboratory, Golden, CO, USA. Introduction: The photosynthetic cyanobacterium Synechocystis sp. PCC6803 has the capacity to integrate light harvesting with charge separation and transfer the electrons to a bidirectional NiFehydrogenase for the photoproduction of H2 from water. However, the H2-production reaction is short-lived in the presence of O2, the latter an inherent byproduct of oxygenic photosynthesis. We propose two approaches to surmount this challenge: (1) to gain more in-depth understanding of the Synechocystis Hox hydrogenase for its improvement; and (2) to genetically transfer and express a foreign O2tolerant hydrogenase into Synechocystis. Both approaches will guide the design of an O2-tolerant system in Synechocystis for sustained H2 production. Methods: The native Synechocystis Hox hydrogenase is a pentameric complex consisting of HoxEFUYH subunits. HoxYH are the catalytic subunits for H2 production, accepting electrons from NAD(P)H mediated by the HoxEFU diaphorase moiety. We are genetically dissecting the 5-subunit Hox complex by generating mutants lacking one or more of the subunits in order to probe their roles both in maintaining complex stability and in hydrogenase activity. Our second approach entails the genetic transfer and expression in Synechocystis a hexameric O2-tolerant NiFe-hydrogenase (CooMKLXUH) from the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus. The Coo hydrogenase is ferredoxin linked; its expression in Synechocystis may improve the overall efficiency of H2 production using a photo-reduced ferredoxin as the electron mediator. Results: We have either acquired or generated Synechocystis mutant lines lacking one or more of the Hox subunits. Western blots confirmed the deletion of the subunits using their respective antibodies. We determined that mutant containing only the HoxY and HoxH subunits retained hydrogenase activity in vitro, using methyl viologen as the redox mediator reduced by sodium dithionite. However, this mutant cannot produce H2 in vivo likely due to the lack of HoxEFU subunits, required for electron mediation from NAD(P)H. Moreover, mutants with deletions in HoxE, F, or U individually or in various combinations still retained 20 to 50% of its in vitro hydrogenase activity, with most loss observed with deletions of the HoxFU subunits. In the second approach, we have successfully transformed and expressed four Rubrivivax hydrogenase and its related genes into Synechocystis hosts lacking native hoxH, using 82
Jens Appel1, Ingo Zebger2, Miguel Saggu2, Friedhelm Lendzian2, Rüdiger Schulz3 and Frauke Germer3. 1
School of Life Sciences, Arizona State University, Tempe, AZ, USA; Max-Volmer-Laboratorium, Technische Universität, Berlin, Germany; 3 Botanisches Institut, Universität Kiel, Kiel, Germany. 2
Introduction: In cyanobacteria two different types of hydrogenases are known. They both belong to the NiFe-hydrogenases and are called uptake and bidirectional due to their physiological characteristics. The cyanobacterial bidirectional hydrogenase is of particular interest, since it is rapidly reactivated in the absence of oxygen and its traits seem to be intermediate between the standard oxygen-sensitive and the oxygen-tolerant NiFe-hydrogenases. In addition, this hydrogenase is well suited for solar-driven biological hydrogen production in cyanobacteria. In this study we developed a rapid and gentle method to purify the bidirectional hydrogenase of Synechocystis sp. PCC 6803 for biochemical as well spectroscopical investigations to characterize its active site. Methods: To overcome the low expression level of the bidirectional hydrogenase and to simplify its isolation a strain containing the psbAII promoter upstream of the hox-operon was constructed. In the same strain the Strep tag II was fused to the C-terminus of hoxF encoding a subunit of the diaphorase part of the enzyme. In the same strain a hyp-gene cluster containing the genes necessary for the posttranslational processing of the hydrogenase was also overexpressed. After isolation, the hydrogenase was subjected to EPR (electron pulse resonance) and FT-IR (Fourier transform infrared) spectroscopy in different redox states. Results: The insertion of the psbAII promoter in conjunction with the Strep tag led to an increase of total hydrogenase activity of two fold. Overexpressing the hyp-genes led to an additional two fold increase. From these strains the hydrogenase could be isolated in a highly pure and active state after a single run of the affinity column. EPR measurements reveal the presence of at least two redox active FeS clusters, but no nickel specific signals. A paramagnetic Ni-species was absent under any conditions. This either indicates that the nickel stays in the diamagnetic Ni(II) state and the redox changes take place at another ligand or that its spin is coupled to a nearby paramagnetic center in the protein. August 9 to 14, 2009 • Montréal, QC, Canada
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In the FT-IR spectra three different signals could be discerned, that are attributed to two cyanides and one carbon monoxide coordinating the iron ion. The FT-IR spectra under different redox conditions are indicative of a NiFe-hydrogenase attaining a Ni-B like state not the Ni-A state when oxidatively inactivated even in the presence of oxygen. For other hydrogenases it is known that they can be rapidly activated when in the Ni-B state but need much more time when in the Ni-A state. Conclusions: The spectroscopic investigations of cyanobacterial bidirectional hydrogenase confirm its intermediary behavior between the anaerobic standard enzymes and the aerobic oxygen tolerant enzymes. With these characteristics it is very well adapted to rapidly changing redox conditions in oxygenic phototrophs. It will be quickly activated under anoxic conditions and able to produce hydrogen by fermentation. It will produce hydrogen as an overflow valve when photosynthesis resumes from oxygen free conditions, but it will also be very rapidly inactivated when photosynthetic oxygen production takes over so as not to waste energy.
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P.001 FUNCTIONAL ANALYSIS OF THE PUH PROTEINS IN RHODOSPIRILLUM RUBRUM. Caroline Autenrieth, Robin Ghosh. Dept. of Bioenergetics, University of Stuttgart, Stuttgart, Germany. Introduction: Our present view of the photosynthetic unit (PSU) of Rhodospirillum rubrum is that it contains a reaction centre (RC; H, L and M subunits) surrounded by a light-harvesting (LH) 1 complex, composed of alpha and beta subunits which aggregate to form a 16fold circular assembly. The polypeptides of the LH1 complex as well as the L and M subunits of the RC are encoded by the puf operon, whereas the H subunit is encoded by the puhA gene of the puh operon. The puh operon encodes a number other putative gene products (puhB, puhC, puhD and puhE) which have been implicated in PSU assembly though their precise role has been unclear so far. In this study we provide new biochemical, functional insight into the role of some of the puh proteins in PSU assembly. Methods and Results: Selected puh proteins were deleted from the R. rubrum chromosome and the phenotype examined. The phenotype, which is unique so far, was quantified functionally using spectroscopic and biochemical methods. The results point to an unsuspected role of the puh proteins in PSU assembly. Conclusions: We will present data as well as a structural hypothesis, that for R. rubrum, the structure of the PSU is more complex than previously thought. In particular, we present a new biochemical insight into the role of the proteins encoded by the puh operon. If our hypothesis is correct, our view of the PSU will have wide-reaching consequences for the interpretations of spectroscopic, cryoEM and AFM data in general.
P.002 THE ROLE OF CYTOCHROME C-554 IN THE ELECTRON TRANSFER PATHWAYS IN THE GREEN SULFUR BACTERIUM CHLOROBACULUM TEPIDUM. Chihiro Azai1, Yusuke Tsukatani2, Jiro Harada3, Ryo Miyamoto4, Toru Kondo4, Hiroumi Murakami4, Shigeru Itoh4, Hirozo Oh-oka1. 1
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043; 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki; 3Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga; 4Division of Material Science, Graduate School of Science, Nagoya University, Nagoya, Aichi; Japan. Introduction: In the green sulfur bacterium Chlorobaculum (Cba.) tepidum (syn. Chlorobium tepidum), the photo-oxidized primary electron donor, P840+, is rapidly rereduced by cytochrome (cyt) cz, one of subunits of the reaction center (RC) complex. Previous studies have revealed two different electron donors to the oxidized cyt cz. In the purified membranes, menaquinol:cyt c oxidoreductase function as the direct electron donor to cyt cz, while in the in vitro reconstitution experiment using the purified RC complex, water-soluble and low molecular-weight (approx. 10,000) cyt c-554 (CT0075) does. However, it has still remained unknown whether the electron transfer scheme from menaquinol:cyt c oxidoreductase to cyt cz via cyt c-554 is operative when all of them co-exist together in vivo. On the other hands, green sulfur bacteria use reduced sulfur compounds (sulfide, thiosulfate, polysulfide and elemental sulfur) as August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts the photosynthetic electron sources. Electrons obtained from the oxidations of these sulfur compounds are transferred to the RC through the photosynthetic electron transfer pathways. Although several in vitro reconstitution experiments carried out in 1970s demonstrated that cyt c-555 isolated from Cba. thiosulfatiphilum, which corresponds to cyt c-554 from Cba. tepidum, could accept electrons from sulfide/thiosulfate oxidation systems, there has been no experimental evidence to support this in vivo. In this study, we constructed three mutants of Cba. tepidum, which were devoid of cyt c-554 (cycA::aadA strain), SoxB (CT1021) (soxB::aacC1 strain), and cyt c-554 and SoxB (cycA::aadA/soxB::aacC1 strain), respectively, to investigate electron transfer pathways coupled with sulfide/thiosulfate oxidations. Results: When purified cyt c-554 was added to the membranes prepared from the cycA::aadA strain, the oxidized cyt cz, immediately formed after flash excitation, was rereduced by both cyt c-554 and quinol oxidoreductase independently; the contribution of the former became larger by increasing its concentration, indicating that the reaction between cyt c-554 and cyt cz obeyed the second-order reaction mode. Unlike the case of the electron transfer reaction from cyt bc1 complex to cyt c2 in purple bacteria, cyt c-554 in the Cba. tepidum never serves as a shuttle-like carrier between menaquinol:cyt c oxidoreductase and cyt cz. The cycA::aadA strain exhibited a decreased growth rate but normal growth yield when compared to the wild type. In accordance with this, the strain was found to oxidize thiosulfate more slowly than the wild type but completely to sulfate as the wild type. This indicates that cyt c-554 is not indispensable for thiosulfate oxidation itself. Furthermore, both the soxB::aacC1 and cycA::aadA/soxB::aacC1 strains did not grow at all in a medium containing only thiosulfate as an electron source. They also grew incompletely even in a medium containing only sulfide when compared to the wild type. This suggests that SoxB is not only essential for thiosulfate oxidation but also responsible for sulfide oxidation. Conclusions: In Cba. tepidum, cyt c-554 serves as an electron carrier between Sox system and cyt cz in the RC. But an alternative electron carrier or electron transfer path should be operating between them, judging from the complete oxidation of thiosulfate in the cycA::aadA strain. The Sox system also seems to be involved in the sulfide oxidation, whose issue would be a future work to be resolved.
P.003 EXPRESSION OF HYDSL HYDROGENASE FROM THIOCAPSA ROSEOPERSICINA IN ESCHERICHIA. COLI. Horcheska Batyrova, Anna Khusnutdinova, Galina Shirshikova, Olga Postnikova, Elena Patrusheva, Aleksander Boutanaev, Anatoly Tsygankov. Institute of Basic Biological Problems, Institutskaya, Pushchino, Moscow Region, Russia. Introduction: The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS contains several Ni-Fe hydrogenases. One of two membrane-bound hydrogenases appears to be very stable against temperature, CO, H2S and proteases. The Ni-Fe hydrogenase HydSL assembly includes the concordant expression of structural genes (hydS and hydL with two additional genes of unknown functional) as well as a number of accessory genes, participating in the maturation process of this enzyme. Structural genes have been cloned by the K.L. Kovacs team (Szeged, Hungary) earlier and their sequences are available in Genbank database. 85
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Final Program and Abstracts The hydSL genes are expressed on the low level in T. roseopersicina what makes difficult both basic and applied investigations of the protein. One possibility to increase HydSL yield might be heterologous expression in a well established system, for instance, such as Escherichia. coli. However, little is known about this possibility. From the other hand, unlike many other hydrogenases, accessory genes of HydSL are dispersed along the T. roseopersicina chromosome what makes difficult their complementation study in E. coli cells. The aim of this research is the expression of hydS and hydL in heterologous system. Methods: In this investigation we started from an attempt to express in E. coli two structural genes hydS and hydL, keeping in mind that some of E. coli accessory genes might complement the lack of their counterparts in T. roseopersicina, as well as the fact that high level of Ni in growth medium might compensate, at least partially, some of T. roseopersicina accessory genes. Two genes have been cloned by means of PCR and artificial operon has been constructed in E. coli pET22b expression vector. After that, all two genes have been expressed in E. coli. Results: An artificial operon from two genes (hydS, hydL) has been created to investigate expression of HydSL hydrogenase from Thiocapsa roseopersicina in E.coli. Proteins were overexpressed. However no hydrogenase activity was found. Detailed description of experiments will be presented. Conclusion: The construction with hydS and hydL was created, introduced in E.coli cells and HydS and HydL proteins were overexpressed.
P.004 THE RHODOBACTER SPHAEROIDES REACTION CENTER ASSEMBLED WITH ZINC BACTERIOCHLOROPHYLL YIELDS EVOLUTIONARY INSIGHTS. Su Lin*†, Paul R. Jaschke§, Haiyu Wang*, Mark Paddock¶, Aaron Tufts†, James P. Allen†, Federico I. Rosell||, A. Grant Mauk||, Neal W. Woodbury*†, J. Thomas Beatty§ *
The Biodesign Institute at Arizona State University, Arizona State University; †Department of Chemistry and Biochemistry, Arizona State University; §Department of Microbiology and Immunology, The University of British Columbia; ¶Department of Physics, University of California, San Diego; ||Department of Biochemistry and Molecular Biology, The University of British Columbia. Introduction: The purple bacterial reaction center RC binds 6 chlorin cofactors: the ‘special pair’ (P), a dimer of bacteriochlorophyll (BChl) a molecules on the periplasmic side of the RC; two monomeric BChls (BA and BB) present on either side of P; and two bacteriopheophytin (BPhe) molecules (HA and HB). When P is excited, an electron is transferred through BA to HA, and then to a quinone. Studies on a variety of species and mutants in which cofactors were changed indicated the importance of the energetics of the cofactors in influencing ET rates. The RC from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides was studied. In this RC, which we call the Zn-RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) binding sites contain Zn-BChl rather than BChl. We used spectroscopic measurements to fully elucidate the chlorin composition at the P, B and H sites, and to measure the efficiency of ET. Methods: Mutant and wild type RCs were isolated and the absorption spectra at room temperature and 10 K were measured, as well as the kinetics of ET in timescales ranging from fs to ms. The 86
electrochemical midpoint of P was determined by potentiometric titration and measurement of changes at the P absorption maximum. Results: Analysis of the room temperature and 10 K absorption spectra of the Zn-RC revealed that the P, B and H sites all contain ZnBChl. Furthermore, the absorption peaks in the visible region indicated that the metals of the Zn-BChls in the P and B sites are pentacoordinated, as is the Mg of BChl in these sites in the wild type RC, whereas the Zn-BChls in the H sites appear to be tetracoordinated. The midpoint potential of P in the Zn-RC was determined to be 515 ± 5 mV, similar to the value of 505 ± 5 mV for the wild type RC. Photobleaching experiments indicated that ET proceeds from P to the quinones, producing a long-lived charge-separated state, as in the wild type RC. The ultrafast kinetics of ET from P to HA in the Zn-RC were found to be very similar to those in the wild type RC, in contrast to an RC in which HA is BChl. Conclusions: The Zn-RC H sites contain Zn-BChl instead of BPhe, and so contains 6 identical chlorins. The Zn-BChl H-cofactor spectral properties in the visible region are proposed to be due to the absence of a 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions that significantly contribute to the redox midpoint potential of H, and the formation of the charge-separated state. In both the purple bacterial and the cyanobacterial PS2 RC, P is a (B)Chl dimer, B sites contain (B)Chl, and H sites contain (B)Phe. In contrast, the cyanobacterial RC in PS1 has six Mg-containing Chls in a similar spatial arrangement, analogous to the Zn-RC, with the A0 Chl in the PS1 RC equivalent to the HA BPhe in the WT RC. The Mg of the Chl electron acceptor A0 in the PS1 RC protein is weakly ligated by the sulfur of a Met side chain, in contrast to the usual His. We speculate that evolution has resulted in two strategies for a high rate of ET in RCs: 1) in the PS2-type of RCs, the primary electron acceptor is a (B)Phe surrounded by protein that excludes (B)Chl and water from the H sites; 2) in the PS1-type of RCs, the primary acceptor is a Chl (A0), but Chl functions well as the primary acceptor because the Mg in the A0 Chl is ligated weakly by a sulphur atom coming from the protein, endowing this Chl with tetracoordination-like electrochemical properties.
P.005 A NEW PROTEIN INVOLVED IN THE OCP-RELATED PHOTOPROTECTIVE MECHANISM IN CYANOBACTERIA. Clémence Boulay, Adjélé Wilson, Diana Kirilovsky. Commissariat à L’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTec-S) et Centre National de la Recherche Scientifique (CNRS) URA 2096, 91 191 Gif-sur-Yvette, France. Introduction: The most characterised photoprotective mechanism in cyanobacteria is named qEcya. Under high light conditions, it diminishes the formation of dangerous oxygen species by increasing energy dissipation into heat and thus, decreases the energy arriving at the reaction centers from the phycobilisomes (PBS, the cyanobacterial external antennae). The mechanism is induced by the absorption of blue-green light by the carotenoid of the soluble photoactive Orange Carotenoid Protein, OCP [1, 2]. The process is accompanied by a reversible decrease (quenching) of fluorescence. When the “quenched” cells are then transferred to darkness or low light intensities, the initial level of fluorescence is recovered within 15-20 minutes. The aim of this study was to discover other proteins involved in this photoprotective mechanism.
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13th International Symposium on Phototrophic Prokaryotes Methods: A mutant unable to recover the lost fluorescence along with several mutants which complemented it were constructed. Their phenotype, with respect to the photoprotective mechanism, was studied by measuring fluorescence kinetics in a pulse amplified modulated fluorimeter (PAM-fluorimeter). Co-purification and coimmunoprecipitation experiments were used to investigate the interaction of a new protein with the OCP. Results: We have demonstrated that a 13kDa membrane protein, which we have named Fluorescence Recovery Protein (FRP), is involved in the PBS fluorescence emission recovery after a strong blue-green light illumination. In the absence of this protein, there is no fluorescence recovery, while overexpression of the same protein accelerates the recovery. When the His-tagged FRP protein was isolated from cells overexpressing this protein, traces of the OCP were co-isolated. Moreover, OCP and FRP co-immunoprecipitated using an antibody against the His-tag present only in FRP. These results strongly suggested that OCP and FRP interact. Conclusion: We have discovered a new protein involved in the OCPrelated-photoprotective mechanism. This protein, named FRP, is essential for fluorescence recovery. [1] A. Wilson, G. Ajlani, J.M. Verbavatz, I. Vass, C.A. Kerfeld, D. Kirilovsky, A soluble carotenoid protein involved in phycobilisomerelated energy dissipation in cyanobacteria, Plant Cell 18 (2006) 992-1007. [2] A. Wilson, C. Punginelli, A. Gall, C. Bonetti, M. Alexandre, J.M. Routaboul, C.A. Kerfeld, R. van Grondelle, B. Robert, J.T. Kennis, D. Kirilovsky, A photoactive carotenoid protein acting as light intensity sensor, Proc Natl Acad Sci U S A 105 (2008) 12075-12080.
P.006 PROBING THE STRUCTURAL CHANGES INVOLVED IN D1 PROTEIN TURNOVER OF PHOTOSYSTEM II IN SYNECHOCYSIS SP. PCC6803. Aparna Nagarajan, Hong-Jin Hwang, Robert Burnap. Dept. Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK, USA. D1 protein subunit is deeply buried within the PSII complex and has a high turnover rate (approximately 30min., depending upon light intensity). The process of turnover involves an efficient process of damage recognition and repair, which is largely unknown. Rearrangements in the structure of the D1 protein and PSII complex are inevitable during this turnover process. We are adopting two technical approaches to detect the structural changes during this process. The first approach involves the use of reagents causing a more general modification of various residues that become exposed to the aqueous phase during the turnover process. Different chemical modification reagents (e.g. carbodiimides) are being explored to probe changes in conformation. These modified PSII subunits can be analyzed by mass spectrometry (MALDI-TOF). We have obtained good coverage for the core PSII proteins with 64% of D1, 57% of CP43 and 47% of MSP full-length sequence. The regions of coverage include the domains that are hypothesized undergo changes. A comparison of peptides before and after treatment could demonstrate the changes in the solvent exposure post modification. The alternative and more specific approach is to detect these structural changes using cysteine-scanning mutagenesis. Analysis of the structure of D1 has allowed us to select a few domains that might be involved in solvent exposure. Based on this structure, a set of mutants have been generated which have all the four native cysteines August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts removed and one cysteine substituted at a specific location. These cysteine residues are probed using thiol reactive reagents like 5iodoacetamido-fluorescein. A cysteine residue that is solvent exposed can react with the probe and can thus be hypothesized as the domain that undergoes a conformational change. These chemically modified domains of D1 protein can be detected by using gel electrophoresis. This system involves the use of PSII particles and will allow us to probe the solvent exposed residues both under native and damage conditions in vitro. Supported by National Science Foundation NSF MCB-0818371
P.007 KINETIC ANALYSIS OF CCMR AND CMPR (LYSR-TYPE TRANSCRIPTIONAL REGULATORS) WITHIN SYNECHOCYSTIS SP. PCC6803. Shawn M. E. Daley1, Marla Carrick1, Robert L. Burnap2. 1
Biochemistry & Molecular Biology; 2Dept. Microbiology & Molecular Genetics; Oklahoma State University, Stillwater, OK, USA.
Aquatic photosynthetic organisms are generally dependent upon active transport of inorganic carbon to supply the Calvin-BashamBenson carbon fixation process. This is an energetically expensive active transport uptake system termed the carbon concentration mechanism (CCM). The control of the high-affinity/low-flux inorganic CCM in cyanobacteria has been of great interest for many years. Two DNA-binding proteins, CcmR and CmpR, are responsible for the transcriptional control of the expression of transport genes for the CCM. CmpR has been shown to be an activator of cmpABCD (Takahashi et al 2004). CcmR has been shown to be a repressor in vivo as established by microarray analysis appearing to control several different genes/operons that are spread throughout the genome of Synechocystis sp. PCC6803 (Wang et al 2004). Therefore the genes/operons under the control of CcmR are referred to as the ‘CcmR Regulon’. This putative regulon contains 5 distinct genes/operons; ccmR, ndhF3 [operon], ndhD5 [operon], sbtA [operon] and ubiX. However, direct physical evidence for the interaction between CcmR and the proposed genes comprising this regulon is limited. This work aims at testing the hypothesis that CcmR indeed interacts with the DNA regulatory region of the genes previously identified by microarray analysis and to provide a more complete molecular characterization of the regulatory features of this putative regulon. For the first time in vitro data supporting direct binding of CcmR to all of the regulon components will be presented. These have been determined by electrophoretic mobility shift assay. The control of this energy intensive system of carbon sequestration has also been of great interest for many years. Recently Nishimura et al (2008) has determined the ligand molecule for CmpR within Synechococcus elongatus PCC7942. Efforts to determine the ligand molecule, using surface plasmon resonance, and its associated kinetic constants (ka, kd, kD) will be reported, for CcmR and CmpR within Synechocystis sp. PCC6803. Supported by US Department of Energy, Energy Biosciences Division DOE DE-FG02-08ER15968
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Final Program and Abstracts
P.008 SUPRAMOLECULAR ORGANIZATION OF PHYCOBILIPROTEINS IN THE CHLOROPHYLL D-CONTAINING CYANOBACTRIUM ACARYOCHLORIS MARINA. Min Chen1, Tom Bibby2, Matthias Floetenmeyer3. 1
School of Biological Sciences, University of Sydney, Australia, National Oceanography Centre, Southampton University, United Kingdom; 3Centre for Microscopy and Microanalysis, University of Queensland, Australia. 2
Acaryochloris marina MBIC11017 is an ecologically important, biochemically interesting and evolutionarily fascinating marine cyanobacterium using the unique chlorophyll, Chl d. In addition to the accessory Chl d-binding light-harvesting protein complexes as its major light harvesting system, which feeds energy to photosystems I and II, it possesses a primitive phycobiliprotein complex as an accessory peripheral antenna system. Using advanced cryo-electronic microscope technologies we have revealed the high resolution details of the organisation of phycobiliprotein structures of Acaryochloris MBIC11017. Interestingly, the new strain isolated from the Salton Sea ( Acaryochloris CCMEE5410) lacks phycobiliproteins and no such supramolecular structure of phycobiliproteins was observed. Cryo-electron transmission-microscopy on native cell-sections of Acaryochloris MBIC11017 shows extensive patches of near-crystalline phycobiliprotein rods that are associated the thylakoid membranes. They are organized as an array of rods along the stromal (or cytoplasmic) side of the thylakoid membranes and divide the thylakoid membranes into an uneven “ribbon” with about 25 nm spaces (for phycobiliprotein region) or a closely stacked region leaving no spaces (less than 10 nm, without phycobiliproteins between the membranes). The dimensions of the individual rods are 10 nm × 25 nm. Aligning the PSII supercomplexes to rod-arrays of phycobliprotein complexes, we suggest that one array of phycobiliprotein rod-complexes lines up with one array of PSII dimer-antenna supercomplexes. Additionally, Acaryochloris possesses a huge genome with size up to 8.3 Mb including nine plasmids from 2.1 kb to 370 kb. The genes encoding phycocyanin related peptides but allophycocyanin α/β subunits are located in the plasmid pREB3. The genes encoding allophycocyanin α/β subunits are located in the main chromosome, and are not clustered together. With the aid of SDS-PAGE analysis we have identified a number of phycobiliprotein polypeptides and the proposed model structure of phycobiliproteins in Acaryochloris agrees with the structure observed by using electronmicroscope. This supramoleuclar photosynthetic structure represents a novel mechanism of organizing the photosynthetic light harvesting machinery. The relationship among photosystems and antenna systems in Acaryochloris will be discussed.
P.009 CRYSTALLOGRAPHIC STRUCTURE OF A LIGHT AND REDOX SENSING PROTEIN MUTANT, AppA17-133 Q63E, FROM RHODOBACTER SPHAEROIDES LOCKED IN PSEUDO-SIGNALING STATE. Vladimira Dragnea, George Feldman, Arun Ignatius, David Giedroc, Carl Bauer. Indiana University, Bloomington, IN, USA. AppA is a light and redox sensing protein and an anti-repressor of photosynthetic gene transcription in Rb. sphaeroides. Structure of the light sensing domain (called Blue-Light- Using-FAD) from several 88
different species has been solved. AppA BLUF domain binds oxidized FAD and is able to undergo a slow photocycle, represented by a 10 nm red-shift in the absorption spectrum of the flavin. Despite the efforts of many groups, the exact mechanism of the photocycle and the structure of its signaling state is still a mystery. Several mutants of AppA were constructed and their influence on AppA photocycle is discussed. Particularly interesting is a Q63E mutant, which exhibits no photocycle, permanent 3 nm red-shift of the flavin absorption spectrum and permanent fluorescence quenching of flavin peak. Acrylamide quenching of tryptophan fluorescence indicated that this mutant is “locked” in the light (signaling) conformation. We have confirmed this conclusion further by recording 2D-NMR spectra of the Q63E mutant, which exhibits almost identical magnetic shifts as AppAlight state. We have solved the crystal structure of AppA17-133 Q63E mutant that represents “light” conformation of this clone.
P.010 HETEROLOGOUS EXPRESSION OF [FeFe]-HYDROGENASES IN ANABAENA SP. STRAIN PCC 7120 FOR IMPROVED HYDROGEN PRODUCTION. Katrin Gaertner1, Ivan Khudyakov2, Sigal Lechno-Yossef3, Hajime Masukawa4, C. Peter Wolk3, and Eric L. Hegg1. 1 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA; 2All-Russia Research Institute for Agricultural Microbiology, Saint Petersburg - Pushkin, Russia 3MSUDOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA; 4Department of Microbiology and Molecular Genetics, Michigan State University, E. Lansing, MI; USA.
Introduction: Problems related to the massive use of fossil fuels are well known. H2 is a promising, clean fuel, but currently the vast majority of industrially produced H2 is generated from fossil fuels and is therefore neither environmentally friendly nor renewable. Some photosynthetic organisms produce H2 from sunlight and water, utilizing the reductant released during water oxidation to reduce protons. Although sunlight and water are abundant and inexpensive, the application of photosynthetic organisms to H2 production on an industrial scale is challenging. The major classes of enzymes capable of synthesizing H2 are nitrogenases (N2ases); [FeFe]-hydrogenases (H2ases); and [NiFe]-H2ases, such as uptake H2ase (Hup) and reversible H2ase (Hox). O2, produced by water-splitting photosynthesis, inhibits or inactivates all known N2ases and H2ases. Unlike N2ases, H2ases require no ATP. [NiFe]-H2ases are less O2-sensitive than [FeFe]-H2ases, but have lower specific activities and turnover rates. Anabaena spp. are filamentous N2-fixing cyanobacteria that contain differentiated, micro-oxic cells called heterocysts that protect N2ases and H2ases from O2. Our goal is to enhance H2 production by Anabaena by heterologously expressing [FeFe]-H2ases in the heterocysts. Methods: We are using standard genetic and molecular genetic techniques to target [FeFe]-H2ases, and the proteins needed for their maturation, to the heterocysts of Anabaena. To obtain optimal expression, these genes from various organisms are being driven by heterocyst-specific promoters (P) starting with Anabaena Pnif with and without amplification (Wolk et al., Mol Microbiol 7: 441, 1993), using integrating and replicating vectors, in Hup- and Hup- Hox- strains. Different combinations of [FeFe]-H2ases and maturation proteins will be tested, beginning with Chlamydomonas reinhardtii hydA and Clostridium acetobutylicum maturation genes hydE, hydF, and hydG (King et al., J Bacteriol 188: 2163, 2006). Results: We have integrated the gene encoding T7 RNA polymerase into the genome of Anabaena in the nif operon, eliminating N2ase August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes activity. The hyd genes will be expressed from PT7 on a replicating plasmid that has been transferred to Anabaena. All H2 production by our Hup- Hox- recipient strains will be due to heterologous expression of an [FeFe]-H2ase. Conclusion: Micro-oxic heterocysts provide a promising environment for the expression of O2-sensitive [FeFe]-H2ases under oxic external conditions. If strains expressing [FeFe]-H2ases in heterocysts produce more H2 than strains containing only the native N2ases, the former strains may provide a route to economically practicable production of H2 from renewable resources. P.011
Final Program and Abstracts mutant indicated a non-essential function. Spectral analysis by EPR indicates the existence of the redox cofactor heme ci in T. elongatus (as reported before for M. laminosus and C. reinhardtii). Due to its transformability, the purification of fully active dimeric cyt b6f complex from T. elongatus now enables an in-depth characterization including site-directed mutants. References: [1] Volkmer, T., Schneider, D., Bernát, G., Kirchhoff, H., Wenk, S-O., Rögner, M., J Biol Chem, 2007, 282, 3730-7 [2] Kurisu, G., Zhang, H., Smith, J.L., Cramer, W.A., Science, 2003, 302, 1009-14 [3] Stroebel, D., Choquet, Y., Popot, J-L., Picot D., Nature, 2003, 426, 413-18
CHARACTERIZATION OF AN ACTIVE CYTOCHROME B6F COMPLEX FROM THERMOSYNECHOCOCCUS ELONGATUS INCLUDING THE NEW SUBUNIT PETP.
[4] Baymann, F., Giusti, F., Picot, D., Nitschke, W., PNAS, 2007, 104, 519-24
Dorothea Gomolla1, Regina Oworah-Nkruma1, Corinna Lüer1, Meike Gendrullis1, Gábor Bernát1, Sascha Rexroth1, Frauke Baymann2, Matthias Rögner1.
P.012
Plant Biochemistry, Ruhr-University Bochum, 44801 Bochum,Germany; 2BIP/CNRS, 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France.
PROTEOMIC ANALYSIS OF KNOCKOUT MUTANTS FOR PLASTOCYANIN AND CYTOCHROME C6 IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803
Introduction: The cytochrome b6f (cyt b6f) complex of oxygenic photosynthesis from the thermophilic cyanobacterium Thermosynechococcus elongatus connects the electron transport between Photosystem 2 and Photosystem 1 (PS1). Each monomer of the dimeric integral membrane complex consists of four major subunits: cyt f (PetA, 33 kDa), cyt b6 (PetB, 24 kDa), the Rieske ironsulfur protein (PetC, 19 kDa) and subunit IV (PetD, 17 kDa). Additionally, there are four small subunits - i.e. PetG, PetL, PetM and PetN with molecular masses ranging from three to seven kDa. Recently, we identified an additional 7.2 kDa subunit of the cyt b6f complex of Synechocystis sp. PCC 6803 (S. 6803), which we named PetP [1].
Manuel Hervás, Ornella Castielli, José A. Navarro, Berta De la Cerda and Miguel A. De la Rosa.
1
Methods: In order to investigate structure-function relationships in a stable and fully active complex we developed a new strategy for the purification of the cyt b6f complex from T. elongatus, involving Histagged cyt f and two chromatographic steps. The purified dimeric complex was analyzed by MALDI-MS and ESI-MS, as well as EPR measurements. Results: The resulting dimeric complex contains all cyt b6f subunits and a 7.2 kDa protein. ESI-MS and MALDI-MS analysis revealed that this protein is encoded by the open reading frame tlr0524 – with high homology to PetP (ORF ssr2998) of S. 6803. Disruption of tlr0524 showed impaired growth especially under high light conditions and a slower electron donation to cyt f and PS1 according to cyt f and P700 absorption kinetics. Crystal structure analysis of cyt b6f from the thermophilic cyanobacterium Mastigocladus laminosus [2] and the green alga Chlamydomonas reinhardtii [3] showed an additional redox cofactor heme ci. This cofactor is located in the Qi pocket of the complex (close to heme bH) and covalently linked to a cystein residue of the cyt b6 protein via a single thioether. Preliminary EPR analysis of heme ci from T. elongatus in presence and absence of the inhibitor 2-n-nonyl-4hydroxyquinoline N-oxide showed similar spectra as obtained with the cyt b6f complex from C. reinhardtii [4]. Conclusions: In summary, we suggest that PetP, which is missing in the crystal structures of M. laminosus and C. reinhardtii, is a new – possibly regulatory – subunit of the cyt b6f complex. Similar to S. 6803 our results showed a direct association of PetP with the electron transport of the cyt b6f complex, although characterization of the PetP-less August 9 to 14, 2009 • Montréal, QC, Canada
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC. Centro Isla de la Cartuja, Américo Vespucio 49, E-41092 Sevilla, Spain. Introduction: Iron and copper participate as essential protein cofactors in photosynthesis and respiration. However, both metals at high concentration promote the generation of reactive oxygen species. Thus, metal homeostasis is crucial in maintaining the adequate concentration of metal ions inside the cell [1]. Plastocyanin (Pc) and cytochrome c6 (Cyt) are two alternative electron transport proteins that can replace each other depending on copper availability [2]. In this work, we describe a comparative proteomic analysis of Synechocystis wild-type (WT) and two knockout mutants for either Pc or Cyt growing with or without copper, thus yielding relevant information on metal homeostasis in cyanobacteria. Methods: Changes in the proteome of Synechocystis have been studied by comparing 2-DE gels of cytoplasmic soluble fractions when cultured with or without copper. Soluble proteins were first resolved by isoelectric focusing on a pH range from 4 to 7, followed by SDSPAGE. Qualitative differences, as well as quantitative comparison of spots, were analyzed using the PDQuest software package (Biorad). The selected spots were further identified by MALDI-TOF. Results: Due to the relevance of copper and iron homeostasis in cyanobacteria, we have studied the effect of copper deprivation on the proteome of Synechocystis [3,4]. Particular emphasis has been made on the combined effect of copper deficiency and deletion of the petE and petJ genes coding for Pc and Cyt, respectively, whose expression is regulated by this metal [2]. The differences in the proteome of WT cells reveal that copper induces not only the over-expression of 19 enzymes involved in the main metabolic pathways, but also the synthesis of the GroEL1 chaperone and of the ATP-dependent ClpP protease induced under stress. On the contrary, the lack of copper enhances the expression of an ABC transporter, which is the major contributor to ferric iron transport across the plasma membrane. When comparing the proteomes of WT and DpetE cells grown in the presence of copper, substantial expression changes are observed in 89
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Final Program and Abstracts 43 proteins involved in the main metabolic pathways. In particular, the changes observed in the mutant concern proteins involved in photosynthesis and enzymes related to regulation of phycobilisomes coupling to PSII. Similarly, the proteomes of WT and DpetJ cells grown in the absence of copper show different expression levels in 29 proteins involved in the main metabolic pathways. Actually, the expression level of proteins involved in carbohydrate metabolism, along with the antenna proteins and enzymes participating in the synthesis of pigments are significantly altered in the mutant cells. Moreover, Synechocystis cells grown with neither Pc nor Cyt suffer from redox stress due to the blocking of the electron transport chains. Accordingly, the over-expression of thioredoxins, peroxiredoxin, superoxide dismutase and the DnaK chaperone, among other proteins, can be observed. Conclusions: The proteomic analyses of Pc and Cyt deletion mutants of Synechocystis grown in the absence or presence of copper, as compared to WT cells, can be used as a powerful tool to understand metal homeostasis in cyanobacteria. References [1] Messerschmidt A et al (eds) (2001) Handbook of Metalloproteins. John Wiley & Sons, Chichester [2] Hervás M, Navarro JA, De la Rosa MA (2003) Accounts Chem Res 36, 798–805 [3] De la Cerda B et al (2008) Brief Funct Genomic Proteomic 6, 322– 329 [4] Castielli O et al (2009) FEBS Lett (in press)
Methods: A genomic library of Gloeothece sp. constructed in EMBL3 was used for screening and sequencing of nif-genes. The gene encoding NtcA (ntcA) from Gloeothece sp. was cloned into the expression vector, pET-28a (+). The vector was transformed into E. coli BL21 (DE3) and recombinant NtcA was purified using a His trap column. An electrophoretic mobility shift assay (EMSA) was performed to determine the NtcA binding site in the upstream region of the nifgenes. Results: The nif-genes were arranged as a continuous cluster spanning approximately 19 kb and organized into eight operons (in order: orf4orf5, nifVZT, nifP, nifBfdxnifSU, nifHDK, nifENX, orf2 and nifW). A typical NtcA-binding sequence was not found at an upstream region of each operon. However, sequences similar to the typical NtcAbinding site were found between nifP and nifBfdxnifSU (TGTN10ACA) that corresponds to the upstream regions of these operons and an upstream region of nifVZT (GTGN10ACA). EMSA showed NtcA bound only to the region between nifP and nifBfdxnifSU. The binding affinity of NtcA to the region between nifP and nifBfdxnifSU was similar to the typical NtcA-binding sequence present in an upstream region of the glnA in Gloeothece sp. The binding of NtcA to the region between nifP and nifBfdxnifSU was enhanced by the addition of 2-OG. Both nifP and nifBfdxnifSU were expressed under nitrogen-limiting conditions in Gloeothece sp. Conclusion: Transcriptional regulation of nifP and/or nifBfdxnifSU is thought to be mediated by NtcA. Enhancement of NtcA binding by adding 2-OG suggests regulation occurs in response to the N-status within the cells according to the level of 2-OG. The data suggests there may be other transcription factor(s) that regulate the transcription of nif-genes other than nifP and/or nifBfdxnifSU.
P.013 TRANSCRIPTIONAL REGULATION OF THE NITROGEN FIXATION RELATED GENES (NIF-GENES) BY NTCA IN A UNICELLULAR CYANOBACTERIUM GLOEOTHECE SP. 68DGA. Tadashi Kawabata1, Yukiko Taniuchi2, Shinya Yoshikawa1, Mitsunobu Kamiya1, Kaori Ohki1. 1
Faculty of Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan; 2Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan. Introduction: The gene set encoding proteins required for the diazotrophic cyanobacteria synthesis of functional nitrogenase (nifgenes) are clustered and organized as several transcriptional units. Expression of the nif-genes (including the structural genes for nitrogenase, nif H, D and K) occurs under nitrogen-limiting conditions. However, it is unclear whether expression of the nif-genes is controlled by a single transcriptional regulator as found in enteric bacteria. A possible candidate for a transcriptional regulator is NtcA that is known as a global nitrogen regulator in cyanobacteria. NtcA is thought to be activated by 2-oxoglutarate (2-OG) whose level increases during nitrogen depletion. A typical binding site for NtcA on the DNA was found to contain the palindromic sequence, GTAN8TAC. In the filamentous cyanobacteria, Anabaena sp. PCC7120, NtcA is required for heterocyst formation where the expression of nif-genes occurs in the late stage of heterocyst differentiation. However, direct interactions between NtcA and nif-genes have not been clear yet. To determine the role of NtcA in transcriptional regulation of nif-genes, we cloned the nif-genes from the unicellular diazotrophic cyanobacteria, Gloeothece sp. 68DGA (Gloeothece sp.); and characterized the binding of NtcA in the upstream region of the nifgenes.
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P.014 THE CBB3 OXIDASE FROM RUBRIVIVAX GELATINOSUS. Bahia Khalfaoui Hassani 2, Ileana Agalidis2, Chantal Astier2, Soufian Ouchane 2, Robert van Lis1, Wolfgang Nitschke1 and Barbara Schoepp-Cothenet1. 1
Laboratoire de Bioénergétique et Ingénierie des Protéines (UPR 9036), Institut de Microbiologie de la Méditerrannée, CNRS, Marseille; 2 CNRS, Centre de Génétique Moléculaire, FRE 3144, Gif-sur-Yvette; 2 Université Paris-Sud, Orsay; 2Université Pierre et Marie Curie, Paris; France. Introduction: Among the three groups of heme/copper O2 reductases, cbb3–type oxidases are the least well-understood. We observed that the β-proteobacterium Rubrivivax (R.) gelatinosus expresses high quantities of a cbb3–oxygen reductase when grown under oxygen-limited conditions. As a result of the high level of enzyme, this species thus lends itself both for the study of the enzyme in membranes and as starting material for biochemical purifications. Methods: The operon coding for the enzyme together with flanking regions containing genes involved in its regulation and maturation were sequenced and analysed. The enzyme was studied in membrane fragments and purified samples by UV/Vis and EPR (including studies on oriented material) spectroscopy and electrochemistry. The two extrinsic c-type cytochrome subunits CcoO and CcoP and sitedirected mutants thereof were heterologously expressed in E. coli. Attribution of EPR spectral features to heme cofactors in the whole enzyme was achieved via a CcoP null mutant. Results: EPR spectral characteristics, redox midpoint potentials and orientations with respect to the membrane of all heme components
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13th International Symposium on Phototrophic Prokaryotes present in the R. gelatinosus cbb3 oxidase were determined. The heme contained in CcoO was found to be spectrally and electrochemically heterogeneous. The spectroscopic results obtained on the product of the ccoO gene and site-directed mutants thereof suggest that during the enzyme’s catalytic cycle, CcoO switches between Met143 and His130 as axial 6th ligand rationalizing the observed redox heterogeneity. This ligand switch is furthermore in line with the absence of a well-defined orientation of the CcoO heme with respect to the membrane as opposed to the situation which we observe for all other hemes of the complex and which are in specific, well-determined orientations. Conclusions: The obtained results together with the observation that both Met143 and His130 are conserved in cbb3 oxidases indicate that the ligand switch is an essential part of the catalytic turnover of this group of O2 reductases. P.015 OPTICAL PROPERTIES OF LIGHT-HARVESTING COMPLEX 2 FROM THERMOPHILIC PURPLE SULFUR BACTERIUM, THERMOCHROMATIUM TEPIDUM.
Final Program and Abstracts concentration of LDAO. Since LH2 complex has the ring structure, the CD signals of B800 and B850 indicate two couplet type CD signals [4]. These results reflected the differences of interaction of BChls a in the B850 and the B800 and suggested that the interaction between BChls a in the B850 became weaker in high concentration LDAO. We also found the blue-shifted LH2 complex was returned to 855 nm by dilution of the concentration of LDAO to 0.05%. Sucrose density gradient of the blue-shifted LH2 complex did not show clearly band. The absorption spectra of the solution of upper and lower area showed blue-shifted LH2 complex and native like LH2 complex, respectively. This result suggested that the size of the blueshifted LH2 complex smaller than that of the native LH2 complex Conclusions: We found that the reversible spectral changes of the LH2 complex from Tch. tepidum were induced by the increasing of the concentrations of detergents. The blue-shifted LH2 complex was smaller than native LH2 complex. The interaction among BChls a of the B850 in blue-shifted LH2 complex were weaker than that in native LH2 complex. Reference [1] M. T. Madigan, Science, 225, 313-315(1985)
Masayuki Kobayashi, Hiroaki Suzuki, Zheng-Yu Wang, Tsunenori Nozawa.
[2] H.Suzuki, Biochim. Biophys. Acta 1767, 1057-1063(2007)
Ariake National College of Technology, Omuta, Fukuoka, Japan.
[4] S. Georgakopoulou et. al., Biophys. J. 82: 2184-2197(2002)
Introduction: In photosynthetic purple bacteria, light energy is absorbed efficiently by antenna complexes and the captured photons are transferred to photosynthetic reaction center. Purple sulfur bacteria have two types of antennas which are core light-harvesting complex 1 (LH1) and peripheral light-harvesting complex 2 (LH2). Thermochromatium (Tch.) tepidum belongs thermophilic purple sulfur bacterium and its optimum growth temperature is the highest of all purple bacteria [1]. In this study, we tried to isolate the LH2 complex from Tch. tepidum and found interested optical properties of that. Methods: Intracytoplasmic membranes (ICM’s) from Tch. tepidum cell were prepared by a sonication method [2]. LH2 complexes were isolated from the ICM’s by treatment with 0.35 % (w/v) lauryl-N,Ndimethyl amine-N-oxide (LDAO) at room temperature for 1 hour. Purification of the LH2 was carried out DEAE-anion exchanged column chromatography. Absorption and circular dichroism (CD) spectra were measured with Shimadzu U-3100 and Jasco J720-W spectrometers, respectively. Results: In near IR region, absorption spectrum of ICM’s from Tch. tepidum has three peaks at 917, 855 and 800 nm. These peaks has been assigned to B915 for LH1-RC complex and B850 and B800 for LH2 complex, respectively [3]. The profiles and peak position of absorption and CD spectra of the solubilized LH2 complex in 0.05% LDAO were very similar to those of native LH2 complex in ICM [3]. These suggest that the solubilized LH2 complex maintains native pigment-protein structure. We observed the changes of absorption and CD spectra at higher concentration of LDAO. The peak position of Qy transition of the B850 band was blue-shifted to 852 nm in 0.1% LDAO, 847 nm in 0.2% LDAO and 841 nm in 0.4% LDAO and the magnitude of that was decreasing according to increasing of the concentration of LDAO. The peak position of Qy transition of the B800 band was not shifted but the intensity of that was increasing by addition of LDAO. The B800 and B850 of LH2 from Tch. tepidum shows positive and negative CD signal from shorter wavelength, respectively [3]. Although the couplet type CD signal of the B850 was changed to one negative CD signal, the couplet type CD of the B800 was unchanged by increasing of the
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[3] T. Nozawa et. al., Biochim. Biophys. Acta 852, 191-197(1986)
P.016 PURIFICATION AND CHARACTERIZATION OF A PHYCOBILISOME SUBCOMPLEX CONTAINING THE LARGE ISOFORM OF FERREDOXIN:NADP OXIDOREDUCTASE. A. Korn, G. Ajlani, B. Lagoutte, A. Gall, P. Sétif. CEA, Institut de Biologie et de Technologies de Saclay, Gif sur Yvette, France The main role of the Ferredoxin:NADP oxidoreductase (FNR) in photoautotrophs is to provide the NADPH for CO2 reduction. In plantroot plastids, a distinct FNR isoform functions in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by carbohydrate oxidation. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene in most cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNRS specifically induced under heterotrophic conditions) is similar in size to the plant FNR (34 kDa) while the other one (FNRL 47 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome (PBS). Mutants have been constructed that contain only one of the two isoforms present in the wild type. The mutants that express exclusively the small isoform and the long isoform attached to the PBS are called FS and MI6, respectively. Following an NADP+/NADPH quantification, significant differences have been found for the NADP+/NADPH ratio in these mutants compared to the wild type. Using a genetically engineered strain of Synechocystis, we successfully purified a native complex composed of a phycocyanin hexamer, the rod-core linker and FNRL (FNRL-PC), in a 1:1:1 stoichiometry. We compared the NADPH-oxidase and NADP+-reductase activities of the two isoforms. Both FNRS and FNRL-PC exhibit similar oxidase activities when using potassium ferricyanide as an artificial electron acceptor, but differences in catalytic constants have been observed when analyzing the Fd mediated reduction of cyt c. This corresponds to a 40 % decrease in the affinity of FNRL-PC to Fd compared to FNRS. 91
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Final Program and Abstracts Furthermore, we found a significant decrease of the reaction rates of the first reduction of FNR by reduced Fd for the complex FNRL-PC compared to FNRS in the absence of NADP+. We also performed multiple catalytic turnover experiments and found that FNRS and FNRL-PC reductions are only limited by electron transfer from reduced Fd. Our results suggest a modified affinity for Fd in the purified FNRLPC complex compared to FNRS, which may be due to steric hindrance, by PC. These differences obtained in the catalytic activities do not explain the different phenotypes of the respective mutants that express only one FNR isoform. Therefore we propose that the activity must be determined by the differential localization of the FNR isoforms, which also probably determine their substrates availability, and their involvement in different pathways of electron flow. Further in vivo studies are underway to elucidate the function of each FNR isoform.
P.017 STRUCTURAL AND FUNCTIONAL ANALYSIS OF A CONSERVED TYROSINE TRIAD IN CYTOCHROME C1. J.A. Kyndt, J.C. Fitch, T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA. Introduction: Cytochrome bc1 (bc1), is a ubiquitous membrane protein found in all chloroplasts and mitochondria of eukaryotes and the cytoplasmic membrane of many respiratory and photosynthetic bacteria. Bc1 serves two functions—to transfer an electron to the soluble electron carrier cytochrome c, and to generate a proton gradient used in photosynthesis and oxidative phosphorylation. The c1 subunit forms the pivotal connection between the bc1 complex and its soluble partners. A triad of tyrosine residues in the c1 subunit of R. capsulatus bc1 (Y152-154) are ideally positioned to be involved in electron shuttling or correlate conformational communication between the Rieske protein, c1 and c2. The role of these residues was investigated by mutagenesis followed by spectral and kinetic analysis. Methods: R. capsulatus bc1 mutants and WT protein were produced in R. sphaeroides ∆fbc (gift from Prof. A. Crofts) with a c1 His tag. All cultures were grown photosynthetically, except for the Y154A complement which could only be grown aerobically in the dark. Proteins were purified on Ni-NTA and eluted in MOPS (pH 7.8) with 20% glycerol, 200mM His, 100mM NaCl, 1mM MgCl2, 0.01 % DM and 15 μg/ml PC. Decylubiquinol was used in the catalytic assays as substrate and the reduction of cytochrome c was measured by monitoring the absorbance change at 550 nm. Results: Mutation of Y153 to Q or A resulted in a spectrally altered cyt bc1. Both absolute and reduced minus oxidized spectra have the Soret peaks slightly blue shifted (by ~ 4 nm). Moreover, the intensities of the cyt c1 α and β peaks are significantly lower than in wild-type cyt c1 upon reduction. Measuring the redox midpoint potential (Em) for Y153Q showed that the Em for the c1 heme in this mutant was lowered by about 115 mV. Since this alone cannot explain the lower intensity of the c1 α and β peaks upon reduction with dithionite, and the fact that there is a slight Soret blue shift, we believe that the mutation of Y153 creates both redox and spin heterogeneity in cyt c1. In addition, the catalytic activity, measured in amount of cyt c reduction per second, is about 10 fold lower for the Y153Q mutant compared to WT. Since Y153 is positioned on the opposite side of the c1 hinge region as the c1 heme, it is likely that mutagenesis at that position creates steric clashes with the hinge region, which will affect the position of the sixth ligand. This was confirmed by structural homology modeling. Mutagenesis of Y to Q at position 152 did not change the spectral and
92
electrochemical properties of c1, and showed WT enzymatic c2 reduction rates. However, changing Y154 resulted in very low yield and non-functional protein. Conclusion: Structural homology modeling and directed mutational analysis of these three tyrosines showed that mutations to Q and A at position 153 clearly affect the electrostatic properties of c1 and the electron transfer rate from quinol to cyt c, presumably through alteration the sixth heme ligand position. Therefore, a model where Y153 has a structural function is more likely than a role as an electron path. The fact that Y152Q has properties very similar to WT indicates that this mutation did not introduce major structural changes in c1 and does not create rate limiting changes in the activity of bc1. Mutagenesis of Y154 displayed a significant destabilization of c1, underscoring its structural importance in bc1.
P.018 EFFORTS TO ENHANCE SOLAR HYDROGEN PRODUCTION BY HETEROCYST-FORMING CYANOBACTERIA. Sigal Lechno-Yossef, C. Peter Wolk. Michigan State University, East Lansing, MI, USA. Introduction: The negative effects of fossil fuel use on our economy and environment make the utilization of sustainable energy sources desirable. A principal such source is sunlight. Its energy may be used by diverse microorganisms to generate H2 from water via photosynthesis by use of nitrogenases (N2ases) or hydrogenases (H2ases). Because these enzymes are oxygen(O2)-sensitive, processes of oxygenic photosynthesis and H2 production are normally separated temporally or spatially. Anabaena spp. and closely related cyanobacteria form specialized cells, heterocysts, in which N2ases and H2ases are protected from O2 by inactivation of O2-producing PSII, accelerated respiration, and a thick envelope of glycolipids and polysaccharide that impedes penetration of O2. Reductant required for N2 fixation and H2 production is generated by photosynthesis in vegetative cells and moves to heterocysts as sugar. We are trying, by modifying Anabaena metabolism, to increase H2 production to a commercially practicable level. In preliminary experiments, in which a short version of the nifH promoter was inserted before hoxYH in an uptake H2ase(Hup)-minus nifHD mutant of Anabaena variabilis strain ATCC 29413, no H2 was produced under oxic conditions. Methods: Our revised approach is to replace N2ase by a native, bidirectional H2ase (Hox) in a Hup-minus derivative of Anabaena sp. strain PCC 7120, in which Hox is encoded by the genes hoxE, F, U, Y, and H that (with other ORFs) are located in two independent transcriptional units separated by ca. 9 kb (1). We are generating a construct in which those two operons are contiguous, together replacing much of nifD, so as to over-express hox, rather than nifD, in heterocysts from the strong nif promoter. We retain nifH because it may provide part of the mechanism to reduce the concentration of O2 in heterocysts (2). The products of genes hydF, C, D, B, and A are required for synthesis of the NiFe cluster of Hox, and of gene hoxW is required for proteolytic processing of HoxH (1). These genes will be expressed on a replicating plasmid from the heterocyst-specific promoters of coxBII and patB (3,4). Abstracts by H. Masukawa, K. Gaertner, J.J. Park and co-authors describe complementary approaches to enhancement of H2 production: modifying N2ase such that it reduces more protons to H2 and fewer N2 to NH3, utilizing not Hox but exogenous Fe-only H2ases, and studying electron donors and pathways in heterocysts.
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13th International Symposium on Phototrophic Prokaryotes Results: To facilitate further genetic modification of the host strain, we have successfully modified a flp-FRT-based technique for removal of antibiotic markers from the genome (5) for use in Anabaena sp. strain PCC 7120. The constructs described above are initiated, and some are quite advanced. Conclusions: It remains to be determined whether the native hox genes, expressed in heterocysts in an oxic milieu, can serve practicably as a source of H2 at the level of a household or a transportation system. Although available strains of Anabaena are convenient experimental objects, it is likely that hitherto unstudied strains may produce H2 more abundantly. References: 1. Tamagnini et al., FEMS Microbiol Lett 31: 692, 2007; 2. Thorneley and Ashby, Biochem J 261: 181, 1989; 3. Valladares et al., Mol Microbiol 4&: 1239, 2003; 4. Wang and Xu, J Bacteriol 187: 8489, 2005; 5. Hoang et al., Gene 212: 77, 1998.
Final Program and Abstracts increased after dark incubation, and is even higher after dark anaerobic incubation. Although the numbers of hits were quite low, a significant increase of transcription of hydrogenase and hydrogenaserelated genes was also observed upon dark incubation. Conclusions: The data obtained in this study demonstrate that both 454 pyrosequencing and SOLiD sequencing of cDNA are appropriate techniques to monitor the transcriptome in Synechococcus sp. PCC 7002. A comparison of four different conditions revealed that under CO2 limitation the transcription of genes involved in CO2 fixation are strongly up-regulated. Transcription of genes coding for components of the photosystems and light harvesting complexes are downregulated upon dark incubation. An interesting finding is the up-regulation of transcription of two genes of pyruvate metabolism upon dark incubation. These transcriptome data provide valuable data for finding strongly regulated promoters for expression systems and give information about metabolic pathways, which is important for future metabolic engineering for biotechnological applications.
P.019 TRANSCRIPTIONAL ANALYSES IN SYNECHOCOCCUS SPECIES PCC 7002.
P.020
Marcus Ludwig, Zhenfeng Liu, Craig A. Praul, Lynn P. Tomsho, Stephan C. Schuster, Donald A. Bryant.
OCCURRENCE OF THE OXYGEN-BINDING SPHAEROIDES HEME PROTEIN AND DIHEME CYTOCHROME C IN RHODOBACTERACEAE.
Dept. of Biochemistry and Molecular Biology, University Park, PA, USA
T.E. Meyer, J.A. Kyndt, M.A. Cusanovich.
Introduction: The cyanobacterium Synechococcus sp. PCC 7002 grows in brackish and marine water, tolerates very high light intensities, and has an extremely short doubling time. Because Synechococcus sp. PCC 7002 is easily manipulated genetically, the organism is a good candidate for biotechnological applications. In this study, we performed transcriptional analyses in Synechococcus sp. PCC 7002 by mRNA/cDNA sequencing. We compared the transcriptomes of cells cultivated under four different conditions in order to find differentially transcribed genes.
Biochemistry Dept., University of Arizona, Tucson, AZ, USA.
Methods: Cells used for transcriptional analyses were cultivated in mineral medium at 38oC, 250µmol photons*m-2*s-1 and continuous bubbling with air containing 1% CO2 (standard conditions). Growth under CO2 limitation was the same except that the air was not enriched with CO2. The cultures were grown to an OD730 of 0.7. In the case of cultures incubated in the dark, the cultures (at OD 0.7) were incubated in dark for 1h with either air or N2 containing 1% CO2 to produce anoxic conditions. cDNA libraries were synthesized after RNA isolation and subjected to 454 pyrosequencing or SOLiD sequencing. The obtained sequences were mapped against the Synechococcus sp. PCC 7002 genome using BLAST (maximum expect value 0.01, minimum identity 78%). The genes with the best hits were counted, and the counts were used for subsequent comparisons. Results: 454 sequencing resulted in about 100000 sequences per condition. Mapping against the genome of Synechococcus sp. PCC 7002 resulted in about 370000 hits in open reading frames and about 2000 in intergenic regions in total. The major fraction (87-96%) of the hits was identified as 16S and 23S rRNA, although procedures had been undertaken to deplete rRNA. Further analyses were only based on the mRNA sequences. The most abundant transcripts resulted from genes coding for the photosystems and light-harvesting complexes. After dark incubation (both aerobic and anaerobic) the transcription level of these genes is significantly lowered. Genes of the CO2 concentration and fixation mechanism are also quite abundant. The transcription level of these genes decreased upon dark incubation, whereas under CO2 limitation these genes showed an increase of transcription. Interestingly, transcription of pyruvate:ferredoxin oxidoreductase and phosphoenolpyruvate synthase is strongly
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Introduction: The oxygen-binding Sphaeroides Heme Protein (SHP) and soluble Diheme Cytochrome c (sDHC) were originally characterized from Rhodobacter sphaeroides, but have since been identified in more than 36 proteobacterial genomes. They usually occur in a three-gene operon along with a membrane-spanning cytochrome b (CytB), which in Rb. sphaeroides is fused with a duplicated mDHC domain (CytB/mDHC). In most species, the SHP operon includes regulatory genes. However, not all strains of Rb. sphaeroides have the SHP operon, it is not associated with regulatory genes, and has not been reported in other species of Rhodobacteraceae. We therefore set out to analyze a variety of Rhodobacter strains for the presence of the SHP operon and determine whether the SHP genes could have been acquired through random gene transfer or are functionally related. Methods: Rhodobacter species were obtained from our own collection, from the DSMZ, Braunschweig Germany, CV Ramana, Hyderabad India, and DK Newmann, Pasadena California and grown photosynthetically on the recommended media. PCR primers were designed based upon the conserved heme binding sites in SHP and DHC. Universal primers were also used to sequence 16S rRNA as a quality control. Results: We found that SHP and DHC are present in 9 out of 10 strains of Rb. sphaeroides, the only negative strain was 2.4.3. We were unable to detect any differences between our own isolates, strains TJ4 and JB15, but they differ from strains 2.4.1 and 2.4.9 by about 30 bases in the 2kb operon. Strain FY differs by about 41 bases and strain KD131 by 56 bases. Strain SCJ differs by about 87 bases. Strains 2.4.18 and IL106 differ from one another by only 5 bases, but are 365 bases distant from strain 2.4.1. As might be expected, there are also significant differences in the translated sequences of the 3 proteins from these two strains, which are 76-82% identical to those of strain 2.4.1, which is about the same extent as observed for distinct species. The SHP operon was also found in Rhodobacter sp. SW2, Rb. changlensis, Rhodovulum adriaticum, Rv. marinum, and Rv. robiginosum. PCR was negative for Rb. capsulatus strains SB1003, 2.3.1, and SP108, Rhodobacter sp. TJ12, Rb. blasticus, Rb. veldkampii, 93
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Final Program and Abstracts Rv. imhoffii, and Rv. sulfidophilum W4 and BSW8, however this does not prove that the genes are absent.
for twitching motility were also present. There are 26 histidine kinases, 43 response regulators, and 40 diguanylate cyclases.
Conclusions: The SHP operon is present in 9 out of 10 R. sphaeroides strains studied and was found in five related species of Rhodobacteraceae. Its occurrence in this family is therefore not due to random gene transfer but must provide a selective advantage. Furthermore, duplication of DHC and formation of the chimera occurred before speciation. SHP is postulated to function as an oxygenase and to share the same role and substrate specificity throughout the Rhodobacteraceae. The function is likely to be different in the related Rhodospirillaceae and in bacteria that have operons with associated sensor kinases and response regulators.
Conclusions: It is now possible to obtain high quality draft genome sequence data at an affordable cost in less than a month and it does not require any special expertise. Most of the expense of genome sequencing comes at the finishing stage, although there is no compelling reason to determine complete sequences. Draft sequences provide most information required and any additional sequence can be readily obtained by walking PCR.
P.021 DRAFT GENOME SEQUENCE OF THE PHOTOSYNTHETIC PURPLE SULFUR BACTERIUM, ECTOTHIORHODOSPIRA VACUOLATA. T.E. Meyer, M.A. Cusanovich. Biochemistry Dept., University of Arizona, Tucson, AZ, USA. Introduction: There are now more than 700 complete bacterial genome sequences, but only five species of non-sulfur purple bacteria and one purple sulfur species completed. By way of contrast, there are more than a dozen green sulfur bacterial genomes and more than 20 distinct cyanobacterial genome sequences. There are also more than 20 species of aerobic anoxygenic phototrophic species that have been sequenced. Although there is tremendous diversity within the purple bacteria, they have been neglected to date with only a handful of species in the pipeline. We have thus initiated sequence determination of the purple sulfur bacterium, Ectothiorhodospira vacuolata (aka Ect. shaposhnikovii). The Ectothiorhodospiraceae are normally found in desert soda lakes that are characterized by alkaline pH, high carbonate coupled with low calcium and magnesium concentrations, and high productivity. When oxidizing sulfur compounds such as sulfide and thiosulfate, elemental sulfur is deposited outside the cells. Ect. vacuolata is unique as the only species in the family to produce gas vesicles. Methods: Ect. vacuolata strain DSM2111 was obtained from DSMZ, Braunschweig Germany and grown photosynthetically on the recommended medium. DNA was extracted with the Qiagen DNeasy kit, sonically sheared (250 base average size), and fragments sequenced in a single run on the Roche 454 sequencer. Sequences were assembled into contigs using Newbler and annotated with Rast. Results: 51 Mb high quality DNA sequence data were obtained and assembled into 121 contigs greater than 500 bases resulting in 3.3 Mb unique sequence (15-fold coverage). The CG content was found to be 63%. Automatic annotation resulted in 2900 coding sequences. The 16S rRNA sequence was in agreement with the published results and all 24 genes previously identified in Ect. vacuolata and Ect. shaposhnikovii were found to be in complete agreement except for the fccB gene for sulfide dehydrogenase which differed by 50%. It is likely that there are two genes for FCSD, only one of which was recovered. The alternative sulfide dehydrogenases, SoxEF and SQR are apparently absent. In the sulfur oxidation pathway, the sulfur carrier SoxYZ and the hydrolase SoxB were clustered, but the SoxAX cytochrome was located on a separate contig. The only possible sulfite dehydrogenase we found was a homolog of YedYZ. A close relative of Ect. vacuolata from Mono Lake was reported to photosynthetically oxidize arsenite to arsenate, but we could only find genes for arsenic resistance, ArrAB and ArsDABC. The GvpANFGSKJ genes for gas vesicle synthesis were recovered. There were 4 clusters of chemotaxis genes and 22 MCPs. The PilABCD and PilGHIJL genes 94
P.022 OPERATION OF THE CYTOCHROME BC1 COMPLEX OF RHODOBACTER CAPSULATUS AT HIGH PH VALUES. Katrin Jahns1, Natalia E. Voskoboynikova1, Maria A. Kozlova2, Armen Y. Mulkidjanian1,2. 1 2
School of Physics, University of Osnabrück, Osnabrück, Germany; Moscow State University, Moscow, Russia.
Introduction: In a dimeric cytochrome bc1 complex an ubiquinol molecule is oxidized in one of the two catalytic centers P and the two released electrons go to different acceptors. One is taken by the mobile domain of the [2Fe–2S] iron–sulphur Rieske protein to be passed further to the c-type cytochromes. The other electron crosses the membrane, via the low- and high-potential hemes of cytochrome b, to reduce a stable semiquinone molecule in one of the two centers N from the opposite membrane side (see refs. [1-3] for reviews). At neutral pH values, a single Zn2+ ion, which can bind close to the center P [4], not only retarded the proton release from this center and the movement of the FeS domain towards cytochrome c1, but also slowed down the oxidation of heme bh and the formation of ubiquinol in center N [5]. This correlation was attributed to the mechanistic coupling between the two quinone-binding centers [1-3,5,6]. Methods: The flash-induced generation of membrane voltage was traced, via spectral shifts of native carotenoid pigments, in vesicular preparations of the inner cellular membranes (chromatophores) of phototrophic α-proteobacteria Rhodobacter capsulatus and correlated with the optically monitored redox changes of the cytochrome hemes as described in ref. [5]. Results: The aforementioned transmembrane coupling between the quinone-binding centers implies that the retardation of events in center N should, reciprocally, affect the events in center P. To check this prediction, we have measured the pH-dependence of flashinduced reactions in the cytochrome bc1 complex, both in the absence and in presence of Zn2+ ions. The difference between kinetic traces, as obtained in the absence and presence of Zn2+ ions, respectively, diminished both under acidic and at alkaline conditions. At acidic pH values, the impact from the addition of Zn2+ ions diminished; most likely, the binding site became protonated with apparent pK of ca. 7.0 and could not bind Zn2+ ions anymore. Under alkaline conditions, in contrast, the oxidation of heme bh slowed down even in the absence of Zn2+ ions. At pH of ca. 9.0, the kinetics of reactions in the cytochrome bc1 complex resembled those measured in the presence of Zn2+ ions at neutral pH. Conclusions: The same kinetic behaviour of the cytochrome bc1 complex could be achieved either by blocking the proton release from center P by Zn2+ ions or via preventing the protonation of ubiquinone in center N by high pH. These observations support our suggestion of a cross-membrane mechanistic/thermodynamic coupling between the quinone-binding sites of this enzyme [1-3, 5, 6].
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13th International Symposium on Phototrophic Prokaryotes References: [1] Mulkidjanian A.Y. (2005) Biochim Biophys Acta 1709, 5-34. [2] Mulkidjanian A.Y. (2006) Biochim Biophys Acta 1757, 415-427. [3] Mulkidjanian A.Y. (2007) Photochem Photobiol Sci 6, 19-34. [4] Giachini L. et al. (2007) Biophys J. 93, 2934-2951. [5] Klishin, S.S., W. Junge, and A.Y. Mulkidjanian (2002) Biochim Biophys Acta 1553, 177-182. [6] Mulkidjanian, A.Y. and W. Junge (1995) in: Photosynthesis: from Light to Biosphere: (P. Mathis, Ed.), Kluwer Academic Publishers, Dordrecht, Vol.II, pp. 547-550.
Final Program and Abstracts and intracellular accumulation of CO2 / HCO3- was comparable to wild-type levels, indicating a capacity for photosynthetic electron transport. Conclusions: Microarray and proteomic analysis indicates CrhR RNA helicase interacts with transcripts involved with maintenance of photosynthetic capacity and protein synthesis. The inability of the crhR mutant to utilize the intracellular CO2 / HCO3- pool implies a defect in photosynthetic carbon assimilation. This is the first time that a RNA helicase has been associated with carbon assimilation in photosynthetic organisms.
P.025 P.023 CYANOBACTERIAL RNA HELICASES: A NOVEL ROLE IN REGULATING CARBON ASSIMILATION. Laura Patterson-Fortin1, Lige Wu1, Meghana Ventakesh2, George S. Espie3, George W. Owttrim1. 1 Department of Biological Sciences, University of Alberta, Edmonton, AB; 2Department of Biology, University of Toronto, Mississauga, ON,; 3 Department of Cell and Systems Biology, University of Toronto, Mississauga, ON; Canada.
Introduction: RNA participates actively in the regulation of gene expression in all organisms. To be functional, RNA molecules must fold into the appropriate secondary structure. RNA helicases are enzymes that alter RNA or RNP structure, thereby participating in the posttranscriptional regulation of gene expression. We have utilized a multidisciplinary approach to study the mechanisms by which alteration of RNA secondary structure by RNA helicases influences gene expression in cyanobacteria. Expression of the helicase, crhR (cyanobacterial RNA helicase Redox), is regulated by the redox potential of the electron transport chain and is therefore enhanced by low temperature (20ºC). Biochemically, CrhR rearranges RNA secondary structure by both unwinding dsRNA and annealing complementary ssRNA. Here we explore the broad ranging effects of a crhR null mutation in the cyanobacterium Synechocystis sp. PCC 6803. Methods: CO2 / HCO3- transport and accumulation was measured using an aqueous inlet mass spectrometer. Photosynthetic O2 evolution was determined with a Clarke electrode. For microarray analysis of RNAs interacting with CrhR, CrhR was immunoprecipated from Synechocystis cells grown at 20ºC for 3 hours. After phenolchloroform extraction the RNA was labeled and hybridized to a Synechocystis microarry consisting of three copies of all identified ORFs. Transcripts detected in all 9 replicates from 3 biological samples were considered positive as potential RNAs interacting with CrhR in vivo. Proteomic analysis was performed using 2-D gels followed by mass spectrophotometric identification of proteins whose levels altered between wild type and crhR mutant cells. Results: Microarray analysis indicates CrhR interacts with a limited number of transcripts encoding genes involved with photosynthetic electron transport, energy metabolism, protein synthesis/degradation and unexpectedly transponsons. Initial proteomic analysis indicates that crhR mutants lack CcmK, a major component of the carboxysome shell. Phenotypically, the crhR mutant grows at near-normal wild-type rates at 30ºC, but not at 20ºC. The reduced rate of growth at 20ºC was correlated with a 4-fold reduction in the rate of photosynthetic oxygen evolution. Interestingly, after brief exposures to 20ºC, photosynthetic capability was restored upon return to 30ºC. In shortterm experiments, mass spectrometric analysis revealed that transport August 9 to 14, 2009 • Montréal, QC, Canada
IDENTIFICATION AND CHARACTERIZATION OF HIGHLY CONSERVED PROTEINS IN THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER SPHAEROIDES. Aaron Setterdahl, Allan Huth, Olga Simmons, Wes Halfacre. Indiana University Southeast, New Albany, IN, USA. Introduction: Genome sequencing of many organisms in recent years has revealed an enormous database of gene sequences that code for many previously unknown proteins (Zhou, Kvikstad et al. 2003). A number of these proteins are highly conserved between many diverse organisms. Sequence analysis of the purple photosynthetic bacterium Rhodobacter sphaeroides reveals several protein sequences that are uncharacterized and are highly conserved among bacteria such as Escherichia coli, Staphylococcus aureus, Xanthomonas axonopodis, Bradyrhizobium japonicum, Rhodopirellula baltica, Gluconobacter oxydans, Pseudomonas syringae, Shigella sonnei, Tenacibaculum, Flavobacterium, Blastopirellula marina, Rickettsiella grylli, Mesorhizobium, Streptomyces ambofaciens, Enterobacter spp., Planctomyces maris, and Klebsiella pneumoniae. Even with detailed annotation of genomes in which sequences of DNA from one organism are compared to other DNA sequences of different organisms, not all genes and their respective gene products can be correctly identified. Numerous instances exist where annotation reveals many genes to be “unknown function” or “hypothetical protein.” Recent studies used microarray analysis to determine the expression of hundreds genes in response to anaerobic-light to aerobic-dark conditions (Arai, Roh et al. 2008). Of the genes analyzed in that study at least 89 genes code for hypothetical proteins. Many of the genes that show sequence similarity and have been assigned a function by sequence comparison have been previously uncharacterized in R. sphaeroides or any other photosynthetic bacteria. Methods: The program Artemis was used to scan the annotated genomic DNA database of R. sphaeroides. Gene sequences that were labeled as unknown function or uncharacterized protein, were BLAST using the non-redundant NCBI database. Conserved protein sequences were targeted for PCR. Genomic DNA of R. sphaeroides is purified and used as a template for PCR. Primers were designed with a specific sequence that will amplify each several genes. Results: PCR has confirmed the presence of the gene sequences of the Iron-containing alcohol dehydrogenase, Putative FAD-dependent glycerol-3-phosphate dehydrogenase, a conserved hypothetical protein, and a Probable FAD oxidoreductase. Gene sequences that were targeted but were unconfirmed by PCR were a putative copper binding protein, a conserved hypothetical protein, probable bacterioferritin, putative iron-regulated protein, and a probable c-type cytochrome.
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Final Program and Abstracts Conclusions: PCR has confirmed the presence of the gene sequences, but more work needs to be done to confirm or discover the function of the gene products. This is a first step in the elucidation of the gene functions. The next step is to systematically clone, express and purify these and many other gene products. The confirmation of these gene identities is paramount to our understanding of these organisms. Many genes have proposed functions, yet still need to be systematically characterized in order to confirm or discover their proper function within R. sphaeroides. References: Arai, H., J. H. Roh, et al. (2008). “Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4.1.” J Bacteriol 190(1): 286-99. Zhou, S., E. Kvikstad, et al. (2003). “Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.” Genome Res 13(9): 2142-51.
P.026 THE INVESTIGATION OF THE ACSF PROTEIN AND PIGMENT BIOSYNTHESIS IN CHLOROFLEXUS AURANTIACUS. Kuo-Hsiang Tang, Jianzhong Wen, Xianglu Li, Robert E. Blankenship. Departments of Biology and Chemistry, Campus Box 1137, Washington University, St. Louis, MO, USA. Introduction: The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have primarily a structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophyll (BChl) c, d and e is not well understood. Methods: In this report, we used spectroscopic, proteomic and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus (C. aurantiacus). Results: We characterized the chlorosome proteins, CsmM, CsmN, and unexpectedly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, under anaerobic growth conditions for C. aurantiacus. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestions. Additionally, realtime (RT)-qualitative PCR studies showed that the gene expression of csmM, csmN, and bchE is up-regulated under anaerobic conditions, but that the transcript level of the acsF gene is not lower in anaerobic growth than in semi-aerobic growth (Tang, K.-H. et al. J. Bacteriol. in press). Finally, we have investigated on the location of AcsF in C. aurantiacus and other photosynthetic bacteria, as well as probed on the biosynthesis of the isocyclic ring formation in BChl a and BChl c. Conclusion: Together, it is clear that even for the most investigated FAP bacterium, C. aurantiacus, the role(s) of AcsF is far away from being understood. Additionally, since no studies have been reported previously for AcsF and BchE in any green bacteria, which have a specialized photosynthetic compartment— the chlorosome, caution is needed to compare the properties and mechanism of action of AcsF and BchE in GSB and FAP with the studies of higher plant, algae, and other bacteria. We are currently investigating these two enzymes with both in vitro and in vivo studies.
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P.027 PURIFICATION AND CHARACTERIZATION OF THE FMO PROTEIN FROM THE NEWLY DISCOVERED AEROBIC PHOTOTROPH, CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUM. Yusuke Tsukatani, Donald A. Bryant. The Pennsylvania State University, PA, USA. Introduction: We have discovered a new thermophilic, aerobic phototroph from the phylum Acidobacteria which had not included photosynthetic members. The discovery of the new acidobacterium Candidatus Chloracidobacterium (Cab.) thermophilum enables us to explore new mechanisms of photosynthesis and may provide new insights into evolution of photosynthesis. Genomic analysis of Cab. thermophilum revealed that this organism possesses genes encoding a reaction center core subunit (pscA), a baseplate protein of chlorosomes (csmA), and a light-harvesting Fenna-Matthews-Olson, so-called FMO protein (fmoA). Thus, Cab. thermophilum appears to do type-1 reaction center-based photosynthesis in the presence of oxygen, whereas green sulfur bacteria and heliobacteria that also have type-1 reaction centers are all strictly anaerobic phototrophs. Here we report the first isolation of the FMO protein from Cab. thermophilum. The FMO protein shows different absorption and fluorescence properties compared to that from green sulfur bacteria. Methods: Membranes prepared from photosynthetically grown cells of Cab. thermophilum were incubated with sodium carbonate and then a supernatant was collected by ultracentrifugation. After dialysis, the supernatant was subjected to anion-exchange DEAE column chromatography. The blue-colored FMO protein was eluted by 500 mM NaCl. Results: The purified FMO protein appeared as a single band upon Coomassie blue-stained SDS-PAGE. The molecular mass predicted from SDS-PAGE was about 44 kDa, which corresponds to the mass predicted by the amino acid sequence of the FMO protein (40.5 kDa). The 44-kDa protein band was confirmed as the FMO protein by peptide mass fingerprinting analysis. Absorption spectrum of pigments extracted from the FMO protein showed that the protein binds bacteriochlorophyll a. The FMO protein showed a large absorption peak at 797 nm and shoulder peaks at around 810 and 824 nm at room temperature. The fluorescence emission spectrum of the FMO protein showed two peaks at around 814 and 827 nm at room temperature. On the other hand, the FMO protein of green sulfur bacteria is known to have a single fluorescence peak at 818 nm at room temperature and the peak is sharpened and shifted to 831 nm at 77K. We will report the low-temperature spectroscopic properties of the FMO protein. We are also characterizing components in membrane preparations and attempting to isolate an active reaction center. Conclusions: We successfully purified the FMO protein from the new aerobic phototroph Cab. thermophilum. The FMO protein was a bacteriochlorophyll a-containing protein like that of green sulfur bacteria. However the FMO protein of Cab. thermophilum had distinctive spectroscopic properties compared to that of green sulfur bacteria. The results suggest that the energy transfer scheme inside the FMO protein of Cab. thermophilum may be significantly different from that of green sulfur bacteria.
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13th International Symposium on Phototrophic Prokaryotes
P.028 GLOBAL PROTEOMICS REVEAL NOVEL NITROGEN UTILIZATION MECHANISM UNDER NUTRITIONAL STRESS BY SYNECHOCYSTIS 6803, A MODEL PHOTOTROPH. Kimberly M. Wegener1, Jon M. Jacobs2, Abhay K. Singh1, Thanura Elvitigala3, Eric A.Welsh1, 4, Nir Keren1, 5, Marina A. Gritsenko2, Bijoy K. Ghosh1, 6, Richard D. Smith2, and Himadri B. Pakrasi1. 1
Department of Biology, Washington University, St. Louis, MO, USA; Pacific Northwest National Laboratory, Richland, WA, USA; 3 Department of Electrical and Systems Engineering, Washington University, St. Louis, MO, USA; 4Pfizer, Inc., St. Louis, MO, USA; 5Dept. of Plant and Environmental Sciences, Silberman Institute of Life Science, The Hebrew University of Jerusalem, erusalem, Israel; 6 Department of Mathematics and Statistics, Texas Tech University, Lubbock, TX, USA. 2
Introduction: Cyanobacteria, oxygenic phototrophic prokaryotes, are the progenitors of the modern chloroplast. They are crucial to global oxygen production and worldwide carbon and nitrogen cycles. Synechocystis sp. PCC 6803 has long been a model cyanobacterium for photosynthesis research. Here we present the first global proteome analysis of an autotrophic photosynthetic bacteria and the most complete coverage of a photosynthetic prokaryotic proteome to date. Methods: Synechocystis 6803 cells were subjected to iron, phosphate, nitrogen, or sulfate depletion and subsequent repletion. Cells were also subjected to environmental stresses including heat shock, cold shock, salt stress, high CO2 (3%) or growth under photoheterotrophic conditions.. Samples were collected at a total of 33 time points across conditions, fractionated, and subjected to proteomic analysis using the Accurate Mass and Time (AMT) tag approach. These results were analyzed statistically to combine technical replicates. Protein fold changes were calculated by comparing levels of an individual protein in a stress condition to those in cells grown in complete BG11. Results: The resulting proteome dataset consists of 22,318 unique peptides, corresponding to 2,369 unique proteins, covering 65% of the predicted proteins. These proteins uniformly represent all functional categories and include proteins encoded by the Synechocystis 6803 chromosome as well as the plasmids pSYSM, pSYSX, pSYSA, and pSYSG (Cyanobase). In contrast, previous proteome studies of Synechocystis 6803 have identified just 1,099 proteins (30% of the predicted proteome). 1,359 of the proteins identified in our study were novel proteomic identifications while 1,010 proteins were common proteins in our study and previously published proteomes. Additionally, we have identified 758 proteins that are currently annotated as hypothetical. These proteins can now be reclassified as unknown proteins, reducing the number of hypothetical proteins in Synechocystis 6803 by over one half. Importantly, quantitative analysis of protein abundance ratio in cultures grown under nutritional stresses revealed that Synechocystis 6803 resorts to a universal mechanism for nitrogen utilization under phosphate, sulfate, iron, and nitrogen depletion. Of the 2,369 proteins identified, 1,233 proteins were differentially regulated under environmental stress conditions. Conclusions: Global proteomics offers a detailed picture into the state of an organism that reveals insight that cannot be garnered from transcriptomic studies alone. Here we present the first global proteome analysis of an autotrophic photosynthetic prokaryote to date. This study offers the most complete coverage of a photosynthetic prokaryotic proteome with quantitative data as to protein response under various critical nutritional stresses. August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts Acknowledgements: This work was supported by National Science Foundation Grants FIBR 0425749 and MCB 0745611 to H. B. P. This work was additionally supported as part of the Membrane Biology Scientific Grand Challenge project at the W. R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s Office of Biological and Environmental Research program (Pacific Northwest National Laboratory).
P.029 INFLUENCE OF THE CAROTENOID NATURE AND OF THE AMINOACIDS AROUND THE CAROTENOID IN THE ORANGE CAROTENOID PROTEIN ACTIVITY IN PHOTOPROTECTION. Adjélé Wilson, Claire Punginelli, Jean-Marc Routaboul, Diana Kirilovsky. School of Biological Sciences (A08), University of Sydney, New South Wales, Australia. Introduction: In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. We studied the effects on the activity of the OCP of changes in the nature of the bound carotenoid and of modifications of the 100% conserved amino-acids surrounding it. Methods: Synechocystis PCC6803 mutants overexpressing the OCP and containing a mutated OCPs, or lacking the crtO gene were constructed. The existence of the photoprotective process was tested by fluorescence measurements using a PAM fluorometer. The OCP protein was isolated and its photoactivity tested.
Results and Conclusions: In the ∆crtO mutant, lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results confirmed that the red OCP is the active form and strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity. Trp288 and Tyr201 forming a hydrogen bond with the carbonyl of the carotenoid are essential for OCP photoactivity and induction of the photoprotective mechanism. They cannot be replaced by other amino-acid without loose of activity. While Trp110 and Tyr 44 can be changed by a Phe without loose activity, they cannot be replaced by Ser suggesting that the interaction of the carotenoid hydroxyl ring with aromatic groups is important for the formation and/or stability of the red form. Trp 277 also is involved in the conversion and /or stabilization of the red form. Finally, while Arg155 is essential for the induction of the photoprotective mechanism is not involved in the light-induced orange to red form conversion.
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P.030
of inactivation by oxygen and that the oxygen-sensitive DPOR, especially L-protein, is protected by some molecular mechanisms in oxygenic cyanobacterial cells.
CHARACTERIZATION OF A NITROGENASE-LIKE PROTOCHLOROPHYLLIDE REDUCTASE IN THE CYANOBACTERIUM LEPTOLYNGBYA BORYANA.
P.031
Haruki Yamamoto, Shohei Kurumiya, Rie Ohashi, Yuichi Fujita.
PPSR AS A HEMIN SENSOR.
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Indiana University, Bloomington, IN, USA.
Introduction: ark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), the determinant enzyme for the greening in the dark, catalyzes the stereo-specific D-ring reduction of Pchlide to convert porphyrin ring to chlorin ring in the bacteriochlorophyll/chlorophyll biosynthesis. DPOR is a three-subunit enzyme of BchL/ChlL, BchN/ChlN and BchB/ChlB, and their amino acid sequences show significant similarity to the nitrogenase subunits NifH, NifD, and NifK, respectively. A series of initial biochemical analysis of DPOR from the anoxygenic photosynthetic bacterium Rhodobacter capsulatus confirmed the nitrogenase-like features. DPOR is distributed widely not only among anoxygenic photosynthetic bacteria but also among oxygenic phototrophs such as cyanobacteria, green algae and gymnosperms, which raises a question how the nitrogenase-like DPOR operates in oxygenic photosynthetic cells. Here we report in-vivo complementation of cyanobacterial DPOR mutants by the expression vectors, reconstitution of DPOR with two components purified from cyanobacterial cells, and the oxygen sensitivity of the DPOR components. Methods: Two mutants YFC2 (∆chlL) and YFB14 (∆chlB) of the cyanobacterium Leptolyngbya boryana strain dg5 were used as the host cells to overexpress the DPOR subunits, ChlL and ChlN-ChlB. We constructed two shuttle vectors for overexpression of Strep-tagged ChlL (pHBL2) and Strep-tagged ChlN-ChlB (pHBNB2) in L. boryana. YFC2 and YFB14 were transformed with pHBL2 and pHBNB2, respectively, by electroporation. These transformants were cultivated in BG-11 supplemented with 20 mM HEPES-KOH; pH7.4, 30 mM glucose and 10 µg ml-1 chloramphenicol. Pigments of cells were extracted in 90 % methanol and the Chl concentration was spectroscopically determined. Crude extracts of these transformants were prepared by sonication in an anaerobic chamber. Strep-tagged ChlL and ChlN protein were purified by Strep-Tactin Sepharose columns. DPOR assays were carried out as described for DPOR from R. capsulatus. Purified components were exposed to air followed by the standard DPOR assay. Results: The ability of chlorophyll biosynthesis in the dark was restored in both transformants, suggesting that both DPOR components are expressed as active forms. Strep-tagged ChlL and ChlN proteins were purified from the crude extracts of the transformants. ChlL protein was purified as a single band. ChlN was co-purified with a protein that was cross-reacted with an anti-ChlB antibody, indicating that ChlN forms a stable complex with ChlB as shown in NB-protein from R. capsulatus. DPOR assay was carried out with the purified proteins. When both proteins, L-protein and NBprotein, were added to the reaction mixture, a marked chlorophyllide (Chlide) formation was observed, while there was no detectable conversion of Pchlide to Chlide in the reaction mixtures containing only one of the purified components. Then we evaluated the oxygen sensitivity of L-protein and NB-protein. While the activity of NBprotein maintained more than 60% of the original level upon 30-min exposure to air, and the L-protein activity rapidly disappeared with a half-life of about less than 3 min upon the exposure to air. Conclusion: The results suggested that L-protein is the primary target 98
Liang Yin, Vladimira Dragnea, Carl Bauer.
PpsR and AppA coordinate light and redox control of photosynthesis gene expression in R. sphaeroides. PpsR is a DNA-binding transcription factor that contains two PAS domains followed by a carboxyl terminal DNA binding domain. Under aerobic conditions, PpsR blocks the transcription of tetrapyrrole biosynthesis and photosynthetic genes that are responsible for biosynthesis of the pigments and the light harvesting II complex (1). AppA is flavin containing photoreceptor that functions as an antirepressor of PpsR by forming an inactive PpsR2-AppA complex (1). In the past year, our results indicate that hemin provides another level of control in this system, showing the first direct link between the biosynthesis of heme with that of the photosystem. In this study we demonstrate that PpsR is a hemin-binding protein containing a 1:1 stoichoimetry of [PpsR]/[hemin]. The presence of hemin significantly inhibited the DNA-binding ability of PpsR, while other tetrapyrrole products did not show any inhibition. Experiments with truncated PpsR suggest that hemin binds to the C-terminal region. Our results suggest that PpsR is likely a hemin sensor with heme controlling the DNA binding activity or PpsR. Most of well characterized hemin-binding proteins have tightly-coordinated hemin to perform many different tasks, such as electron transfer, oxygen storage and gas molecule sensing (2). However in recent years several heme sensors, have been identified from bacteria (3) to mammals (4) that are able to sense the free hemin in vivo. Our study of heme binding by PpsR will provide new insights about hemin sensors. 1. Masuda, S., and Bauer, C. E. (2002) Cell 110, 613-623 2. Gilles-Gonzalez, M. A., Gonzalez, G., Perutz, M. F., Kiger, L., Marden, M. C., and Poyart, C. (1994) Biochemistry 33, 8067-8073 3. Qi, Z., Hamza, I., and O’Brian, M. R. (1999) Proc Natl Acad Sci U S A 96, 13056-13061 4. Hu, R. G., Wang, H., Xia, Z., and Varshavsky, A. (2008) Proc Natl Acad Sci U S A 105, 76-81
P.032 EXCITATION ENERGY TRANSFER IN FILAMENTS AND HETEROCYSTS FROM NOSTOC PUNCTIFORME. Tanai Cardona, Ann Magnuson. Department of Photochemistry and Molecular Science, The Ångström Laboratories, Uppsala University, Uppsala, Sweden. The filamentous cyanobacterium Nostoc punctiforme sp. ATCC 29133 is able to differentiate a type of cell specialized in N2 fixation, a heterocyst. Heterocysts undergo dramatic changes associated with nitrogen starvation and the protection of the oxygen sensitive nitrogenase enzyme, like the partial degradation of phycobilisomes and the inactivation of Photosystem II. It is poorly understood whether heterocysts are capable or not, to control the distribution of excitation energy to Photosystem I and maximize N2 fixation under different light conditions. Using fluorescence spectroscopy, we compared the responsiveness of filaments and isolated heterocysts to green and red August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes light illumination. It was found in both cell types that 5 minutes green illumination triggered the quenching of a 680 nm emission peak corresponding to fluorescence from the terminal emitter of the phycobilisome and the appearance of a fluorescence peak at 750 nm corresponding to the trimerization of Photosystem I. We concluded that the quenching of the terminal emitter might be due to direct energy transfer from the phycobilisome core to the Photosystem I trimer. It is suggested that heterocysts possess a functional, possibly modified phycobilisome capable of distributing excitation energy to and away from Photosystem I to optimize cyclic photophosphorilation.
P.033 TRANSCRIPTION OF A “SILENT” CYANOBACTERIAL PSBA GENE IS INDUCED BY MICROAEROBIC CONDITIONS. Cosmin Ionel Sicora1,2, Felix M. Ho3, Tiina Salminen4, Stenbjörn Styring3, Eva-Mari Aro1. 1
Department of Biology, University of Turku, FI-20014, Finland; Biological Research Center Jibou, 455200, Romania; 3Molecular Biomimetics, Department of Photochemistry and Molecular Science, Uppsala University, SE-75120 Uppsala, Sweden; 4Department of Biochemistry and Pharmacy, Åbo Academy University, Finland. 2
Introduction: Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Methods: The main investigative methode used was Real-time RT-PCR for the estimation of expression of each individual gene in the family using specific primers. We also used molecular modeling to compare the structural differences between different forms of D1 and flash fluorescence to estimate the functional changes between isoforms. Results: Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities
Final Program and Abstracts Conclusion: The presence of an anaerobically induced gene in three unrelated cyanobacteria, makes us to conclude that a new form of D1 is present and important to cellular adaptation to specific conditions.
P.034 IDENTIFICATION AND CHARACTERIZATION OF BILIN LYASES FOR PHYCOERYTHRIN I BIOSYNTHESIS IN MARINE SYNECHOCOCCUS. Yasmin M. Vasquez, Avijit Biswas, and Wendy M. Schluchter. Department of Biological Sciences, University of New Orleans, New Orleans LA, USA. Introduction: Cyanobacteria have evolved photosynthetic lightharvesting complexes called phycobilisomes. The main component of the phycobilisome is phycobiliproteins. In marine Synechococcus, one of the most abundant phycobiliproteins is phycoerythrin (PE). PE is composed of α and β subunits that have the linear tetrapyrrole phycoerythrobilin (PEB) and/or phycourobilin (PUB) attached to specific cysteine residues via thioether linkages. We are interested in identifying bilin lyases involved in attaching the PEB chromophores to PE I subunits in marine Synechococcus. Methods: Putative bilin lyases from Synechococcus sp. WH8020 (CpeU, CpeY, CpeZ) or from Synechococcus sp. WH8102 (CpeS) were amplified by PCR and individually cloned in different Duet vectors. Bilin biosynthetic genes for making PEB from heme (pebS and ho1) were cloned together in pACYC (kind gift of Dr. Nicole FrankenbergDinkel). The cpeA and cpeB genes were cloned into the pMAL vector (fusing each to the gene encoding the maltose binding protein). The cultures were initially grown at 37ºC until the OD at 600 nm was between 0.5-0.6, then they were cooled down to 18ºC and induced with 0.5 mM IPTG for 15-18 hours for protein expression. The purification of MBP-CpeA and MBP-CpeB was accomplished using an amylose resin affinity column. Results: We achieved successful cloning of all the genes, which was confirmed by sequencing. Bilin lyase as well as phycobiliprotein subunit expression was found to occur optimally at 18 ºC with IPTG for a period of 15 hours. Soluble subunits CpeA and CpeB were purified with amylose resin column. SDS-PAGE confirmed bands at 57.1 kD corresponding to CpeA with maltose binding protein tag and a band at 59.1 kD corresponding to CpeB with the maltose binding protein tag. SDS-PAGE also confirmed bands for CpeZ, CpeY, CpeS and CpeU at 20.6 kD, 47.4 kD, 20.8 kD and 22.7 kD respectively. Solubility of these bilin lyases is currently being analyzed. Once conditions are optimized to obtain soluble lyases, these will be tested for bilin addition activity in in vitro reactions with PEB. Conclusions: CpeZ, CpeY, CpeS and CpeU bilin lyases are hypothesized to participate in PEB addition to the α and β phycobiliprotein subunits for PE I. Further work will focus on cloning the suspected lyases with a histidine tag in pCDF vector. The histidine tag will allow for further purification of the lyases using a nickelnitrilotriacetic acid (Ni-NTA) affinity column. Purified proteins will be used to carry out in vitro reactions to confirm the specificity of the bilin lyase for specific cysteine residues on the α and β PEI subunits.
between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also presented.
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P.035
P.036
USE OF BARLEY STRAW FOR THE CONTROL FRESHWATER CYANOBACTERIAL BLOOMS. Rob Iredale, Japareng Lalung, David Adams.
DIFFERENTIAL RESPONSES OF EPIPHYTIC AND PLANKTONIC TOXIC CYANOBACTERIA TO ALLELOAPTHIC ACTIVITY OF THE SUBMERGED MACROPHYTES STRATIOTES ALOIDES.
University of Leeds, Leeds, West Yorkshire, United Kingdom.
Abdulrahman M. Al-Shehri, Zakaria Mohamed.
Introduction: The potential of barley straw to control nuisance growths of cyanobacteria in freshwater has been well documented in a number of laboratory and field studies, but its mode of action is still unclear. In light of this, a study was designed to investigate the effect of straw preparation and decomposition conditions on treatment efficacy, in the hope that the collected data would allow us to comment on current theory. Additionally, the relative straw-sensitivity of different cyanobacteria, some of which were freshly isolated, was determined, and we attempted to tentatively identify some of the compounds leached from straw during the early stages of decomposition.
Department of Biological Sciences, College of Science, King Khalid University, Abha, Saudi Arabia.
Methods: A specially constructed decomposition chamber containing a combination of natural spectrum and UV fluorescent tubes was used to vary the light conditions under which straw was decomposed. Laboratory bioassays were used as a means of testing organism response to the presence of straw and straw liquor, with all assays conducted in triplicate. Solid phase extraction and reverse-phase HPLC were used in order to identify compounds in straw leachate, and flow cytometry was employed as a means of estimating chlorophyll a per cell for a strain of Microcystis. Results: We found varying sensitivity of different cyanobacteria strains to the effects of straw, consistent with previous studies. Fine chopping of fresh straw resulted in instant activity even at low concentrations, whereas fresh, whole straw exhibited little activity. We have shown that microbial activity is essential for straw to become active, and that straw decomposed under UV light appeared to be more active than batches decomposed under natural spectrum light alone. The addition of catalase to UV-decomposed straw bioassays reduced the inhibitory action of straw, suggesting hydrogen peroxide may play a role in the activity of straw. Flow cytometry data showed that Microcystis cells exposed to straw for 48 hours contained around 35% of the chlorophyll a content of cells not exposed to straw. HPLC spiking experiments suggested that a mixture of low molecular weight aromatic compounds were leached from the straw in the first week of decomposition. Conclusions: It is currently accepted that the activity of straw is at least partly attributed to the solubilisation of lignin by microorganisms, possibly yielding a cocktail of various smaller aromatic compounds. Our findings support this theory, both in terms of our HPLC detection of compounds which could have originated from lignin, and the fact that fresh, finely chopped straw was inhibitory to growth, whereas fresh whole straw was not. This implies that the breakdown of lignin might be the rate-limiting step determining the onset of activity, which can be enhanced by the combination of lignin disruption and increased surface area resulting from fine chopping of the straw. The possibility of hydrogen peroxide playing a role in the activity of straw has been previously hypothesised, but this study has provided the first experimental data that this may be the case. Data from this study have also highlighted the potential use of fresh, finely chopped straw as a cyanobacterial growth control agent, and larger scale studies, possibly involving the use of chopped and whole straw in combination should be considered in the future. Although many questions remain regarding its exact mode of action, barley straw remains the leading candidate for environmentally sound control of cyanobacteria in freshwater. 100
Introduction: Cyanobacteria are prevalent bloom-forming algae, commonly occurring in most water bodies, such as lakes and reservoirs. The cyanobacterial bloom can lead to the deterioration of water quality through production of toxins that pose a risk to animal and human health. Therefore, control and prevention of the growth of such harmful organisms is a significant goal in environmental science. Bioremediation using macrophyte allelochemicals for algal bloom control has been widely accepted because of its higher environmental safety and the ability of macrophytes to restore and renew the damaged aquatic ecosystem. The present study investigates whether epiphytic and planktonic toxic cyanobacteria respond similarly to allelopathic activity of the submerged macrophyte stratiotes aloides. Methods: Changes in growth, toxin production, and activities of alkaline phosphatase (APA) and antioxidative enzymes (Glutathione Stransferase (GST) & Glutathione peroidase (GPX) of toxic epiphytic (Merismopedia tenuissima & Leptolyngbya boryana) and planktonic (Anabaena variabilis) of cyanobacteria were studied in a batch experiment over a 15-day exposure to different concentrations of the 50 % aqueous acetone extract of the submerged macrophyte Stratiotes aloides. Results: The results showed that epiphytic and planktonic species of cyanobacteria exhibited differential responses to the allelopathic activity of this macrophyte. Stratiotes aloides extract reduced the growth, alkaline phosphatase activity and toxin production of planktonic cyanobacterim, with an increase in lipid peroxidation, glutathione and activities of antioxidative enzymes (GST & GPX) of this alga. In contrast, this extract increased the growth and toxin production in epiphytic cyanobacteria, but it did not significantly affect lipid peroixdation or the activities of APA and antioxidative enzymes. Conclusions: The macrophyte Stratiotes aloides has an allelopathic inhibition on planktonic cyanobacteria only, but can stimulate the growth and toxin production of epiphytic cyanobacteria attached to its surface. Therefore, this study suggests that macrophytes stimulating the growth of toxic epiphytic cyanobacteria should be avoided as a biological control of harmful cyanobacteria in water resources.
P.037 LEAD(II) UPTAKE BY THE CYANOBACTERIUM SYNECHOCYSTIS SP. STRAIN PCC 6803 IMMOBILIZED IN CALCIUM-ALGINATE BEADS. L.F. Allison, J-H. Yau, N. T. Flynn, M.M. Allen. Wellesley College, Wellesley, MA, USA. The presence of unsafe levels of lead(II) in water systems is a problem worldwide as lead is known to cause irreversible health problems. Bioremediation, the use of living organisms to remove pollutants, has emerged as an efficient and less expensive method for environmental decontamination. The cyanobacterium Synechocystis sp. strain PCC 6803 was immobilized in calcium-alginate beads in order to enable easy removal of contaminated bacteria from the environment. Alginate is a polymer derived from brown algae, which has shown potential to bind cations. Beads were prepared by dropping varying concentrations of alginate and cells through a syringe needle into 0.1 August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes M calcium chloride. Lead accumulation experiments were conducted for 4 hours in 200 mL of 500 ppm lead; the concentration of lead in solution was monitored with an ion-selective lead electrode. The amount of lead removed from the system was measured relative to the dry weight of the beads and reported as mg Pb2+ per gram dry weight of alginate. In order to examine the effect of immobilization on the cyanobacteria, 5% EDTA (pH 9) was used to degrade the calciumalginate. Cells were immobilized in beads and grown in BG-11 medium with 100 ppm lead; the calcium-alginate was then degraded and optical density at 750 nm was measured. It was demonstrated that the cells exposed to lead in beads grew at the same rate as free cells in BG-11 medium with no lead. The cells were observed to remain in clusters after the alginate was degraded. Rapid aggregation was also observed for free cells inoculated into a 500 ppm lead solution. These results suggest the cyanobacteria may be forming a biofilm when stressed in the calcium-alginate beads and when exposed to lead. The ability of calcium-alginate to absorb lead from solution was evaluated; beads prepared with 4% (w/v) alginate and without cells were capable of removing an average of 340±30 mg Pb2+ per gram. When Synechocystis sp. strain PCC 6803 cells were embedded in the beads, the maximum accumulation of lead for all cell samples measured was 479 mg Pb2+ per gram; this sorption was produced by cells with an optical density at 750 nm of 1.0 in the exponential phase of growth. Stationary phase cells at the same optical density were found to remove 466 mg Pb2+ per gram. However, cells in the exponential and stationary phases of growth, when tested at higher optical densities of 2.0 and 4.0, had decreased lead uptake by 110 to 170 mg Pb2+ per gram. Beads embedded with killed cyanobacteria were able to absorb lead at levels approximately equal to the 4% beads without cells. The average uptake for the dead cells in 4% alginate was 330±15 mg Pb2+ per gram. Thus, cyanobacteria have been shown to remove lead from solution at levels significantly higher than calcium-alginate beads without cells. Both the growth phase and density of the cells at the time of uptake appear to significantly affect the amount of lead the cells are capable of removing from solution.
P.038 USED OF ARBUSCULAR MYCORRHIZAL FUNGI AND PSEUDOMONAS PUTIDA IN PHYTOTOXICITY TESTS OF SEWAGED SOIL. M. Attia, Namet M. Awad, Azza Sh. Turky. Agricultural Microbiology Dep. National Research Centre, Dokki, Cairo, Egypt. A pot experiment, with tomato plants, was carried out to judge the use of bio-indicators for phytotoxicity in sewaged soil. A sandy soil heavily fertilized with compost was used for comparison. Results show that mycorrhizal root colonization of tomato plants and total mycorrhizal spore numbers were significantly low in sewaged soil compared to organic fertilizer amended soil. More dense population of Pseudomonas spp., bacterial counts as well as high dehydrogenase activity was retrieved in the rhizosphere of tomato plants grown in soil amended with organic fertilizers compared to those grown in sewaged soil. Relatively different CO2 release potentials were showed among different treated soils (from about 15 to about 72 mg CO2 100g-1 within 7 days). Soils biofertilized with both microorganisms liberated more CO2 than non-biofertilized ones. Microbial respiration looks as though unaffected even at heavy metal concentrations. AM fungi and Ps. putida elevated soil bio-activity regime as a result of improving soil quality, as indicated by the dehydrogenase activity, microbial density, CO2 evolution, as well as percentage of mycorrhizal infection and spore numbers. August 9 to 14, 2009 • Montréal, QC, Canada
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P.039 USING METABOLIC NETWORKS TO EVALUATE PATHWAY CONSTRAINTS OF BIOENERGY PRODUCTS IN SYNECHOCYSTIS SP. PCC 6803. K.L. Tibbles, S. Bryan, N.J. Burroughs. Warwick Systems Biology Centre, Coventry, United Kingdom. A stoichiometric metabolic network of Synechocystis sp. PCC 6803 was constructed from genomic data and coupled with models for respiration, photosystem electron transport and bioenergy product generation- hydrogen, ethanol and bio-diesel, building on the previous models of Shastri & Morgan, 2005 and Fu, 2008. By determining the elementary models of the system we examined the optimal pathways for growth and bioenergy product generation. This allowed determination of essential genes for production of these products and their commonality with requirements for growth, thereby ascertaining the biotechnological constraints of synechocystis for bioenergy utilisation. Further, we determined which of these 3 products is theoretically the most efficient as regards light energy capture.
P.040 NOVEL PIGMENT PROFILES OF AEROBIC ANOXYGENIC PHOTOTROPHS INDUCED BY HEAVY METAL STRESS. Julius T. Csotonyi, Vladimir Yurkov. Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada. Aerobic anoxygenic phototrophs (AAP) are obligately aerobic bacteria that can nonetheless supplement their energy requirements with light, using the pigment BChl a, which is functionally incorporated into a reaction center and light harvesting complexes. Many AAP possess constitutive high level resistance to several toxic oxyanions of heavy metals or metalloids – collectively, metal(loid)s – such as Se, Te or V. The reason for their high level resistance is incompletely understood, but the involvement of photosynthetic pigments has been hypothesized. To gain an understanding of the response of their pigment systems to heavy metal duress, AAP from hypersaline supralitoral marine microbial mats, hypersaline springs and hydrothermal systems were cultured in the presence of high concentrations of K2TeO3 (up to 1000 µg/ml), Na2SeO3 (up to 1000 µg/ml) and NaVO3 (up to 10000 µg/ml). Spectrophotometry was performed on pigments extracted with acetone/methanol (7:2, v/v). From Erythrobacter litoralis strain T4, extracted pigments were further purified by thin layer chromatography for identification. AAP expressed a diversity of altered pigment profiles under heavy metal stress, including decreased or enhanced BChl and carotenoid synthesis, and the appearance of novel pigments not expressed in metal-free cultures. In the presence of tellurite, Erythrobacter litoralis strain T4 accumulated a putative precursor to BChl a, tentatively identified by spectrophotometric analysis as Mg-protoporphyrin IX or its monomethyl ester. Identification of accumulated intermediates of pigment synthesis pathways helps to reveal the nature of the response of AAP to heavy metal stress. Elucidation of their molecular defense strategies will be invaluable to the development of waste management techniques in heavy metal industries.
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P.041 HYDROGEN PHOTO-PRODUCTION FROM VARIOUS DARK FERMENTATION EFFLUENTS BY TWO RHODOPSEUDOMONAS PALUSTRIS STRAINS. Alessandra Adessi, Lucia Bianchi, Luca Collina, Michela Palomba, Roberto De Philippis. Department of Agricultural Biotechnology, University of Florence, Piazzale Cascine 24, I 50144 Firenze, Italy. Introduction: Among the biological H2 production processes so far tested or still under study, the sequential fermentations carried out by chemo- and photo-heterotrophic bacteria seems to be the most promising one, owing to the possibility given by the combination of the two types of fermentation to approach the theoretical maximum yield of 12 mol of H2 produced per mol of glucose consumed. However, in spite of the theoretical advantages of the integration of the two processes, only few experimental data are available on the use of the combined H2 production system. The aim of this work was to test the H2 photo-evolution performances of two Rhodopseudomonas palustris strains growing on three different spent media deriving from dark fermentations carried out by chemoheterotrophs. Methods: Rp. palustris strains 42OL and AV33 were grown in 250 ml bioreactors under anaerobic conditions, 30°C, light intensity of 200 µmol (photons) m-2 sec-1. The media utilized for photo-fermentation were: (1) TDF, constituted by the effluent of the dark fermentation of glucose carried out by the thermophilic bacterium Thermotoga neapolitana DSM 4359 (2.1 g L-1 acetate and 0.3 g L-1 lactate). (2) MDF, constituted by the effluent of the dark fermentation of glucose carried out by the mesophilic bacterium Paenibacillus polimixa ISSDS-851 (0.8 g L-1 acetate and 2.7 g L-1 lactate). (3 VDF, constituted by the effluent of the dark fermentation of vegetable residues carried out by the autochthonous microflora present on the residues (2.1 g L-1 acetate and 7.8 g L-1 lactate). Results: A first set of experiments showed that the addition of ferric citrate to the effluents is necessary in order to have H2 production. Both R. palustris strains showed good performances with VDF: a mean H2 production rate of 15.6±1.0 ml H2 (L*h)-1, with a substrate conversion of 43%, and a mean H2 production rate of 10.7±2.6 ml H2 (L*h)-1, with a substrate conversion of 44%, were obtained with 42OL and AV33, respectively. Photo fermentation of the other two effluents showed a lower degree of substrate conversion in H2 as a consequence of the considerable growth observed with both substrates. Indeed, with TDF the highest H2 production rate, obtained with strain 42OL, was about 4 ml H2(L*h)-1, with a substrate conversion of 16.5%, while with MDF the highest H2 production rate, obtained with strain AV33, was about 2 ml H2(L*h)-1, with a substrate conversion of 7.1%. NMR analyses of TDF and MDF substrates showed the presence of large amounts of N-containing compounds, which caused the utilization of fatty acids as carbon sources for cell growth instead of as electron donors for the synthesis of H2. Conclusions: Photo-fermentation of vegetable residues or of effluents derived from previous dark fermentations is a promising process, giving the possibility to recover energy from organic wastes by coupling the production of H2 with waste disposal, but needs to be optimized, in particular in the presence of N-containing compounds which cause the shift of the microbial metabolism from H2 production to cell growth. 102
Acknowledgments The Authors would like to thank the Italian Ministry of University and Research (MIUR) (FISR Project IDROBIO) and Ente Cassa di Risparmio di Firenze (Firenze Hydrolab Project), which partially supported this research, and Dr Agata Gambacorta, ICB-CNR, Pozzuoli, Italy, and Dr Giulio Izzo, ENEA - Casaccia, Roma , Italy, who kindly provided the spent media of Thermotoga neapolitana and Paenibacillus polimixa, respectively.
P.042 NEW MOLECULAR MARKERS FOR QUICK IDENTIFICATION OF THE HEPATOTOXIN-PRODUCING CYANOBACTERIAL STRAINS BELONGING TO GENUS MICROCYSTIS. Bogdan Drug 1,2, Martin Welker3, Bogdan Fren iu1, Adriana Bica1,2, Cristian Coman1,2, Nicolae Drago 1,2. 1
Babe -Bolyai University, Cluj-Napoca, Romania; 2Institute of Biological Research, Cluj-Napoca, Romania; 3Technische Universität, Berlin.
Introduction The cyanobacteria belonging to genus Microcystis are well-known for their capacity to produce hepatotoxins, also known as “microcystins”. These are secondary metabolites of peptide nature, and they are nonribosomally synthesized by a multi-enzymatic complex, consisting of peptide synthetases and poliketide synthases. The microcystins characteristics make their identification and characterization very difficult, and thus, the cyanobacterial blooms may be extremely dangerous for the humans or animals that interact with the water in which these blooms are developed. The cells destruction by chemical means does not represent a proper solution, as long as the toxins are usually found only inside of the cells, thus being released if the cells are lysed. That is why the most suitable solution to this problem is to find ways to identify the toxic cyanobacterial blooms during their initial phases, in order to limit the risk of intoxication. The purpose of this study was to find suitable molecular markers able to provide the strict and quick detection of the cyanobacterial strains with toxic potential of genus Microcystis. Methods The hepatotoxic potential of the strains was analyzed by PCR amplification of certain genomic regions considered responsible for the synthesis of the enzymes involved in toxin production. The results achieved by this molecular method were verified through certain toxicity tests that consisted in the intraperitoneal injection of mice with cyanobacterial suspension concentrated by centrifugation. The strains toxicity was also verified by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization) using a mass spectrometer. In order to achieve a correlation between PCR results and toxicity tests we tried to discriminate the amplicons by DGGE (Denaturing Gradient Gel Electrophoresis). Eventually, we have sequenced the PCR amplicons, and based on the sequences multiple alignments we have designed new primer pairs able to generate amplicons only in those strains which produce hepatotoxins. Results The intraperitoneal injection of mice with cyanobacteria has emphasized the presence of toxicity in 5 of the 24 analyzed strains. The MALDI-TOF MS spectra have also displayed the presence of microcystins in these 5 strains, while cyanopeptolins and other secondary metabolites were detected in other strains. The primers used for the amplification of certain genomic regions correlated with the toxic potential were found in several articles, but during PCR they have generated amplicons in 7 strains, not only in the 5 toxic ones. The DGGE profiles could not differentiate these 7 amplicons according to the strain toxicity. The sequences multiple alignments allowed us to design new primer pairs, which were specific for the 5 toxic strains.
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Conclusions During this study we have observed that the oligonucleotide primers found in various articles did not generate amplicons during PCR in our toxic strains, as expected, but also in other two non-toxic strains. The DGGE technique has not proved to be proper to discriminate between amplicons according to strains toxicity.
design and construction of functional modules that can be assembled into circuits with predictable behaviour. The final goal is the construction of the cyanobacterial chassis accommodating the characterized synthetic modules will allow a sustainable photobiological H2 production, and the parts and devices produced will become available for other biotechnological applications.
Based on the sequences multiple alignments, we have obtained new primer pairs which are able to amplify certain regions of the gene cluster responsible for toxicity, these being highly specific for the toxic strains. One of these 5 strains belongs to species Microcystis wesenbergii, which was thought to be the only species of genus Microcystis which does not produce microcystins.
References:
P.043
1,2
1,3
P.044 CHANGES IN MICROCYSTINS AND OTHER OLIGOPEPTIDES PROFILES IN THE CYANOBACTERIUM RADIOCYSTIS FERNANDOI EXPOSED AT THREE DIFFERENT LIGHT INTENSITIES. Alessandra Giani, Daniel de Albuquerque Pereira.
PARTS AND MODULES FOR H2 PRODUCTION AND CONSTRUCTION OF A CYANOBACTERIAL CHASSIS. 1,2
[1] http://www.biomodularh2.org
1
Daniela Ferreira , Filipe Pinto , Catarina C. Pacheco , Miguel Lopo , Pedro Moradas-Ferreira1,3 & Paula Tamagnini1,2. 1
IBMC - Instituto de Biologia Molecular e Celular; 2Faculdade de Ciências, Departamento de Botânica; 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto, Portugal. Introduction: The BioModularH2 project (FP6, NEST-2005-Path-SYN, Contract 043340) aims at designing reusable, standardized molecular building blocks that integrated into a chassis will result in a photosynthetic bacterium containing engineered chemical pathways for competitive, clean and sustainable hydrogen production [1]. The chassis is being constructed using as basis the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synthetic parts and modules related to oxygen consumption and hydrogen production were designed, and are being constructed/synthesized. Methods: Design and construction of standardized vectors compatible with the BioBrick™ format. The vectors are being used to remove redundant parts (e.g. genes) from the bacterial genome and/or to integrate synthetic parts/devices. Generation of mutants through double homologous recombination, using the constructed standard vectors. Mapping of the Synechocystis chromosomal neutral sites using bioinformatics tools and RT-PCRs. Design and construction of synthetic parts and assembly of modules following the BioBrick™ system philosophy. Characterization of parts and devices in the cyanobacterial chassis, using reporter genes and measuring enzyme activities. Results: The chassis preparation started with the removal of redundant parts, like the deletion of genes encoding the Synechocystis native bidirectional hydrogenase. In addition, the Synechocystis genome was screened for putative neutral sites, which will facilitate the integration of the synthetic parts/devices (e.g. an efficient heterologous hydrogenase). The construction of optimized synthetic modules/devices requires the preliminary characterization of parts, such as promoters, transcription factors, RBSs and terminators, which is currently being performed. Furthermore, several oxygen consuming devices were designed and synthesized, and are in the process of being tested. One of these devices will be chosen to provide a microaerobic/anaerobic environment required for the optimal expression of a heterologous hydrogenase.
Department of Botany, Institute of Biological Sciences, Universidade Fedearl de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil. Introduction: Cyanobacteria are known to produce various kinds of bioactive oligopeptides. The most common peptides are microcystins, cyanopeptolins, anabaenopeptins and microviridins. There are several hypotheses about the functions of these metabolites, which includes protection against grazing, allelophatic effects, quorum sensing and internal metabolism, but some of these functions might be only of secondary importance. The comprehension of the factors affecting the production of these secondary metabolites may give some clues about the ecological and physiological functions of these compounds. Methods: In this study, the influence of light intensity on the pattern of oligopeptide production of a Radiocystis fernandoi strain was tested. The strain used in this work was isolated from a reservoir located in the southeastern region of Brazil. This species usually forms blooms in eutrophic aquatic systems. The experiments were done in triplicates and consisted of three treatments, each one at a different light intensity: 25 µmol.m-2.s-1, 65 µmol.m-2.s-1 and 95 µmol.m-2.s-1. Experiments lasted 10 days and growth conditions were 12h light: 12h dark photoperiod at 20°C. At the end of the experimental time, cultures were freeze dried and the peptides extracted with 75% methanol and purified with C18 SPE cartridges. The peptides were quantified by HPLC (Waters Alliance) and the main peaks were collected and analyzed in a MALDI-TOF Bruker Autoflex III mass spectrometer. Results: Seven different peptides were identified, 1 cyanopeptolin (Cy-1071), 1 microviridin (Mv-1709) and 5 microcystins (Mc-RR, dMcYR, Mc-YR, Mc-FR and Mc-WR). The production of Mc-YR, Mc-FR, Mc-WR and the sum of all five microcystins were highest at 25 µmol.m2 -1 .s , lowest at 65 µmol.m-2.s-1 and intermediate at 95 µmol.m-2.s-1. The production of Mc-RR was intermediate at 25 µmol.m-2.s-1, higher in the 65 µmol.m-2.s-1 treatment and lower at 95 µmol.m-2.s-1. The Mv-1709 was produced in the greatest amount at 65 µmol.m-2.s-1 and in lower amounts in the other two treatments. The production of the seven peptides together was highest in the 25 µmol.m-2.s-1 treatment and lower in the other two treatments. The production of Cy-1071 and the dMc-YR showed no significant differences among the treatments. Conclusions: It is possible that differences observed between the MCRR and other microcystins could be explained by genetic regulation and/or aminoacids and enzyme activities modulated by different light intesities. The fact that the different classes of peptides do not present similar responses to environmental factors, such as light, suggests that these compounds may have different functions.
Conclusions: The characterization of the different parts will allow the August 9 to 14, 2009 • Montréal, QC, Canada
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P.045 ENGINEERING BIODEGRADABLE PLASTICS USING EXTRACELLULAR MATRIX IN THE TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE.
blended control, indicating that the composite has the strength for practical usages as bioplastics. The composites have water-absorbing capacity and can be used as a container for either fresh vegetables or dry medicament. N. commune EPSs enhance biodegradability of the composites in soil and it maybe an advantage for the disposal of the plastics.
K. Inoue-Sakamoto1, T. Ogawa2, T. Sakamoto3. 1 Department of Applied Bioscience, College of Biotechnology and Chemistry, Kanazawa Institute of Technology (K.I.T.), Ohgigaoka, Nonoichi; 2Department of Applied Chemistry, College of Biotechnology and Chemistry, K.I.T., Ohgigaoka, Nonoichi; 3Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa; Japan.
Introduction: The terrestrial cyanobacterium Nostoc commune is widely distributed on earth and is adapted to various external conditions by acquiring tolerance to desiccation, ultraviolet light and extreme temperatures. N. commune forms colonies in which the filamentous cells are embedded in extracellular matrix. The major component of extracellular matrix in N. commune is extracellular polysaccharides (EPSs). EPSs absorb large amount of water rapidly and help the cells to maintain biological activities including photosynthesis and respiration. The hydroscopic or absorbent ability of EPSs can be applied to non-hydroscopic materials so the composites are used as a package to regulate moisture level inside the package. Alternatively, the composites can be used for agricultural soil cover sheet as a biodegradable sheet to enhance degradation of the composite by microorganisms in soil. In this study, colonies of N. commune or isolated EPSs were blended with plastics to test the capacity of water in the composites, and the physical properties of the composites were investigated. Methods: The colonies of N. commune naturally grown in the field were collected, washed and air-dried. The dried colonies were crushed in a kitchen mixer and then powdered in a frozen pulverizer. The powder with diameter less than 100 µm were selected with a sieve and blended directly with pellets of polybuthylene succinate (PBS) or low density polyethylene (LDPE) in weight ratio of 10:90, 15:85 and 20:80 by using a compression molding machine and an extruder in combination. EPSs were prepared from N. commune colonies by extraction with acetone and diluted acetic acid and precipitation by ethanol. EPSs were freeze-dried, powdered and blended with PBS or LDPE as described above. The sample sheets of the composite were used for the analyses. For mechanical properties, tensile stress and tensile strain at break were measured by a tensile tester. For the water absorption capacity, the samples were immersed in deionized water and the increase in weight of the samples was measured. For the biodegradability assay, the samples were buried in wet soil and their surfaces were periodically observed by scanning electron microscopy.
P.046 ONE-STEP PLASMID CONSTRUCTION FOR GENERATION OF KNOCK-OUT MUTANTS IN CYANOBACTERIA. J.H. Jacobsen1, D. Paiva2, N.-U. Frigaard1, Y. Sakuragi2. 1
Department of Biology, Faculty of Science, University of Copenhagen, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark. 2
Introduction: Cyanobacteria include some of the most efficient phototrophic organisms known, featuring doubling times of less than 4 hours at exponential growth for certain strains. They are therefore ideal for biomass and bioenergy production through metabolic engineering, including gain-of-function and loss-of-function mutagenesis. The recently reported uracil-specific excision reagent (USER) cloning method1, 2, previously applied on plants and fungi, allows highly efficient ligase- and restriction-enzyme-independent construction of knock-out vectors. In the present work we adapted the “four fragment USER-cloning method”1, for utilization in cyanobacteria in order to streamline the construction of vectors for loss-of-function mutagenesis. Methods: Synthetic oligonucleotides encoding the USER-cloning cassette, comprising a BamHI restriction site, flanked by PacI and Nt.BbvCI restriction sites, was inserted into pUC19. Antibiotic resistance cassettes suitable for selection in cyanobacteria were inserted at this unique BamHI restriction site within the USER-cloning cassette. Digestion of the resulting USER-cloning vectors with PacI and Nt.BbvCI generates two fragments, the vector backbone and a fragment containing the antibiotic resistance gene, respectively. Regions flanking the genes encoding glycogen phosphorylase (glgP) and photomixotrophic growth related protein (pmgA) from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 were amplified with uracil-containing oligonucleotides and annealed with the digested vector and directly used for Escherichia coli transformation. Constructs were confirmed by PCR and restriction map analysis. The final constructs were used to transform Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803.
Results: The surface of sheets made from plastic composites with N. commune powders were rougher than that of control, showing that adhesion between plastics and N. commune powder is poor and the two polymers are incompatible. The tensile stress of pure LDPE polymer was about 10 MPa, whereas the stresses of LDPE composites were 6-9 MPa. The composites absorbed 1.2-1.5% (w/w) of water and the tensile stresses of the water-absorbed composites were 4-5 MPa, which was approximately 50% of control. The surface of the composite sheets kept in soil for several months had more irregular structure than that of untreated control, suggesting that the polymer from N. commune absorb water and enhance degrability of the composites.
Results: A set of eight different plasmids for “four fragment USERcloning” has been constructed, each construct containing one of the antibiotic resistance conferring genes aadA (SmR,SpR), aacC1 (Gmr), ermC-cat (Emr,Cmr), nptII (Kmr,Neor), inserted in either parallel or antiparallel orientation. Knock-out vectors for insertional deletion of glgP or pmgA in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 have been constructed in the plasmid vectors containing aacC1 and aadA for glgP and pmgA, respectively. Restriction digestion patterns of selected clones confirmed the presence of the correct inserts in all the clones, demonstrating the high efficiency of the cloning strategy. Transformants from cyanobacterial transformation with glgP and pmgA deletion constructs have been obtained, and homozygous mutants are currently being selected. The results from analysis of glycogen contents, thus biomass capacity, of the mutants will be presented.
Conclusions: The composites were successfully made from plastic pellets and powders of N. commune colonies or EPSs. The plastic composites still remained over 50% of the tensile stress of non-
Conclusions: The USER-cloning method provides a fast and reliable strategy for generation of knock-out mutants in the cyanobacterial species Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803
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13th International Symposium on Phototrophic Prokaryotes through single step construction of knock-out vectors. References: 1 Frandsen, R.J., Andersson, J.A., Kristensen, M.B., Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Molecular Biology (2008), 9. 70 2 Nour-Eldin, H.H., Hansen, B.G., Norholm, M.H., Advancing uracilexcision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Research (2006), 34. e122
P.047 MAXIMIZING HYDROGEN PRODUCTION IN RUBISCOCOMPROMISED MUTANTS OF NONSULFUR PURPLE PHOTOSYNTHETIC BACTERIA. Rick A. Laguna, F. Robert Tabita. Department of Microbiology, The Ohio State University, Columbus, OH, USA. Introduction: The Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway allows for the reduction of CO2 into cellular carbon under aerobic chemoautotrophic and anaerobic photoautotrophic growth conditions. By contrast to its use as a major carbon source, during photoheterotrophic growt CO2 is primarily used as an electron acceptor. The CBB cycle thus performs the important function of maintaining the redox balance of the cell under these growth conditions. Ribulose 1,5-bisphosphate carboxylase-oxygenase is the key enzyme of the CBB pathway that catalyzes the reduction of CO2. Over the years we have shown that nonsulfur purple (NSP) photosynthetic bacteria possess an array of metabolic and regulatory capabilities that allow for the utilization of alternative redox sinks when the primary electron sink, CO2, is nullified via the inactivation or deletion of the RubisCO genes. For example, in many instances the derepression of nitrogenase synthesis occurs under normal repressive conditions. Such gain-of-function adaptive mutant strains have been obtained from Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, and Rhodopseudomonas palustris, whereby such strains balance their redox potential via nitrogenase-catalyzed reduction of protons to hydrogen gas. Over the years we have shown that nitrogenase-derepressed mutant strains produce copious quantities of hydrogen gas by virtue of using the nitrogenase enzyme complex exclusively as a hydrogenase. NifA is the transcriptional activator of the nif (nitrogen fixation) operons. In general nifA expression is regulated by the cascade of proteins that encompass the nitrogen fixation regulon (Ntr), while post-translational regulation of NifA occurs through protein-protein or protein-metabolite mediated contact. Method: To determine if a mutation occurred in NifA of RubisCOdeletion nitrogenase derepressed mutant strains, the nifA genes were sequenced. Next, the mutant NifA protein was inserted into a wildtype background to determine if derepression of the nitrogenase complex occurred. Hydrogen production values were recorded from the RubisCO-deletion nitrogenase derepressed mutant strains. Results: A single point mutation in the NifA protein was identified in the RubisCO-deletion nitrogenase derepressed mutant strains of R. capsulatus, R. sphaeroides, and R. palustris. A mutant NifA protein appeared to be responsible for derepression of the nitrogenase complex in R. palustris. In R. sphaeroides an additional unidentified mutation appears to be involved in the derepression of the nitrogenase complex. Interestingly, no such nifA mutation was found in a nitrogenase-derepressed strain of a RubisCO knockout strain of R.
August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts rubrum. The RubisCO-deletion strain of R. palustris produced the most hydrogen compared to the other mutant strains. Conclusion: These results suggest that some NSP bacteria utilize different mechanisms to derepress nitrogenase synthesis in CBBcompromised mutant strains. Inactivating competing redox balancing processes, such as the CBB pathway, plays a crucial role in maximizing hydrogen production in nitrogenase-derepressed strains. Further studies are directed at understanding the molecular mechanisms that allows the cbb and nif systems to compete for reducing equivalents and become subject to reciprocal control.
P.048 SCREENING OF SOIL CYANOBACTERIA FROM VIETNAM FOR ANTIBACTERIAL ACTIVITY. Le Thi Anh Tuyet1, Sabine Mundt2. 1
Department of Natural Sciences, Hong Duc University, Thanh Hoa, Vietnam; 2Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Germany. Introduction: A large number of interesting metabolites of wide chemical diversity has been isolated from cyanobacteria and compounds with antibacterial, antiviral, antifungal and cytotoxic activity have been detected. Currently we are investigating biologically active chemical substances from cyanobacteria with the objective of finding new antibacterial compounds which could be a source of new lead structures for development of antibacterial drugs. As only a few cyanobacterial species from Vietnam have been screened for antibacterial activity , in the present study 11 cyanobacterial strains isolated from acidic soils of paddy and cotton fields in Vietnam belonging to the genera Nostoc, Anabaena, Calothrix, Scytonema, Oscillatoria and Westiellopsis were cultured and tested. Methods: All the organisms were grown under controlled laboratory conditions in Greifswald University, Germany. The biomasses were lyophilised and extracted with n-hexane, methanol, and water, respectively. Culture media were extracted with ethyl acetate. All crude extracts were tested for antibacterial activity against Bacillus subtilic ATCC 6051, Staphylococcus aureus ATCC 6538, E.coli ATCC11229, and Pseudomonas aeruginosa ATCC 22853 in the agar diffusion test system using 6mm diameter paper disc containing 2 mg extract per disc. An inhibition zone of 8 mm or more was considered as good antibacterial activity. Ampicilline was used as control. Results: Antibacterial evaluation demonstrated that 91 % of methanol extracts exhibited activity to Bacillus subtilis and Staphylococcus aureus, whereas the maximum inhibition zones of 19mm were presented by methanol extracts of Westiellopsis sp. VN and Calothrix elenkinii. 45 % of the methanol extracts inhibited the growth of E.coli, while 18 % of methanol extracts possessed antibacterial activity against Pseudomonas aeruginosa. 17 % of n-hexane extracts inhibited the growth of Gram-positive bacteria and 6 % of n- hexane extracts exhibited activity against Gram- negative bacteria. No aqueous extract showed antibacterial activity. 75% of the ethyl acetate extracts of culture medium exhibited significant activity against Gram-positive bacteria (a maximum inhibition zone of 25mm was exhibited by Westiellopsis sp. VN) and 50 % of ethyl acetate extracts inhibited growth of Gram-negative bacteria. Of the 11 cyanobacteria investigated, at least one of the prepared extracts from biomass or cultivation medium showed antibacterial activity. Among the active extracts, the best antibacterial activity was detected for methanol and ethyl acetate extracts of Westiellopsis sp. VN and methanol extract of Calothrix elenkinii. 105
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Final Program and Abstracts Conclusions: This screening proves that Vietnamese cyanobacteria are a promising source of new antibacterial substances. Based on these results Westiellopsis sp.VN and Calothrix elenkinii were selected for bioassay-guided isolation of the active compounds. Structural elucidation of the substances is in progress.
these mutants exhibited significantly higher H2 production rates than did the parental ∆hupL strain.
P.049
P.050
SITE-DIRECTED MUTAGENESIS OF ANABAENA SP. PCC 7120 NITROGENASE TO INCREASE PHOTOBIOLOGICAL H2 PRODUCTION.
CYANOBACTERIA - A PROLIFIC SOURCE OF NEW PHARMACEUTICALS.
Hajime Masukawa1, Kazuhito Inoue2,3, Hidehiro Sakurai3,4, and Robert P. Hausinger1,5. 1
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA; 2Department of Biological Sciences and 3Research Institute for Photobiological Hydrogen Production, Kanagawa University, Hiratsuka, Kanagawa, Japan; 4Department of Biology, School of Education, Waseda University, Shinjuku, Tokyo, Japan; 5Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA. Introduction: Hydrogen gas (H2) produced photobiologically by cyanobacteria that use sunlight as the sole energy source and water as the ultimate electron donor has the potential to become a large-scale, environmentally-friendly, renewable energy commodity. H2 is generated by hydrogenases and, as an inevitable by-product of N2 fixation, by nitrogenases. The best studied nitrogenase consists of two separable metal-containing proteins, the Fe protein (dinitrogenase reductase encoded by nifH) and the MoFe protein (dinitrogenase encoded by nifDK). The latter contains the FeMo cofactor (FeMoco) at which substrate is bound and reduced. Mutagenesis of Azotobacter vinelandii MoFe protein has shown that selected amino acid substitutions in the vicinity of FeMoco allow for effective proton reduction, while eliminating or greatly diminishing N2 fixation. We have examined whether modifications of residues near FeMoco of the cyanobacterial MoFe protein would allow electron flow through nitrogenase to be directed preferentially toward proton reduction to increase the rate of photobiological H2 production. Methods: The nifHDK genes, with the insertion element within nifD excised, were obtained from Anabaena sp. (sometimes called Nostoc sp.) PCC 7120 genome and a non-replicating vector expressing nifHDK was constructed. Site-directed mutations were introduced into nifD. To express the mutated version of nifHDK and eliminate the wild-type version, the nifHDK genes were deleted within a ∆hupL, uptake hydrogenase-negative, variant of Anabaena sp. PCC 7120 as the parental strain and the vector was integrated into the nifHDK deletion mutant chromosome by homologous recombination. Results: We performed site-directed mutation to replace six target residues, all predicted to lie within 5 of FeMoco, with different residues for a total of 49 variants. H2 production and nitrogenase (acetylene reduction) activities of the site-directed mutants were determined and several mutants were shown to exhibit significantly higher H2 production rates in the presence of N2 than did the parental ∆hupL strain. Additional targets for site-directed mutagenesis were based on the proposed hydrophobic substrate channel that connects FeMoco to the protein surface in the X-ray crystal structure of A. vinelandii MoFe protein. Corresponding residues were predicted in the cyanobacterial enzyme for this region. Four hydrophobic residues in the putative channel were replaced with bulky or charged residues in an attempt to inhibit access of N2 to FeMoco. Thirteen single and six double site-directed mutants were constructed; however, none of 106
Conclusion: Particular mutations of certain residues close to the FeMoco prosthetic group were shown to lead to increased partitioning of electron flow toward proton reduction relative to acetylene reduction.
Sabine Mundt1,2, Elmi Zainuddin1, Thi Ngoc Ha Bui1, Susann Kreitlow1, GeroldLukowski2, Rolf Jansen 3, Victor Wray3, Manfred Nimtz3. 1
Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald; Institute of Marine Biotechnology e.V., Greifswald; 3Helmholtz Centre for Infection Research, Braunschweig; Germany.
2
Introduction: In the last decades screening programs have revealed that cyanobacteria are potential sources of new active substances, but the rich resources of German lakes, the Baltic Sea and Asian countries are hardly screened so far. Due to recent development in bacterial and viral resistance and the increase in incidence of tumour diseases worldwide we focused on screening of antibiotic and cytostatic/cytotoxic activity of such hardly tested cyanobacterial strains and isolation and structural elucidation of the active substances. For practical use of biomass or isolated substances the form of application is very important. Methods: Laboratory cultures were established and biomass and cultivation medium were extracted with solvents of different polarity. Lipophilic and hydrophilic extracts have been tested in agar-plate diffusion test for antibacterial and antifungal activity, for antiviral activity against Influenza virus A and Herpes virus Typ1 and for cytotoxic/cytostatic activity against MCF7 (breast cancer cell line), 5637 (urinary bladder) and FL (amnion) cell lines. Bioassay-guided isolation of the active compounds was done by column chromatography including HPLC. Structure was elucidated by analysis of ESI-MS-MS, ESI-TOF-MS, 1D (1H and 13C) and 2D (COSY, TOCSY, ROESY, NOESY, HMQC and HMBC) NMR spectra and amino acid analyses. The preparation of micro- and nanoparticles was done according to PCT/DE 03/00747. A pre-suspension of the biomass and a surfactant-water mixture was produced by stirring followed by high pressure homogenization. Results: Separation of the n-hexane extract of Limnothrix redekei HUB 051 (Müggelsee, Germany) resulted in the identification of three unsaturated fatty acids, α-linolenic acid, coriolic acid and αdimorphecolic acid showing antimicrobial activity in vitro. From the methanol extract of the filamentous Nostoc strain CAVN 10 from a soil sample collected in North Vietnam four new compounds, carbamidocyclophanes, with cytotoxic activity against tumor cell lines have been isolated. Separation of the methanol extract of a Microcystis strain (Plön, Germany) led to identification of two new cyclic peptides with activity against Influenza virus A. Application of micro- or nanoparticles prepared from the biomass of an Anabaena strain Bio 33, isolated from the Baltic Sea, prevents the dermal colonisation of MRSA. Conclusions: Our first results show that so far hardly investigated cyanobacteria from the Baltic Sea, German lakes and from Asian countries are sources of structural new active compounds with potential therapeutically value. Especially for external application micro- and nanoparticles are a promising application form.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
P.051
P.052
GENES PUTATIVELY INVOLVED IN THE SYNTHESIS OF CYANOBACTERIAL EXOPOLYSACCHARIDES.
IDENTIFICATION OF PROTEINS INVOLVED IN THE CHROMIUM REDUCTION IN SYNECHOCYSTIS DERIVATIVE MK-TR OBTAINED BY GAMMA IRRADIATION.
Sara Pereira1,2, Ângela Brito1,2, Andrea Zille1, Pedro Moradas-Ferreira1,3 & Paula Tamagnini1,2. 1
2
IBMC - Instituto de Biologia Molecular e Celular; Faculdade de Ciências, Departamento de Botânica; 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS); Universidade do Porto, Porto, Portugal Introduction: Many cyanobacterial strains possess outside their outer membrane, additional surface structures, mainly of polysaccharidic nature, that are usually referred to as exopolysaccharides (EPS). Even though EPS are common in cyanobacteria, their biosynthetic pathways and the factors that regulate these processes are far from being fully understood. However, the mechanisms involved in the synthesis of EPS are relatively conserved throughout bacteria, involving three classes of proteins: (i) enzymes involved in the biosynthetic pathways of nucleotide sugars (ii) glycosyltransferases, and (iii) those required for the oligosaccharide or polysaccharide extension and processing. The sugar activation/modification enzymes and the glycosyltransferases are strain dependent, whereas the proteins involved in the polymerization, chain length control and export are generally conserved among bacteria [1]. Methods: Genome sequences were retrieved from GenBank and computer-assisted sequence comparisons were performed using BLASTp, cDART (NCBI), ClustalW (EMBL-EBI), and SMART. Transcription profiles of the genes were evaluated by RT-PCR using Cyanothece sp. CCY 0110 cells grown under different physiological conditions that are known to affect EPS production: presence/absence of combined nitrogen and different light regiments. Carbohydrate content of the cultures was measured using the Dubois method [5]. Results: An in silico analysis of genome sequences performed in three morphological distinct types of cyanobacteria (unicellular, filamentous and filamentous heterocystous) revealed the presence of genes encoding proteins that, in other organisms, are involved in the last steps of EPS production. In cyanobacteria those genes, namely wza, wzb, wzc, wzx and wzy are frequently clustered and often occurring as multiple copies. Using this information a model was proposed for the polymerization and export of cyanobacterial EPS. Additionally, preliminary results revealed that the transcripts levels of wzb (encoding a tyrosine phosphatase that putatively regulates polysaccharide chain-length by dephosphorylation of Wzc) and wzy (encoding a polymerase) are higher in cells grown in absence of combined nitrogen. Conclusions: Although several genes encoding proteins putatively involved in the EPS polymerization and export were identified in cyanobacterial genomes, their physical organization differs from what is observed in other microorganisms, where the genes are frequently clustered and transcribed as one or two operons [2,3] suggesting that the regulation of the synthesis and export of EPS is more complex in cyanobacteria. References: [1] Pereira et al (2009) FEMS Microbiol Rev (accepted for publication) [1] De Vuyst L et al (2001) Int Dairy J 11: 687-707 [1] Whitfield C (2006) Annu Rev Biochem 75: 39-68
August 9 to 14, 2009 • Montréal, QC, Canada
Saiqa Razi, Shahida Hasnain, Byung-Gee Kim. University Of The Punjab, Lahore, Punjab, Pakistan. Introduction: Synechocystis species, AHZ-HB-MK (DQ 381960) isolated from the local chromium contaminated industrial wastewater, able to reduce 68% toxic hexavalent Chromium (Cr VI), was used in this study. Cultural characteristics of this strain have been determined (Hameed and Hasnain, 2005) and the plant-microbe interaction experiments with this strain proved that it is important in the bioremediation of toxic Cr (VI) from the soil and in the improvement of growth parameters of Triticum aestivum grown under Cr (VI) stress (Faisal et al., 2005). To produce mutants of this strain with better chromate reduction abilities, the strain was irradiated with a 60Co source in a dose range of 0.5, 1, 2, 5, 10 and 20 Grays (Gy) at different stages of growth i.e. after 5, 15 and 30 days of incubation. Derivatives reducing < 90% Cr VI were obtained with low dose irradiation in cultures irradiated at earlier growth stage (Razi and Hasnain, 2006). Biochemical analysis including the estimation of soluble proteins, peroxidases, carotenoids and auxin showed that low radiation also caused stimulatory effects on all of these parameters at earlier stage of growth (Razi and Hasnain, unpublished data). Maximum reduction potential i.e. 94.93% was obtained at dose 2 Gy in cultures irradiated after 5 days of incubation. This derivative was labeled as Synechocystis MK-TR. In the present study, proteomic study of this selected derivative was done in order to identify the proteins involved in the chromium reduction. Methods: After three days of Cr VI exposure, crude extracts from treated and untreated cells were compared at the proteome level using one-dimensional SDS-polyacryalmide gel electrophoretic approach. Proteins were extracted from 100 ml of cultures by repeated freeze-thawing, sonication and vortex mixing (Nayak et al., 2007). From cell extracts, the concentration of proteins was measured by the Coomassie Blue protein assay at 595 nm utilizing bovine serum albumin as a standard (Bradford, 1976). Ten out of total twenty protein bands were upregulated in this derivative under Cr VI stress. These ten protein bands were cut from 1D-SDS polyacryalmide gels and were subjected to in-gel trypsin digestion (Shevchenko et al., 1996). The eluted peptides were dissolved in 0.1% formic acid and loaded on C18 columns. Samples were analyzed by a combination of a nano-HPLC/microelectrospray ionization on a LCQ Deca mass spectrometer (ThermoFinnigan, San Jose, CA). MS-MS spectra were matched with the genome sequence of the model strain Synechocystis PCC 6803 CyanoBase http://bacteria.kazusa.or.jp/cyanobase/Synechocystis/index.html (Kaneko et al., 1996), Results: Seventy-four peptides were identified from these ten selected protein bands. In the long list of these identified peptides we found sll0170, a heat shock protein, slr2076 a 60kD chaperonin and sll0416 identified as 60kD chaperonin 2 and GroEL2. Whereas sll0170 is also a heat shock protein 70, slr1198 was identified to be an antioxidant protein and slr1516 was annotated as superoxide dismutase. Conclusion: This can be assumed from these findings that these proteins were induced due to radiation-stress and are associated with General Stress-proteins family including Heat-shock proteins (HSP). They helped this strain to cope with the osmotic and heat stress when treated with gamma radiation. When this strain was subjected to 107
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Final Program and Abstracts chromium stress these proteins gave stronger expression and again helped the strain to overcome the osmotic stress in this case induced by chromium. The radiation dose of 2 Gy might have caused mutations in the genes of this derivative resulting in the overproduction of these stress proteins.
P.053 ENHANCEMENT OF GROWTH AND YIELD OF TOMATO BY APPLICATION OF PURPLE NONSULFUR BACTERIA. Hong-Gyu Song, Kang-Hyeong Lee. Kangwon National University, Chuncheon, South Korea. Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strain Rhodopseudomonas sp. KL9 maximally produced 5.56 mM/min/mg protein and 67.2 μM/min/mg protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. Germination percentage of tomato seed, total length and dry weight of germinated tomato seedling increased by 30.2%, 71.1% and 270.8%, respectively compared to those of the uninoculated control 7 days after inoculation of strain KL9. A greenhouse test was also carried out to examine the effects on tomato growth of application of purple nonsulfur bacterium Rhodopseudomonas sp. The shoot length of tomato plant inoculated by Rhodopseudomonas sp. KL9 increased by 34.6% compared to that of control in 8 weeks of cultivation. During the same period, this strain increased 120.6 and 78.6% of dry weight of shoot and root of tomato plants, respectively. The formation ratio of tomato fruit from flower was also raised by inoculation of KL9. In addition, Rhodopseudomonas sp. KL9 treatment enhanced the fresh weight and lycopene content in the harvested tomato fruits by 98.3 and 48.3%, respectively compared to those of the uninoculated control. When the effect on the indigenous bacterial community and fate of the inoculated Rhodopseudomonas sp. KL9 were monitored by denaturing gradient gel electrophoresis analysis, its application did not affect the native bacterial community in tomato rhizosphere soil, but should be repeated to maintain its population size. This bacterial capability may be applied as an environment-friendly biofertilizer to cultivation of high quality tomato and other crops including lycopenecontaining vegetables and fruits.
P.054 UNUSUAL CAROTENOIDS FROM SOME NEWLY DESCRIBED PURPLE BACTERIA. 1
2
3
Shinichi Takaichi , Ch. Sasikala , Ch. V. Ramana . 1
Department of Biology, Nippon Medical School; 2Center for Environment, Institute of Science and Technology, J. N. T. University; 3 Department of Plant Sciences, School of Life Sciences, University of Hyderabad. Introduction: Anoxgenic phototrophic purple bacteria belong to the Proteobacteria, and more than 170 species have been described. These bacteria produce many different carotenoids, which are essential for photoprotection and light-harvesting. Base on the structures of the carotenoids and the characteristics of the carotenogenesis enzymes, the normal pathway of carotenogenesis is the normal spirilloxanthin pathway, and is found from around half species. When one enzyme of the normal spirilloxanthin pathway is lacking or is present with reduced activity, the carotenoid composition of the bacterium will be expected to change (unusual spirilloxanthin, spheroidene, and carotenal pathways) (Takaichi (2009) Distribution and 108
Biosynthesis of Carotenoids, In The Purple Phototrophic Bacteria, pp.97-117). Further, Ramana group have recently found and described more than 20 species of purple bacteria. We found some unusual carotenoids from these newly described purple bacteria. Methods: New purple bacteria were described in Int. J. Syst. Evol. Microbiol., and cultured anaerobically under light. Carotenoids were extracted from the lyophilized cells and purified. They were identified based on absorption spectra, retention time on C18-HPLC, MS, and 1HNMR analyses. Results: Phaeospirillum chandramohanii JA145 (Anil Kumar et al. IJSEM in press) contained 10% (mol% of total carotenoids) lycopene, 77% hydroxylycopene, 11% hydroxylycopene glucoside and 2% dihydroxylycopene diglucoside; “Phs. oryzae” JA317 contained 12% lycopene, 81% hydroxylycopene and 7% hydroxylycopene glucoside; Phs. molischianum DSM120 contained 17% lycopene, 77% hydroxylycopene, 5% hydroxylycopene glucoside and 1% dihydroxylycopene glucoside; Phs. fuluvum contained 11% lycopene, 84% hydroxylycopene, 5% hydroxylycopene glucoside and 1% dihydroxylycopene glucoside. Carotenoid glycosides in purple bacteria are rare, and dihydroxylycopene diglucoside is only found from Halorhodospira. This may be due to no activity of CrtD, and additionally these have glucosyl transferase. Roseospira visakhapatnamensis JA131 (Chakravarthy et al. (2007) IJSEM 57: 2453-2457) contained 3% lycopene, 82% 3,4dehydrorhodopin, 1% anhydrorhodovibrin, 4% hydroxyspirilloxanthin and 10% spirilloxanthin. This may be due to low activity of CrtF. Since all of other four species of Roseospira showed almost compatible absorption spectra of the cells, the major carotenoid may also be 3,4dehydrorhodopin. Conclusion: The main carotenogenesis pathway of purple bacteria is the normal spirilloxanthin pathway, and unusual spirilloxanthin pathways are also found. We found genus specific unusual carotenoid compositions, and this might be due to absence or low activity and presence of certain novel enzyme.
P.055 HYDROGEN PHOTOPRODUCTION BY SUSPENSION OR IMMOBILIZED PURPLE PHOTOSYNTHETIC BACTERIAL CULTURES USING PRODUCTS OF DARK FERMENTATION OF STARCH AND POTATO. Darya N. Tekucheva1,3, Tatyana V. Laurinavichene1, Maria L. Ghirardi2, Michael Seibert2, Anatoly A. Tsygankov1. 1
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, Russia; 2National Renewable Energy Laboratory, Chemical and Biosciences Center, Golden, CO, USA; 3 South Federal University, Rostov-on-Don, Russia. Introduction. Transformation of organic wastes to pure water and H2 is an important societal goal. This project is a preliminary study of the steps required to integrate dark fermentative waste treatment with light-dependent H2 production into a single process. Methods. Fermentative effluents (FEs) after starch and potato treatment in the dark were characterized and tested as substrates for subsequent light-dependent H2 production. Before testing, the FEs were neutralized, centrifuged and sterilized. H2 photoproduction and the efficiency of volatile fatty acid (VFA) utilization by the purple bacteria (PB) were characterized in suspension and immobilized cultures using different FE dilutions. Results. Acetate and butyrate were the predominant products of both starch and potato fermentations. Concentrated FEs inhibited both August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes growth and H2 production of PB due mainly to the high VFA content. FEs after potato fermentation contained a variable amount of NH4+, which is detrimental for short-term H2 photoproduction mediated by the nitrogenase enzyme. Furthermore, during long-term experiments in the presence of N compounds, a portion of the VFAs is converted to biomass, thus lowering the efficiency of H2 production. Moreover, at ≥2.5% FE, light limitations occur due to high biomass concentration promoted by the availability of N compounds in the FE. In thin-layer photobioreactors (PhBR) with higher irradiation, a higher percentage of the FE could be treated, and 10% FE provided a volumetric H2 output of approx 2.7 L L-1 PhBR, which corresponded to a potential value of 27 L L-1 FE. PhBRs containing PB immobilized on a glass-fiber matrix can also avoid light limitation. The operational characteristics of such a PhBR under a continuous H2-production regime were examined using artificial media with different concentrations of N-sources and electron donors. (lactate, acetate, and mixed VFAs) or real FEs (at different dilutions). H2 photoproduction was stable for ~3.5 months. The FEs obtained from the fermentation of 400 g L-1 of potato biomass (total VFA ~400 mM) maintained H2 photoproduction at ~60 ml h-1 L-1 of PhBR volume even at concentrations of 1.25% FE in water. The potential total volumetric H2 output recalculated for 100% FE was ~36 L L-1 FE. If the dark fermentations were performed at low substrate concentration (i.e., at 5% starch) and the accumulated VFA concentration was not high (~27 mM total), the photofermentation stage could be successfully operated without preliminary FE dilution. Conclusions. Our results indicate that the integration of dark fermentative treatment of starch or potato biomass and light dependent H2 production by PB using FEs is possible in principle. However, some critical problems will need to be solved. This work was supported by the Russian Program of Basic Research RAS, RFBR, and the US DOE Hydrogen, Fuel Cell, & Infrastructure Technologies Program.
P.056 Metabolic Engineering of Glycogen Metabolism to Enhance the Production of Glucose Derivatives in a Cyanobacterium Synechococcus sp. PCC 7002. Yu Xu, Donald A. Bryant. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA. Introduction: Glycogen is a major storage form for carbon and reducing power in cyanobacteria. Slower or interrupted glycogen assimilation could provide more glucose or glucose derivatives in an industrially appealing way for biofuel production. Accumulation of these substances could also facilitate the biosynthesis of some valuable metabolites such as sucrose, lactate, H2 etc., by either converting the carbon product directly or using the reducing equivalents of glucose, or by both ways. Another promising application is to co-cultivate a cyanobacterium with a heterotrophic bacterium, in which system the cyanobacterium provides feedstock for the heterotroph and the latter produces a valuable end product(s). In this report, we use a marine-type cyanobacterium Synechococcus sp. PCC 7002 as the model system to achieve the goals above, by interrupting several key enzymes involved in glycogen biosynthesis. Methods: Linearized DNA fragments that contain a suitable antibiotic resistance gene marker flanked by upstream and downstream homologous regions of the targeted gene were constructed and used August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts to transform WT cells. Desired deletion mutants were selected and segregated by selection with specific antibiotics. Glucose concentrations in different cell fractions were measured by a glucose assay kit provided by Sigma. Photosynthetic O2 evolution rates were measured with a Clark-type electrode. Cell ultrastructure was observed by TEM. Results: Two glycogen synthase genes glgA1 and glgA2, a 1,4-alphaglucan branching enzyme gene glgB, and a glucose-1-phosphate adenylyltransferase gene glgC were successfully deleted in Synechococcus sp. PCC 7002 to create the following mutants: glgA1, glgA2, glgB, glgC, glgA1 glgA2, glgA1 glgB, glgA2 glgB and glgA1 glgA2 glgB. All mutant alleles segregated completely. High-light adapted (HLA) strains of each mutant are also selected. Preliminary data show no obvious difference in O2 evolution rates in those HLA mutants compared to HLA WT cells. HLA mutants of glgA1 glgA2, glgA1 glgA2 glgB, and particularly glgC, were found to have less insoluble glycans. They excrete more glucose into the growth medium than wild type, although excretion levels were still low. The ultrastructure of HLA glgA1 glgA2 shows noticeably altered ultrastructure, which could result from high levels of glucose derivatives in cells. Conclusion: These mutants should have comparable photosynthesis rates as the WT, indicating a stable carbon and energy input in these mutants. However, they are defective in depositing carbon and reducing equivalents into glycogen. Therefore, more glucose or glucose derivatives should accumulate in cells or be excreted. Further analyses are in progress to identify these glucose derivatives and quantify their amounts compared to the WT cells.
P.057 PHOSPHOLIPID FATTY ACID ANALYSIS OF THE EUPHOTIC MICROBIAL COMMUNITY OF LAKE TUSCALOOSA. P.O. Akinwole, E. Lefevre, M.J. Powell, R.H. Findlay. Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, USA. Introduction: Euphotic microbial communities are metabolically important in aquatic system, since they carry out primary production and are critical to the cycling of both organic and inorganic carbon. We studied the seasonal variation of a euphotic microbial community to determine the factors related to its structural dynamics. Method: Integrated water samples were collected fortnightly from the euphotic zone of Lake Tuscaloosa; a man-made system in the westcentral Alabama, southeastern USA, during the period February 2008 through March 2009. Euphotic microbial biomass and community structure were determined using the total phospholipid phosphate (PLP) and fatty acid techniques respectively. Results: Lake Tuscaloosa is a warm dystrophic monomictic lake that was strongly stratified from April to November 2008. Secchi disk transparency depth was inversely correlated with total euphotic microbial biomass (r2= 0.6). Euphotic microbial biomass, measured as PLP, ranged from 5.63 (during the period of warm water) to 20.18 nmol PLP L−1 (during the period of cold water). Phospholipid fatty acids (PLFA) profiles were dominated by polyenoic fatty acids. Principal component analysis of PLFA profiles showed seasonal pattern of change in the euphotic microbial community. There was a significant shift from aerobic bacterial and phototrophic microeucaryotes during period of cold water (Winter mixing) to anaerobic bacteria and heterotrophic microeucaryotes during the period of warm water when the lake was thermally stratified. Maximum
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Final Program and Abstracts contribution to total microbial biomass by microeucaryotes occurred during the period of cold water while maximum contribution by prokaryotes occurred during the period of warm water and during Winter flood events. Conclusions: This study suggests that euphotic planktonic microbial communities show regular variations in structure that are similar to those previously observed in shallow-water marine and freshwater sediment communities.
paper contributes to outlining the ecostrategies of different species, and probes below the species level to show that we have been attempting to understand the dynamics of a “strain flock” rather than an individual genotypic organism. Genetic tracing has provided us insights that were not accessable by microscope alone.
P.059
P.058
ADAPTATION REACTIONS OF SIDEROPHILIC CYANOBACTERIA TO HIGH AND LOW LEVELS OF ENVIRONMENTAL IRON: IMPLICATIONS FOR BIOSPHERE HISTORY.
GENE TRACING OF SEASONAL SUCCESSION OF CYANOPROKARYOTES IN MISSISQUOI BAY, LAKE CHAMPLAIN.
I.I. Brown1, D. A. Bryant2, K.L. Thomas-Keprta1, S.A. Sarkisova1, E.E. Foraker1, W.J. Clarke1, G. Shen2, D.H. Garrison1, D.S. McKay1.
Hongmei Jing, David Bird, Nathalie Fortin, Rocio Aranda-Rodriguez, Charles Greer, Serge Paquet, Catalina Gonzalez Rueda.
1
Université du Québec à Montréal, Montreal, Quebec, Canada.
Introduction: Of all extant environments, iron-depositing hot springs may constitute the most appropriate natural models (Pierson and Parenteau, 2000) for analyzing the ecophysiology of ancient cyanobacteria (CB) that may have emerged in association with hydrothermal activity (Brown et al., 2007) and elevated levels of environmental Fe (Rouxel et al., 2005). Elevated environmental Fe2+ posed a significant challenge to the first oxygenic phototrophs because reduced Fe2+ induces toxic Fenton reactions (Wiedenheft et al., 2005). Ancient CB could have also experienced stress during occasional migrations from the Fe2+-rich ocean to the basaltic land that was almost devoid of dissolved Fe2+; therefore, the study of adaptation reactions of siderophilic CB, which inhabit iron-depositing hot springs, to varying levels of environmental Fe may shed light on the paleophysiology of ancient oxygenic prokaryotes.
Introduction: Shallow Missisquoi Bay, in the northern part of Lake Champlain, suffers from serious cyanobacteria blooms. For several years now, the public has been advised to stay away from recreational use of the bay for most of the summer. Following suggestions from the literature, we looked for taxonomic and genotypic changes that we could associate with toxin presence, in order to understand successional events and identify driving factors. We followed the taxonomic and genotypic dynamics, along with changes in nutrient concentrations and weather variations, to look for linkages. Methods: Two stations, one pelagic and the other littoral and downwind, were sampled weekly over the growing season from May to November in 2006 and 2007. The seasonal dynamics of cyanobacterial populations were followed by taxonomic microscopy and denaturing gradient gel electrophoresis (DGGE). Bands were excised and sequenced. Two primers were used, one (CYA) that detects most Cyanobacteria, and another (MIC) that is more restrictive and detects mainly toxic species, such as Anabaena, Aphanizomenon and Microcystis. Results: There was generally a close correspondence between taxonomic species richness in the Cyanobacteria, and DGGE band richness with the general cyanobacterial primer. DGGE band cluster analysis revealed two very distinct cyanobacterial communities, one associated with summer months (July to September) and the other developing in fall and reappearing the following spring. We suggest that this pattern is indicative of 1) niche dynamics and 2) two ecological stategies: species that are constantly present at a low and stable level, and species that appear in summer, bloom and become dominant, then disappear. Both microscopy and DGGE sequencing showed that strains of Microcystis were dominant during the summer bloom period in 2006. The greatest number of Microcystis genotypes and morphotypes were detected during the annual radiation maximum at the summer equinox, before the biomass maximum of the bloom was reached. Higher intrageneric diversity of Microcystis was detected by DGGE than by microscopy. Major storm events were always associated with important changes in community makeup in this large shallow lake Although more genotypes of M. elabens, M. viridis and M. wesenbergii were present than were genotypes of the dominant M. aeruginosa, the succession of two major M. aeruginosa genotypes accounted for the observed Microcystis biomass dynamics. The potential toxic Microcystis genotype was present throughout the whole growing season. Conclusions: Phytoplankton ecology is in its infancy. Little to nothing is known of the factors that lead to cyanobacterial dominance in a given lake, nor especially to the dynamics of individual species. This
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NASA Johnson Space Center, Houston, TX; 2The Pennsylvania State University, University Park, PA; USA.
Methods: Siderophilic CB (Brown et al., 2007) were cultivated in media with different concentrations of added Fe3+. Basaltic rocks were used as a source of Fe and trace elements in some cases. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive spectrometry (EDS) techniques were used for studying Fe mineralization and rock dissolution processes. Fluorescence spectroscopy was used to estimate the contents of chlorophyll-protein complexes. Results: Five siderophilic isolates (Chroogloeocystis siderophila, JSC1, JSC-3, JSC-11, and JSC-12) precipitated Fe-bearing phases on the exopolymeric sheaths of their cells when Fe3+ concentration was ~ 400 – 600 µM (high Fe). The same Fe3+ concentration was optimal for the culture proliferation rate (Brown et al., 2005; Brown et al., 2007). Higher concentrations of Fe3+ repressed the growth of some siderophilic CB (Brown et al., 2005). No mineralized Fe3+ was observed on the sheath of freshwater isolates Synechocystis sp. PCC 6803 and Phormidium aa strain. Scanning TEM and thin-window EDS revealed intracellular Fe-rich phases within isolates JSC-1, JSC-3, and JSC-11. The elemental composition of the Fe-rich precipitates indicates P, Fe, and O as the major elements with minor amounts of Al and Ca. The PSI/PSII ratio is higher in JSC-1 and JSC-3 isolates than in CB without any detectable ability to mineralize Fe. SEM-EDS studies of the interaction of siderophilic CB with Fe-rich minerals and rocks revealed the ability to leach ilmenite, olivine, TiO, FeS, , and ferrosilicates, perhaps because the CB studied can secrete 2-oxo-glutarate and malate that possess chelating properties. The 7.8-Mb draft genome sequence of Cyanobacterium sp. JSC-1 has been determined and is currently being closed and polished; these data will hopefully reveal the molecular mechanisms of Fe mineralization and Fe-rich minerals by siderophilic CB. Conclusions: The results suggest that colloidal Fe3+ is transported in August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes the CB cytoplasm most likely through an ABC-type Fe3+ transport system (Braun et al., 2004). The prevalence of PSI over PSII in some species of siderophilic CB as well as the ability of siderophilic CB to mineralize Fe within their cytoplasm may indirectly support the hypothesis that PSI in CB can be involved in Fe2+ oxidation (Cohen, 1984; 1989) and could be considered a protective mechanism against oxidative stress induced by high levels of environmental Fe2+ and ultraviolet radiation. The ability to leach Fe-rich minerals could have supported the expansion of ancient CB onto basaltic land.
P.060 A CLONAL POPULATION OF CHLOROBIUM DOMINATES THE ANOXIC BACTERIAL COMMUNITY IN A SWISS MOUNTAIN LAKE. Raymond P. Cox1, Lea H. Gregersen2, Donald E. Canfield3, Kirsten Habicht3, Mette Miller1 and Niels-Ulrik Frigaard2. 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 2Department of Biology, University of Copenhagen, Copenhagen, Denmark; 3Institute of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense, Denmark.
Introduction: Lake Cadagno is a meromictic lake in the Swiss Alps. The anoxic bottom water contains about 2 mM sulfate and the sulfide produced by bacterial activity supports a large population of phototrophic sulfur bacteria at and below the chemocline. Historically the dominant anoxygenic phototrophs were purple sulfur bacteria but since 2000 these have been replaced by green sulfur bacteria (GSB). We have investigated this GSB population using a multigene sequencing approach. Methods: DNA was extracted from filtered water samples from different depths and partial gene sequences obtained by PCR, cloning and sequencing. The water samples were also used for turbidity measurements and pigment analysis by HPLC with diode-array detection. Results: Analysis of a 16S rRNA clone library obtained with general bacterial primers showed that at least 50% of the recovered sequences, and more than 99% of the GSB sequences, were essentially identical and also identical with 16S rRNA from the sequenced genome of Chlorobium clathratiforme DSM 5477. The three 16S rRNA genes on this genome show a 2:1 variation in one position, and a similar variation was observed in the recovered sequences. The clonal nature of the GSB population was confirmed by partial sequences from a protein-coding gene (fmoA) and from the csmCA region containing both coding and non-coding regions. The sequence differences which were observed (less than 1 base variation per 1000 bases) were within the range expected due to sequencing artefacts from cloning of PCR products.. The clonal population was present both at the chemocline and deeper in the water column where shading by GSB in the upper layers precludes significant phototrophic growth. Conclusions: We conclude that the currently dominating GSB has arisen since 2000 from a single cell or a small clonal population. The change might have been due to mutation, invasion or a change in the environmental conditions.
Final Program and Abstracts
P.061 CANDIDATUS CHLORACIDOBACTERIUM THERMOPHILUM REVEALS A NEW NICHE FOR CHLOROSOME-CONTAINING PHOTOTROPHS. Amaya M. Garcia Costas1, Zhenfeng Liu1, Christopher Klatt2, Lynn Tomsho1, Stephan C. Schuster1, David M. Ward2, Donald A. Bryant1. 1 Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA; 2Dept. of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT; USA.
Introduction: Candidatus Chloracidobacterium (Cab.) thermophilum is a thermophilic heterophototroph isolated from the phototrophic mats of Octopus Spring in Yellowstone National Park. It is the only known phototroph in the bacterial phylum Acidobacteria, and it has not yet been isolated from any other environment. The light-harvesting apparatus of Cab. thermophilum resembles that of green sulfur bacteria, and it consists of the vesicle-like chlorosomes as antennae structures as well as the Fenna-Matthews-Olson (FMO) protein and a type 1, homodimeric reaction center. Unlike the strictly anaerobic green sulfur bacteria, Cab. thermophilum grows under oxic conditions. In order to further our understanding of the metabolic capabilities of Cab. thermophilum, as well as the distribution and role of this organism in natural environments, we have conducted biochemical analyses of its chlorosomes and have complemented these studies with genomic, metagenomic and metatranscriptomic analyses. Methods: Chlorosomes of Cab. thermophilum were isolated on sucrose gradients and analyzed by high pressure liquid chromatography (HPLC), absorption and fluorescence spectroscopy, electron microscopy and SDS-PAGE. The Cab. thermophilum genome was sequenced using 454 technology. Gap closing was done by Sanger sequencing of PCR products, and contigs were assembled with Phred/Phrap/Consed. For metatranscriptome analyses, total RNA was extracted from mat samples that had been collected at four different times during a diel cycle, converted into cDNA, and sequenced by pyrosequencing. RNAs were identified by blastN analyses, initially using the genomes of seven organisms known to occur in the mats and subsequently the GenBank NR database. Results: The chlorosomes of Cab. thermophilum contain unique pigments and proteins not present in other chlorosomes. The bacteriochlorophyll (BChl) c is methylated at the C-8 and C-12 positions as occurs in the green sulfur bacteria, but the BChl c is esterified with a mixture of alcohols, of which the major species is octadecanol. Interestingly, the chlorosomes of Cab. thermophilum exhibit redox-dependent fluorescence emission which suggested the presence of quinones in the chlorosomes. We have identified menaquinone-8 as the major quinone in chlorosomes. The Cab. thermophilum genome consists of two circular chromosomes, 2.7 and 1.1 Mb, respectively, and contains genes involved in phototrophy, photoprotection, and other Acidobacteria-specific genes. The genome encodes a complete aerobic respiratory chain, and genes involved in carbon fixation appear to be missing. This gene composition suggests that Cab. thermophilum is a photoheterotroph. Preliminary metatranscriptome analyses have revealed that the genes for the photosynthetic apparatus are highly expressed at dusk and dawn. Conclusions: Based on our combined biochemical and genomic sequencing studies we propose that Cab. thermophilum has a unique niche in the environment defined by aerophilic/microaerophilic conditions, tolerance of high light, and ability to harvest light in low light environments. Traditionally, chlorosomes have been associated
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Final Program and Abstracts with low-light, anoxic conditions. However, Cab. thermophilum appears to occupy a temporally defined, low-light niche in an otherwise highly oxic and highly illuminated environment.
P.063
P.062
ON THE OCCURRENCE OF MICROCYSTIN PRODUCING CYANOBACTERIA IN MURCHISON BAY OF LAKE VICTORIA, UGANDA.
PH-DEPENDENT HYDROGEN SULFIDE SPECIES MAY CONTROL COMPOSITION OF HOT SPRING MICROBIAL MATS.
Sigrid Haande, Thomas Rohrlack, Pål Brettum, Robert Ptacnik, Bente Edvardsen, Anne Lyche-Solheim, Unn-Hilde Refseth, Petter Larsson.
Zorigto Namsaraev1,2, Bair Namsaraev3,4, Vladimir Gorlenko1.
NIVA, Oslo, Norway.
1
2
Winogradsky Institute of Microbiology RAS, Moscow, Russia; Center for Protein Engineering, University of Liège, Liège, Belgium; 3Institute of General and Experimental Biology Siberian Branch RAS, Ulan-Ude, Russia; 4Buryat State University, Ulan-Ude, Buryat Republic, Russia. Introduction. Increasingly more data suggest that early life lived at an environmental temperature similar to those of today’s hot springs. At temperatures lower 73-77 °C and pH higher 4.5 hydrotherms are dominated by microbial mats formed by phototrophic bacteria. Castenholz have shown that with the increase of temperature above ~55 °C natural populations of cyanobacteria cannot tolerate high sulfide concentrations. It was concluded that at these temperatures and concentration of sulfide higher 1 mg/L mats are formed exclusively by anoxygenic phototrophic bacteria (APB). We studied alkaline (pH up to 9.8) non-mineralized (salinity less 1 g/L) hydrotherms of Baikal rift zone (Southern Siberia, Russia) and found that cyanobacteria could develop at much higher concentrations of sulfide than it was thought before (up to 5.9 mg/L at 62 °C). Methods. Microscopy, spectrophotometry, radioisotopy, cultivation and cultivation independent methods. Results. Cyanobacteria belonging to genus Phormidium were dominant and formed so called “translucent” mats up to 13 cm thick. The productivity of mats was relatively high: maximal content of chlorophyll a is 892 mg m–2, maximal rate of oxygenic photosynthesis 3.5 g C m–2d–1. APB Chloroflexus aurantiacus was found and isolated from mats, but it was not dominant. This fact can be explained by a decrease of sulfide toxicity in increase of pH. Other sulfidic hydrotherms worldwide follow the same rule. For example, at 60 °C and pH lower than 7 the majority of H2S molecules are not dissociated and are capable of penetrating easily through a cell membrane. The community dominated by APB Chloroflexus aurantiacus develops (Iceland, Italy and Yellowstone). Less toxic HSion starts to dominate at pH higher than 7 and cyanobacteria appear in the mat. They form a layer under top layer of APB Chloroflexus aurantiacus which protects cyanobacteria from the influence of sulfide dissolved in water above the mat (Iceland and Kamchatka). At pH higher than 8.5 all sulfide molecules are transformed to a hydrosulfide ion and, as it have been shown by us, cyanobacteria dominate in the microbial community. Conclusions. Our results show that distribution of different types of phototrophic communities in sulfidic hydrotherms can be explained by pH-dependent hydrogen sulfide species.
Introduction: One of the major threats to Lake Victoria is eutrophication and an increasing proliferation of cyanobacteria. The main concern is that many cyanobacterial species have the ability to produce toxic compounds, among them the commonly reported hepatotoxic microcystins, which can cause considerable hazards for animal and human health. Murchison Bay is a 30 km long embayment in the north western part of Lake Victoria, and the inner part of the bay serves as drinking water supply for the nearby city Kampala, the capital of Uganda. The bay is also the recipient of both industrial and municipal wastes, sewage effluents and surface runoff from the city, and is heavily eutrophicated. This study aimed to examine the abundance of cyanobacteria and microcystins in Murchison Bay and to identify the main microcystin producing species, as well as to test for possible correlation between microcystin concentration and environmental factors. Methods: Sampling (n=25) of physical, chemical and biological parameters in Murchison Bay was done at two stations, representing the semi-enclosed innermost part of the bay and the wider outer part of the bay, every second week in the period from April 2003-March 2004. Vertical profiles of water temperature, oxygen and electrical conductivity were measured, and the recorded secchi depths were used to estimate euphotic depth. The nutrients analysed were total phosphorous, phosphate, total nitrogen, nitrate, total organic carbon and silicon. Clorophyll-a was analysed and phytoplankton countings were performed. The presence of microcystins was analysed by Liquid chromatography (LC-MS), and the identification of the microcystin producing strains was performed by real-time PCR, using species specific primers. Correlations between microcystin concentration and various environmental parameters were analysed by Spearman’s rank correlations. Results: There was a considerable loading of nutrients to Murchison Bay with high concentrations of total phosphorous (>90µg/L) and total nitrogen (>1100µg/L) in the inner part of the bay. There was a rapid decrease in conductivity and nutrient concentrations from the innermost part of the bay to the outer part of the bay. The phytoplankton community was dominated by a number of cyanobacteria, among them known microcystin producing species of the genera Microcystis, Anabaena and Anabaenopsis. The proportion of N-fixing species like Anabaena sp. was higher in the outer part of the bay whereas Microcystis sp. was more abundant in the inner part of the bay. Microcystins (MC-RR, -LR, -YR) were detected during the whole study period, on average 1.1 µg L-1 in the inner part of the bay and 0.6 µg L-1 in the outer part of the bay. Based on qPCR analysis, Microcystis aeruginosa was identified as the main microcystin producer in Murchison Bay. Nutrient availability and light climate seemed to be the most probable influencing environmental factors influencing the microcystin production in Murchison Bay, but the microcystin concentrations could not be predicted by any given environmental factor alone, by the biovolume of cyanobacteria or of a certain cyanobacterial species. Conclusions: This study showed that Murchison Bay was heavily eutrophicated and that there was a presence of microcystin producing
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13th International Symposium on Phototrophic Prokaryotes cyanobacteria in the bay. The microcystin concentrations in Murchison Bay were at times higher than the WHO recommended limit of 1 µg L-1 MC for drinking water, possessing a health risk for lake water users.
P.064 DIVERSITY AND NITROGENASE GENE EXPRESSION OF DIAZOTROPHIC CYANOBACTERIA IN THE BALTIC SEA. Henning Johansen, Kirsten Isensee, Maren Voss, Klaus Jürgens. Leibniz Institute for Baltic Sea Research, Rostock, Germany. Introduction: Low phosphorous concentrations in the water column are important for the development of heterocystous cyanobacteria blooms recurring in summer in the Baltic Sea, a brackish water body in Northern Europe. These blooms significantly contribute to the nitrogen input into the system and consist mainly of the genera Aphanizomenon and Nodularia. While nitrogen fixation rate measurements indicate a distinct diel cycle, there are contradicting findings from the concentration of the nitrogenase enzyme during day. However, there is a general lack of knowledge about nitrogenase gene expression (nifH) and the coupling between gene activity and nitrogen fixing activity in the Baltic Sea so far. Here we present data on both gene expression and rate measurements, giving a comprehensive insight into regulation of nitrogen fixation of filamentous cyanobacteria. Methods: Surface water samples (3 m) were taken at a LaGrange drift station in the Bornholm Basin, Baltic Sea, in July 2008. The diversity of the nitrogen fixing community was studied using both cell counts and fingerprint analyses (Denaturing Gradient Gel Electrophoreses) of the nifH gene and its transcripts. Concentrations of gene copies and transcripts were determined by a quantitative PCR assay using primers specific for heterocystous cyanobacteria. Rate measurements of nitrogen fixation and primary production were done using stable isotope labelling (15N2 and 13C). Results: Sequencing of excised bands from nifH fingerprint analyses showed highest identity to strains Nodularia sp. KAC13, Aphanizomenon sp. KAC15 and Anabaena sp. PCC7120, being in accordance with microscopic cell counts. The analyses of cyanobacterial nifH transcripts indicated the presence and activity of the genera Anabaena, Aphanizomenon and Nodularia during a complete diel cycle. This is in accordance with the nitrogen fixation rate measurements showing a lowered rate but uptake of molecular nitrogen in darkness whereas the pattern of carbon fixation is following the solar radiation pattern. Results of the nifH gene expression measurements indicate an uncoupling of maximum nitrogen fixation rates and maximum transcripts. Conclusions: This study is the first to show nifH gene expression in natural bloom conditions in the Baltic Sea throughout the whole diel cycle. Therefore, an uncoupling of nitrogen fixation rate measurements and gene expression can be suggested. A tentative explanation is the enhanced regeneration of the nitrogenase enzyme during the night, while nitrogen fixation activity is more closely coupled to light energy during the day. In case our further studies confirm these findings the gene expression may not directly be linked to nitrogen fixation.
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Final Program and Abstracts
P.065 MAKING SENSE OF MARINE SYNECHOCOCCUS DIVERSITY, THE KEY TO UNDERSTANDING THEIR UBIQUITOUS ECOLOGICAL DISTRIBUTION. Sophie Mazard, Martin Ostrowski, Dave Scanlan. Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom. Introduction: At the global scale the marine photosynthetic community is dominated by two closely related genera of cyanobacteria, Prochlorococcus and Synechococcus. Their success is underpinned by a high degree of genetic and genomic variation, such that at least 16 different evolutionary lineages of Synechococcus have been identified, each occupying distinct spatial and temporal niches in the ocean. Our overall aim is to understand what factors define particular niches (e.g. light, temperature, nutrients, metals) and to correlate these factors with community structure. One way to achieve this is by monitoring how the genetic composition of photosynthetic communities changes in different ocean regimes linked with targeted metagenomics using high-throughput sequencing of DNA and RNA extracted from natural populations. Analysis of preliminary results revealed that our ability to interpret the data is limited by the low taxonomic resolution and low throughput of community analyses based on standard markers (16S rRNA and ITS). Methods: In this work, we describe the development of a fast, reliable and efficient method of assessing community composition at high taxonomic resolution using the petB locus. In preparation, we designed a Multi Locus Sequence Analysis (MLSA) scheme targeting 7 housekeeping genes for marine Synechococcus based on fully sequenced genomes. Metagenomics and transcriptomics studies were carried out on flow-sorted Synechococcus from contrasting stations on oceanographic cruises of the Atlantic (AMT18) and the Mediterranean Sea (BOUM). Results: A high resolution taxonomic framework for marine Synechococcus was produced with our MLSA scheme on more than 120 cultured isolates maintained in the Warwick culture collection. Phylogenetic reconstructions based on single and concatenated loci were congruent and comparable with those obtained with general phylogenetic makers (16S rDNA, 16S-23S ITS). However, sequencing of the single locus petB allowed us to obtain a resolution differentiating up to 55 coherent taxonomic units as compared to a maximum of 16 for 16S rDNA and ITS. Furthermore, the reliability of the petB alignment (cf. ITS) makes this locus suitable for assessing community structure with high-throughput and rapid analyses of environmental libraries. Indeed, preliminary results from environmental libraries highlighted ecologically important clades and sub-groups at a much higher resolution than previous studies. Conclusions: Earlier work performed using classical markers emphasized broad trends in the ecological distribution of marine Synechococcus. The establishment of a high resolution phylogenetic framework helps us to fine tune the geographical partitioning of this genus in situ, and will facilitate the uncovering of specific adaptation mechanisms of the numerous phylotypes observed. In concert with indepth genomic study of this genus, this might prove to be the key to understanding its complex but successful ecological distribution throughout the world’s oceans.
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Final Program and Abstracts
P.066 ALGAECIDAL EFFECT OF RHODOCOCCUS ON MICROCYSTIS. Chi-Yong Ahn, Young-Ki Lee, Song-Gun Kim, Hee-Sik Kim, Hee-Mock Oh. Environmental Biotechnology Research Center, KRIBB, Daejeon, Republic of Korea. Rhodococcus sp. is one of the most common bacteria in the reservoirs of Korea. Rhodococcus sp. is also frequently found with Microcystis, a major bloom-forming cyanobacterium. Two strains of Microcystis were isolated in Daechung and Wangsong reservoir, Korea, during blooming season (from July to October, 2007). Eleven strains of coexiting bacteria were also isolated from mucilage of cultivated Microcystis strains. They were identified by 16S rRNA gene sequences. Two of them were designated as Rhodococcus sp. KWBRH2 and KWB-RH5. Rhodococcus strains exerted an inhibitory effect on the growth of Microcystis aeruginosa NIES 843, UTEX 2388 and isolated M. aeruginosa. The cell-free culture supernatant of Rhodococcus strains also showed prominent algaecidal activity. To identify the causal compounds, supernatants of Rhodococcus strains were dialyzed with a MW 8,000 and MW 2,000 membranes. The suspected extracellular substances had molecular weights below 2,000 Da. The small molecules were further analyzed by HPLC, LC-MS and enzyme treatments. The extracellular material from Rhodococcus strains could be a more effective bloom extinguisher, compared to generally known algaecidal bacteria, because it does not require bacterial cell itself for bloom control.
P.067 CHARACTERISATION OF CYANOPHAGE RESISTANTANCE IN THE MARINE CYANOBACTERIUM SYNECHOCOCCUS SP. WH7803. Edward Spence, Nicholas Mann. University of Warwick, Coventry, United Kingdom. Introduction: Cyanophage are viruses which specifically infect cyanobacteria. This project focuses on the interaction of the cyanophages S-PM2 and S-RSM42 with their host, the marine cyanobacterium Synechococcus sp. WH7803. The main aim of this project is to identify the host receptor(s) and characterise naturally occurring phage resistant mutants of Synechococcus sp. WH7803 which have been isolated. The phage resistant mutants have been characterised in order to determine what physical changes have occurred to their cell surface, in order to provide resistance and what impact this has on their fitness. Previous research with the freshwater cyanobacterium Anacystis nidulans has shown that the lipopolysaccharide (LPS) component is necessary for the adsorption of the cyanophage AS-1. Methodology: Spontaneous resistant mutants were isolated from regrowth of survivors from completely lysed cultures, following cyanophage infection. Single colonies were isolated from pour pates and maintained in liquid culture under selection with the addition of cyanophage lysate. The outer membrane protein component was isolated by Triton X-100 precipitation and profiled by gradient SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide was isolated using hot phenol/water extractions and profiles were obtained with SDS-PAGE using isolated LPS or using whole cell digests with protease K, visualised with silver staining. Results: The results indicated that the LPS component of the resistant mutants had an altered profile suggesting changes to the oligosaccharide or variable region. The outer membrane protein 114
components remained unchanged in the resistant mutants. The resistant mutants also had a reduction in their growth rates, in comparison to the wild type under standard conditions. An altered pigment content was also observed suggesting alteration to the photosynthetic components particularly to the phycobilisome. Conclusion: It seems likely that the LPS is the major receptor molecule for the cyanophages S-PM2 and S-RSM42 adhesion to their host Synechococcus WH7803. Outer membrane proteins do not seem to have a role in these cases for cyanophage attachment. There is also a cost of resistance relating to the growth rate of the resistant mutants and changes to the pigment content. This suggests that genetic changes resulting in the alteration of the cell surface receptor may be as a result to changes of a regulatory component. This may explain additional pleiotrophic phenotypic changes observed, relating to pigment composition and a reduction in the growth rate in the cyanophage resistant mutants.
P.068 THE ROLE OF PHOTOTROPHIC MICROORGANISMS IN BIOLOGICAL SOIL CRUSTS FROM THE MIDTRE LOVÉN GLACIER FORELAND. Laura Tiano1,2, Luigi D’Acqui1, Claudio Sili1, Marc Staal3, Silvia Turicchia4, Matthias Zielke5, Stefano Ventura1. 1
CNR–Institute of Ecosystem Study, Sesto Fiorentino, Italy; 2University of Florence, Master in Environmental Biology, Italy; 3Department of Biology, University of Copenhagen, Denmark; 4University of Siena, PhD School in Polar Science, Siena, Italy; 5Department of Marine Biotechnology, Norway College of Fishery Science, University of Tromsø, Norway. Introduction: Global change models predict that the speed and magnitude of climate warming will be greatest at the highest latitudes, with raised mean temperatures, particularly during winter, and altered patterns of precipitation (IPCC 2001). These predicted changes are under way in many parts of the high Arctic, as it can be seen from the decreasing extent and thickness of the sea ice and the process of glacial ‘retreat’. Glacial regression makes new ground surface available to biotic colonization. Low temperatures, short growing seasons, the effects of permafrost, limited moisture and nutrients availability limit the growth of the vegetation, leading to different successions from those observed elsewhere. In these polar environments, pioneering organisms such as cyanobacteria, green algae, lichens, mosses and heterotrophic bacteria are typically the first to colonize the substrate as biological soil crusts (BSCs). Cyanobacteria are often found in great variety in all the high arctic zones, and in several ecosystems they are the dominant microorganisms in terms of biomass and productivity (Vincent, 2000). Nitrogen fixation by cyanobacteria compensates for the lack of nitrogen in arctic soils (Lennihan et al., 1994) and facilitates the subsequent colonization of these habitats by other microorganisms and plants (Bliss and Gold, 1994). This study describes community assembly on typical high Arctic glacier forelands, with a focus on the phototrophic components of the BSCs, to gain a broader understanding about dynamics and development of these fragile systems and their rates of recovery following disturbance. Methods: Sampling sites were located close to Ny-Ålesund, Svalbard, on the foreland of the Midtre Lovén glacier. Seven locations with different types of crust were chosen, and an integrated approach was August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes applied, carrying out both morphological and molecular analysis of environmental samples. A general insight into the phototrophic microorganisms colonizing the crusts was gained with microscopy observations; a deeper understanding of the communities’ structure is expected with molecular fingerprints (ARISA, T-RFLP). Also physical, chemical and granulometric characteristics of soil were analyzed to investigate the influence of those parameters on the development of the crusts. Furthermore, respiration rate, nitrogen fixation and production were measured, thus adding more information on the main activities performed by the primary colonizers of these surfaces.
Final Program and Abstracts sp., Phormidium autumnale, Phormidium priestleyi, Stigonema sp. Cultivation studies showed the importance of the incubation temperature for the composition of enrichment cultures, with an increased proportion of unicellular cyanobacteria at lower temperatures. Conclusion: Our first results show that the Western part of the Sör Rondane Moutains harbour a high and unique diversity of cyanobacteria. Precautions should be taken to avoid introduction of non-native species in the area of the research station.
P.070 SYNTHESIS OF BACTERIOCHLOROPHYLL-F.
Results: The molecular characterization of the soil crusts is still in progress, the morphological analysis by microscopy allowed us to identify several species of cyanobacteria and algae, as Nostoc sp., Phormidium sp., Leptolyngbya sp., several diatoms, etc.
Hitoshi Tamiaki, Jun Komada, Michio Kunieda, Kazuhiro Fukai, Taichi Yoshitomi, Jiro Harada, Tadashi Mizoguchi.
Nitrogen fixation rate, C/N concentration and granulometric characteristics of soil, confirmed the low nutrients availability of the high arctic ground and the extremely slow pedogenesis of these soils.
Introduction: Green sulfur bacteria are a family of anaerobic photoautotrophic bacteria living. These bacteria have elliptic shaped light-harvesting apparatus, called chlorosomes, that are attached with the inner side of their cytoplasmic membrane. Chlorosomes are unique photosynthetic antennae, where characteristic chlorophyllous pigments, bacteriochlorophyll(BChl)s-c, d and e self-aggregated to form large oligomers without any assistance of peptides. In contrast, the other antennae are prepared by complexation of pigments with peptides. In chlorosomal aggregates, specific interaction between the 31-OH, Mg and 13-C=O is observed. All the chlorosomal chlorophylls have such functional groups and three BChls mentioned above are distinguishable from substituents on the C7- and C20-positions. BChlc has methyl groups at the C7- and C20-positions, and BChl-d is the 20-demethyl form of BChl-c. On the other hand, BChl-e has a formyl group at the C7-position and a methyl group at the C20-position. The 20-demethylated form of BChl-e is named as BChl-f, but it has not been detected in any natural bacteria yet. Here we report the synthesis of BChl-f and its physical properties comparing with BChls-c, d and e extracted from natural photosynthetic green bacteria.
Conclusions: We expect to draw conclusions about the role of phototrophic microorganisms in these high arctic biological soil crusts as soon as all the results, collected from the different type of analysis, will be matched together and organized under a statistical approach.
P.069 CYANOBACTERIAL DIVERSITY OF THE SÖR RONDANE MOUNTAINS NEAR THE NEW “PRINCESS ELISABETH” STATION (ANTARCTICA). Zorigto Namsaraev1,2, Rafael Fernandez1, Marie-José Mano1, Patricia Simon1, Annick Wilmotte1. 1
Centre for Protein Engineering, University of Liège, Liège, Belgium; Winogradsky Institute of Microbiology RAS, Moscow, Russia.
2
Introduction: During austral summer 2008, the new Belgian “Princess Elisabeth” research station has been constructed on a granite ridge near the Utsteinen nunatak in the Western part of the Sör Rondane Mountains. This pristine area has not yet been studied at the biological level, and exhaustive baseline data are needed to obtain information about the initial state of the environment. Moreover, the Sör Rondane Mountains could be a possible refuge during the Last Glacial Maximum. Cyanobacterial communities in this area could harbour species that survived the population bottleneck of glaciations. Methods. Samples were taken in several locations in the austral summers of 2007 (before the construction start) and of 2009 (BELSPO projects ANTAR-IMPACT and BELDIVA). A polyphasic approach was used to study of cyanobacterial diversity. It included a molecular study using 16S rRNA gene sequences, an isolation of cyanobacteria in culture and a microscopic observation of environmental samples and isolated cultures. Results: Preliminary results showed a relatively high biodiversity in the studied area. Cyanobacteria were mostly associated with gravel and in some cases with rocks, whereas lichens were dominant on rock surfaces. Molecular studies showed a quite high degree of endemism and genotypes never recorded before. 92% of the studied samples shared at least one OTU, concordant with a mobility of species between nearby habitats. Microscopical observations showed the presence of at least 11 morphotypes of cyanobacteria: Asterocapsa sp., Aphanocapsa sp., Chroococcus sp., Coleodesmium sp., Cyanothece aeruginosa, Leptolyngbya sp., Nostoc commune, Nostoc August 9 to 14, 2009 • Montréal, QC, Canada
Department of Bioscience and Biotechnology, Kusatsu, Shiga, Japan.
Methods: Naturally occurring chlorophyll(Chl)-b was extracted from spinach with organic solvents. By modifying Chl-b, methyl bacteriopheophorbide-f was obtained according to the previous report [1]. The methyl ester was transferred to the farnesyl ester and successive magnesium-metallation gave BChl-f possessing 8-ethyl and 12-methyl groups. BChls-c, d and e were extracted from cultured green bacteria, Chlorobium tepidum, vibrioforme and phaeovibrioides, respectively. BChls-c, d, e and f were analyzed by reverse phase high performance liquid chromatography (RP-HPLC). Results: RP-HPLC analysis of farnesylated BChls-c, d, e and f indicated that they were eluted in the order of BChl-f < BChl-e < BChl-d < BChlc (31R, 8-Et, 12-Me = R[EM]): ODS column and aqueous MeOH. The 20-methylation and 7-reduction retarded their elution. Their visible spectra showed that the 20-methylation red-shifted both Qy and Soret peaks in their monomeric states and that the 7-oxidation induced the Qy and Soret peaks to move to shorter and longer wavelengths, respectively. Moreover, the ratio of Qy/Soret peak intensities decreased to less than half by the oxidation. Conclusion: We first synthesized BChl-f from easily available Chl-b. Farnesylated R[EM]BChl-f was eluted more quickly than the corresponding BChls-e (20-methylation) and d (7-reduction) in RPHPLC. Based on the analysis, the HPLC peaks of BChl-f were searched in the extracts from cultured green bacteria, but we could not detect it. [1] H. Tamiaki, M. Kubo and T. Oba, Tetrahedron, 56, 6245-6257 (2000). 115
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P.071 QUORUM SENSING IN CYANOBACTERIAL BLOOM. Young-Ki Lee, Chi-Yong Ahn, Hee-Sik Kim, Jung-Kee Lee, Won-Gon Kim, Hee-Mock Oh. Environmental Biotechnology Research Center, KRIBB, Daejeon, Republic of Korea. Microcystis aeruginosa is a ubiquitous cyanobacterium that causes ecological and economic damage to freshwater ecosystems by bloom formation. Cyanobacterial blooms are visually unpleasant and they produce malodorous compounds (geosmin, MIB) and toxic metabolites (microcystin), which pose more serious problems for animals and humans. Recent studies focused on cell to cell communications in ecogenomics. Cell density-dependent communication has been known as “quorum sensing”. To investigate the relationship between quorum sensing and bloom, we isolated Microcystis sp. Mi0601 and Microcystis sp. KW from surface scums during bloom season (from July to October, 2007) in Daechung reservoir and Wangsong reservoir, Korea. Both of them were identified as M. aeruginosa by 16S rRNA gene and cpc-IGS region. Concentrated cell-free extracts were bioassayed with Agrobacterium tumefaciens NT1 (traR, tra::lacZ749) and separated by thin-layer chromatography. Two signal molecules were detected in TLC plate but they were not general signal molecules, such as C4-, C6-, C10homoserine lactones. Extracted signal molecules were analyzed by LC-MS and NMR. The signal molecule from each strain exhibited the same molecular weight. NMR analysis indicated that they were composed of two unknown materials. A strong correlation was found between Microcystis growth and accumulation of signal molecule. This implies that quorum sensing plays an important role in the onset of cyanobacterial bloom.
Methods: Nine collection strains including Allochromatium vinosum (strain DSMZ 183 and DSMZ 185), Lamprocystis roseopersicina (DSMZ 229), Thiocapsa roseopersicina (DSMZ 217), Marichromatium purpuratum (DSMZ 1591), Thiocystis violaceae (DSMZ 207), Thiocystis minor (DSMZ 178) Chlorobium limicola f. thiosulfatophilum (DSMZ 249), were used to test a basal medium specific for purple and green sulfur bacteria proposed by Pfennig and Trüper (1981) and changing the electron donors like (alone and mixed): a) S2-, b) S2-+S2O32-+SA, c) Sx2- d) Sx2-+SA e) cysteine+SA, f) methionine+SA, g) SA h) cysteine and i) methionine. Liquid culture media (Na2S as electron donor) specific for phototrophic sulfur bacteria was enriched with water samples from Tampachachoco Lagoon and positive cultures were used to inoculate Petri dishes containing the next electron donors: a) S2-, b) S2-+S2O32+SA, c) Sx2- d) Sx2-+SA. In all cases, anaerobic conditions were gotten using gas pack bags and the cultures were incubated at room temperature and light as energy source (incandescent=IL and fluorescent=FL). Red colonies were isolated in cultures growing in medium d (Sx2- and SA). Pure culture of these bacteria were verified by optical microscopy and their phonotipic identity was done by the use of pigment and fatty acid composition (by gas-chromatography) as well as cellular morphological properties.
P.072
Results: All the collection strains grew well in the medium d (S2+S2O32- ) specially on IL (incandescent light) but DSMZ 249 only grew with FL (fluorescent light) and both strains were able to grow. Acetate alone and mixed with cysteine were used only for strain DSMZ 235 and DSMZ 183 (IL and FL); But DSMZ 1591, all purple sulfur bacteria assayed used Sx2- (IL). Except DSM235 (IL) and DSMZ 185 (IL and FL) all the strains were not able to use acetate as electron donator. It was possible to obtain five pure cultures of phototrophic purple sulfur bacteria using the media d (Sx2-+SA). The strains were also able to grow well in media b (S2-+S2O32-+SA) and d ( Sx2-+SA. Sulfur globules into the cells, the presence of Bchl a, as well as the octadecenoic (C18:1), hexadecenoic (C16:1) and hexadecanoic (C16:0) as major fatty acids, related all the strains isolated as members of the Chromatiacea family.
ISOLATION OF ANOXYGENIC PHOTOTROPHIC SULFUR BACTERIA FROM TAMPAMACHOCO LAGOON (VERACRUZ, MÉXICO) USING DIFFERENT ELECTRON DONORS AND AGAR PLATE CULTURES.
Conclusions: A combination of electron donors could be an alternative to isolate and to obtain pure cultures of marine phototrophic sulfur bacteria in agar plate cultures incubated in gas pack bags.
María Teresa Núñez-Cardona1, Jordi Mas2, Marina Luquin3, Martha Signoret4.
P.073
1,4
Universidad Autónoma Metropolitana-Xochimilco, 1Laboratory of Ecología Microbiana; 4Plancton y Bioenergética, Distrito Federal, México; 2,3Departament de Genetica i Microbiologia, Universidad Autónoma de Barcelona, Spain. Introduction: Phototrohic sulfur bacteria (PSB) usually live in aquatic systems. Actually there is a limited number of strains described if we compare with the chemoorganoheterotrophic bacteria. Maybe it is because PSB are anaerobic microorganisms, and the use of organic compounds as carbon and electron donors, for growing is limited. It has been shown that PSB are able to use different containing sulfur compounds as electron donors like H2S (in laboratory cultures as Na2S=S2-), polysulfides (Sx2-), thiosulfate (S2O32-), cysteine, methionine, and acetate (sodium acetate=SA) and also that is possible to incubate them in gas pack bags. With the aim to isolate and getting pure cultures of anoxygenic phototrophic sulfur bacteria it was assayed a basal medium adding different electron donors alone and mixed in agar plate cultures. Fatty acids and pigment composition as well as morphological properties were used to characterize the strains isolated from water samples collected at Tampamachoco Lagoon (Veracruz, México) 116
TOWARDS TO INTEGRATION OF BACTERIAL LIGHT-DEPENDENT HYDROGEN PRODUCTION AND ELECTRICITY GENERATION. Evgeny Shastik1,3, Tatyana Laurinavichene1, Evgeny Minakov1, Oleg Voronin2, Nikolay Zorin1, Arkady Karyakin2, Anatoly Tsygankov1. 1 Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region; 2Chemical Faculty, Lomonosov Moscow State University, Moscow; 3South Federal University, Rostov-on-Don, Russia.
Introduction. For future H2-based economy many issues should be addressed. Human-friendly and cheap H2 production and transformation of H2 energy into electricity are among them. Purple bacteria are promising H2 producers since they use sunlight and simple organic compounds to evolve H2 gas with high rates. A fuel cell (FC) application to transform hydrogen energy to electrical one seems to be perspective. Existing FC’s with electrodes based on noble metal catalysts (e.g. platinum) are not able to work in microbiological media, and an enzymatic catalyst is the best alternative for biological systems. Particularly, hydrogenase (H2ase) from Thiocapsa roseopersicina was shown to be stable at high temperature, and in the presence of CO, August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes H2S, and proteases. A direct H2ase bioelectrocatalysis on technological carbon materials used in energetic would be optimal and profitable. The aim of this study was to explore a possibility to combine bacterial H2 production with hydrogen electrode in one space. Methods. Reactors with H2 producing bacteria (including immobilized purple bacteria), hydrogen enzyme electrodes (carbon textile with polymerized different promoters) with immobilized H2ases from T. roseopersicina and D. baculatum, and combination of them were used in this study. Results. Before the integration of hydrogen producing reactors with bacteria and hydrogen enzyme electrode, some preliminary research on bacterial reactors and H2 electrode have been done. It was shown that the photobioreactor with immobilized purple bacteria (Rhodobacter capsulatus and Rba. sphaeroides) under continuous synthetic medium flow stably produced H2 with average rate app. 1.5 ml h-1 ml-1 matrix for more than 5000 hours. Operational stability of H2 electrodes in K-P buffer saturated by H2 was studied for more than 500 h. pH-dependence of H2 electrode with different H2ases was studied. Temperature profiles of H2 electrodes with different H2ases with polyviologen and neutral red as promoters was studied. It was shown that the temperature dependence of the current was dependent on the voltage applied to the electrode. At low (5-20 mV) overvoltage the temperature dependence was maximal whereas at high (150-200 mV) overvoltage the temperature dependence was very low. The dependence of activation energy on the overvoltage of the electrode will be discussed. The stability of H2 electrode in the medium going from the bioreactor was shown to be the same as in KP buffer. Finally, the stability of H2 electrode integrated into the bioreactors with H2 producing bacteria was studied, detailed results will be presented, and problems appeared during the integration of bacterial H2 production with H2 enzyme electrodes will be discussed. Conclusion. The integration of H2 producing bacteria with H2 enzyme electrode in one space is possible. However, many problems should be solved before this system shows its practical efficiency. This work was supported by the Russian Program of Basic Research RAS
Final Program and Abstracts Methods: In order to study functions of the subdomains in SID of Hik33 firstly we attempted to replace SID of a phosphate-deficient sensor SphS with the SID from Hik33 to express chimera sensor containing SID of Hik33 and transmitter domain of SphS (Hik33nSphSc). SphS regulates expression of proteins for the phosphate-acquisition, including an alkaline phosphatase and highaffinity phosphate transporters. When SphS was replaced with the chimeric sensor Hik33n-SphSc, it altered expression pattern of the SphS-regulon under the environmental conditions, which might be perceived by the Hik33 but not under the phosphate-deficiency. Then, we deleted the subdomains in the SID of Hik33n-SphSc and examined effects on the expression of the phoA gene for alkaline phosphatase to identify the important subdomain to perceive the stimuli. Results: SphS is activated under the phosphate-deficient condition and upregulates expression of the phoA gene. However, the strain expressing Hik33n-SphSc expressed the phoA gene under the standard growth condition and expression levels of phoA were repressed under high salt and cold stress conditions, which might be perceived by Hik33. These results indicated that Hik33n-SphSc might be active under the standard conditions and once the signals were accepted, it might become inactive. Deletion of the periplasmic loop did not affect the ability of Hik33n-SphSc, i.e., the phoA gene was expressed under the standard conditions and repressed under the stressed conditions. However, deletion of PAS domain lost the responsiveness to the stress, i.e., the phoA gene was constitutively expressed. And simultaneous deletion of HAMP and PAS domains lost activity of expression of the phoA gene regardless of the growth conditions, suggesting that the HAMP domain might be essential for the kinase activity. Conclusions: A chimeric sensor Hik33n-SphSc indicated that SID of Hik33 might activate the transmitter domain under the normal growth conditions. The PAS domain in the SID might play important roles in perception of the stimuli.
P.075 RNASE P FROM THE CHROMATOPHORE OF PAULINELLA CHROMATOPHORA. Agustin Vioque, Pilar Bernal, Leonor Puerto-Galan.
P.074 FUNCTIONAL ANALYSIS OF DOMAINS FOR THE SIGNAL PERCEPTION OF HIK33 IN SYNECHOCYSTIS SP. PCC 6803. Yohei Shimura, Satoshi Kimura, Yoshihiro Shiraiwa, Iwane Suzuki. Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan. Introduction: Two-component systems, which generally consist of a sensory histidine kinase and a response regulator, are major signaling pathways in most of bacteria. In general, histidine kinases possess a conserved transmitter domain associating kinase activity at the Cterminus and a unique signal input domain (SID) at the N-terminus. The SIDs in the histidine kinases are thought to play important roles in perception of the specific stimuli and regulation of the kinase activity of the transmitter domain, however, the actual molecular mechanisms of signal perception are not clarified yet. Hik33, namely NblS ortholog in the cyanobacterium Synechocystis sp. PCC 6803, regulates gene expression under cold, high light, oxidative, high salt and hyperosmotic stress conditions. It has two transmembrane helices, a periplasmic loop, a HAMP and a PAS domain in its SID. In this study, we examined functions of the subdomains in SID of Hik33 in perception of the stimuli. August 9 to 14, 2009 • Montréal, QC, Canada
Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla and CSIC, Sevilla, Spain. Introduction: Ribonuclease P (RNase P) is required for the generation of the mature 5’ end of tRNAs from precursors. RNase P is present in all domains of life as a ribonucleoprotein with a catalytic RNA subunit (P RNA). The only exceptions found so far are in human mitochondria and higher plant chloroplasts, where RNase P seems to be a protein enzyme lacking an RNA subunit. In several algae the plastid genome contains a gene homologous to the bacterial P RNA gene (rnpB). In contrast with the bacterial P RNA, plastids P RNA has no significant activity in vitro in the absence of protein, and only very weak activity could be reconstituted with a bacterial protein subunit (P protein) and the P RNA from Cyanophora paradoxa. Plastid P RNA seems to be unable to fold into a functional structure for pre-tRNA substrate binding. Paulinella chromatophora is a protist that contains an obligate cyanobacterium endosymbiont (chromatophore) whose genome has been recently sequenced. This genome is highly reduced compared with free-living cyanobacteria and could represent an early step in the integration of a cyanobacterium into a eukaryotic cell, a process that is at the origin of chloroplasts. Therefore, we have characterized the chromatophore RNase P, and compared its structure and function with 117
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Final Program and Abstracts both cyanobacterial and plastid RNase P, under the hypothesis that its properties could illustrate about the process of P RNA loss of function in plastid evolution. Methods: We have identified in the chromatophore genome the rnpA (encoding the P protein) and the rnpB genes and have undertaken their functional and structural analysis. The rnpB gene has been cloned and the P RNA has been obtained by in vitro transcription. Its RNase P activity has been analyzed either in the absence or presence of a cyanobacterial P protein. The structure and folding of the P RNAs has been analyzed by native gel electrophoresis and sensitivity to lead-induced cleavage. Results: Chromatophore P RNA has a predicted secondary structure similar to other cyanobacterial P RNAs. Chromatophore P RNA has reduced activity in the absence of protein, but it can reconstitute a fully functional holoenzyme with cyanobacterial P protein. Comparison of reaction rates in the presence or absence of the protein subunit indicate that chromatophore RNase P is more dependent on the protein subunit for activity than free living cyanobacterial RNase P. The pattern of lead induced cleavage of chromatophore P RNA is similar to the pattern observed with Anabaena 7120 P RNA, but the mobility of the P RNA on native gel electrophoresis indicates that this RNA has a reduced ability to fold into a functional native compact structure. Conclusions: The chromatophore P RNA has functional and structural properties that suggest that it might represent an early step in the process of increased dependence on protein for folding and activity prevalent in present day plastids.
P.076 THE GENOME SEQUENCE OF THIODICTYON SP. AND ITS SIGNIFICANCE FOR THE CAROTENOGENESIS OF OKENONE. Kajetan Vogl, Niels-Ulrik Frigaard, Mauro Tonolla, Lynn Thomso, Qi Ji, Stephan C. Schuster,Donald A. Bryant.
proximity to the gene cluster. Conclusions: The carotenogenesis gene cluster revealed a so far unknown duplication of the crtD gene. Since this duplication was not observed in other phototrophic bacteria, it seems to be possible that the second crtD gene has another function than desaturating the 3,4 and/or 3’,4’ bond (Albrecht et al. 1997), and its function is correlated with okenone production. In the past the function of certain carotenoid genes were successfully examined by heterologous expression in Eschericha coli (Maresca et al. 2007). In combination with the approach of heterologous gene expression in E. coli and Chlorobaculum tepidum, the genome sequence of Thiocystis sp. will allow the elucidation of the carotenogenesis pathway for okenone. References Albrecht M, Ruther A and Sandmann G (1997) Purification and biochemical characterization of a hydroxyneurosporene desaturase involved in the biosynthetic pathway of the carotenoid spheroidene in Rhodobacter sphaeroides. J Bacteriol 179:7462-7467 Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W and Lipman DJ (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res 25:33893402 Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA and Bowden SA (2005) Biomarker evidence for green and purple sulphur bacteria in a stratified Palaeoproterozoic sea. Nature 437:866-870 Madigan MT and Jung DO (2009) An Overview of purple bacteria: systematics, physiology, and habitats. In: Hunter CN, Daldal F, Thurnauer MC and Beatty JT (eds) The Purple Phototrophic Bacteria, pp 1-15. Springer Netherlands Maresca JA, Graham JE, Wu M, Eisen J and Bryant DA (2007) Identification of a fourth type of carotenoid cyclase in photosynthetic organisms. Proc Natl Acad Sci USA 104:11784-11789
Penn State University, University Park, PA, USA.
P.077
Introduction: Anoxygenic phototrophic purple bacteria are a major group of phototrophic microorganisms which occur mainly in aquatic environments. The group of purple bacteria consists of purple nonsulfur bacteria (alpha- or beta- proteobacteria) and purple sulfur bacteria (gammaproteobacteria). Within the group of purple sulfur bacteria, over 25 genera are recognized (Madigan and Jung 2009). These bacteria have two pathways for carotenogenesis, the spirilloxanthin pathway and the okenone pathway. The carotenoid okenone is of special interest, because okenone and its derivative okenane serve as the only known hydrocarbon biomarker for purple sulfur bacteria (Chromatiaceae) used to reconstruct geologic history (Brocks et al. 2005). However, so far no genome sequence of an okenone-producing purple sulfur bacterium is available and the biosynthetic pathway of okenone is not understood in detail.
THYLAKOID MEMBRANE REDUCTION AFFECTS THE PHOTOSYSTEM STOICHIOMETRY IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Methods: Thiodictyon sp. strain Cad 16 genomic DNA was isolated using standard protocols. Then 454 pyrosequencing was performed and contigs harboring carotenoid genes were identified with BLASTN (Altschul et al. 1997) and annotated manually with MacVector. Results: The following homologs of carotenogenesis genes are present in the okenone producing purple sulfur bacterium: geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI), hydroxyneurosporene synthase (crtC), two gene copies of C-3’,4’ desaturase (crtD), hydroxyneurosporene-O-methyltransferase (crtF), gamma-carotene desaturase (crtU) and lycopene cyclase (crtY), The crtY, crtU, crtD1, crtD2 and crtC are clustered together. CrtE, crtI and crtB are in close 118
Eva Fuhrmann, Dirk Schneider. University Freiburg - Department of Biochemistry and Molecular Biology, Freiburg, Baden-Wurttemberg, Germany. Introduction: Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is incompletely understood today. The “vesicle inducing protein in plastids 1” (Vipp1) was found to be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Since the exact physiological function of VIPP1 is still mysterious, we generated a Synechocystis sp. PCC 6803 Vipp1 depletion strain to characterize the physiological effects of the Vipp1 depletion in cyanobacteria more in detail. Methods: To inactivate the Vipp1 gene a kanamycin resistance cassette was introduced into the vipp1 gene (sll0617) from Synechocystis sp. PCC 6803. Wild type (wt) and mutant cells were analyzed by electron microscopy as well as by absorbance and 77 K fluorescence emission spectroscopy. Photosystem (PS) II and PS I activity was determined by oxygen evolution and consumption measurements, respectively. Membrane protein complexes were characterized by sucrose density gradient centrifugation as well as by blue native polyacrylamid gelelectrophoresis (BN-PAGE). Results: Although the vipp1 gene was disrupted in a few chromosomal copies, no completely segregated strain obtained even August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes after two years on selective medium. Nevertheless, based on immunoblot analyses the total Vipp1 content in the mutant strain was significantly decreased when compared to wt cells. Electron microscopy revealed an average of four to six layers of thylakoid membranes present in wt cells whereas in the mutant strain only two to three layers were observed. Under increasing light intensities the chlorophyll content per cell decreases, and, furthermore, it is always lower in the merodiploid cells at any light intensity compared to wt cells. Sucrose density gradient centrifugation and BN-PAGE analyses revealed that the amount of PS I trimers is significantly reduced in membranes isolated from the mutant cells. Furthermore, the cellular concentration of chlorophyll binding proteins is significantly lowered in the merodiploid cells. By 77 K and UV/VIS spectroscopy we observed that the ratio of PS I to PS II has changed in the mutant strain. While the PS II activity is not significantly altered the activity of PS I was determined to be decreased in the mutant relative to the wt strain. However, the mutant cells still appear to be able to operate the electron transfer chain at wt rates. Conclusion: We show that in the cyanobacterium Synechocystis sp. PCC 6803 the synthesis of active PS I depends on the amount of thylakoid membranes present per cell. Down-regulation of the amount of active PS I appears to be a general adaptation mechanism in cyanobacteria and Vipp1 could be directly involved in such mechanisms, resulting in changes of the PS I to PS II ratio in response to e.g. increasing light intensities. The depletion of the vipp1 gene product resulted in a specific decrease of functional PS I in Synechocystis, whereas the amount of functional PS II was not significantly altered. In contrast, a Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain1. Therefore, it has been suggested that depletion of Vipp1 in Arabidopsis affects thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes2. 1
Kroll et al (2001) Proc Natl Acad Sci USA 98: 4238–4242
2
Aseeva et al (2007) Plant Physiol Biochem 45: 119–128
P.078 THE EVOLUTIONARY PATH TO TERMINAL DIFFERENTIATION AND DIVISION OF LABOR IN CYANOBACTERIA. Valentina Rossetti, Bettina E Schirrmeister, Marco V. Bernasconi, Homayoun C Bagheri. University of Zurich, Zurich, Zurich, Switzerland. Introduction: The transition from unicellular to multicellular organisms is not a well understood process in evolution. A common trait often associated with multicellularity is cellular differentiation, which is a separation of tasks through the division of labor. However, the division of labor does not necessarily have to be constrained to a multicellular setting. In this study, we focus on the possible evolutionary paths leading to terminal differentiation in cyanobacteria. Methods: We develop mathematical models for two developmental strategies. One, of populations of terminally differentiated single cells surviving by the exchange of common goods. Second, of populations exhibiting terminal differentiation in a multicellular setting. We test the evolutionary stability of the two strategies with respect to resistance against disruptive mutations (i.e. “cheater” mutants). We also assess the effects of selection on the optimization of the ratio of vegetative August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts (carbon fixing) to heterocystous (nitrogen fixing) cells, which in turn leads to the maximization of the carrying capacity for the population density. In addition we compare the performance of differentiated populations to undifferentiated ones that temporally separate tasks in accordance to a day/night cycle. We then compare some predictions of our model with phylogenetic relationships derived from analyzing 16S rDNA sequences of different cyanobacterial strains. Results: In line with studies indicating that group or spatial structure are ways to evolve cooperation and protect against the spread of cheaters, our work provides theoretical and phylogenetic evidence that differentiated single-celled populations of cyanobacteria are not stable. The compartmentalization afforded by multicellularity is required to maintain the vegetative/heterocyst division and for selection to optimize the carrying capacity. Conclusions: In cyanobacteria, multicellularity is a necessary condition for both the evolutionary stability of terminal differentiation and for the optimization of the division of labor. Furthermore, in regimes of long daylight periods, terminally differentiated species perform better than undifferentiated species that follow the day/night cycle; indicating that terminal differentiation can be an evolutionary advantage in high irradiance regions. Conversely, undifferentiated species have an advantage in regimes of short daylight periods.
P.079 DIVERSITY OF PHOTOTROPHIC BACTERIA IN MICROBIAL MATS OF LAGUNA CHAXA AND LAGUNA TEBENQUICHE AT THE SALAR DE ATACAMA, CHILE. Cristina Dorador, Andrea Gärtner, Yaneisi Vazquez, Carolina Cubillos, Vera Thiel, Johannes F. Imhoff. Departamento de Acuicultura, Universidad de Antofagasta, Avenida Universidad de Antofagasta, Antofagasta, Chile. Introduction: The Salar de Atacama is located in the Atacama Desert (Chile) at an altitude of 2300 m above sea level. In the interior of the salar there are several small shallow ponds that exhibit salt concentrations >100 mS cm-1. Intensive solar radiation and fluctuating daily temperatures between day and night are typical features of these environments. It has been proposed that phototrophic bacteria should play an important role in the primary production of saline lakes in the Atacama Desert; however their diversity and functionality are under investigated. The diversity of phototrophic bacteria was focused in two main lakes of the Atacama Desert which exhibit microbial mats on their shore. Methods: We investigated the diversity of specific phototrophic bacterial in samples collected from Laguna Chaxa and Laguna Tebenquiche at the Salar de Atacama. DNA was extracted from homogenized samples of microbial mats and was amplified using described specific 16S rRNA gene primers for the groups Chromatiaceae, Chlorobi, Cyanobacteria and Roseobacter clade. Microbial diversity was examined through DGGE and clone libraries of the 16S rRNA gene. Results: Microbial mats were found at the shore of Laguna Tebenquiche and Laguna Chaxa. Mats from Laguna Tebenquiche exhibited 1-3 layers compared with mats of Laguna Chaxa which had between 3-4 layers. The target groups were recorded from all samples analysed. Analyses of DGGE band patterns and clone libraries revealed that the lakes supported different microbial communities. Conclusions: Phototrophic bacteria occupied an important part of the microbial diversity of inland lakes of Salar de Atacama. The Roseobacter clade, initially described from marine systems, 119
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Final Program and Abstracts represented an important component of the diversity of phototrophic bacteria from inland lakes of the Salar de Atacama. Future studies will include activity assays to determine the role of the phototrophic bacteria in the primary productivity of evaporitic basins in northern Chile.
P.080 CYANOBACTERIA IN THE PCC: WHO IS RELATED TO WHOM? Muriel Gugger1, Alexandra Calteau2, Rosmarie Rippka1, Thérèse Coursin1, Thierry Laurent1, Jennifer Tambosco1, Corinne Cruaud2, Frederik Gavory2, Jean Weissenbach2, Nicole Tandeau de Marsac1. 1
Unité des Cyanobactéries, Institut Pasteur, Paris ; 2CEA / Institut de Génomique / Génoscope / Laboratoire de Génomique Comparative, Evry, France. Introduction: Cyanobacteria are oxygenic phototrophic prokaryotes that exhibit a wide range of morphological and physiological features and colonize strikingly different ecosystems. The global picture of cyanobacterial interrelationships is mainly based on complete or partial 16S rDNA sequences of non-axenic isolates for which little information may be available, or on environmental samples representative of very specific ecosystems. Recently, the evolution of this phylum has been re-examined based on cyanobacterial genome sequences, but so far is highly biased towards marine picocyanobacteria (50 % of the genomes). Methods: The PCC (Pasteur Culture Collection of Cyanobacteria) presently includes almost 800 axenic strains originating from diverse ecosystems and representing about 60 major morphotypes. They exhibit widely differing physiological characteristics, and some produce hepato- or neurotoxins. To overcome the paucity of information associated with many of the available 16S rDNA sequences, either with respect to knowledge of the organisms or because of insufficient sequence length, the 16S rRNA genes of 709 PCC strains were sequenced over their full length to establish more precisely their phylogenetic relationships. Results: The sequence analyses confirmed the monophyly of the heterocystous cyanobacteria, and the intermixed relationships of the other morphotypes. Although many genera are polyphyletic, several sustained genetic clusters were recognized that contain PCC reference strains, previously proposed based on mean DNA base composition and phenotypic characters. Moreover, PCC strains hitherto insufficiently characterized can now be assigned to appropriate genetic clusters within the cyanobacterial phylum. In addition, relatives from other culture collections or those only known from environmental sequences could be recognized. Conclusions: The new 16S rDNA sequence database of the PCC provides a more complete reference frame for the genetic diversity and interrelationships of cultured and uncultured cyanobacteria, and should help to place the phenotypic properties of the organisms into an evolutionary context.
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P.081 MOLECULAR BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF ODOR PRODUCING CYANOBACTERIA FROM DRINKING WATER RESERVOIRS. Frank Ludwig, Sabine Hacker, Andy Weiss, Hetvi R. Gandhi. Isolde Röske Institute of Microbiology, Dresden University of Technology, Dresden, Germany. Introduction: Recently, taste and odor problems (TO problems) could be observed frequently. In the most cases the odor is described as an earthy-musty smell, which may be caused by the volatile sesquiterpene geosmin, which has a very low odor threshold concentration of 4 ng/l (Young et al. 1996). Besides actinomycetes, cyanobacteria are mostly responsible for the release of odorous substances as geosmin and 2-methylisoborneol (2-mib) into the water. Because the drinking water should be free from flavor and smell, TO problems lead to higher costs. Therefore, we have investigated the occuring cyanobacteria from three different drinking water reservoirs. Methods: Material from the phytobenthic mats of the Cranzahl, Klingenberg and Saidenbach (all in Saxony, Germany) drinking water reservoirs was used to prepare single trichomes of cyanobacteria. From the obtained cultures the isolated dna were used to amplify genes like the rbcL and geoA. These partial gene sequences and furthermore the sequence area between the 16S rDNA and 23S rDNA could be used for the characterization of the isolates. The development of degenerated primer pairs for the amplification of the cyanobacterial genes like rbcL and the geoA were the basis for the successful molecular biological determination of the isolated strains. Supplementary we have conducted biochemical analyses with selected cultures for the confirmation that these isolates are capable to produce odorous substances as geosmin or/and 2-mib. Finally phylogenetic trees were established with the obtained nucleotide sequences for the depiction of the existing diversity. Results: Through the successful development of a new method to isolate pure cultures of cyanobacteria from very heterogeneous environmental samples we were able to obtain a lot of taxonomically very different cyanobacterial cultures. This could be affirmed by the nucleotide sequences of the 16S-23S rDNA, rbcL and the geoA genes. The biochemical analyses shows also the important potential of cyanobacteria to produce and to release odor to the ambient biocenosis. Experiments with different light and nutrient conditions have demonstrated that the pattern of the produced odor is variable. Conclusions: The achieved results show the great diversity of the occurring cyanobacteria in different drinking water reservoirs. Through the biochemical analyses it can be confirmed that cyanobacteria are a major originator of TO problems. It is very interesting that especially the light quality has an influence of the synthesized odorous substances. Because it is very difficult to cultivate cyanobacteria and to identify factors which affect the odor production, the management of the drinking water reservoirs is very interested to find out parameters which can indicate that odorous substances will be released into the water at a great scale.
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
P.082
P.083
THE TWIN ARGININE TRANSLOCATION SYSTEM IN SYNECHOCYSTIS PCC6803.
POLYFACETIC STUDY OF NON PURPLE SULFUR BACTERIA ISOLATED FROM THE SOUTHERN GULF OF MEXICO WATER SAMPLES.
Anja Nenninger1,3, Gemma Warren2, Conrad W. Mullineaux1 and Colin Robinson3. 1
Queen Mary University of London, London; 2MOAC, Doctoral Training Centre, University of Warwick, Coventry; 3University of Warwick, Coventry; United Kingdom Introduction: Targeting and translocation of newly synthesised proteins is a crucial process in living calls, using a range of mechanism at different membrane systems. The transport mechanism studied here is the twin arginine translocation (Tat) system. Discovered in the early 1990s in plant thylakoids it has since been shown to operate in the plasma membrane of a wide variety bacteria. Cyanobacteria represent a unique subject for Tat studies. They are considered to be the progenitors of plant chloroplasts with an oxygenevolving photosynthetic apparatus very similar to higher plants at an abundant internal network of thylakoid membranes; and as Gramnegative bacteria they have a cell envelope consisting of an outer membrane, a periplasmic compartment, and a plasma membrane. Sequencing of the Synechocystis PCC6803 genome has shown that a single set of tat genes is present, with a homologue of cptatC/tatC and two paralogues of tha4/tatA. Methods: The plasmid described in Spence et al. (2003) was used as template for QuikChange® Site-directed Mutagenesis and the following mutants of the twin arginine motif were obtained: RK, KR, KK. Translocation of GFP in Synechocystis was observed using a confocal microscopy. (Spence E. et al., 2003, Mol. Micro. 48, 1481-89) Results: In Synechocystis GFP tagged with a Tat signal sequence (from E. coli TMAO reductase; torA-GFP) is found exclusively in the periplasm. GFP fluorescence can be seen contiguous to the thylakoid membrane system corresponding with exported periplasmic GFP. Disrupting the twin arginine motif leads to accumulation of clearly visible GFP aggregates inside the plasma membrane. The analysis of numerous cells for the different mutations has shown that these aggregates are primarily located in the thylakoid region, but they can as well be found in the cytoplasm. Conclusions: Proteomic studies on Synechocystis identified several potential Tat substrates. The twin arginine is part of the signal sequence in most Tat substrates in plants and bacteria. However, the importance of this motif varies between the two systems. In plants substitution of either arginine led to a complete block in translocation, whilst bacterial Tat system only shows a full block in translocation when both arginine residues were replaced. Substitution of the arginine residues in Synechocystis shows an extreme level of stringency very similar to the situation observed in plants. This absolute requirement for an intact twin arginine motif is unique among bacteria studied to date. With the endosymbiotic theory now widely accepted for the origin of plastids in higher plants and phylogenetic studies clustering the cyanobacterial tatC (sll0194) with the chloroplast cptatC the results described here emphasise the relationship of cyanobacteria and plant chloroplast on a functional level for the first time.
August 9 to 14, 2009 • Montréal, QC, Canada
María Teresa Núñez-Cardona1, Verónica Pacheco-González2, Jaime García-Mena3, Martha Signoret4. Universidad Autónoma Metropolitana-Xochimilco, 1,2Laboratory of Ecología Microbiana; 4Plancton and Bioenergética; 3Centro de Investigación y de Estudios Avanzados-Zacatenco, Departamento de Genética y Biología Molecular, Distrito Federal, México. Introduction: Studies about diversity of phototrophic bacteria (PB) using laboratory cultures are scarce if we compared with the application of molecular techniques like nucleic acids hybridization in situ and the gene bank. These molecular tools have lead to the conclusion that the most abundant marine microbial groups are as yet uncultivated and probably they play a significant role in marine biogeochemical cycling. Nevertheless pure laboratory cultures are used as reference and as models too. The goal of this investigation was to isolate and to identify nonpurple sulfur bacteria from a 100 m depth water sample collected in the Gulf of Mexico. Methods: Conventional and molecular analyses were used to characterize and to identify the isolated strains. Water samples were collected at 100 m depth and were used to enrich glass vials containing van Niel medium, specific for growth of nonpurple sulfur bacteria. Strains were purified by the agar shake technique. All the cultures were incubated at room temperature (20-28 °C) and exposed to light (1.3-1.8 Klux). Morphological, pigment composition (in vivo analysis) and the ability to use different chemical compounds as electron donors in van Niel basal medium, were used to characterize the isolated strains. They were identified by the analysis of the 16S rDNA. This one was achieved and amplified by PCR technique and the use of Unifor and Unirev primers. Results were analyzed by electrophoresis in an agarose gel (1.0%) and the DNA sequences were gotten. Results: Pure cultures (13 strains) of nonsulfur bacteria were obtained after serial dilutions on semisolid agar. Cell morphology of these corresponded to small budding Gram negative rods, with a little difference in size (2-3 μm). In vivo pigments analyses revealed the presence of bacteriochlorophyll a and spirilloxanthin as major accessory pigment in all wild strains. It was assayed 20 different compounds to know the capability of the strains to use them as electron donors. Sodium and magnesium acetate as well as glycerol have been used for all the strains assayed, 12 used yeast extract and eleven to the pyruvate and propionic acid, sucrose, maltose, lactose, by other hand, succinate. methionin, mannitol and glycine were used only by 5, 6 and 7, strains, respectively. A BLAST and phylogenetic analyses based on 16S rDNA gene sequence revealed that four strains were related with Rhodobium bactotapetarum, three to Rhodopseudomonas spp and other three were identified as members of Rhodopseudomonas julia. It was observed differences in the use of the electron donors among the strains of the same specie. Conclusions: With the enrichment technique used it was possible to detect, isolate and to get pure cultures of phototrophic bacteria from marine water samples. The presence of bacteriocholorophyll a and the 16S rDNA analysis identify them as members of purple non sulfur bacteria
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P.084 DETECTION OF CYANOBACTERIA WITH CLASSICAL MOLECULAR METHODS IN THE SEDIMENT OF A DRINKING WATER RESERVOIR. Susanne Schumann1, Heidemarie Horn2, Tim Köhler3, Frank Ludwig1, Isolde Röske1, Kerstin Röske2. 1
Institute of Microbiology, Dresden University of Technology, Dresden; Taxony Academy of Sciences, Leipzig; 3Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, Marburg; Germany.
genera of purple sulphur bacteria (Chromatiaceae). Allochromatium was the most common genus 161 (50%) followed by Thiocystis 50 (15.53%), Thiocapsa 40 (12.42%), Thiodictyon 38 (11.8%) and Lamprocystis 33 (I0.25%) from the total number of isolates during the whole period of study. These genera represented by six speces Allochromatium vinosum and A. warmingii; Thiocystis violacea; Thiocapsa roseopersicina; Thiodictyon elegans and Lamprocystis roseopersicina. Physico–chemical parameters of water samples were determined.
2
Introduction: Cyanobacteria were able to colonize almost all habitats and are special important in the water because they have the ability to do photosynthesis. So they make a great contribution to primary production (Martínez-Alonso et al., 2004). Through formation of mats on the water surface, cyanobacteria can be able to reduce the development of organisms in deeper water layers. Methods: Sediment samples of different sampling sites of the dinking water reservoir Saidenbach (Saxony, Germany) and the pre-reservoir Forchheim were examined to follow up the deposition of cyanobacteria. To analyze the seasonal changes of the cyanobacteria composition in this habitat cultivation methods as well as PCR with established primer sets for cyanobacteria were applied. These partly well-known primer were combined with Cya564F, a new one to sequence the 16S rDNA to 23S rDNA of the cyanobacteria cultures which provides the basis of an alignment and cluster analysis. This results were compared with a phylogenetic tree which was established on the basis of the sequencing of the cyanobacterial phycocyanine gene (cpc). In addition a new primer system specific for species of the genus Synechococcus was developed and tested by PCR and cloning. Results: With the applied methods it was possible to detect differences and parallels in the emerging of cyanobacteria during the examination period. The amplification with the new primer system designated ProCR demonstrated clear variation in the occurrence of species of the genus Synechococcus. In spring, all samples were positive but in the further examination period it showed partly negative results. These findings could be confirmed by microscopy and cultivation. The alignment of the whole 16S rDNA of about 30 cyanobacterial pure cultures corroborates the belief that there is an increase of differences in the rear sequence sections. Conclusions: The received results show seasonal changes in the deposition of cyanobacteria of the examined drinking water reservoir. Furthermore differences between the sampling sites Saidenbach and Forchheim could be demonstrated. The first molecular biological analysis show the necessity of the development of new primer sets to sequence the whole 16S rDNA. So a reliable analysis of the isolated cyanobacterial cultures becomes possible.
P.085 ECOLOGICAL AND PHYSIOLOGICAL STUDIES ON PURPLE SULFUR BACTERIA (CHROMATIACEAE) AT ASWAN HIGH DAM LAKE. Mohamed S. A. Shabeb 1, Nahla S. Abd-El Azim1, Ahmed A. M. A. Shoreit 2. 1
Botany Department, Faculty of Science, Aswan South Valley University, Aswan, Egypt; 2Botany Department, Faculty of Science, Assiut University, Assiut, Egypt 322 isolates of purple sulfur bacteria were isolated from different localities of study in Aswan High Dam Lake. These comprise five 122
P.086 FUNCTIONAL GENE APPROACH ON COMMUNITIES OF PHOTOTROPHIC PROKARYOTES IN HIGH ALTITUDE WETLANDS IN NORTHERN CHILE. Vera Thiel, Marcus Tank, Cristina Dorador, Johannes F. Imhoff. IFM-GEOMAR, Leibniz Institute of Marine Sciences (at the University of Kiel), Kiel, Schlewig-Holstein, Germany. Introduction: The Chilean Altiplano located 2300 m above sea level harbours a set of extreme habitats characterized by high UV-radiation, partly extreme saline conditions and high diurnal temperature variations. These unexpectedly quite productive habitats are dominated by microbial life and phototrophic bacteria are assumed to have a major impact. However, key processes of primary production have not yet been identified and little is known about the diversity and composition of the phototrophic prokaryotic communities. To gain insight into the diversity of assumed key players involved in primary production in Chilean Altiplano salares, we studied the phototrophic bacterial communities of different locations around the Salar de Atacama in the Chilean Altiplano characterized by salinities varying from 54 mS cm-1 to 261 mS cm-1. Due to the polyphyletic character of the physiological groups of phototrophic bacteria, 16S rRNA gene based molecular studies often fail to cover the diversity of the phototrophic community. Thus, in order to specifically study the phototrophic prokaryotes of the habitats, we used molecular genetic analyses with group specific primers for functional genes in addition to isolation methods. Methods: We sampled several colourful microbial mats along two lakes of the Salar de Atacama (Laguna Chaxa and Laguna Tebenquiche). Different liquid media were inoculated for enrichment and isolation of phototrophic bacteria. Pure cultures were obtained by serial dilution in agar shakes and identified by 16S rRNA gene sequencing. For the molecular approach genomic DNA was extracted and the specific functional genes fmoA, pufLM and bchY amplified using PCR method. Clone libraries were constructed and the community composition was established on the basis of the functional gene sequences. Results: Enrichment and purification in different media lead to the isolation and identification of representatives of the green sulfur and purple sulfur bacteria as well as purple nonsulfur bacteria. Using functional genes members of different phototrophic bacterial groups were detected in all samples. The clone libraries lead to an estimate of the diversity of phototrophic prokaryotes in the different samples. The diversity and composition of the phototrophic prokaryotic communities varied between the different samples and between the two lakes. Conclusion: This study is the first report on the diversity of phototrophic prokaryotes in the Chilean Altiplano salares determined by functional gene approach and isolation methods. We demonstrate the suitability of the used functional genes, fmoA, pufLM and bchY for August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes studying phototrophic communities as well as the unique phototrophic communities of the different habitats around the Salar de Atacama.
P.087 EVOLUTION OF THE CYANOBACTERIUM NOSTOC –PLANT SYMBIOSES. Akiko Tomitani. Institute of Biogeosciences, Japan Agency for Marine-Earth Science & Technology, Yokosuka, Kanagawa, Japan. Cyanobacteria are oxygenic photosynthetic bacteria, many of which have ability to fix nitrogen. Filamentous cyanobacteria of the genus Nostoc produce differentiated cells such as heterocysts (specialized cells for nitrogen fixation) and hormogonia (transient motile filements), and many of them are known to establish symbiotic association with various plants (bryophytes, pteridophytes, gymnosperms, angiosperms) and algae and supply fixed nitrogen to the hosts. To investigate how Nostoc has evolved the capability to infect a wide range of plants, molecular-phylogenetic analyses together with coculturing experiments were performed. Phylogenetic analyses of free-living and symbiotic Nostoc species suggest that symbiotic competency may have evoloved polyphyletically within the genus. Co-culturing assays using 10 Nostoc strains and a host bryophyte indicate that their ability of hormogonia formation may not correspond to plant-infection efficiency. Continued molecular-biological and physiological study of diverse Nostoc species will provide a clue to understand mechanisms and evolution of the cyanobacterium-plant symbioses.
Final Program and Abstracts endemic to Antarctica and three others constitute a previously undiscovered diversity. A multivariate analysis run with data from various samples revealed that the OTU composition is geographically structured as each region has its more or less unique flora. Moreover, salinity seems to play an important role on the composition of the cyanobacterial communities in these samples. Conclusions: The three new OTUs included sequences from the Belgian Base, Transantarctic Mountains and East Antarctica which suggests a flux of microorganisms between these regions. On the other hand, differences in cyanobacterial composition between geographically close lakes were observed. These might be underlain by several reasons, such as differences in limnological properties (inferring different histories and ecological processes between regions), or rather the result from dispersal limitation among cyanobacteria. Also, samples originating from coastal lakes seem to be more diverse (average number of OTUs) than others, this may be due to the difference in the meteorological conditions (oceanic versus continental climates). More detailed studies and refined molecular analysis (Real Time Quantitative PCR, clone libraries) should allow us to assess the real cyanobacterial diversity in antarctic lakes and the importance of geographical and environmental factors shaping the microbial communities.
P.089 IN THE FRAME OF THE BELSPO PROJECT B-BLOOMS2, WE HAVE OBSERVED THE PRESENCE OF MICROCYSTINS BY ELISA IN BLOOMS OF WORONICHINIA IN ONE BRUSSELS POND. Annick Wilmotte, Yannick Lara, Alexandre Lambion, Anatoly Peretyatko, Ludwig Triest, Geoffrey Codd.
P.088
University of Liege, Liege, Belgium.
BIOGEOGRAPHICAL DISTRIBUTION AND DIVERSITY OF CYANOBACTERIA IN ANTARCTICA, A POLYPHASIC APPROACH.
So far, Woronichinia naegliana was isolated only two times according to the literature (Rajaniemi et al., 2005; Willame et al., 2006) and the isolates were not toxic. However, the cultures were quickly lost, illustrating the difficulties in obtaining and keeping strains of this genus. Therefore, there is an important lack of knowledge and molecular data. As an alternative, we propose to analyze genotypes of environmental single colonies.
Annick Wilmotte1, Pedro De Caralho Maalouf1, Frédéric Zakhia1, Arnaud Taton1, Rafael Fernandez-Carazo1, Aaike De Wever2, Elie Verleyen2. 1
Centre d’Ingénierie des Protéines, B6, Université de Liège, Liège; Department of Biology, Research group of Protistology and Aquatic Ecology (PAE), Gent University, Gent; Belgium. 2
Introduction: In high latitude ecosystems, cyanobacteria are of particular interest because they often represent the predominant phototrophs. The cyanobacterial diversity from microbial mats in antarctic lakes was investigated using microscopic and molecular approaches. This enables us to assess the relative importance of ecological versus historical factors in explaining the geographical distribution of cyanobacteria in Antarctica. Methods: Samples from a wide geographic scale were chosen in order to represent a range of environmental conditions and to evaluate the influence of lake characteristics on the cyanobacterial diversity. In the framework of the AMBIO project (“Antarctic Microbial BIOdiversity: the importance of geographical and ecological factors”, www.ambio.ulg.ac.be), microscopic identification and morphological characterization were complemented with molecular tools (Polymerase Chain Reaction, Denaturating Gradient Gel Electrophoresis, sequencing, clone libraries). This polyphasic approach aims to reveal the cultivated and non-cultivated diversity of microorganisms.
Methods: Woronichinia were directly isolated under a binocular from a fresh sample. As the DNA content of one single colony limits the number of PCR reactions that can be carried out, we have developed a new approach using Whole Genome Amplification with Phi29 polymerase to allow for the Multi Locus Sequences Analysis of a single colony. Subsequent PCR reactions were performed with cyanbacterial specific primers. Results: For the first time, we have obtained the sequences of rpoC1, rbcLX and rRNA-ITS from 4 single colonies of the genus Woronichinia (identified by microscopy). About 12 PCR reactions were successfully performed on one single colony. mcyE genes were not detected by PCR. Conclusion: This approach allows to work with a small amount of DNA, and represents a concrete answer to the lack of molecular data on non-cultivable or difficult to isolate cyanobacteria.
Results: Preliminary results from the 16S rDNA sequence analysis allowed the construction of phylogenetic tree and revealed the presence of 23 Operational Taxonomic Units (OTU). Five OTUs are August 9 to 14, 2009 • Montréal, QC, Canada
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P.090 HOW DOES CYANOBACTERIAL PHOTOSYNTHESIS CHANGE WITH SALINITY AND TEMPERATURE IN HYPERSALINE MICROBIAL MATS LOCATED AT DIFFERENT INTERTIDAL POSITIONS? Raeid M. M. Abed. College of Science, Biology Department, Sultan Qaboos University, Al Khoud, Sultanate of Oman. Intertidal cyanobacterial mats of the Arabian Gulf are exposed to continuous fluctuation in temperature and salinity, daily and seasonally. The temperature ranges between 18-55°C and salinity varies between 4-25% depending on the mat’s tidal position. Using microsensors, we studied the short-term temperature effects and the effects of salinity fluctuation on gross photosynthesis (GP), net photosynthesis (NP) and light respiration (LR) in three submerged mats from a transect on an intertidal flat. Areal rates of GP and NP increased with temperature and maximum rates were detected at 45°C. The photosynthetic zone decreased from 3mm to 1.75mm with increasing temperature. Above 50°C, photosynthesis was completely inhibited, probably due to high sulfide concentrations. The areal LR rates between 25°C and 45°C did not change significantly but showed a decreasing trend in the photosynthetic zone. This trend suggested that the coupling between photosynthesis and LR was apparently broken. The response of GP, NP and LR in lower, middle and upper tidal mats to various salinities (65, 100, 150 and 200‰) was compared. GP and LR at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP and LR rates at salinities >100‰. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different temperatures and salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.
P.091 DISCOVERING THE METABOLIC BOTTLENECKS OF FUEL PRODUCTION: LIQUID CHROMATOGRAPHY MASS SPECTROMETRY (LC-MS) ANALYSIS OF THE FERMENTATIVE METABOLOME OF THE HYDROGEN-EVOLVING CYANOBACTRIUM SYNECHOCOCCUS SP. PCC 7002.
the KEGG pathway database (www.genome.jp/kegg/pathway.html) and NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). Analysis of autofermentation of Synechococcus was conducted by re-suspension of cells in the appropriate medium, sealing to light & air, purging with argon gas, and performing an optimized chemical extraction method on samples in triplicate at selecting time points post-anaerobisis. A chemical assay for total internal reducing sugars was performed on remaining cell material, and extracellular nitrate and metabolic end products were measured by chemical assay and NMR of the fermentation medium, respectively. Analysis of metabolite extracts was conducted using reversed-phase ion-pairing chromatography coupled directly to multiple-reaction monitoring (MRM) detection using an Agilent 6410 Triple Quadrupole mass spectrometer. Results: A hypothetical fermentative reaction network was constructed for Synechococcus based on whole genome information. An analysis method for these individual metabolites was developed using online chromatography coupled to mass-spectrometry, in which compoundspecific mass decomposition channels were optimized for each target metabolite and chromatography examined for optimal resolution and sensitivity of the suite of target compounds. Using this method, an anaerobic extraction protocol for Synechococcous samples employing fast cell filtration and cold chemical extraction was optimized for the target metabolome. This chemical/analytical procedure was applied to monitor the metabolic response of Synechococcous during autofermentation. As predicted, direct LC-MS detection of pyridine nucleotides and adenosine phosphates indicate increased redox poise and decreased cellular energy charge during fermentation. The differential effect of nitrate availability on metabolite pools during auto-fermentation was subsequently tested, and the roles of nitrogen as a both an electron sink (and competitor with hydrogenase) as well as a precursor to biosynthesis examined. Conclusions: A robust method for the metabolomic analysis of cyanobacterial fermentation has been developed using the model organism Synechococcus sp. PCC 7002. This approach has been employed to quantitatively monitor the intracellular response of this phototroph to auto-fermentative conditions, as well as the specific effect of nitrate on hydrogen evolution and fermentative metabolism.
P.092 EXAMINATION OF ALLOPHYCOCYANIN AND PHYCOERYTHRIN SUBUNIT BIOSYNTHESIS.
Nick Bennette, Kelsey McNeely, John Eng, G. Charles Dismukes.
Avijit Biswas, Shervonda Williams, Monica Kronfel, Yasmin Vasquez, Richard Alvey, Donald Bryant, Wendy Schluchter.
Princeton University, Princeton, NJ, USA.
Univeristy of New Orleans, New Orleans, Louisiana, USA.
Introduction: Many cyanobacteria possess the capability to evolve hydrogen fermentatively, deriving the requisite protons and electrons from water in a potential method of clean and renewable energy production. Unfortunately, much of the research regarding the production of hydrogen from cyanobacteria has dealt with internal cellular metabolism as a “black box” rather than a complex network of individual biochemical reactions in which electron and proton flux to hydrogen production may be bottlenecked at many different points. In order to resolve the internal cellular dynamics leading to hydrogen evolution, detection of the involved metabolite pools as well as resolution of their intervening fluxes is required. The objective of this research is to quantify the time-dependent intracellular metabolic response of a model hydrogen-evolving cyanobacterium, Synechococcus Sp PCC 7002, to effectors of H2 rate and yield, specifically the effect of extracellular nitrate.
Introduction: Cyanobacterial phycobilisomes are composed of the brilliantly-colored, water-soluble, highly fluorescent phycobiliproteins in addition to the linker proteins which hold the structure together. Each phycobiliprotein is composed of two subunits, α and β, and each subunit has between one and three linear tetrapyrrole prosthetic group(s) called bilin(s) attached at Cys residues via a thioether bond. In cyanobacteria, there are four different naturally occurring bilins with the most prevalent being phycocyanobilin (PCB) and phycoerythrobilin (PEB). For most phycobiliproteins, enzymes called bilin lyases are responsible for catalyzing the attachment of the bilin to the appropriate Cys residue. This study determined the bilin lyases involved in allophycocyanin subunit biosynthesis, evaluated the substrate specificity of two different types of bilin lyase; CpcE/F and CpcSU toward addition of PEB instead of PCB, and expressed putative bilin lyases to be tested for attachment of bilins to Phycoerythrin II (PEII) subunits.
Methods: Genetic analysis of Synechococcus was performed using 124
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes Methods: An in vivo heterologous expression system using E. coli was developed by co-expression of genes encoding the phycobiliprotein substrate, the biosynthetic enzymes of the prosthetic group, and the enzymes which catalyze the attachment of bilin. The phycobiliprotein subunit genes were fused downstream of a gene encoding a tag (e.g. Histidine tag or Glutathione S-transferase tag). The purified products were evaluated by absorbance and fluorescence spectroscopy, and the protein and bilin content were assessed after SDS-PAGE. For some experiments, in vitro assays were used to test activity where all recombinant proteins were expressed separately. Results: A multi-plasmid heterologous system was developed and used to create holo-allophycocyanin in E. coli cells. We also used this system to show that the heterodimeric CpcS/CpcU from Synechococcus sp. PCC 7002 is required for attachment of PCB to the allophycocyanin subunits ApcF and ApcD. For the ApcE linker protein (LCM99), we show that the amino-terminal allophycocyanin-like domain has autocatalytic bilin lyase activity and is also similar in sequence to CpcS and CpcU proteins. We also show that the CpcEF bilin lyase can efficiently attach PEB to CpcA using the in vivo heterologous system, enabling us to create unique phycobiliproteins. The CpcSU bilin lyase was not as efficient at attaching PEB over PCB to CpcB in this system. We have successfully overproduced soluble Synechococcus WH8020 HT-MpeB (β-phycoerythrin II) and HT-MpeA (α-phycoerythrin II) by coexpression with the E. coli GroES chaperone. The putative bilin lyase subunits MpeU, MpeV, MpeY, CpeU, and CpeS have been overproduced separately. Most of them are insoluble, but we are attempting to renature them from inclusion bodies. Results from these studies will be discussed. Conclusions: This in vivo heterologous system was developed in order to facilitate to test the activity of many different bilin lyases, to test the ability of known bilin lyases to attach different chromophores to create unique phycobiliproteins, and to produce large amounts of holophycobiliproteins. For some bilin lyases that are insoluble in E. coli, we may be able to renature them and test for bilin lyase activity in vitro.
P.093 GENETIC ANALYSIS OF CHLOROBACULUM TEPIDUM SULFUR ISLANDS. Ernest O. Bonsu1,2, Lisa Waidner2, Thomas E. Hanson1,2,3 . Department of Biological Sciences1, Delaware Biotechnology Institute2 and College of Marine and Earth Studies3, University of Delaware, Newark, DE, USA. Introduction: Chlorobaculum tepidum is an anaerobic phototrophic bacterium that utilizes reduced sulfur compounds such as sulfide, elemental sulfur and thiosulfate as electron donors during anoxygenic photosynthesis. Chlorobaculum tepidum is a model system for the study of anaerobic sulfur oxidation pathways as it grows rapidly, is genetically amenable, and its genome has been completely sequenced. In the C. tepidum genome sequence many predicted sulfur oxidation genes are grouped in clusters called Sulfur Islands. The overarching goal of this project is to refine models of anaerobic sulfur oxidation in C. tepidum by the genetic analysis of the Sulfur Islands. The analysis of two specific mutant strains will be discussed. Methods: In-vitro transposon mutagenesis (IVTM) was used to generate mutations in regions of the C. tepidum chromosome containing the sulfur islands. Mutant C. tepidum strains showing distinct growth or sulfur oxidation phenotypes were then characterized in detail to determine what genes were affected by the transposon insertion. Pulsed field gel electrophoresis, PCR, RT-PCR, DNA August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts sequencing and rescue cloning of DNA flanking transposon insertions have all been utilized to define the genotypes of interesting mutant strains. Further characterization of the genes affected is often necessary by creating mutant strains carrying a single gene inactivation. Results: Two strains are the focus of the current study. Mutant strain C5 was incapable of growth with thiosulfate as the sole electron donor. The genotype of strain C5 (∆CT0867CT0876::TnOGm) is complex, a deletion-insertion event that eliminated all or part of nine genes in a section of Sulfur Island I. By comparison of the deleted region with other anaerobic sulfur oxidizers, gene CT0872 was hypothesized to be responsible for the thiosulfate growth defect. The phenotype of strain CT0872::TnOGm is consistent with this prediction while the inactivation of CT0873 and CT0874 had no significant effect. Strain C3 grows slowly under all conditions tested and accumulates extremely high levels of extracellular sulfur globules during growth. PFGE data indicated that mutant strain C3 carries two TnOGm insertions localized in two distinct regions of the chromosome that are consistent with insertions in Sulfur Islands I and II, both of which carry copies of the dsr genes that are crucial for elemental sulfur oxidation in purple sulfur phototrophic bacteria. Conclusion: Detailed analysis of genes deleted in strain C5 has implicated the CT0872 gene product in thiosulfate oxidation. The identification of genes inactivated in strain C3 will provide important clues to the specific gene products required for elemental sulfur oxidation in C. tepidum.
P.094 THE STOMATIN, PROHIBITIN, FLOTILIN AND HFLK/C DOMAIN HOMOLOGUE SLR1768 IS REQUIRED FOR THYLAKOID BIOGENESIS IN THE CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803. Samantha Bryan, Edward Spence, Conrad Mullineaux. Queen Mary, University of London, London, United Kingdom. Introduction: Nearly all cyanobacteria possess thylakoid membranes, the single known exception being the atypical cyanobacterium Gloeobacter, which houses all its photosynthetic complexes in the plasma membrane. Thylakoids are intricate internal membrane systems, where all the photosynthetic complexes that catalyse the light induced oxidation of water to molecular oxygen are contained. Biogenesis and regulation of the thylakoids is still an open problem, with only two known regulators, Vipp1 and Alb3 indentified to date. Methods: A slr1768 knockout was generated using the pGEM T-easy vector and REDIRECT technology. Whole cells were scanned for absorption in a UV 500 spectrophotometer. Photochemical efficiency was determined using 77K fluorescence emission spectra, and chlorophyll a fluorescence. Maximum photochemical efficiency was calculated as (FM`dark – F0)/ FM`dark. Both wild-type Synechocystis sp. PCC 6803 and ∆slr1768 strains were collected and fixed, ultrathin sections were then negative stained and viewed using a JEOL 1220 transmission electron microscope. Results: By comparing growth and the photosynthetic efficiency of the slr1768 mutant with the wild-type, we found that slr1768 has a conditional phenotype; specifically under highlight conditions (130 µmol m-2 s-1) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density. The chlorophyll a pigment content decreases with the loss of thylakoids, although the photosynthetic efficiency is unaffected.
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Final Program and Abstracts Conclusions: This is the first example of a gene that affects thylakoid biogenesis with a phenotype conditional on light intensity. Our results demonstrate that Slr768 has a leading role in acclimatisation, linking light damage with maintenance and biogenesis of the thylakoids.
P.095 LIGHT VARIABILITY ILLUMINATES NICHE-PARTITIONING AMONG MARINE PICOCYANOBACTERIA. Douglas Campbell, Zoe Finkel, Andrew Irwin, Christophe Six. Mount Allison University, Sackville, NB, Canada. Prochlorococcus and Synechococcus picocyanobacteria are dominant contributors to marine primary production over large areas of the ocean. Phytoplankton cells are entrained in the water column and are thus often exposed to rapid changes in irradiance within the upper mixed layer of the ocean. An upward fluctuation in irradiance can result in photosystem II photoinactivation exceeding counteracting repair rates through protein turnover, thereby leading to net photoinhibition of primary productivity, and potentially to cell death. We show that the effective cross-section for photosystem II photoinactivation is conserved across the picocyanobacteria, but that their photosystem II repair capacity and protein-specific photosystem II light capture are negatively correlated, and vary widely across the strains. The differences in repair rate correspond to the light and nutrient conditions that characterize the site of origin of the Prochlorococcus and Synechococcus isolates, and determine the upward fluctuation in irradiance they can tolerate, indicating that photoinhibition due to transient high-light exposure influences their distribution in the ocean.
P.096 CHROMATIC PHOTOACCLIMATION EXTENDS UTILISABLE PHOTOSYNTHETICALLY ACTIVE RADIATION IN THE CHLOROPHYLL D-CONTAINING CYANOBACTERIUM, ACARYOCHLORIS MARINA. Zane Duxbury, Martin Schliep, Raymond J. Ritchie, Anthony W. D. Larkum, Min Chen. School of Biological Sciences, University of Sydney, Australia. Chromatic photoacclimation and photosynthesis were examined in two strains of Acaryochloris marina (MBIC11017 and CCMEE5410) and in Synechococcus PCC7942. Acaryochloris contains Chl d, which has an absorption peak at ca 710 nm in vivo. Cultures were grown in one of three wavelengths (525 nm, 625 nm and 720 nm) of light from narrow-band photodiodes to determine the effects on pigment composition, growth rate and photosynthesis. No growth occurred in 525 nm light. Synechococcus did not grow in 720 nm light because Chl a does not absorb effectively at this long wavelength. Acaryochloris did grow in 720 nm light, although strain MBIC11017 showed a decrease in phycobilins over time. Synechococcus and Acaryochloris MBIC11017 showed a dramatic increase in phycobilins when grown in 625 nm light. The various photopigments were acclimated in response to the light spectral conditions. Photoacclimation and the Qy peak of Chl d can be understood in terms of the ecological niche of Acaryochloris, which grows in habitats enriched in near infra-red radiation.
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P.097 ROLE OF RUBISCO AND RUBISCO LIKE PROTEINS IN SULFUR METABOLISM IN RHODOSPIRULLUM RUBRUM. Swati Dey, Jaya Singh, F. Robert Tabita. Departments of Microbiology, Environmental Sciences Graduate Program and the Plant Cellular Molecular Biology, The Ohio State University, Columbus, Ohio, USA. Introduction: With the discovery of the type IV RubisCOs or the RubisCO- like proteins (RLP), a new family of proteins was discovered that was found to be structurally similar to the large subunit of RubisCO, though RLP was unable to catalyze CO2 fixation like RubisCO. RLPs from organisms like Bacillus subtilis, Geobacillus kaustophilus and Microcystis aeruginosa have been shown to be involved in an enolase reaction of a methionine sulfur salvage pathway. We have been studying the functional role of RLP in various organisms such as Chlorobium tepidum, Rhodopseudomonas palustris, and Rhodospriullum rubrum and its relation to RubisCO. It was shown that RLP from R. rubrum utilizes methylthioadenosine (MTA, an intermediate of known methionine salvage pathways) as sole sulfur source. The RLP from R. rubrum catalyzes a novel isomerization reaction distinct from that catalyzed by the B. subtilis-type RLP of the methionine salvage pathway. Interestingly R. rubrum RubisCO was also shown to weakly catalyze an enolase reaction similar to the RLP of B. subtilis. In the present study, we found that R. rubrum RubisCO is involved in the sulfur salvage pathways under anaerobic condition apart from its normal CO2 fixing ability. Methods: Using MTA as a sole source of sulfur, growth experiments were performed under both aerobic and anaerobic conditions to illustrate the function of RLP and the dual function of RubisCO in R. rubrum after inactivation of the RLP gene. Complementation studies were used to confirm these results. In addition, RubisCO activity assays and western immunoblots also confirmed the presence of RubisCO in mutant strains lacking RLP that were capable of simultaneously fixing CO2 and using MTA as a sole source of sulfur. Results: Studies show that R. rubrum strains with an inactivated RLP gene were able to grow with MTA as a sole source of sulfur under anaerobic conditions. R. rubrum RLP gene deletion strains were unable to grow under aerobic conditions. Complementation of RLP gene supported aerobic growth in these strains. Western blots showed the expression of RLP under aerobic conditions and RubisCO under anaerobic conditions in various mutant strains. Conclusion: These studies showed that under aerobic growth conditions RLP is involved in the salvage of sulfur in R. rubrum. A different and novel substrate is utilized by RLP under aerobic growth conditions when MTA is used as a sulfur source for growth. However, under anaerobic conditions, using MTA as sole sulfur source, it is not RLP, but RubisCO that catalyzes a B. subtilis type reaction using 2,3diketo-5-methylthiopentanyl -1- phosphate as substrate. In addition, this work illustrates the dual role of RubisCO as an enzyme required for CO2 fixation as well as sulfur salvage. Hence, the RubisCO family not only is involved in carbon metabolism but this enzyme may be used simultaneously to catalyze an important reaction of sulfur metabolism in vivo.
August 9 to 14, 2009 • Montréal, QC, Canada
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Final Program and Abstracts
P.098
P.099
CHARACTERIZATION OF BILIN LYASES FOR R-PHYCOCYANIN IN SYNECHOCOCCUS SP. WH8020
MOLECULAR COMMUNICATION BETWEEN CYANOBACTERIA AND THEIR HOST PLANTS: ROLE OF CHEMOTAXIS IN SYMBIOTIC COMPETENCY.
Tierna Dragomani, Avijit Biswas, Wendy M. Schluchter. Department of Biological Sciences, University of New Orleans, New Orleans, LA, USA.
Paula Duggan, Dave Adams.
Introduction: Cyanobacteria utilize phycobilisomes composed primarily of phycobiliproteins to maximize capture of light energy. In marine Synechococcus, various strains exhibit distinctive phycobiliprotein compositions adapted to capitalize on the wavelengths of available light. Specific attachment of phycobilins endows each phycobiliprotein with characteristic capacities for light absorption and emission. Rarely, this attachment may be autocatalytic, but more often it is catalyzed by bilin lyases that attach phycobilins at conserved cysteine residues on the phycobiliprotein subunit. The E/F type bilin lyase was the first one characterized; each CpcE and CpcF subunit contains conserved “E-Z” motifs, and together, the heterodimer catalyzes bilin attachment at Cys-84 of the α-subunit of the phycobiliprotein, phycocyanin (PC). The rpcE and rpcF genes are similar to cpcE and cpcF, respectively and are located downstream of the rpcA gene in Synechococcus sp. WH8020. Recently, RpcG from Synechococcus sp. WH8102, a fusion of RpcE and RpcF, was shown to have phycoerythrobilin (PEB) lyase and isomerase activity (Blot et al., 2009; J. Biol. Chem. 284: 9290-9298). The purpose of this study is to test whether rpcE and rpcF in strain WH8020 encode an E/F heterodimeric lyase that catalyzes attachment of PEB to Cys-84 of α-Rphycocyanin (R-PC).
Introduction: Cyanobacteria, such as Nostoc punctiforme, are able to live in symbiotic association with a wide range of host plants. Prerequisites for successful infection of the host tissues include the formation of motile filaments known as hormogonia and the production of a plant-derived chemical(s) that attracts these filaments towards the sites of infection within the host plant. There is evidence of two-way communication between plants and their prokaryotic partners regulating the infection process and the events involved in the establishment of a functional dintrogen-fixing association. Moreover there is increasing evidence to suggest that chemotaxis-like signal transduction systems may be central to these processes. The genome of N. punctiforme contains five loci of genes encoding elements resembling chemotaxis genes of other bacteria. We have focused our attention on one locus, which appears to have been acquired from a horizontal transfer event and is unique amongst the other clusters in that it contains a putative CheR methyltransferase and CheB methylesterase, which are central to chemotactic adaptation in other bacterial systems and enable the bacterium to respond to new stimuli in a background containing constant levels of chemoattractants and/or chemorepellents.
Methods: In Synechococcus sp. WH8020, the cpe/ mpe operons encoding phycoerythrin and R-Phycocyanin genes have been sequenced (Wilbanks and Glazer, 1993; J. Biol. Chem. 268:12261235). Oligonucleotide primers were developed to amplify the rpcA gene (α-R-PC) from WH8020 chromosomal DNA, and this product was ligated into the pET Duet vector so that its sequence was fused to the reading frame encoding a Hexa-histidine tag. The rpcE and rpcF genes were cloned into the pCDF Duet vector (individually and together). A pACYC Duet plasmid contained genes (ho1, pebS) encoding enzymes known to convert heme to biliverdin and ultimately to PEB (generous gift of Dr. Frankenberg-Dinkel). Plasmids will be introduced into chemically competent E. coli strain BL21(DE3) and plated onto Luria-Brutani agar containing the appropriate antibiotics. Transformants will be cultured and protein production will be induced with IPTG. Each plasmid will be tested individually in cells for protein production to determine optimal growth temperature conditions for each set of proteins. Results: The rpcA plasmid construct resulted in the production of soluble His-tagged RpcA which was purified by metal affinity chromatography and verified by SDS-PAGE. We have successfully demonstrated that CpcE and CpcF can attach PEB to HT-RpcA. However, we are currently testing conditions for expression of soluble RpcE and RpcF. Results from these studies will be presented. In addition, we will test whether RpcE and RpcF form a heterodimer.
Faculty of Biological Sciences, Leeds, Yorkshire, United Kingdom.
Methods : Nostoc genes NpR0244 (cheB) and NpR0248 (cheR) were inactivated by insertional mutagenesis and recombinant strains were characterised in terms of chemotactic motility and their ability to infect the host bryophyte Blasia pusilla. Electron microscopy was used to examine the levels of cell surface piliation of hormogonia differentiated by the mutant strains compared with those of the wildtype. Results: Both the NpR0244 (cheB) and NpR0248 (cheR) mutants failed to infect the host plant even after protracted periods of co-culture with the host B. pusilla. Chemotaxis assays revealed that the mutant strains lack the wild-type positive chemotactic behaviour towards exudates released from nitrogen-starved B. pusilla tissue. Wild-type piliation phenotypes were observed with both the cheB-like NpR0244 and the cheR-like NpR0248 mutants implying that, unlike some other bacterial taxis systems, these genes are not involved in the biosynthesis of the pilus-like appendages present on the surface of hormogonia. Conclusions: Our data provide molecular evidence that the chemotaxis-like elements studied here function in the establishment of the Nostoc-Blasia symbiosis, most probably through the disruption of the normal chemotaxis responses that occur towards host-derived signals and that cyanobacteria, like other bacteria, also exhibit an adaptation response.
Conclusions: Soluble expression of RpcA was achieved, and it was a suitable substrate for bilin addition by another closely related bilin lyase CpcE/CpcF (from Synechocystis sp. PCC 6803). Assays with RpcA, RpcE and RpcF will be performed if soluble protein can be obtained.
August 9 to 14, 2009 • Montréal, QC, Canada
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P.100 RESPONSES OF CHLOROBACULUM TEPIDUM TO ELEVATED SULFIDE. Brian J. Eddie, Leong-Keat Chan and Thomas E. Hanson. College of Marine and Earth Studies and DBI, Newark, DE, USA. Introduction: Chlorobaculum tepidum (syn. Chlorobium tepidum) is a green sulfur bacterium (GSB) that has become a model system for phototrophic sulfur oxidation due to the relative ease of culture and genetic manipulation. While it preferentially uses sulfide as an electron donor, C. tepidum is inhibited by sulfide concentrations in excess of 8 mM, a much higher sulfide tolerance than originally reported for this strain. To understand mechanisms of sulfide tolerance, we seek to identify sulfide regulated genes and assess their role(s) in C. tepidum‘s physiology Methods: C. tepidum was grown either on thiosulfate as the sole electron donor or with varying concentrations of sulfide followed by a comparison of cellular properties and the proteome of cultures by 1D SDS-PAGE. In separate experiments, C. tepidum actively growing on thiosulfate as the sole electron donor was provided with varying doses of sulfide and the adaptation of the cultures to this shock was monitored by quantifying sulfur compound turnover, cellular properties and changes in the proteome. In addition, samples were taken for subsequent analysis by a modified version of the mRNA-Seq protocol normally applied to larger sized eukaryotic genomes. This protocol has been developed to allow inexpensive multiplexing of samples from prokaryotic genomes to scale the power of next generation sequencing techniques to smaller genome sizes. Results: C. tepidum cultures grown with varying sulfide concentrations showed a clear alteration in the major in vivo absorption peak of Bchl c and several polypeptides were significantly more abundant in high sulfide cultures. A high concentration sulfide shock (8 mM) resulted in complete growth inhibition, but it is not known whether this was due to sulfide poisoning or pH inhibition. Milder doses of neutralized sulfide (1.6mM) did not result in growth inhibition or cause significant alterations in the protein profile of the culture. Two independent samples from this experiment were analyzed by the mRNA-seq protocol and produced millions of sequences that could be aligned to the C. tepidum TLS genome. Analysis of the reproducibility of the modified mRNA-seq protocol and multiplexing technique will be presented as will cell property and protein data from additional sulfide shock experiments. Conclusions: This study is the first to investigate the response of C. tepidum TLS to sulfide and is one of the first to use mRNA-seq to investigate a prokaryotic transcriptome. Preliminary results indicate that the modified mRNA-seq protocol applied to C. tepidum identifies actively transcribed regions of the genome in a less annotation dependent fashion than can be achieved with current microarray technology. This included the identification of non-coding RNA’s, which are receiving increased attention as regulatory elements in prokaryotes.
P.101 IN VITRO ASSEMBLY OF THE CARBOXYSOMAL BICARBONATE DEHYDRATION COMPLEX. Charlotte De Araujo, Swan Cot, George Espie. University of Toronto, Mississauga, ON, Canada. Introduction: Cyanobacteria possess a carbon dioxide concentrating 128
mechanism (CCM) that greatly enhances photosynthetic efficiency. This adaptive mechanism minimally consists of multiple membrane transport systems for inorganic carbon and intracellular inclusions known as carboxysomes, where Rubisco is located. The structural and functional basis through which carboxysomes enhance CO2 fixation remains unclear. Based on yeast two-hybrid analysis Cot et al. (J. Bacteriol. 2008,190: 936) hypothesized that the proteins, CcaA, CcmM and CcmN interact to form a multiprotein bicarbonate dehydration complex (BDC) within the carboxysome which vectorially channels CO2 to Rubisco, thereby enhancing fixation. Methods: ccaA, ccmM and ccmN (slr1347, sll1031 & slr1032) from Synechocystis sp. PCC6803 were cloned and over-expressed using standard techniques. Carbonic anhydrase activity was determined by mass spectrometry. Results: Mass spectrometric assays revealed that neither recombinant CcmM nor CcmN possessed HCO3- dehydration activity alone. Overexpression of CcaA using the pET-15b E.coli expression system produced active protein capable of the catalytic interconversion of HCO3- and CO2. To examine the role of the BDC in the supply of CO2, attempts were made to reconstitute it in vitro using affinity purification techniques. We found that N-terminal 6xHis-tag-CcaA retained catalytic activity when bound alone to an affinity matrix. In contrast, a complex of matrix-bound T7-tag-CcmM + 6x-His-CcaA was catalytically inactive as was the T7-tag-CcmM + flag-CcmN + 6x-HisCcaA. Western blot analysis verified the presence of all 3 proteins within the affinity matrix. However, matrix-bound 6x His-CcaA + T7CcmM complexes and His-CcaA + T7-CcmM + flag-CcmN exhibited CA activity, suggesting that the orientation of CcaA was an important determinant in maintaining catalytic activity. In buffered solution at pH 8, CcaA activity was reduced by 20 % in the presence of CcmM, but not BSA. Conclusions: Initial results show that CcmM-CcmN-CcaA complexes are formed by affinity tag binding to a matrix and subsequent protein/protein interactions. The orientations of the proteins in relationship to one another are critical in maintaining CA activity. When the CcaA active site at the N-terminus of the protein is exposed to CcmM, activity was eliminated, suggesting that the binding of CcmM modulates CcaA activity. Potentially, small differences in the nature of the CcmM – CcaA interaction within carboxysomes may play a role in controlling the supply of HCO3- to CcaA and in regulating CcaA dehydration activity.
P.102 THE CYANOBACTERIAL RIBOSOMAL-ASSOCIATED PROTEIN LrtA MODULATES TRANSLATION IN SYNECHOCYSTIS SP. PCC 6803. Carla V. Galmozzi, Francisco J. Florencio Bellido, M. Isabel Muro Pastor. Instituto De Bioquímica Vegetal Y Fotosíntesis. Universidad De SevillaCsic, Seville, Seville, Spain. LrtA is a protein encoded by a light-repressed transcript in cyanobacteria. The gene coding for LrtA was identified in Synechococcus sp. PCC 7002 and showed expression dependent on darkness (Samartzidou y Widger, 1998; Tan et al., 1994). LrtA is related to a family of proteins present in several bacteria. In Escherichia coli two proteins belonging to this family, termed YfiA and YhbH, have been shown to be associated with the 30S ribosome subunit (Agafonov et al., 1999; Maki et al., 2000). YfiA has also been shown to inhibit translation in vitro (Agafonov et al., 2001) suggesting a role of this family of proteins in translation control related to stress adaptation. However, in vivo data that demonstrate a function of these August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes proteins in translation have not yet been reported. The lrtA gene product also displays sequence similarity to a spinach plastid-specific ribosomal protein (PSRP-1), which is present in the chloroplast stroma either unbound or associated with the 30S ribosomal subunit (Yamaguchi et al., 2000). Genes homologous to lrtA are present in all the cyanobacterial sequenced genomes, but their function remains unknown. The analysis of the Synechocystis sp. PCC 6803 genome showed the existence of an open reading frame (sll0947), the product of which displayed strong similarity to the previously identified Synechococcus sp. LrtA protein. In Synechocystis it has been shown that the amount of SigB sigma factor increased in darkness and contributes significantly to the up-regulation of lrtA in these conditions (Imamura et al., 2003). In addition, proteomic analysis of salt-stress proteins in the plasma membrane of Synechocystis resulted in the identification of LrtA protein induced under these conditions (Huang et al., 2006). We analyzed the transcript levels of the Synechocystis lrtA gene and the LrtA protein. In agreement with the Synechococcus homologous gene, the level of the Synechocystis lrtA transcript is up-regulated under dark conditions. However, the level of LrtA protein remains constant upon light-dark transitions. Using extracts from Synechocystis fractionated by sucrose gradient we show that LrtA is a ribosome associated protein present in 30S and 70S ribosomal particles, suggesting a role of this protein in translation control. We have constructed an lrtA-deleted Synechocystis strain, which was shown to be viable in laboratory conditions, indicating that lrtA is a dispensable gene in this cyanobacterium. Furthermore, lrtA mutants present decreased sensitivity to tylosin and erythromycin, two inhibitors of protein synthesis, again connecting the role of LrtA with translation. In addition, we analyzed in vivo incorporation of [35S]methionine into proteins in both wild-type and ∆lrtA strains. We found that at least two proteins were diferentially synthesized in WT versus ∆lrtA cells. We will carry out proteomic analysis comparing wild-type and ∆lrtA strains, in different culture conditions or stresses, to identify changes provoked by the absence of the LrtA protein. We expect to identify proteins whose synthesis is in some way regulated by LrtA. These results may allow us to establish a role of this protein in translational adaptation. References: Agafonov, D.E., Kolb, V.A., Nazimov, I.V., y Spirin, A.S. (1999) A protein residing at the subunit interface of the bacterial ribosome. Proc Natl Acad Sci U S A 96: 12345-12349. Agafonov, D.E., Kolb, V.A., y Spirin, A.S. (2001) Ribosomeassociated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep 2: 399-402. Huang, F., Fulda, S., Hagemann, M., y Norling, B. (2006) Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803. Proteomics 6: 910-920. Imamura, S., Asayama, M., Takahashi, H., Tanaka, K., Takahashi, H., y Shirai, M. (2003) Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria. FEBS Lett 554: 357-362. Maki, Y., Yoshida, H., y Wada, A. (2000) Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli. Genes Cells 5: 965-974. Samartzidou, H., y Widger, W.R. (1998) Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002. Plant Physiol 117: 225-234. Tan, X., Varughese, M., y Widger, W.R. (1994) A light-repressed transcript found in Synechococcus PCC 7002 is similar to a chloroplast-specific small subunit ribosomal protein and to a August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts transcription modulator protein associated with sigma 54. J Biol Chem 269: 20905-20912. Yamaguchi, K., von Knoblauch, K., y Subramanian, A.R. (2000) The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast). J Biol Chem 275: 28455-28465.
P.103 INVESTIGATING THE MOLECULAR BASIS FOR CYANOBACTERIAL EXCAVATION IN A BORING CYANOBACTERIUM (MASTIGOCOLEUS/FISCHERELLA). Q. Gao, E. Ramirez-Reinat, F. Garcia-Pichel. School of Life Sciences, Arizona State University, Tempe, AZ, USA. Introduction: Microorganisms are the most common, widespread and environmentally significant among carbonate borers. Boring cyanobacteria, as photosynthetic microbes, have been a part of the geological reworking of carbonates since at least 1500 Myr ago. Their action has played important roles in geological cycles, and they are important to the fisheries industry, as they can be a pest of mullusks. Despite this, the boring mechanism of any of these cyanobacteria remains unknown. Concurrent physiological experiments in our laboratory have recently shown that active pumping of calcium ions (through cellular uptake, trans-cellular transport and extrusion processes) is at the base of the boring ability, likely involving P-type ATPases. To rigorously identify the putative cellular mechanism for this unique phenomenon, we attempted to find genes encoding for calcium-transporting P-type ATPases in a model cyanobacterium (Mastigocoleus/ Fischerella), so that expression analyses could be carried out. Our study provides the first glance at the molecular mechanism for microbial excavation. Methods: The genes sequences for calcium related P-type ATPases from a variety of cyanobacteria were retrieved from the NCBI database and Cyanobase. 40 such putative calcium-related P-type ATPase genes were identified as having both a calcium binding motif and an ATP-binding motif, and classified into two groups according to independently obtained consensus phylogeny of the cyanobacteria from which the genes were obtained. Group-specific sequence alignments were used to design degenerate primers matching conserved sequence regions corresponding to the phosphorylating and ATP-binding motifs. These primers were used to clone calciumtransporting P-type ATPases from Fischerella/Mastigocoleus genome through PCR amplification of genomic DNA. The Purified PCR products were cloned into pCR®4 vector through invitrogen TOPO TA Cloning system. Six individual clones were sequenced to verify each PCR product. The corresponding, deduced protein sequences were compared with known proteins in the databases by available blast methods. Results: Using genomic DNA from two known calcium P-type ATPasecontaining strains, Synechocystis sp. PCC 6803 and Nostoc punctiforme, we could show that our primers amplify P-type ATPases from cyanobacteria, and were specific for P-type ATPase genes, since they yielded a single product of the predicted size. Using genomic DNA of boring Mastigocoleus/Fischerella as template, each primer pair yielded product of the expected size, about 155 bp and 1098 bp, resepctively. Cloning and sequencing of the two products, proved that they belonged to two distinct P-type ATPase genes, one resembling most that of Nostoc punctiforme and another one most closey related to one from Synechocystis sp. The sequence analysis of the two PCR products indicates there exist at least two putative calcium-binding P-type ATPases in Mastigocladus/Fischerella genome. 129
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Final Program and Abstracts Conclusions: Our experiments confirmed the feasibility of the genomic scanning by PCR method in the detection of calcium related P-type ATPases genes. This result, more importantly, provides molecular evidence for the existence of multiple putative calcium transporter genes in Fischerella, which could support our proposed mechanism for boring activity. Although this experiment is a first step in our exploration, cloning of calcium P-type ATPase paves the way for the further investigation of the molecular mechanism of cyanobacterial excavation. Ongoing studies attempt to address the expression of each of these genes under boring vs. free-living conditions, and the localization of this activity in these morphologically complex cyanobacteria.
P.104 THE GLOBAL RESPONSE TO UVA STRESS IN NOSTOC PUNCTIFORME ATCC 29133 ASSESSED BY A TEMPORAL DNA MICROARRAY STUDY. F. Garcia-Pichel1, Q. Gao1, J. C. Meeks2, V. Stout1, T. Soule1,3. 1
School of Life Sciences, Arizona State University, Tempe, AZ; 2Section of Microbiology, University of California, Davis, CA; 3Environmental Biotechnology, Savannah River National Laboratory, Aiken, SC; USA. Introduction: UVA radiation (320-400 nm) is an important component of the solar spectrum reaching the ground. Unlike short-wavelength ultraviolet, UVA does not directly damage biological materials. Its harmful effects come about through photosensitizers, molecules that create reactive compounds upon photons absorption and in the presence of oxygen. Cyanobacteria, because they contain high level of photosensitizing pigments, and are exposed to internally generated oxygen, are particularly prone to UVA-mediated damage. Despite this, gene expression studies have focused in the response to UVA have not been presented. To identify some of the cellular mechanisms used to cope with this part of solar radiation, we examined the temporal global stress response to an up-shift in UVA the cyanobacterium Nostoc punctiforme ATCC 29133 as a model organism using whole genome DNA microarrays. Our study provides the first global UVA stress response analysis for any phototroph. Methods: Nostoc cultures grown under white light were split into 2 series, one kept under white light, and the second exposed to supplemental UVA radiation, over the course of four days. At 24 hr intervals, triplicate cultures were harvested from each treatment. Total RNA was extracted, reverse-transcribed, and labeled with fluorescent dyes. Labeled cDNAs were hybridized to microarray slides and raw images were processed for analysis. Genes with at least a statistically significant 2-fold differential expression level between the UVAtreated and untreated samples were considered responsive to UVA and were used to construct gene lists for analysis. Results: The study detected statistically robust signals for differential regulation in response to UVA in 573 out of the 6903 genes probed on the array. Most of the up-regulated genes, 299, were uncategorized, and 8% (82) of all of the genes known to be dedicated to adaptive metabolism were upregulated. The most common category of downregulated genes was those associated with light-harvesting pigment biosynthesis, likely to minimize photosensitizer density. Among the expected responses, we could detect the up-regulation of several antioxidant enzymes, likely to help the cells cope with reactive oxygen species, and the up-regulation of genes for the biosynthetic pathway of the UVA sunscreen, scytonemin. Most intriguingly, we found that by far, the strongest and most sustained up-regulation involved a group of contiguous genes of unassigned metabolism, but widely distributed among cyanobacteria, that on plasmid A. 130
Conclusions: Our experiments confirmed the importance of several well known adaptive strategies to cope with UV: decrease in photosensitizers, increase in antioxidant systems, and synthesis of UVA sunscreens. It also revealed that we know virtually nothing about the vast majority of genes involved in this response. Overall, our results suggest that UVA plays an important role in the physiology of Nostoc, but also that the UVA cyanobacterial stress response is obviously far from being well understood. Some of the genes identified here provide new avenues for exploration.
P.105 THE BRANCHPOINT FOR BACTERIOPHEOPHYTIN BIOSYNTHESIS IN RHODOSPIRILLUM RUBRUM. Robin Ghosh1, Hartmut Grammel2, Jörg Hammel1, Rudolf Saegesser1, and Khaled Abou-Aisha1. 1 Dept. of Bioenergetics, University of Stuttgart, Pfaffenwaldring 57, D70550 Stuttgart, Germany, 2Max-Planck Institute for the Dynamics of Complex Technical Systems, Magdeburg, Germany.
Introduction: The pathway for bacteriochlorophyll a (BChla) biosynthesis has now been well-characterized in purple photosynthetic bacteria, particularly in the species Rhodobacter capsulatus and Rhodobacter sphaeroides. By contrast, the biosynthesis of bacteriopheophytin a (BPha) appears to have been largely ignored. In R. rubrum, the chemical structure of BPha is useful for tracing the pathway of biosynthesis, as BChla contains a geranyl-geranyl chain, wheras BPha contains a phytyl chain. In this study, by a combination of organic separation and spectroscopy we have deduced the probably branch point of BPha biosynthesis from the main BChla pathway. Methods and Results: A number of photosynthetically incompetent mutants in genes for pigment synthesis in R. rubrum were generated by both random as well as Tn5 mutagenesis. The lesions were identified by both Southern hybridization as well as complementation analysis. Cells were grown semi-aerobically in the dark using a unique medium (M2SF) which allows up-regulation of photosynthetic genes to levels normally only observed under low light photoheterotrophic conditions. Growth in this medium causes R. rubrum to produce very large quantities of pigments, thereby simplifying subsequent chemical analysis. The pigments were separated in organic solvents and analyzed by absorption, fluorescence and mass spectroscopy. This analysis allowed us to deduce the probable locus of the BPha shunt. Conclusions: The removal of Mg2+ from the BChla-precursor occurs at an unexpected locus.
P.106 HGDD, A TOLC-LIKE OMF INVOLVED IN HETEROCYST DEVELOPMENT. Alexander Hahn, Peter Staron, Iris Maldener, Enrico Schleiff. Goethe Universitat, Cluster of Excellence, Center of Membrane Proteomics, Frankfurt, Hessen, Germany. Protein secretion of the type I system and multi-drug efflux in Gramnegative bacteria are based on a tripartite secretion complex. This complex usually contains an inner membrane ABC-transporter or a proton driven efflux pump, a membrane fusion protein (MFP) and an outer membrane factor (OMF) of the TolC-family. Together they form a single channel tunnel to secret proteins and toxic substances from the cytoplasm or the inter membrane space. The cyanobacterium Anabaena sp. PCC 7120 has only one TolC–like protein. We identified its expression in all cells of the filament, but it was found to be August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes indispensable only for heterocyst development. The phenotype of the deletion strain parallels that of the knock out of the devBCA operon encoding for an ABC-transporter. Hence, it is concluded that DevBCA together with the identified TolC-like protein is involved in the assembly of the heterocyst specific glycolipid-layer (HGL-layer) and is therefore termed HgdD. As HgdD is the only TolC present in the outer membrane of Anabaena sp. PCC 7120 an interaction with the multitude of inner membrane located ABC-transporter is expected, a hypothesis which is currently investigated.
P.107 CYANOBACTERIOCHROME CCAS REGULATES PHYCOERYTHRIN ACCUMULATION IN NOSTOC PUNCTIFORME THAT PERFORMS GROUP II CHROMATIC ADAPTATION. Yuu Hirose1, Mitsunori Katayama2, Rei Narikawa3, Masahiko Ikeuchi1,3. 1
Department of Biological Science, Graduate School of Sciences, the University of Tokyo; 2College of Industrial Technology, Nihon University; 3Department of Life Sciences (Biology), the University of Tokyo; Japan. Introduction: Cyanobacteriochromes are unique phytochrome-related photoreceptors that undergoes reversible conversion between violet/yellow, blue/green or green/red absorbing forms with photoisomerization of a covalently bound linear tetrapyrrole (Ikeuchi and Ishizuka, 2008). Cyanobacteria utilize phycobilisome (PBS) as light harvesting antenna. Typical light harvesting proteins of PBS are phycoerythrin (PE) and phycocyanin (PC), which absorb green light (GL) and red light (RL), respectively. Certain cyanobacterial species are able to raise PE/PC ratio under GL and reduce under RL, which has been called “complementary chromatic adaptation”. Some acclimate only PE accumulation (group II) while the others acclimate both PE and PC accumulation (group III). In Fremyella diplosiphon that performs group III chromatic adaptation, a cyanobacteriochrome gene, rcaE, is suggested to induce the expression of PC genes under RL, although another GL receptor was implicated to regulate the expression of PE genes (Kehoe and Gutu, 2006). Our recent studies suggested that another cyanobacteriochrome CcaS regulates the expression of PBS linker gene, cpcG2, in Synechocystis sp. PCC 6803 that possesses PC but not PE. CcaS shows reversible photoconversion between GL and RL-absorbing forms and GL-activated phosphorylation to the cognate response regulator CcaR (Hirose et al, 2008). Interestingly, CcaS and CcaR orthologs are clustered with PE genes (cpeC, cpcG2 and cpeR) in the genome of Nostoc punctiforme ATCC 29133 that performs group II chromatic adaptation. In this study, we disrupted ccaS, ccaR and the clustered PE genes in N. punctiforme and examined their response to GL and RL. Material and methods: N. punctiforme cells were grown in liquid BG11 medium under 20 μE m-2s-1 of GL (peaked at 530 nm) or RL (650 nm). Absorption spectra of fully acclimated cells were measured by spectrophotometer with an end-on photomultiplier. To create ccaS, ccaR and PE gene mutants, each gene was replaced with a neomycin cassette from pSCR9 as described (Cohen and Meeks, 1997). RNA was extracted by acidic phenol. Northern blotting analysis was performed according to standard protocol using radio-labeled probes. Results: We found that PE was hardly detected in wild type cells grown under RL, while PE content was high in cells grown under GL. In a ∆ccaS mutant, some amounts of PE accumulated under both GL and RL, suggesting that ccaS is essential not only for PE accumulation under GL but also for repression under RL. In a ∆ccaR mutant, PE hardly accumulated under both GL and RL, suggesting that CcaR is transcriptional activator for PE accumulation. Northern blotting August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts analysis indicated that the clustered PE genes, cpeC, cpcG2, and cpeR, are co-transcribed under GL but not RL in wild type. ∆cpeC/∆cpeG2/∆cpeR triple mutant was also defective in PE accumulation under both GL and RL, suggesting that the induction of these genes are essential for PE accumulation. From these results, we conclude that CcaS regulates PE accumulation by the transcriptional regulation of cpeC, cpcG2, and cpeR, via GL-activated phosphorylation and probably RL-activated dephosphorylation of CcaR. Conclusion: In complementary chromatic adaptation, the green light receptor for PE accumulation has not yet been identified. In this study, we found that cyanobacteriochrome CcaS and response regulator CcaR regulates PE accumulation via the transcriptional regulation of the clustered PE genes in N. punctiforme.
P.108 CIRCADIAN TRANSCRIPTIONAL REGULATION IN THE DARK WITHOUT CYCLIC KAI GENE EXPRESSION IN SYNECHOCOCCUS. N. Hosokawa1, T. Hatakeyama1, H. Iwasaki1,2. 1 Department of Electrical Engineering and Bioscience, Waseda University; 2PRESTO, Japan Science and Technology Agency, Wakamatsu, Shinjuku, Tokyo; Japan.
Most of organisms exhibit daily cycles, driven by endogenous circadian clocks. The unicellular cyanobacterium, Synechococcus elongatus PCC7942 is an obligate photoautotroph and known as the simplest model organism for circadian biology. In S.elongatus, most of genes exhibit circadian expression rhythms, which are regulated by three clock genes, kaiA, kaiB and kaiC, under continuous light (LL) conditions. Interestingly, when cells are transferred to continuous dark (DD) conditions from hour 12 in the light, kaiA and kaiBC genes are rapidly downregulated to the zero level, while the KaiC phosphorylation cycle persists in the dark even in the presence of excess transcription/translation inhibitors. Thus, the basic oscillation is generated via post-translational process (Tomita et al., 2005). When we performed DNA microarray analysis, expression of most of genes on the genome was also dramatically and rapidly suppressed in the dark, whereas a minor subset of genes was upregulated. These dark-induced genes required de novo transcription under DD. Then, we examined if such gene expression profiles under DD were affected by the Kai-based clock even in the absence of de novo kai-gene expression. We found the magnitude of dark-induction in some genes was dependent on time when the cells were transferred from light to DD. Moreover, expression profiles of such genes in DD were dramatically altered in the kaiABC-null mutant strain. Thus, in contrast to our previous model, the Synechococcus clock regulates transcriptional outputs even in the dark.
P.109 CHARACTERIZATION OF UNIQUE PHOTOCHEMICAL PROPERTIES OF CYANOBACTERIOCHROME TePixJ. T. Ishizuka1, A. Kamiya2, H. Suzuki3, T. Noguchi3, T. Kohchi4, K. Inomata2, M. Ikeuchi1. 1
Department of Life Sciences (Biology), The university of Tokyo; Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University; 3Institute of Materials Science, University of Tsukuba; 4Graduate school of Biostudies, Kyoto University; Japan. 2
Introduction:Cyanobacteria harbor many putative GAF-containing 131
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Final Program and Abstracts photoreceptors that may bind a linear tetrapyrrole as a chromophore (cyanobacteriochromes) in addition to typical phytochromes. One of them, TePixJ of Thermosynechococcus elongatus BP-1 is essential for phototaxis. Previously, we reported novel properties of the GAF domain of TePixJ (denoted TePixJ_GAF) that was expressed in Synechocystis PCC 6803. Purified TePixJ_GAF showed reversible photoconversion between the 433nm (Pb form) and 531nm-absorbing forms (Pg form). In contrast with Pr and Pfr forms of Synechocystis phytochrome Cph1 carrying phycocyanobilin (PCB), Pb and Pg of TePixJ_GAF are extremely blue-shifted (approximately 150~200nm). Here, we studied the unique photochemical properties of TePixJ. Methods: in vitro reconstitution: synthetic PCB was added to a reconstitution buffer containing TePixJ_GAF apoprotein prepared from E. coli. The mixture was incubated in the dark at 50ºC, which is close to the optimum growth temperature of T. elongatus. in vivo coexpression: TePixJ_GAF apoprotein and two enzymes (Ho1, PcyA), which synthesize PCB from heme, were co-expressed in E. coli cells to allow in vivo assembly of the holoprotein. Denaturation analysis: The Pb*, Pb, Pg* and Pg forms of TePixJ_GAF and Pr and Pfr of Cph1 were denatured with 8 M urea/HCl pH 2.0 at room temperature in the dark. Native holo-TePixJ_GAF (~20 mg/ml) was subjected to FTIR analysis. Results: Previously, we reported that molecular mass of the chromophore of TePixJ was identical to PCB but was different in spectral properties. Moreover, we carefully compared TePixJ with Cph1 after denaturation with acidic urea. The spectral properties of TePixJ chromophore were clearly different from those of Cph1 PCB, but were very similar to those of phycoviolobilin (PVB), an isomer of PCB. In the early phase of in vitro reconstitution, free PCB was covalently incorporated with concomitant accumulation of photoconvertible holoprotein between Pb-like blue-absorbing form (Pb* form) and Pg-like green-absorbing form (Pg* form). Further incubation at 50ºC resulted in conversion to spectral properties similar to the native TePixJ. Concomitantly, PCB was isomerized to PVB as revealed by denaturation analysis. The holocomplex prepared from in vivo co-expression included both PCB and PVB as a chromophore. When this holocomplex was further incubated, additional isomerization from PCB to PVB was detected. There is conserved cysteine residue (Cys494: TePixJ numbering) among blue-green photoreversible cyanobacteriochromes. Mutation of Cys494 resulted in assembly of a red-absorbing form which did not exhibit photoconversion. Moreover, FTIR spectroscopy of the native TePixJ_GAF revealed green light-induced crosslinking of a free SH group. These results suggest that the light-induced crosslinking of a cycteine residue (probably Cys494) to the PVB takes place in the assembly of Pb that is extremely blue-shifted compared with the typical phytochrome. Conclusions: As a first step, TePixJ apoprotein and PCB form an intermediary photoactive Pb*. Secondly, subsequent isomerization from PCB to PVB proceeds during in vitro reconstitution. The extreme blue shift of TePixJ can be explained by reversible adduct formation between the chromophore and Cys494. Thus, we can conclude that the GAF domain of TePixJ is sufficient for both lyase, isomerase and photoconversion activities.
P.110 CELL LINEAGE ANALYSIS ON HETEROCYST PATTERN FORMATION IN ANABAENA SP. PCC 7120. S. Iwamori1, H. Asai1, K. Kawai1, S. Shoji1, H. Iwasaki1,2. 1
Department of Electrical Engineering and Bioscience, Waseda University, Japan; 2PRESTO, Japan Science and Technology Agency, 132
Wakamatsu, Shinjuku, Tokyo; Japan. hetR and patS genes have been reported as an activator and a repressor of heterocyst formation, respectively, in the filamentous cyanobacterium, Anabaena sp. PCC 7120. It is proposed that hetR activates its own transcription and patS gene expression, while the PatS peptide inhibits the HetR function. This type of combination of the negative and positive feedback loops with a possible diffusible inhibitor is reminiscent of the Turing instability dynamics, while its possibility has not yet been well validated experimentally. For better understanding of spatio-temporal dynamics underlying the heterocyst patterning, we have developed a monitoring/culturing system that enabled us to observe morphological changes, hetR expression profile, and chlorophyll fluorescence from individual filaments during the course of heterocyst development. Moreover, micro-liquid chamber arrays have been developed to analyze multiple Anabaena filaments simultaneously. Cell lineage analyses demonstrated that initial distributions of hetR::gfp signals and chlorophyll activity signals at nitrogen-step-down were not correlated to the resulting distribution of developed heterocysts, supporting a random and non-deterministic selection of initial heterocyst positions. We also observed cells that differentiated into heterocysts without cell division after nitrogen-stepdown, suggesting that cell division of mother cells is not an essential requirement for heterocyst differentiation. We will also report that the hetR expression profile exhibits a complicated, transient and possibly competing dynamics even before determination of initial heterocyst positions.
P.111 THE PHYSIOLOGICAL ROLE OF THE CBBRRS TWO – COMPONENT SYSTEM IN RHODOPSEUDOMONAS PALUSTRIS. Gauri. S. Joshi, F. Robert Tabita. Department of Microbiology, The Ohio State University, Columbus, OH, USA. Introduction: Rhodopseudomonas palustris is a metabolically versatile nonsulfur purple photosynthetic bacterium. The unique feature of the R. palustris cbbI CO2 fixation regulon is the presence of a twocomponent system (referred to as the CbbbRRS system) between the master transcriptional regulator CbbR and genes encoding form I RubisCO (cbbLS). CbbR belongs to the LysR family of regulators. The CbbRRS system is an atypical two-component system consisting of a sensor kinase and two response regulators with no apparent DNA binding domains on any of these proteins. Recent physiological studies have shown that the CbbRRS system plays a regulatory role in modulating the expression and activity of form I RubisCO only during photoautotrophic (CO2) growth and not during photoheterotrophic (benzoate) growth. The distinctive presence of this two component system and its role in modulating form I RubisCO expression is unique to R. palustris as similar systems have not been described in closely related NSP bacteria, Rhodobacter spaheroides, Rhodobacter capsulatus, and Rhodospirillum rubrum. The observations stemming from the physiological studies with photoautotrophically grown cells led to the hypothesis that the CbbRRS proteins (especially the response regulators CbbRR1 and CbbRR2) influence the interactions of CbbR at the cbbI promoter and thereby influence form I RubisCO expression. Methods: A bacterial two hybrid analysis (qualitative and quantitative) was performed to test whether the response regulators (CbbRR1 and CbbRR2) interact with CbbR in vivo. In this Escherichia.coli-based system, interaction between a pair of proteins results in transcriptional activation of a selectable (His3- aadA)/quantitative (lacZ) reporter gene August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes by recruitment of RNA polymerase at the promoter. The strength of the interaction between the protein pair determines the magnitude of transcriptional activation. In vitro gel shift assays and chemical crosslinking are additional approaches adopted to demonstrate the DNA – protein and protein – protein interactions. Results: The bacterial two hybrid system enabled the identification of a protein – protein interaction between the transcriptional regulator CbbR and CbbRR1, response regulator 1 of the CbbRRS system. Site directed mutagenesis of the phosphoacceptor residues of CbbRR1 (D54N and H171D) did not affect its interaction with CbbR significantly suggesting that phosphorylation of CbbRR1 may not be important for the interaction. Gel mobility shift analyses revealed that the affinity of CbbR for the cbbI promoter was specifically enhanced in the presence of CbbRR1, while the presence of CbbRR2 in the complex decreased the mobility of the DNA – protein complex. Chemical crosslinking of CbbR with the response regulators CbbRR1 and CbbRR2 is underway to further support the in vivo protein – protein interaction data. Conclusions: The results of the bacterial two hybrid analysis indicate that the CbbRRS two component system potentially exerts its regulatory effect on the cbbI promoter (form I RubisCO expression) by interacting with the master transcriptional regulator CbbR. The binding of CbbR to the cbbI promoter is stabilized in the presence of CbbRR1 as observed in gel shift assays, further strengthening the in vivo data. The interactions of the response regulators with CbbR represent additional transcriptional control beyond that provided by CbbR alone, for fine tuning the expression of form I RubisCO in R. palustris.
P.112 PHOTOTROPISM OF CYANOBACTERIA. Mitsunori Katayama1, Mari Kobayashi2, Masahiko Ikeuchi3. 1
Department of Liberal Arts and Basic Sciences, Nihon University; 2 Tokyo Institute of Technology, School and Graduate School of 3 Bioscience and Biotechnology; Department of Life Sciences (Biology), The University of Tokyo; Japan. Introduction. Phototropism is directional growth in response to directional light, which is widely observed in eukaryotes such as fungi, algae, and higher plants. In eukaryotes, blue light commonly induces phototropic response. On the contrary, phototropism was barely reported for prokaryotes. We recently found out phototropic response in filamentous cyanobacterium Rivularia sp. IAM M-261 (Rivularia M261). To characterize phototropism in cyanobacteria, its spectral dependence and the distribution among cyanobacteria were investigated. Methods. Microscopic examination of filamentous cyanobacteria collected from environmental samples and culture collections was made to survey species exhibiting phototropsm. Spectral dependence of the phototropic response was examined in the three Calothrix species and in the one Fischerella species by irradiation with series of monochromatic light (400 nm to 750 nm) provided by light emitting diode. Phototropic response was quantified by measurement of average angles of curvature of multicellular filaments. Results. Phototropism was observed in some species that belong to Calothrix, Tolypothrix, Scytonema and Fischerella. Rivularia M-261 was thought to be classified into Calothrix based on the 16S rDNA sequence. As for spectral dependence of the phototropic response, Rivularia M-261, Calothrix sp. PCC 7715 and Calothrix sp. PCC 7102 exhibited positive phototropic response to blue light (460 nm). On the other hand, one Fischerella sp. exhibited positive phototropic
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Final Program and Abstracts response to blue light (460 nm) and far-red light (750 nm). Conclusions. Cyanobacteria belongs to diverse group (subsection IV and subsection V) were shown to exhibit phototropic response. Blue light commonly induced phototropic response in Calothrix and Fischerella likewise eukaryotes. In addition to this, one Fischerella species exhibited phototropic response to far-red light. Most of species exhibiting phototropism grow on surface of soil or rock suggesting that phototropism is advantageous for terrestrial growth in cyanobacteria.
P.113 EFFECTS OF GROWTH CONDITIONS AND THE COMPOSITION OF GAS PHASE ON PHOTOBIOLOGICAL HYDROGEN ACCUMULATION IN THE HYDROGENASE MUTANT OF NOSTOC SP. PCC 7422. Masaharu Kitashima, Hajime Masukawa, Hidehiro Sakurai, Kazuhito Inoue. Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, Japan. Introduction: We are aiming at developing nitrogenase-based photobiological hydrogen production utilizing cyanobacteria with water as the terminal electron donor. In the previous report (Yoshino et al., 2007), we reported that the uptake hydrogenase gene disrupted (∆hup) mutant of Nostoc sp. PCC 7422 had high hydrogen production activity and accumulated hydrogen to about 30% (v/v) under a starting gas phase of Ar + 5% CO2. In order to attain a higher level of accumulation of hydrogen concentration, we have studied the effects of nitrogen and CO2 gas concentration of the first (heterocyst induction) and the second (hydrogen production period) gas phase on hydrogen accumulation. Methods: The culture media were either BG11 or BG110 (NaNO3 omitted) medium. The starting culture of the ∆hup mutant was grown photoautotrophically at 26°C by bubbling air under the 12 hour light12 hour dark cycle. In the light period, cultures were illuminated by fluorescent light at photosynthetically active radiation (PAR) of about 100 µmol photons m-2 s-1. The cells in the exponential growth phase were washed with and suspended in BG110 medium at a chlorophyll α concentration of about 3 µg ml-1, and 15 ml samples were transferred into 25-ml Fernbach flasks equipped with butyl rubber stoppers. Gas composition of the first phase was Ar + 5% CO2 + varied concentration of N2 (1 - 80%). After photoautotrophically cultivating for 2 to 4 days, the gas phase was changed to Ar + 5% CO2 + varied concentration of N2 (0.5 - 80%) for the second phase. For determination of accumulated hydrogen and oxygen, 50 µl each of gas sample was withdrawn from flasks at about the middle of the light phase and analyzed by gas chromatography (Molecular Sieve 13X column). Nitrogenase activity was assayed by reduction of acetylene to ethylene. Results: When the duration of in the first phase was two days, the nitrogen concentration at 1-20% did not greatly affect the hydrogen accumulation in the second phase, but 80% nitrogen significantly decreased the accumulation. With the first phase of three days, the hydrogen accumulation by 5% nitrogen culture was lower than that by 1% nitrogen culture. After two days under 1% nitrogen, the nitrogen concentration was changed (the second phase). As for the hydrogen accumulation of the culture (0-13 days), 0.5% nitrogen gave slightly higher concentration than 1%. At 5% and 20% nitrogen, the accumulation was much lower than the formers. After about 8 days under 0.5% nitrogen, the hydrogen concentration became about 50% (v/v) with oxygen concentration of about 20-25%. The profiles of 133
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Final Program and Abstracts nitrogenase activity could largely account for the hydrogen accumulation profiles. These results indicate that a large part of hydrogen was produced with water as the terminal electron donor under the experimental conditions used. Conclusions: By optimizing the gas composition of the culture, the genetically improved mutant strain of cyanobacterium cells are able to accumulate hydrogen to about 50% catalyzed by nitrogenase reaction, mostly using water as the terminal electron donor. Reference: F. Yoshino, H. Ikeda, H. Masukawa, H. Sakurai, High photobiological hydrogen production activity of a Nostoc sp. PCC 7422 uptake hydrogenase-deficient mutant with high nitrogenase activity, Mar. Biotechnol., 9: 101-112 (2007)
metabolism. Our results will be supplemented with and tested against high-quality data, aiming to iteratively improve and refine the current annotation. Our stoichiometric model is intended to serve as a first step towards a comprehensive computational description of cellular metabolism in unicellular autotrophs and can be extended to a genome-scale level in further studies.
P.115 CIRCADIAN SYSTEM IN THE HETEROCYST-FORMING CYANOBACTERIUM, ANABAENA SP. PCC 7120. H. Kushige1, M. Matsuoka1, H. Iwasaki1,2.
P.114 CHARACTERIZING THE PRIMARY METABOLISM OF SYNECHOCYSTIS SP. PCC6803: NETWORK RECONSTRUCTION, THERMODYNAMIC CONSTRAINTS AND FLUX-BALANCE ANALYSIS. H. Knoop, Y. Zilliges, W. Lockau and R. Steuer. Institut fuer Biologie, Humboldt Universitaet Berlin, Germany. Introduction: Cyanobacteria, are the only prokaryotes which possess the capability to carry out photosynthesis, similar to higher plants. Therefor it is not surprising, that cyanobacteria attract growing attention in various areas of research and for economic purposes. Among the diverse strains, Synechocystis sp. PCC6803 is a widely used model organism for the analysis of regulatory networks, metabolic pathways as well as photosynthetic processes. With a rich compendium of genomic, biochemical and physiological data available, the cyanobacterium Synechocystis sp. Strain PCC 6803 can serve as an ideal example for the reconstruction of an integrative computational model that describes the relevant metabolic processes in the cell. Methods and Results: We present a draft reconstruction of the primary metabolism of Synechocystis sp. PCC6803. Our reconstruction is based on multiple data sources and extensive manual curation. Integrating information from the genome database (CyanoBase), several pathway databases, as well as information from the primary biochemical literature. Our reconstruction comprises the main pathways of central and intermediate metabolism, including the Calvin cycle, glycolysis, the TCA cycle, the pentose phosphate pathway, oxidative phosphorylation and amino-acid synthesis. Based on the reconstruction, for which also thermodynamic constraints were taken into account, we systematically evaluate and describe the metabolic capabilities of Synechocystis sp. PCC6803 during phototrophic growth. In more detail, we used flux balance analysis to calculate the robustness of the model and to check the sensitivity of specific pathways. Additionally essential genes, correlated reaction sets and potential metabolic bottlenecks were identified, as well as several gaps and inconsistencies in current annotations. In addition, kinetic parameters were added to the model at interesting metabolic branch points, which could play a key role for biotechnological engineering. Conclusion: Owing to the complexity of even comparatively simple metabolic maps, a detailed and systematic evaluation of the properties and metabolic capabilities of organisms necessitates a computational approach. Our reconstruction of the primary metabolism of Synechocystis sp. PCC6803 allows for such a systematic computational evaluation of metabolic routes. The reconstruction significantly improves upon the current genome annotation and provides a resource for targeted manipulation of
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Department of Electrical Engineering and Bioscience, Waseda University, Japan; 2PRESTO, Japan Science and Technology Agency, 22 Wakamatsu, Shinjuku, Tokyo, Japan. Cyanobacteria are the simplest organisms to exhibit circadian rhythms, while studies on the mechanism and physical properties of the clock regulation have been exclusively performed in the unicellular Synechococcus. Since Anabaena is one of the simplest multicellular organisms which harbor both pattern formation with cell differentiation and circadian rhythms, it provides an excellent model system to analyze circadian functions and mechanisms which cannot be addressed in unicellular species. i) Is there any difference in the kaiABC clock gene functions between Synechococcus and Anabaena? Note that KaiA homologs in filamentous species lack two-third of the protein that are well conserved among unicellular species. ii) Is there any difference between the clock systems in vegetative cells and heterocysts? iii) Are clocks in neighboring cells synchronized to each other? Note that in unicellular Synechococcus cell-cell communication has been validated to be negligible in terms of circadian synchronization. iv) Is heterocyst differentiation/patterning modified by the circadian clock? As an initial step to address these questions, we performed DNA microarray analysis to reveal the genome-wide circadian expression profile. Surprisingly, none of Anabaena kai genes show significant expression rhythms, while we found ~600 clockcontrolled genes. In contrast, in Synechococcus the kaiBC operon expression shows the highest amplitude cycle. Moreover, although clock-controlled genes in Synechococcus peaked exclusively at subjective dawn or dusk, that in Anabaena peaked more widely throughout the circadian cycle. Thus, there is striking difference in circadian outputs as well as the clock gene expression profile between the two species. For better understanding the clock function, we have disrupted the kai genes in Anabaena. Transcriptomic and physiological properties in the mutants will be also reported.
P.116 EXAMINING THE ROLE OF THE CBBX PROTEIN IN RHODOBACTER SPHAEROIDES. Amanda K. Luther, Rick A. Laguna, F. R. Tabita. Department of Microbiology, The Ohio State University, Columbus, OH; USA. Introduction: Carbon dioxide is fixed into organic carbon via the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway during photoautotrophic and chemoautotrophic growth of nonsulfur purple photosynthetic bacteria. However during photoheterotrophic growth, the primary function of CO2 fixation is to maintain cellular redox balance. Rhodobacter sphaeroides belongs to the alpha subdivision of proteobacteria. R. sphaeroides possesses two distinct cbb operons, cbbI and cbbII. Each operon encodes enzymes for the August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes CBB cycle, including ribulose 1,5-bisphosphate carboxylaseoxygenase (RubisCO), the enzyme that catalyzes the reduction of CO2. These operons are located on two distinct genetic elements and the regulation of the cbbI and cbbII operon differs upon carbon availability. During photoautotrophic growth, both operons are maximally expressed, although the cbbI operon attains a greater level of expression as compared to the cbbII operon. When conditions are changed to photoheterotrophic growth, down-regulation of each operon occurs. However, under this condition cbbII expression is greater than cbbI expression. In R. sphaeroides the cbbXYZ operon is located immediately downstream of the cbbI operon. With the exception of CbbZ (which functions as a phosphoglycolate phosphatase), the physiological functions of proteins encoded by genes of this operon have yet to be determined in R. sphaeroides. However, a deletion of the cbbX gene resulted in a long lag during photoautotrophic growth conditions compared to wild-type. It was recently shown that Cyanidioschyzon merolae, a unicellular red alga, encodes a CbbX protein that functions as a transcriptional regulator of the cbbLS gene that encodes RubisCO. We are currently examining whether the CbbX protein encoded by R. sphaeroides possesses such function. Methods: To gain a greater understanding of CbbX protein function in R. sphaeroides, the following experiments were performed: We compared the R. sphaeroides CbbX protein in terms of genomic context, primary amino acid sequence, and domain architecture to the CbbX protein of C. merolae. We examined the RubisCO activity and expression of the cbbI and cbbII operons in the cbbX gene deletion strain of R. sphaeroides as compared to the wild-type strain under different growth conditions and carbon sources. RubisCO activity was determined by a standard RubisCO assay, and cbbI and cbbII expression were determined by immunoblot analysis of the RubisCO proteins encoded by each operon. Results: The R sphaeroides cbbX gene deletion strain appeared to increase RubisCO activity as compared to the wild-type strain under photoautotrophic growth conditions. The increased activity appeared to be a result of increased RubisCO protein as determined by immunoblot analysis. Under photoheterotrophic growth conditions with malate as the carbon source, RubisCO activity was the same as the wild-type. However, when acetate was used as the carbon source, there was an apparent loss of regulation of the cbbII operon in the cbbX gene deletion strain, as compared to the wild-type. Conclusion: Based on the results thus far attained, it appears that the CbbX protein in R. sphaeroides may function as a regulator of one or both of the cbb operons. Further experiments are under way to determine the exact role that this protein may play in regulating these operons in R. sphaeroides.
P.117 CHARACTERIZATION OF CAROTENOID CLEAVAGE DIOXYGENASE IN ANOXYGENIC PHOTOTROPHIC BACTERIUM, RHODOPSEUDOMONAS PALUSTRIS. Isamu Maeda, Atsushi Inaba, Kazuyuki Yoshida. Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan. Introduction: Carotenoid oxygenases catalyze formation of apocarotenoids such as retinoids and the precursors of some phytohormones through oxidative cleavage at specific double bonds of carotenoids. Carotenoid cleavage dioxygenases (CCDs) belong to a subgroup of carotenoid oxygenases, and have been found in several higher plants and two cyanobacterial species. Although their substrate
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Final Program and Abstracts specificities and cleavage patterns have been characterized in detail, none of CCDs in anoxygenic phototrophic bacteria have been experimentally characterized. In this study, therefore, Rhodopseudomonas palustris CCD (RpsCCD) was chosen to analyze the activity of anoxygenic phototrophic bacterial enzyme. The in vivo and in vitro cleavages of carotenoids by RpsCCD were analyzed using lycopene, produced in a recombinant E. coli strain, and b-apo-8’carotenal as the substrate, respectively. Methods: Pantoea ananatis crtE, crtB, and Pantoea agglomerans crtI were introduced to E. coli JM109, resulting in lycopene-producing E. coli (pHYcrtEIB). Rps. palustris ccd was inserted in pMG103 to construct pMG-Rpsccd. Apocarotenoid production was performed with cultivation of E. coli (pHYcrtEIB, pMG-Rpsccd) and E. coli (pHYcrtEIB, pMG103) for 24 hours. RpsCCD produced in E. coli BL21 (DE3, pMG-Rpsccd) was used in the in vitro analysis. The crude protein extract was incubated with all-trans-b-apo-8’-carotenal (96% purity, Fluka) in the presence of 0.1% (v/v) Triton X-100. Apocarotenoids were directly extracted with hexane/diethyl ether (1:4, v/v). The organic phase was analyzed with GC-MS. Results: A specific peak was detected in the chromatogram of E. coli (pHYcrtEIB, pMG-Rpsccd) extract. The mass spectrum showed the parent-ion mass of 154, and corresponded to that of citrol, the reduced form of citral. The data suggests that citral is produced through the 7,8 (7’,8’) double bond cleavage of lycopene by RpsCCD and then reduced to citrol in E. coli cells. The in vitro analysis using b-apo-8’carotenal showed two specific peaks with the parent-ion masses of 284 and 164. The mass spectra corresponded to those of retinal (m/z 284) and apo-8’,15’-apocarotene-dial (m/z 164). The data demonstrates the 15,15’ double bond cleavage of bapo-8’-carotenal by RpsCCD. Conclusions: Dioxygenase activity was confirmed using recombinant RpsCCD protein. This is the first report that has identified cleavage patterns of an anoxygenic phototrophic bacterial enzyme.
P.118 ANABAENA SP. STRAIN PCC 7120 GENE ALL0187 IS DEVELOPMENTALLY REGULATED AND ESSENTIAL FOR DIAZOTROPHIC GROWTH AND HETEROCYST MORPHOGENESIS. Rodrigo A. Mella-Herrera*, M. Ramona Neunuebel, James W. Golden* Department of Biology, Texas A&M University, College Station, TX; *Current address: Division of Biological Sciences, University of California, San Diego, CA, USA. Introduction: Under diazotrophic growth conditions, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 undergoes development to form a pattern of about one heterocyst every ten to twenty vegetative cells along filaments. The heterocyst differentiation process involves genetic, physiological, and morphological changes to produce the micro-oxic environment necessary for nitrogen fixation. These changes include remodeling of the cell wall and deposition of two heterocyst-specific extracellular envelope layers. The all0187 gene is predicted to encode a protein containing a LytR domain, which is known to be associated with regulation of cell wall maintenance. We investigated all0187 to determine if it is involved in heterocyst differentiation or in diazotrophic growth.
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Methods: Anabaena (Nostoc) sp. strain PCC 7120 and its derivatives were grown in BG-11 or BG-110 (lacking sodium nitrate) medium at 30°C with white-light illumination of approximately 75 µM photons m−2 s−1. Standard protocols were used for cloning, E. coli transformation, PCR, and northern RNA blot analysis. Microarrays experiments were performed using Anabaena PCC 7120 RNA samples hybridized to a custom microarray slide containing 768 probes. Light and fluorescence microscopy were performed on a Zeiss Axioplan II microscope with a 40´ objective and green fluorescent protein (GFP)specific emission (518 ± 13 nm) filter sets. Nitrogenase activity was measured with an acetylene reduction assay. Results: Insertional inactivation of all0187 caused a partial cell division defect of vegetative cells grown in nitrate-containing medium. In nitrogen-free medium, mutant filaments formed abnormally long heterocysts and were unable to grow diazotrophically, but vegetative cell septation appeared normal. Septum formation between heterocysts and their flanking vegetative cells was incomplete, leaving one or both poles of the heterocysts partially opened. We investigated whether these morphological defects led to inactivation of nitrogenase due to lessened protection against oxygen. Acetylene reduction assays for nitrogenase activity showed that the mutant strain retained approximately seventy percent of the wild-type activity; this observation shows that heterocysts of the all0187 mutant strain are partially functional. Northern RNA blot analysis showed that all0187 expression was upregulated by 8 h after nitrogen step-down and fluorescence microscopy of a Pall0187-gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts by 9 h after nitrogen step-down, whereas vegetative cells maintained a lower level of fluorescence. Conclusions: We hypothesize that the diazotrophic growth defect of the all0187 mutant is caused by the inability of the heterocysts to transport fixed nitrogen to the neighboring vegetative cells.
P.119 FUNCTIONAL ANALYSIS OF THE ISCR-LIKE TRANSCRIPTION FACTOR, INVOLVED IN EXPRESSION OF THE PSAAB TRANSCRIPT IN SYNECHOCYSTIS SP. PCC 6803. T. Midorikawa1, K. Matsumoto1, R. Narikawa2, M. Ikeuchi1,2. 1
Department of Biological Science, Graduate School of Sciences; Graduate School of Arts and Sciences; University of Tokyo, Japan.
2
Introduction: Since the light environment varies depending on location, time, and weather, it is important for the oxygenic phototrophs to acclimate to these environmental changes. It has been established that the regulated accumulation of photosystem I (PSI) is critical for long term acclimation to the light conditions in cyanobacteria. To this end, expression of psaA and psaB, which encode the PSI reaction center subunits, is tightly regulated in the acclimation processes. In the course of our screening of transcriptional regulators in Synechocystis, we found that an iscR-like regulator Slr0846 is critical for maintenance of normal chlorophyll accumulation. Here we studied the target genes of Slr0846 by DNA microarray analysis, gel shift assay and primer extension analysis. We also studied PSI/PSII ratio of the slr0846 disruptant under various light conditions.
with CyanoCHIP version 1.6. The N-terminal poly histidine-tagged Slr0846 prepared from E. coli was used for gel shift assay.
Result: Full segregation of the ∆slr0846 mutant was achieved. It is suggested that Slr0846 is not essential for this strain. DNA microarray analysis revealed that the expression level of psaAB was downshifted in the ∆slr0846 mutant, which was grown under the standard light conditions. Direct binding of Slr0846 protein to a promoter of psaAB was studied by gel shift assay. Further dissection revealed that Slr0846 bound to a far upstream region in the promoter. Primer extension analysis showed that transcription from the two known start sites was almost equally affected in the mutant. These results suggest that Slr0846 is a transcriptional activator for psaAB. The ∆slr0846 mutant exhibited much lower chlorophyll contents and PSI/PSII ratio than wild type. The mutant also showed the growth sensitive to high light. Notably, the difference in the PSI/PSII ratio between wild type and the mutant was attenuated under the PSI light conditions. These findings suggest that the activity of Slr0846 may respond to various light conditions. Conclusion: In this study, we demonstrated that the Slr0846 binds to a far upstream region in a promoter of psaAB and acts as a transcriptional activator. It may contribute to the optimal regulation of the psaAB expression under various light conditions.
P.120 ALR2269 – THE OMP85 IN ANABAENA SP. PCC 7120. Kerstin Nicolaisen, Constance Vollmer, Iwo Tews, Enrique Flores, Enrico Schleiff. Goethe University Frankfurt, Frankfurt, Germany. The outer membrane of gram-negative bacteria protects the organism against environmental influences and allows interactions with its surrounding. Proteins of the Omp85 class embedded in the outer membrane function as “chaperones” for the insertion of the outer membrane proteome required for the function of this barrier. Omp85 are polypeptide transporting β-barrel proteins (1), which are also found to transport polypeptides across the outer envelope of chloroplasts (1). It is thereby an essential class of proteins in endosymbiotically derived organelles and bacteria. In Anabaena sp. PCC 7120, the closest relative to the ancestor of chloroplasts (2), Alr2269 functions as Omp85-like protein (3). Therefore we studied its role as transporter (3), analyzed its properties in protein recognition (5) and are investigating the possibilities to complement other proteins of the Omp85 class with Alr2269 and vice versa (6). References: 1. Schleiff, E., Soll, J. (2005) EMBO Rep. 6, 1023-1027 2. Bredemeier, R. et al. (2007) J.Biol.Chem. 282,1882-1890 3. Ertel, F. et al. (2005) J.Biol.Chem. 31, 28281-28289 4. Nicolaisen, K. et al. (2009) Mol. Microbiol. submitted 5. Wunder, T. et al. (2007) BMC Evol. Biol. 7: 236 6. Wunder, T. et al. (2009) Endocyt. Cell Res.
Methods: The original motile strain of Synechocystis sp. PCC 6803 showing positive phototaxis was used as wild type. The slr0846 gene was disrupted by insertion of the chloramphenicol resistance gene. The low temperature chlorophyll fluorescence at 77 K was monitored for estimation of the PSI/PSII ratio. DNA microarray analysis was done
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P.121 ANALYSIS OF BIOSYNTHETIC PATHWAY OF CHL A ESTRIFIED WITH ∆2,6-PHYTADIENOL IN CHLOROBIUM TEPIDUM BY CONSTRUCTING DISRUPTION MUTANTS OF CT1232 AND CT2256 GENES. JiroHarada1,SyoheiMiyago2,TadashiMizoguchi1,HitoshiTamiaki1, HirozoOh-oka2. 1
Final Program and Abstracts [2]T.Mizoguchi,J.HaradaandH.Tamiaki,FEBS Lett.,580:6644-6648 (2006). [3]J.Harada,S.Miyago,T.Mizoguchi,C.Azai,K.Inoue,H.Tamiaki andH.Oh-oka,Photochem. Photobiol. Sci. 7:1179-1187(2008). [4]T.Mizoguchi,M.Isaji,J.Harada,K.WatabeandH.Tamiaki,J. Porphyr. Phthal. (in press).
P.122
DepartmentofBioscienceandBiotechnology,FacultyofScienceand Engineering, RitsumeikanUniversity;2DepartmentofBiological Sciences,GraduateSchoolofScience,OsakaUniversity;Japan.
ACID STRESS RESPONSIVE GENES, SLR0967 AND SLL0939, ARE DIRECTLY INVOLVED IN LOW-PH TOLERANCE OF CYANOBACTERIUM SYNECHOCYSTIS SP. PCC 6803.
Thegreensulfurbacteria contain threekindsof(bacterio)chlorophyll [(B)Chl]pigments,BChl aP,ChlaPD andBChlcF.BChlscF arelocatedin largelight-harvestingapparatuses,chlorosomes,wherelightenergyis capturedandmigratedtoFMOproteinviabaseplateandfinally convergedonP840,aspecialdimerofBChlaP,inthereactioncenter. BChlsaP alsoserveasantennapigmentsassociatedwithCsmAprotein inbaseplate,FMOproteinandthereactioncentercomplex.Within thereactioncentercomplex,ChlsaPD aresupposedtofunctionasa primaryelectronacceptoraswellasaccessorypigments.ChlaPD has thesamechlorinπ-system asChlaP inhigherplantsandcyanobacteria exceptforalonghydrocarbonchainattheC-17position; theformer isesterifiedwith∆2,6-phytadienolandthelatteriswithphytol[1].A precursorofphytolisgeranylgeraniol,whichisstepwiselyreducedby geranylgeranylreductase(GGR)[2].However,thereactionprocessto produce∆2,6-phytadienylesterisstillunknown.Inthegenomeof Chlorobium tepidum,twoparalogousgenes,CT1232 andCT2256, exhibitsequencesimilaritytoGGRgenesofcyanobacteriaand/or purplebacteria,andarepresumedtobeinvolvedinhydrogenationof geranylgeranylgroup.
HisatakaOhta1,2,AtsushiMoriyama1,YukoKubo1,YousukeShibata1, YouheiHaseyama1,YukaYoshino1,TakehiroSuzuki1,MasahikoIkeuchi3, IsaoEnami1,ShuseiSato4,YasukazuNakamura4,SatoshiTabata4.
Inordertoclarifythebiosyntheticprocess of∆2,6-phytadienylester, weconstructeddisruptionmutantsof thesegenesusing an insertional inactivationmethodandanalyzedtheirpigmentcompositions and photosyntheticcompetences [3,4].TheCT2256 mutantaccumulated ChlaGG andBChlaGG esterifiedwithgeranylgeraniol,indicatingthat CT2256was involvedintheproduction ofboth ∆2,6-phytadienyland phytylgroups.Themutantstillgrewphotosyntheticallyalthoughits growthratedrasticallydecreasedtolessthanahalfcomparedtothat ofthewildtype.Thiscouldbeexplainedbysomehindranceofthe energytransferfromchlorosomestothereactioncentercomplex, becausetherelativelyhighfluorescenceemissionfromchlorosomesin themutantwasobserved.However,theCT1232 mutantshowedno apparentphenotype comparedtothewild type.The purple bacteriumRhodobacter capsulatus mutantdefectiveinthebchP gene waspartiallycomplementedwiththeCT2256 gene byaconventional conjugationmethod; BChlaP wassynthesizedinthemutantin additiontoaccumulatingotherintermediates. Furthermore, cyanobacterialandpurplebacterialGGRgenes,whicharechlP and bchP genes,respectively,wereincorporatedintotheCT2256 mutant genomebyreplacingthedisruptedsiteoftherelevantgene. Thetwo strains,thusobtained,partiallyrestoredareductionactivityto producephytolandotherintermediatesfromgeranylgeraniol.Allof theabovedatasuggestthattheCT2256mightdiscriminatethe differenceofringstructuresbetweenchlorin andbacteriochlorin and produceBChlaP andChlaPD fromBChlaGG andChlaGG,respectively. References [1]M.Kobayashi,H.Oh-oka,S.Akutus,M.Akiyama,K.Tominaga,H. Kise,F.Nishida,T.Watanabe,J.Amesz,M.Koizumi,N.IshidaandH. Kano,Photosynth. Res.,63:269-280(2000).
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1 Department ofBiology,Faculty ofScience,TokyoUniversity of Science, Shinjuku; 2TissueEngineeringResearchCenter,Tokyo UniversityofScience,Noda;3Department ofLifeScience,Universityof Tokyo,Meguro,Tokyo;4KazusaDNAResearchInstitute,Kisarazu, Chiba;Japan.
Introduction: DatingfromthePrecambrianera,cyanobacteriahavea longhistoryofadaptingtotheEarth’senvironment.Byevolving oxygenviaphotosyntheticreactionssimilartothoseofplantsand greenalgae,theseprokaryoteswereessentialtotheevolutionofthe presentbiosphere. Theycontinuetomakealargecontributiontothe equilibriumoftheEarth’satmospherebyproducingoxygenand removingcarbondioxide. Tosurviveinextremeorvariable environments,cyanobacteriahavedevelopedspecificregulatory systems,inadditiontomoregeneralmechanismsequivalenttothose ofotherprokaryotesorphotosynthesisbacteria (1). Severalspeciesof cyanobacteriaserveasmodelorganismsforelucidatingboth functionalandregulatoryaspectsofphotosynthesis (2). Aboveall, Synechocystis sp.PCC6803wasthefirstphotosyntheticorganismfor whichacompletegenomesequencebecameavailable(3), andDNA microarrayshavebeenusedtoexaminegeneexpressioninresponse tovariouskindsofstresssuchasosmotic,salinity, andhighlightstress (4-7). Acidrainis oneofthemostseriousofenvironmentalstresses.It causesacidificationoflakesandstreamsandcontributestodamage ofplants, algae, andcyanobacteria inmanypartsoftheworld. Rhizotoxicityinacidsoil,whichinvolvestheactionofAl3+ hasbeen wellinvestigated(8). Nevertheless, littlehasbeendonetoelucidate thebasicsetofadaptationsnecessaryforacidtoleranceinplants, algae, orcyanobacteria. DNAmicroarrayanalysisofSynechocystis sp.PCC6803 cells revealed that acidstressinducedtheexpressionof putativestress-related proteins,suchaschaperones (slr0093[dnaJ],sll1514[hspA],and sll0170[dnaK)]), regulatoryfactors(sll0306[sigB] andsll2012 [sigD)]), andproteinsof unknownfunction(9).Amongtheup-regulatedgenes withunknownfunction,slr0967 andsll0939 continuouslyincrease7 and16-foldafter4hofacidstressandareupregulated byosmotic andsaltstresses(6).Interestingly,thesetwogenesarelocated adjacentlyontheSynechocystis sp. PCC6803 genome(Fig.1). Inthisstudy,weexamined thephysiologicalfunctionofcyanobacteria genes using mutant cellsinwhicheachgenewasdisruptedbya kanamycin-resistance cartridgegene. Basedonphenotypes ofthe mutantsandreal-time quantitativeRTPCR analysis ofthetranscripts of thetwogenes, theexpressionprofileoftheslr0967 deletionmutant onacidstresswascomparedwiththatofwild-typecells.
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Final Program and Abstracts Methods: Culture conditions- Synechocystis sp.PCC6803was cultivatedin BG-11medium(10) bufferedwith10mMTESNaOH,pH 8.0, orin10mMMESNaOHpH6.0(long-termacidstresscondition), at30°C. Cultureswerebubbledwith3%CO2-containingair and illuminatedwith30µmolphotonsm−2 s−1 fromincandescent bulbs. CellgrowthwasmeasuredusingaPharmaciaspectrophotometerat 730nm (AmershamPharmaciaBiotech,Piscataway,NJ,USA). Generation of insertion mutants- Mutantsimpaired inselectedgenes weregeneratedbyreverse genetics.Thecodingsequencesand neighboring sequenceswereamplifiedbyPCR.Approximately 2kbof PCRproductswereclonedintopUC19 (Toyobo,Osaka,Japan).The primersforamplificationwere designedusingthecompletegenome sequenceof Synechocystis (3).Sequencesthat containedappropriate restrictionsiteswereselected toimprovecloningof fragments. The kanamycin(Km) resistance gene(kmr)isolatedfrom plasmidpUC4K (AmershamPharmacia)wasinsertedinto uniquerestrictionsitesofthe encodingsequences. Transformantswereinitiallyselectedona medium containing10μgKmmL−1 (WakoPureChemical,Osaka, Japan),whereasthe segregationofcloneswasperformedby restreaking(atleastthreetransfers)ofprimary clonesonplates supplementedwith50μgKm mL−1. Duringthecultivationofmutants, Kmwasaddedtotheliquidmedia. Generation of overexpressing mutants-Overexpressingmutantstrcslr0967 andtrc-sll0939 ofSynechocystis 6803 weregeneratedusing pTrc-slr0967andpTrc-sll0939 plasmids.Theplasmids wereintroduced intothe cmr gene from pLysS (Novagen,Gibbstown,NJ,USA) and the trcpromoterwasintroducedfrompTrcHisB (Invitrogen,Carlsbad, CA,USA) intotheupstreamoftherespectivegenesasdescribedby Kameietal.(11). Short-term acid stress conditions-Exponentially-growing cellswere acid-stressedbycentrifugingthecell culturesandresuspendingthe cellpelletsinapH adjusted BG-11mediumusing10mMglycine(pH 3.0)buffers insteadofTESbuffer for8h. Serialdilutionsofthecells werespotted ontonormalBG-11 plates aftertreatment andcultured for7 days at30°C.Experimentswere performedinduplicateatleast threetimes. RNA isolation and quantitative real-time RTPCR-TotalRNAwas isolatedusingtheRNeasy Minikit(Qiagen,Hilden,Germany)as described byHiharaetal. (7).Forthereversetranscriptase (RT) reaction,100ngRNAwasincubatedwitha mixtureofPCRreverse primersfor10minat70°C priortoadding100USuperscriptIIRT (Gibco-BRL,Carlsbad,CA,USA). TheRTreactionwasperformedat 42°Cfor1hand terminatedbyincubatingthecellsat72°Cfor10 min. APerfectRealTimekit(TakaraBio,Otsu,Shiga,Japan)wasused according tothemanufacturer’sinstructions. DNA microarray analysis- Cellswereharvestedfrom10mLofculture bycentrifugationat4,000×g for5minat25°Candthenbroken immediatelywithaMini-BeadBeater(Biospec,Bartlesville,OK,USA). TotalRNAwasisolatedusingtheRNeasyMinikit(Qiagen)as describedby Hiharaetal. (7). ASynechocystis DNAmicroarray (CyanoCHIP)wasobtainedfromTakara(Kyoto,Japan).Thismicroarray covered3,079ofthe3,168openreadingframes(ORFs) of Synechocystis,excludingORFstransposases.Conditionsforthe synthesisofCy3-labeledandCy5-labeledcDNAs,hybridization,and washingwereaspreviouslydescribed(Hiharaetal.2001). Image acquisitionwithaScanArray4000(GSILumonics, Watertown,MA, USA)wasperformedwiththeautobalance-autorange feature. With thisfeature,thesensitivityoftheinstrument canbeautomatically adjustedbychangingthelaserpowerand photomultipliergain settingssothatthesignaliswithin90% ofmaximumtoprevent saturation.Therawdataobtainedwith theScanArray4000were 138
13th International Symposium on Phototrophic Prokaryotes analyzedwithQuantArrayversion2.0 software(GSILumonics,Tokyo, Japan). Thefluorescenceintensityofeach spotforbothCy3andCy5 imageswasquantified,andlocalbackground fluorescencelevelswere subtracted. Cy3andCy5imageswere normalizedbyadjustingthe totalsignalintensitiesofthe twoimages. Theresultsshownare averagesof4–6 biologicallyindependentexperiments. Results: Characterization of the deletion mutants of slr0967 and sll0939 Wild-typeSynechocystis sp.PCC6803wastransformed with slr0967 andsll0939 that hadbeeninterruptedwithcassetteconferring resistance tokanamycin. Toexaminesegregation ofthesegenes withintheSynechocystis genome, we performedaPCRanalysisusing DNAofwild-typeand the∆slr0967 and∆sll0939 mutantcells.PCR with genomic DNAofwild-typecells asatemplateamplified respective 0.4 kbDNAfragmentsforslr0967 andsll0939,whereas PCRwithDNAfrom∆slr0967 and∆sll0939 mutantcellsyieldeda fragmentof 1.6 kb(results notshown). Theseresultsindicatedthat slr0967 andsll0939 genesin∆slr0967 and∆sll0939 mutantcellshad beendisruptedbytheinsertion ofthekmr gene. InnormalBG-11mediumatpH8.0,allstrainsexhibitedasimilar photoautotrophicdoublingtime(Fig.2A),suggesting thatdeletionof thesegenesdidnotaffect theirgrowthinnormalconditions. In contrast, intheacidstressed conditionatpH6.0,thegrowthofall mutant cells was slightlybutsignificantlyinhibited comparedwiththat ofwild-typecells(Fig.2Band2C). Thesll0939 andthedoubleslr0967 andsll0939 mutants were moresensitive toacidstressthanthe ∆slr0967 cells(Fig.2B) indicatingthatslr0967 andsll0939 areinvolved inacidtoleranceofSynechocystis cells. Overexpression of slr0967 and sll0939 Overexpressionofthesegenes failedtoenhancetheacidtoleranceafter culturingatpH6.0for7 days(Fig. 3A).Wethencharacterized theexpressionofthesetwo genesatthelevelofthetranscriptbyquantitativeRT-PCR usingtotal RNAisolatedfromacidtreatedwild-typecellsandtrc-slr0967andtrcsll0939 mutantcells. Therelativeexpressionof slr0967 and sll0939 promotedbytrc waslowerthanthatofwild-typecellsduringa 4h acidstressbuthigherthanthatfromnon-treatedwild-typecells(Fig. 3CandD).Accordingly,survivability ofthemutantsbyacidstress treatmentatpH3.0for1–12hwastested (datanotshown).Asa result,wefounda significantdifferencein survivalbetweenwild-type andtrc-mutantsthat weretreatedfor 8h(Fig. 3B).Thetrc-mutants became moretoleranttoacidstressthanwild-typecells. Quantitative RT-PCR analysis of slr0967 and sll0939 in mutual deletion mutants. Thesetwogenes arelocatedadjacent tothe operonandare codedbyopposite strands ontheSynechocystis genome.Toexamine theregulatoryrelationshipbetweenthesetwogenes whosedeletion mutants weresensitivetoacidstresscomparedwiththewild-type,the abundanceofmutual genes inthedeletionmutants was measured in normalgrowthandacidstress conditions by culturing thematpH 3.0 for30min. Asshown inFig. 3,therewasincreasedexpressionof slr0967 andsll0939 inwild-typecellsbyacidstressafter30 min; however,therewas nosignificant alterationinslr0967 expression evidentinthe sll0939 mutant.Therewasaslightup-regulationof sll0939 in the normalgrowthcondition,but deletionofsll0939 didnot affectslr0967 expressioninthe acidstressed condition.Incontrast, sll0939 expressionwasconsiderably affectedbydeletionofslr0967 in bothnormalandacidstressed conditions.Therelativelevelsofthe sll0939 transcriptwereonly3%and4%ofwild-typecells, respectively. Theseresultsindicatethatslr0967 regulatessll0939 expressionin Synechocystis inbothnormalandacidstressed conditions.
DNA micro array analysis of ∆slr0967 mutant. Weexaminedthe genome-wideexpressionofgenesin∆slr0967 mutantcellsusingDNA microarraystodeterminewhether slr0967 isinvolved intheexpression August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes ofotheracidstressresponsivegenes. Table1showsgenes thatwere 3-foldup-regulatedbyslr0967 deletion comparedto wild-typecells. Thesegenescanbeclassifiedinto twogroups.1) Genesthatwerenot repressed inmutantcellsbutwere3-foldormorerepressed inwildtypecellsunder acidstress,and2)Genesthatwereup-regulated morethan3-foldinmutantcellsbutnotinwild-typecells. Thefirst groupcontainsgenesforproteins involvedinCO2 uptake, CO2 fixation, andbicarbonatetransport(sll1732 [ndhF3],sll1733 [ndhD3], sll1734 [cupA],slr0040 [cmpA], slr0041 [cmpB], slr0042, slr0043, slr0044, andslr1512 [sbtA]) (12-16). Aspreviouslydescribed(9),the bicarbonatetransportsystemmighthavebecomeredundantforwildtypecellsunderacidstressbecausemostofthebicarbonateexistsin theformofCO2 thatdiffusesthroughthecellmembranesfreely. It maybethatthesegeneswerenotup-regulatedinmutantcellsfor survivalbuttherepressionmachineryofthesegeneswasvirtually haltedduetolackofslr0967. Thesecondgroupcontainsgenes encodingstressregulatedproteins(ssl0452 [nblA],sll0247 [isiA], slr0373,slr0374, slr0376, and slr0228 [ftsH]).Degradationofthe cyanobacterialphycobilisomelight-harvestingantennaisageneral acclimationresponsethatisobservedundervariousstressconditions suchasN-,S-,andP-starvation(17-20). IntheSynechocystis strain PCC6803genome,twotandemcopiesofnblA arepresent.The expressionofssl0453 (nblA2), whichislocateddownstreamofssl0452 (nblA1),wasnotaffectedbyslr0967 deletion(datanotshown). The isiA geneencodesaproteinthatissimilartothephotosystemII chlorophyll-bindingproteinCP43andisinducedunderiron-stressed conditions(21,22).Theslr0373,slr0374, and slr0376 genesconstitute an operon thatisresponsivetovariousenvironmentalstressesbut theirfunctionsareunknown(23,24).AnFtsH proteaseplaysan essentialroleintheturnoverofthereactioncenterD1proteinin Synechocystis PCC6803underheatstress aswellaslightstress conditions(25,26).SLR0967 deletionincreasesdeathduetoacid stress,resultinginincreaseinthenumberofstressresponsivegenes beingup-regulatedinmutantcells.Genes whoseexpressionwere upregulatedinwild-typecellbyacidstresswerealmostalldown regulated bydeletionofslr0967 (datanotshown). Table2showsgenesthatwere3-foldormorerepressedbyslr0967 deletionthanthoseinwild-typecells. Thesegeneswerealsodivided intotwogroups:1) Geneswhoserepressionwasobserved inmutant cellsbutnotinwild-typecellsbyacidstress,and2)Geneswhoseupregulationdecreasedinmutantcellsbut were 3-foldormore up-regulatedinwild-typecells.Genesencodingthephycobilisome proteins(sll1577 [cpcB], sll1578 [cpcA], sll1579 [cpcC2], sll1580 [cpcC1], slr0335 [apcE], slr1986 [apcB], slr2067 [apcA], slr2051 [cpcG1]),photosystemIsubunits(slr1834 [psaA], ssl0563 [psaC], slr0737 [psaD], sll0819 [psaF], and slr1655 [psaL]) andchaperone proteins(slr2075 [GroES] andslr2076 [GroEL1]) thatwererepressedby deletionofslr0967.Allakhverdievetal.(27,28).demonstratedthat saltstressandhyperosmoticstresshaveinhibitoryeffectsonthe electron-transferactivityofphotosystemI.Repressedexpressionof genesforphotosystemIandphycobilisomesmightbeimportantfor maintenanceofacertainlevelofphotosyntheticactivity.Moreover,it seemspossiblethat decreasedabundanceofFe-requiring photosystemIcomponentsisaccompaniedbyirondeficiency,which maybecausedbyacidstress.Itisunknownwhychaperoneexpression wasrepressesedasthesegeneswereonlymarginally up-regulatedby acidstressinwild-typecells. High-lightinducibleproteinssr2595 (HilB) (29)andthetwocomponentresponseregulatorslr1214 (rre15) showed remarkablydecreasedexpressionfollowing slr0967deletion. Conclusions: Inthisstudy,wefoundthatacidresponsiblegenes slr0967 andsll0939 aredirectlyinvolvedinacidtoleranceofthe cyanobacteriaSynechocystis sp.PCC 6803, andslr0967 mayregulate August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts theexpressionofgenesthatrespondtoenvironmentalstresses. The physiologicalfunctionsofthesegeneshavestillnotbeenidentified. Sequenceanalysisrevealedthatsll0939hastwotransmembrane segmentsandslr0967hastowpentapeptidesequences sequences. Interestingly,preliminarydatafromatwohybridyeastanalysis revealedthatsll0939interactswithslr1596(pxcA), whichisaproteinin thecytoplasmicmembraneinvolvedinlight-inducedprotonextrusion (datanotshown).Thedeletionmutantofslr1596 isunabletogrowat acidicpH(30). Thesedatasuggestthatsll0939 mayalsofunctionwith slr1596forlight-inducedprotonextrusion.However,deletionof sll0939 andslr0967 isnotaffectedbylight-inducedprotonextrusion (datanotshown). Up-regulationofslr1214 (rre15) considerably decreased by deletionofslr0967. Itwasthought thatslr1214 affected acidstresstolerance,butdeletionofthegenedid notaffectgrowthat pH6.0(31).Therefore,wearenowintensivelystudyingthe relationshipoftheseproteins. Tofurtherunderstandthefunctionof slr0967andsll0939,wehaveinvestigated theproteinexpressionof slr0967 andsll0939 inE. coli cellsusingpETvectors,buttheirfunction isstillunknown.IPTGinductionofthesegenesinE. coli causecell lysesandthecellsareunabletorecovertheseproteins.Weare interestedintheregulatorymechanismsofacid-responsiblegenes; therefore,analysisofthepromoterregionofslr0967 andsll0939 shouldbeinvestigatedintensively.
P.123 CELL-SPECIFIC TRANSCRIPTOME ANALYSIS IN ANABAENA VARIABILIS ATCC 29413 GROWN PHOTOTROPHICALLY, PHOTOHETEROTROPHICALLY, AND HETEROTROPHICALLY. Jeong-JinPark1,SigalLechno-Yossef2,HajimeMasukawa1, C.PeterWolk2,3,ClaireVieille1. 1
DepartmentofMicrobiologyandMolecularGenetics;2MSU-DOE PlantResearchLaboratory;3DepartmentofPlantBiology,Michigan StateUniversity,EastLansing,MI,USA. Introduction: Anabaena variabilis ATCC29413isapotential candidatefortheproductionofhydrogen(H2)asacleanrenewable energycommodity.A. variabilis filamentsarecomposedofvegetative cellsthatperformoxygenicphotosynthesis,andofheterocyststhatfix nitrogen(N2)inanoxicenvironment.A. variabilis producesH2 asabyproductofN2 fixation.Tounderstandhowelectronsarechanneledto H2 productioninA. variabilis underdifferentgrowthconditions,we arecomparinggeneexpressioninphototrophic(inthepresenceof light),photoheterotrophic(inthepresenceoflightplusfructose),and heterotrophic(withfructoseinthedark)culturesusingRNAextracted separatelyfromvegetativecells,heterocysts,andwholefilaments. ThesestudiesshouldhelpusidentifygeneswhoserolesinH2 productionareaffectedbycarbonandenergysources,andregulatory mechanismsthatinter-relatethosegenes. Methods: A.variabilis ATCC29413wasgrowninAA/8mediumat 30°CinFernbachflasksunderwhitefluorescentlightorinthedark. Heterotrophicculturesweresupplementedwith5mMfructose. Heterocystsandvegetativecellswereisolatedasdescribed(Peterson andWolk,PNAS,1978,75:6271),withvariations.RNAwasextracted fromeachtypeofcellusingAmbionRiboPure”!-Bacteriakits.RT-qPCR reactionswereperformedwithprimersspecifictotheA. variabilis 16s rRNA,rnpB,rbcL, ftsZ, nifK,andfdxN genes,using16srRNAorrnpB asinternalcontrolsfordatanormalization.cDNAwaspreparedby usingSuperscriptIIreversetranscriptaseandrandomprimers.RTqPCRwasperformedusingSYBRgreen(DuandArvidson.Infect. Immun. 2006,74:2767).Microarrayexperimentsarebeingperformed withRocheNimbleGen60-merarrays.Nitrogenaseactivity,withC2H2
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Final Program and Abstracts assubstrate,and H2 productionwerequantitatedbyacombinationof gaschromatographywith,respectively,aflameionizationanda thermalconductivitydetector,. Results:Comparedtophototrophicgrowth,photoheterotrophic growthonfructoseincreasedA. variabilis‘specificH2 productionrate 30-fold,andnitrogenaseactivitytwo-fold.Culturesformicroarray studieswereharvestedfourdaysafterinoculation.Cellpurification andcell-specificRNAextractionwereconfirmedbymicroscopic examinationandRT-qPCRwithcell-specificgenes,respectively.We areusingcDNAmicroarrayexperimentstostudycell-specificgene regulationinresponsetochangesingrowthconditions,from phototrophictophotoheterotrophicandtofullyheterotrophic.To groupgenesaccuratelythatshowsimilarexpressionpatternsineach growthconditionorineachcelltype,expressiondatawillbeclustered usingaself-organizingtreealgorithmandvisualizedasheatmaps.We expecttobeabletocategorizetypesofresponsesasconnected directlyorindirectlytoH2 production. Conclusions: Wewillreportmicroarraystudiesofcelltype-specific geneexpressioninafilamentouscyanobacterium.Weexpectthatour resultswillanswerpreviouslyunansweredquestionsoncell-specific generegulationandH2 metabolism.
physiologicalstress. Duringtheadaptationphasetonitrogenlimitationwhenformationof additionalheterocystsisinducedforassimilationofatmospheric nitrogen,theintracellularCYNcontentofAphanizomenon strains increasesuptotwicetheamountfoundincellsadaptedtonitrogen saturation.Incontrastinbatchcultureswithlimitingfactorsotherthan nitrogen,intracellularCYNdecreasescontinuouslyandCYNreleaseis substantiallyhigherthaninnitrogenlimitedcultures. Conclusions: ThevariationofthetotalCYNconcentrationinstrains fromtemperatelakesrespondstoenvironmentalvariablesinarange similartotherangedescribedpreviouslyforthewellinvestigated cyanobacterialpeptidetoxins(microcystins). WeconfirmedthatCYNreleasecouldnotonlyberelatedtocelllysis andproposeafurtheractiveprocesswhichseemstoberegulatedby variousenvironmentalfactors.Incipientnitrogenlimitationreduced CYNreleasefromcells,whileincontrastotherphysiologicalstress conditions,particularlyphotoinhibitionenhancedrelease. Riskassessmentthusneedstotakeintoaccountthatlargeamountsof dissolvedCYNmayoccurinwaterbodiesevenwhilecyanobacterial bloomsareintact.
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P.125
FACTORS INFLUENCING PRODUCTION AND RELEASE OF THE CYANOBACTERIAL TOXIN CYLINDROSPERMOPSIN.
VITAMIN-E DEFICIENCY RESULTS IN OVER-ACCUMULATION OF GLYCOGEN IN THE CYANOBACTERIUM SYNCHOCYSTIS SP. PCC6803.
KarinaPreußel,JuttaFastner,IngridChorus. Umweltbundesamt(FederalEnvironmentalAgency),Berlin,Germany. Introduction: Thecyanobacterialsecondarymetabolite cylindrospermopsin(CYN)isaninhibitorofeukaryoticprotein synthesis.Unlike thewellexaminedcyanobacterialpeptidetoxins (microcystins),CYNoftenoccurswithahighproportionofextracellular dissolvedtoxininwaterbodiesaffected.ThedissolvedCYNfraction hasimportantimplicationsbothforaquaticecosystems andforhuman health:asCYNinhibitsanubiquitousprocessingrowthand maintenancemetabolismoforganisms,thedissolvedtoxinmay impactawiderangeofaquaticorganisms(includingplants) independentlyoftheirtrophicrelationshipstocyanobacteria. ItissofarunknownifthehighamountsofdissolvedCYNinpersisting cyanobacterialbloomsandinstationaryphasesofbatchculture experimentsarisefrompassiveleakageandsimultaneousenrichment duetoitspoorbiodegradability,orifspecificenvironmentalfactors mayenhanceanactiveCYNreleasefromcells. Ourinvestigationsaimatimprovingtheunderstandingofthefactors whichinfluenceCYNproductionandreleaseespeciallytowards humanhealthriskassessment. Methods: Semicontinousaswellasbatchcultureexperimentswith differentCYNproducingcyanobacterialstrainswereperformedto measureCYNproductionandreleaseundervariouscultivation conditions. Ninedifferentcombinedlight-temperatureconditionsaswellasthe influenceofnitrogenandphosphoruslimitation weretested. IntracellularandextracellularCYNcontentswereanalysedbyLCMS/MSandHPLC-PDA. Results: BiovolumerelatedCYNcontentsvariedatmaximumbya factorof2.7inAphanizomenon strainsattheconditionstested,with temperatureasthefactorcausingthestrongesteffects.Extracellular CYNconcentrationsamountto11–26%oftotalCYNbutmay increaseupto58%underconditionswhichwerepostulatedtoactas 140
LisaRosgaard, YumikoSakuragi. DepartmentofPlantBiologyandBiotechnology, FacultyofLife Sciences,UniversityofCopenhagen;VKRResearchCentre“Proactive Plant”;StrategicResearchConsortium“Fuel-for-Life”,FacultyofLife Sciences,UniversityofCopenhagen;Denmark. Introduction: Photoautrotrophiccyanobacteriaareoneofthemost importantprimaryproducersonEarth.Theyareresponsiblefornearly 70%ofCO2 assimilationintheaquaticenvironmentand understandingoftheircarbonmetabolismisthereforeveryimportant (1). ThisprojectisfocussedoninvestigatingtheroleofvitaminEandits roleincarbonmetabolisminthemodelcyanobacteriumSynechocystis sp. PCC 6803.Thepreviousworkhasdemonstratedthatthe Synechocystis mutantslr1736- whichisdeficientinvitaminE,exhibit ca.20%increasedphotosyntheticcapacitiesandincreasedtotalsugar content (2).WehypothesizethatvitaminEfunctionsasregulatorof photosynthesisandcarbonmetabolism,inadditiontoitsantioxidant activity (2). WewanttounderstandthemechanismbywhichvitaminE regulatesthecarbonmetabolismandinthisworkweexaminedthe carbohydratecontentoftheslr1736- mutant. Methods:Thetotalsugarcontentwasdeterminedbythephenolsulfuricacidmethodasdescribedin(3)andthetotalhexosecontent wasdeterminedusinganthroneinsulfuricacidaccordingto(4). Glycogenwasextractedbyethanolprecipitationfromliquidcultures ofwildtypePCC6803andtheslr1736- mutant.Theglycogenextract washydrolyzedbyamyloglucosidaseandtheamountofglucose releasedwasquantifiedbycolorimetricdeterminationusingaglucose oxidaseandperoxidasecoupledassay. Results: Analysisoftheslr1736- mutantshowedthatamountoftotal sugarswas40%highercomparedtothewildtype.Thecontentoftotal hexoses andglycogen wasalsodetermined;theslr1736- contained approximately30%morehexosesandca.70%moreglycogenas comparedtothewildtype.Theincreaseintotalhexosecontentwas largelyattributedtotheincreaseinglycogen. August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes Conclusion: Theresultsconfirmthepreviousresultsshowing increasedtotalsugarcontentintheslr1736- strain.Theincreasein hexosecontentfoundintheslr1736- mutantcouldbeattributedtoan increaseinglycogencomparedtothewildtype.Thissuggestsa connectionbetweenvitaminEandglycogenmetabolismandfurther worktoelucidatethemechanismofglycogenaccumulationinslr1736isinprogressandwillbepresented. BryantDA(2003).Proc.Natl.Acad.Sci.USA17:9647-9640. SakuragiY,MaedaH,DellaPennaD,BryantDA(2006).PlantPhysiol. 141:508-52. Masuko,T.,Minami,A.,Iwasaki,N.,Majima,T.,Nishimura,S-I.,lee,Y. (2005).AnalyticBiochem.39:69-72. Fry,S.C.(1988).In:TheGrowingPlantCellWall.Wilkins,M(Ed).pp. 64-101.LongmanScientific&Technical,Harlow,UK.
P.126 OVEREXPRESSION OF PKNE (ALR3732) INHIBITS HETEROCYST DEVELOPMENT IN ANABAENA SP. STRAIN PCC 7120. SushantaKumarSaha,JamesW.Golden. DivisionofBiologicalSciences,MolecularBiologySection,University ofCalifornia,SanDiego,CA,USA. Introduction: Proteinkinasesplayakeyroleinsignalingpathwaysby modifyingtheactivityoftheirtargetproteins.Thenitrogen-fixing filamentouscyanobacterium Anabaena PCC7120containsseveral eukaryotic-typeserine/threonineproteinkinases.Reportedincreases ofintracellularCa2+ concentrationduringheterocystdifferentiation promptedustodeterminethespatiotemporalexpressionoffour putativeCa2+ dependentserine/threoninekinases,andthenfurther characterizetheroleofpknE inheterocystdevelopment. Methods: Anabaena strainswereconstructedtocontainGFP reporters,andtoknockoutandoverexpresspknE.Strainswere characterizedbystandardandfluorescencemicroscopy,determination ofgrowthcharacteristics,andwesternimmunoassayofproteins. Results: Weconstructedreporterstrainscontaininggfp reporter fusionswiththeupstreamintergenicregionsofthefourkinasegenes all2334,alr3732 (pknE),all4668,andall4838.Allofthesekinasegenes hadlowlevelsofGFPreporterexpressionwhengrownonBG-11 medium.Uponnitrogenstep-down,theGFPfluorescencewas increasedindifferentiatingcellsfromthepromotersPpknE andPall4668, notchangedfromPall2334,andverylowthroughoutthefilamentfrom Pall4838.WefurthercharacterizedpknE becauseitshowedstrongupregulationofGFPreporterexpression indifferentiatingcellsafter nitrogenstep-down.ThepknE genewasinactivatedbysingle recombination.Theresultingstrainwasnearlynormal,buthadshorter filamentswithslightlyhigherheterocystfrequencycomparedtothe wild typeafternitrogendepletion.OverexpressionofpknE fromits nativepromoteronamulticopyshuttleplasmidcompletelyinhibited heterocystformation,andthestrainwasunabletogrowonnitrogendepletedmedium.However, overexpressionfromthe copper-induciblepetE promoter,whichisexpressedinvegetative cells, didnotinhibitheterocystdevelopmentbutcaused a24hdelay inheterocystdifferentiation.ThePpetE-pknE strainalsoformedgranular pleomorphicvegetativecellsafter4daysofgrowthandhaddefective celldivisionresulting4to10cell-lengthslongcells onBG-11medium. WeexaminedexpressionofthehetR geneinpknE mutantand overexpressionbackgrounds.InthepknE mutantbackground,normal patternedexpressionofthePhetR-gfp reporterwasobservedduring heterocystdevelopment.Inthe strainoverexpressingpknE fromits ownpromoter,PhetR-gfp reporterexpressionwasinhibited;andwestern August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts blotimmunoassayshowed nodetectableHetRproteininthis strain. BecauseofhetR positiveautoregulation,thepknE overexpression phenotypecouldbecausedbyablockeitherupstreamor downstreamofHetR.Ingeneticepistasisexperiments,wefoundthat theintroductionofpknE onamulticopyplasmidintothreestrainsthat produce ahigherfrequencyofheterocysts,AMC451(∆patS), AMC1289(PpetE-hetRR223W), andAMC1537(∆hetR containinghetRHis6), causedcompleteinhibitionofnewheterocystformationafter nitrogenstep-down.Therefore,itappearsthatoverexpressionofpknE blocksheterocystdifferentiationdownstreamofHetR. Conclusions: ThepknE geneisdevelopmentallyregulated,andits properexpressionisrequiredforbothnormalvegetativecellgrowth andcelldivision,andfornormalheterocystdevelopment.
P.127 BIOCHEMICAL STUDIES OF THE PROTEINS ENCODED BY SOX GENES INVOLVED IN THIOSULFATE AND SULFIDE OXIDATION FROM THE GREEN SULFUR BACTERIUM CHLOROBACULUM TEPIDUM. HidehiroSakurai,TakuroOgawa,ToshinariFurusawa,MichikoShiga, RyoheiNomura,DaisukeSeo,NaomiHosoya-Matsuda,Kazuhito Inoue. Res.Inst.Photobiol.H2Production,KanagawaUniv.,Hiratsuka, Kanagawa,Japan. Introduction:ThegreensulfurbacteriumChlorobaculum tepidum, utilizesvarioussulphur compoundssuchassulfide,thiosulfateand elementalsulphur aselectrondonorsforphototrophicgrowth.Thesox (sulfur oxidizingsystem)geneclusterofthisbacteriumconsistsof soxF2XYZAKBW genes.Wehavepurifiedandcharacterizedthree proteins(thecoreTOMES,thiosulfateoxidizingmulti-enzymesystem) indispensableforinvitrothiosulfateoxidation.Wehavealsopurified SoxF2,andstudieditseffectsonthiosulfateoxidizingactivityofthe coreTOMESaswellasitssulfide oxidationactivities. Methods: C. tepidum cellswerephotoautotrophicallygrownwith sulfide andthiosulfateaselectrondonors,andproteinswerepurified toapparenthomogeneityasdescribedelsewhere(Ogawaetal.,2008, Ogawaetal.,inpreparation). Results:FromC. tepidum cells,wehavepurifiedthreefactors indispensableforthiosulfate-dependentreductionofsmall,monohemecytochrome(cyt)c-554:SoxYZ, SoxBandSoxAXK(thecore TOMES).Thelastcomponentistrimericprotein(alsocalledcytc-551) composedofmono-hemecytSoxA,mono-hemecytSoxX,andthe colour-lessproductofthehypotheticalORFCT1020 (soxK).TheSoxA, XandKwereseparatelyexpressedinEscherichia coli.Inthepresence ofothertwocoreTOMESfactors,therecombinantSoxAandSoxX showedalowbutdiscernibleactivityofthiosulfate-dependentcyt c554reduction.FurtheradditionoftherecombinantSoxKgreatly increasedtheactivity,andthetotalactivitywasashighasthatofthe nativeSoxAXKcomplex.TherecombinantSoxKparticipatedin formationofatightcomplexwithSoxAandSoxX,andisreferredtoas SAXB(SoxAX bindingprotein).ThehomologuesofSAXB gene(soxK) arefoundinroughlyaboutonethirdofthiosulfateoxidizingbacteria whosesox geneclustersequenceshavebeendepositedsofar, rangingoverChlorobiciae, Chromatiaceae, Hydrogenophilaceae, etc. EachofthededucedSoxAandSoxXproteinsofvariousbacteria constitutegroupsthataredistinctfromthosefoundinbacteriathat apparentlylacksoxK genehomologues. WehavepurifiedSoxF2asamonomericflavoprotein.Thecore TOMEScatalysedoxidationofboththiosulfateandsulfitewithvarious
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Final Program and Abstracts cytochromesaselectronacceptors,andSoxF2stimulatedtheformer andinhibitedthelatterreaction.SoxF2alonecatalysedoxidationof sulfidewithaKm valueofabout2μMwithvariouscytochromesas electronacceptors. Conclusions: ThecomponentsofthecoreTOMESofC. tepidum are basicallysimilartothosefoundinvariousthiosulfateoxidizingbacteria suchasthepurplebacteriumAllochromation vinosum andthe facultativeorganotrophicbacteriumParacoccus pantotrophus. However,wehavefoundthatSoxAXproteinadditionallybindsanew subunitSoxKwhichisabsentinsomeofthem. AlthoughsoxFgeneisfoundinthesox geneclusterofvarious bacteria,itsbiochemicalfunctioninthiosulfateoxidationhasnotbeen demonstratedbyinvitroexperiments.Wehaveshownforthefirst timethatitstimulatesthiosulfateoxidizingactivityofthecoreTOMES byreconstitutedsystem. References: Ogawa,T.etal.,SoxAXbindingprotein,anovelcomponentofthe thiosulfate-oxidizingmultienzymesysteminthegreensulphur bacteriumChlorobaculum tepidum.J.Bacteriol.190:6097-6110 (2008). Ogawaetal.,(inpreparation).
sequencesofAnabaena sp.PCC7120SPPSPS, andSuS-A, respectively.TheresultingHis-taggedfusionproteinsshowedSPP, SPS,andSuSactivity.Inaddition,cellfreeextractsfromM. aeruginosa werechormatographedthroughanionexchangecolumnandthe enzymeactivitieswereassayedintheelutedfractions.Whenwe studiedthebiochemicalpropertiesoftheenzymes,wefoundthatSPS specificitywassimilartothatofothercyanobacterial SPSs,and interestingly,SuSactivitycouldonlybemeasuredintheSuccleavage direction.ExpressionanalysesbyRT-PCRandNorthernblotting indicatedthatthethreegenesaretranscribedduringstandardculture conditionsofthecyanobacterium. Conclusions: ThisisthefirstreportshowingthatbothSucsynthesis andcleavagetakeplaceinapotentialtoxiccyanobacterium. Remarkably,weshowedthatSuSisalsopresentinaunicellular,nonN2-fixingstrain.ThefactthatthisenzymeactsonlyintheSuccleavage directionsuggeststhatitmightcorrespondtoanovelclassofSuS. Furtherstudiesonthisenzymemayshedsomelightintoitsreaction mechanismandintotheroleofSucinMicrocystis cells. References: CuminoAC,MarcozziC,BarreiroR,SalernoGL.PlantPhysiol.143, 1385-1397(2007) CurattiL,GiarroccoLE,CuminoAC,SalernoGL.Planta228,617-625 (2008)
P.128 SUCROSE METABOLISM IN MICROCYSTIS AERUGINOSA PCC 7806. MaríaA.Kolman,LauraE.Giarrocco,CorinaM.Berón,GracielaL. Salerno. CEBB-MDP,CIB,FIBA,MardelPlata,Prov.BuenosAires,Argentina. Introduction: Sucroseisoneofthemostcommonnon-reducing disaccharidesinNature.Ithasbeenextensivelystudiedinplants, whereitisthemaintransportphotosyntheticproduct,asourceof carbonandenergy,andamoleculeassociatedwithenvironmental stressandsignaltransduction.Duringthelastdecade,Sucmetabolism wasdemonstratedincyanobacteria.SucissynthesizedthroughatwosteppathwayinvolvingSPS (U/ADP-glucose:D-fructose-6-phosphate 2-α-D-glucosyltransferase)and SPP(sucrose-6F-phosphate-phosphohydrolase),eitherinunicellularorfilamentouscyanobacteriumstrains. However,SuccleavagebySuS(A/UDP-glucose:D-fructose2-α-Dglucosyltransferase),hasonlybeenreportedinfilamentous heterocyst-formingcyanobacteria,whereSucwasdemonstratedto playacrucialroleinN2 fixationandinpolysaccharideproduction. Objective: TheaimofthisstudywastoinvestigatetheroleofSucin Mycrocystis aeruginosa,a unicellularnon-N2 fixingstrain, wellknown asoneofthemostcommonbloom-formingcyanobacteriainfresh waterenvironments. Methods: M. aeruginosa PCC7806wasculturedinBG11medium. DNAfragmentscorrespondingtoSucmetabolismcandidategenes werePCRamplified.Theopenreadingframes(orfs)wereligatedinto thepRSETvector.TherecombinantHis–taggedproteinswere expressedinEscherichia coli BL21(DE3)pLysS.Proteinswerepurified byNi2+ affinitychromatography,whileproteinextractsandpurification, enzymeactivitiesandexpressionanalyseswereperformedasreported previouslyforAnabaena [Cuminoet al.2007;Curattiet al.2008]. Results: WesearchedintheM. aeruginosa genomeforsequences homologoustothoseoffunctionallycharacterizedgenescodingfor SPS,SPPandSuS.Weretrievedthreecontiguousorfs inthecontig 328: IPF_1566, IPF_1564,andIPF_1565whosededucedamino-acid sequencesareabout 55%,53%,and72%identicaltotheprotein 142
P.129 REGULATION OF AUTOTROPHIC AND HETEROTROPHIC METABOLISM IN SYNECHOCYSTIS SP. STRAIN PCC 6803 BY TWOCOMPONENT SYSTEM CLUSTERS ON BOTH THE CHROMOSOME AND A PLASMID. Louis.A.Sherman,SowmyaNagarajan. DeptartmentofBiologicalSciences,PurdueUniversity,WestLafayette, IN,USA. Introduction: Synechocystis sp.strainPCC6803(Synechocystis)isa freshwatercyanobacteriumwitha3.57Mbchromosomeand7 plasmids,including3over100kb.Thechromosomecontains80 ORFs(>2.5%)thatcodeforTwoComponentSystem(TCS)proteins thathelpthecelladapttoenvironmentalchanges.Weareinterested in theTCSinathree-geneclusterthatcontainsahistidinekinase (Hik31),aDNA-bindingresponseregulator(Rre34),andanupstream hypotheticalprotein(Uhp).Thesegeneswerestronglyupordownregulatedunderseveralgrowthandstressconditionsinthewildtype (WT)andvariousmutantsinmicroarraystudies.Importantly,thereare duplicate(>95%identical)operonsofthisTCSonthechromosome (sll0788-sll0790)andonthe plasmidpSYSX(slr6039-slr6041).No detailedstudyofplasmid-encodedgeneshasbeenperformedin Synechocystisandthepresenceofthesetwoclusters(withdifferent promoters)raisesimportantquestionsastothefunctionand differentialregulationoftheparalogs.ThisistheonlyduplicatedTCS inSynechocystis, andtheonlyclusterpresentonboththe chromosomeandaplasmid,among16sequencedcyanobacteria. Methods: Wehaveconstructeddeletionmutantsthatlackallthree genesintheputativeoperonorhik31 alone,oneitherthe chromosome ortheplasmid,andonboththechromosomeandthe plasmid.Methodologyincludedgrowthunderdefinedphysiological conditions,carefulmeasurementsofcelldoublingandmorphology, spectralanalysisandmicroarrays. Results:Wereportdifferencesinmorphology,pigmentcontentand growthbetweenthecultures.Initialphenotypeanalysissuggeststhat thechromosomaloperonisinvolvedinnegativecontrolofautotrophic August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes events,whereastheplasmidoperonisinvolvedinpositivecontrolof heterotrophicevents.First,thedeletionoftheentireplasmidoperon andthedeletionofbothoperonsresultedinstrainsthatwerenotable togrowinthepresenceof5mMglucoseunder12hL-12hD conditions.Secondly,theplasmidoperonmutantgrowsbetteron continuouslightthanunder12hLDconditions.Thirdly,thedouble hik31 mutantdemonstratedsignificantalterationsinpigment compositionwhengrowninthepresenceofglucoseunder moderatelyhighlight(150-200µmolesphotonsm-2s-1).Fourth,the doubleoperonmutantdemonstratedthepoorestgrowthunderall conditionsstudiedtodate.Finally,thechromosomeoperonmutant growsbestundermanyconditions,includingphotoautotrophicallyin highlightandmixotrophicallyinlowlight. Conclusions:Theseresultssuggesttheimportanceofgenesonboth thechromosomeandtheplasmidontheregulationofmajor metabolicprocesses.Inaddition,allthreegenesintheoperon appeartobefunctionallyimportant.Theuniquenatureofthese operonsandtheintriguingphenotypesofthemutantsindicatethat thissystemisavaluablemodelforthestudyofregulation.Supported byagrantfromtheDOE.
P.131 PROTEOME DYNAMICS IN NOSTOC SP. PCC 7120 AND NOSTOC PUNCTIFORME ATCC 29133. KarinStensjö1, SawYenOw2,TanaiCardona1,ArnaudTaton3,Ann Magnuson1,PeterLindblad1,PhillipC.Wright2. 1 DepartmentofPhotochemistryandMolecularScience,TheÅngström Laboratories,UppsalaUniversity,Uppsala,Sweden; 2Biological& EnvironmentalSystemsGroup,DepartmentofChemicalandProcess Engineering,TheUniversityofSheffield,Sheffield,UnitedKingdom; 3 CenterfortheStudyofBiologicalComplexity,Virginia CommonwealthUniversity,Richmond,VA,USA.
Introduction Nostoc sp.PCC7120andNostoc punctiforme ATCC 29133aremulticellularcyanobacteria capableoffixingatmospheric nitrogen.Thenitrogenfixingprocesstakesplaceinheterocysts; specializedcellswithalteredmetabolismtomediatetheN2-fixing process.Oneofouraimsistofurthertheunderstandingofthe metabolicbalancebetweentheheterocystsandthephotosynthetic CO2 fixingvegetativecellsthroughtheuseofquantitativeshotgun proteomics. Methods Relativecomparisonsbetweentheproteomesofenriched heterocysts,vegetativecellsandcompletefilamentswereachieved usingaquantitativeproteomicapproach.A4-plexor8-plexisobaric peptidetaggingtechnology(iTRAQ) workflowcoupledwith chromatographicpre-fractionationandtandemmassspectrometry wasused.Reliabilityinourdatasets isexemplifiedbybiological replicatesforallsamples. Results Morethan500proteinsfromeachstrainwereidentifiedwith confidentquantifications.Observationsprovidedbypurified heterocystanalysisenabledtheelucidationofthedominantmetabolic processesbetweentherespectivecelltypes.Approximately20%of theidentifiedproteomeshowedsignificantdifferentialregulation. Key indicatorslikethenitrogenaseenzymecomplexand proteinsofthe oxidativepentosephosphatecyclewereasexpected moreabundant inheterocystsofbothstrains.
Final Program and Abstracts Conclusions Byusingpurifiedheterocystsweareabletopresenta quantitativeinvestigationwithmuchhigherdetailrelatingNostoc sp. PCC7120andN. punctiforme heterocystspeciescomparedtoinour earlierinvestigation,providingresolutionsunachievablebywhole filamentproteomics.Complementarydatafromthetwo cyanobacterialstrainsexhibitsimilarmetabolictrends.Findings obtainedfromthisstudycontributetotheunderstandingofheterocyst specificmetabolismimportantfordownstreamresearchforbio-fuel productionfromN2 fixingheterocystouscyanbacteria. P.132 USING 23NA AND 31P NMR SPECTROSCOPY TO STUDY PH AND NA+ HOMEOSTASIS IN SYNECHOCYSTIS SP. STRAIN PCC 6803. JessicaLiTse,HaticeGizemYayla,MaryMAllen,NancyHKolodny. WellesleyCollege,Wellesley,MA,USA. Cyanobacterialiveinmanyextremeconditions,includinghypersaline, hyperosmotic,andofrecentinterest,increasinglyacidicenvironments causedbypollutionandindustrialwaste.Cellshavebeenshownto survivein,andadaptto,acidicconditionsbyraisingthepHoftheir environment[1],butthemechanismsthattheyusetoregulatetheir internal(pHin)andexternal(pHex)pHarespeculative.Wehypothesize thatthecyanobacteriauseNa+/H+ antiporterstoregulatetheirinternal pH.ThemodelorganismSynechocystis sp.strainPCC6803contains fiveputativeantiportergenes,whichcodefortransmembraneproteins thatactivelytransportNa+ intothecellcytoplasmandH+ outofthe cell,orvice-versa,dependingonthepHoftheenvironment [2].A BrukerAvance400MHzNMRspectrometerwasusedtodetect phosphorussignalsinsolution,suchasinternalandexternalinorganic phosphatetodeterminepHin andpHex,respectively,sugarphosphate, and -, -,and -ATP.Sodiumsignalsinsolution,bothinside(Na+in) andoutside(Na+ex)thecyanobacteria,weredetectedusing23NaNMR spectroscopy.SincetheNa+in andNa+ex peaksresonateatthesame frequency,ashiftreagent,Na4HTmDOTP,wasusedtoresolvethe Na+ex fromtheNa+in peakbyshiftingtheNa+ex peakdownfield.23Na and31PNMRspectrawereacquiredforcontrolcellsbufferedin minimalBG-11+Na2CO3 medium(pH≈10)andacid-stressedcells bufferedin60mMPIPESmedium(pH≈6.5).Aninitial23NaNMR spectrumwasacquiredafteradditionofNa4HTmDOTP,followedbya timedseriesof31PNMRspectratodeterminethepHin ofthecellsand afinal23NaNMRspectrum.23NaNMRspectrawereanalyzedto determinetheratiooftheintegralofNa+ex totheintegralofNa+in.The chemicalshiftsoftheinternalinorganicphosphatepeakshowthat althoughthecellsamplesweresuspendedindifferentpH environments,thecellswereabletomaintainapHin ofabout7.2.The pHex ofcontrolcellsdecreasedovertime,whilethepHex oftheacidstressedcellsincreased,suggestingthatpHregulationdoesoccur. Additionofexternalinorganicphosphateincontrolcellsproduced spectrainwhichtheinternalandexternalinorganicphosphatepeaks werecompletelyresolved,whichwasnotseeninspectraofacidstressedcells.Inboththecontrolandacid-stressedcellsamples,using 2.2mMNa4HTmDOTP,theintegralofNa+ex totheintegralofNa+in decreasedovertimebetweeninitialandfinal23NaNMRspectra acquisition,suggestingthattheamountofNa+in increasedovertime. [1]HuangJ.J. et al.(2002)Arch.Microbiol. 177,486–493. [2]WangH-L et al.(2002)Mol.Microbiol. 44,1493–1506.
Moreinterestingarethefindingsofproteins,suchasthioredoxins involvedinredoxregulationandsuperoxidedismutasesinvolvedin cellprotectiveprocesseswithadifferentabundanceinheterocystsas comparedtovegetativecells.
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P.133 WHICH IS THE INTRINSIC FORM OF PHOTOSYSTEM II, A MONOMER OR A DIMER? MaiWatanabe1,MasakoIwai2,ReiNarikawa1,MasahikoIkeuchi1. 1
UniversityofTokyo,DepartmentofLifeSciencesBiology;2Tokyo UniversityScience,FacultyScienceandTechnology,Departmentof AppliedBiologicalScience;Japan. Introduction: PhotosystemII(PSII)isamembraneproteincomplex consisting ofatleast20proteinsubunits,manypigmentsandlipids. Todate,bothdimericandmonomericPSIIcomplexeshavebeen preparedfromawiderangeofoxygenicphototrophs.However,the oxygenevolutionactivityofPSIIwasoftenhigherinthedimerthanin themonomer.Thus,itiswidelybelievedthatthePSIIcomplexmay functionasadimerinthethylakoidmembrane.Thecrystalstructures ofthedimericPSIIfromthermophiliccyanobacteriahavebeen determineduptotheresolutionof2.9Å.Here,wereportconversion betweenthedimericandmonomericcomplexesofPSIIbytreatment ofadetergentandgenedisruption. Methods: ThethylakoidmembranesfromThermosynechococcus elongatus weresolubilizedwithvariousconcentrationsofn-dodecyl-bD-maltoside(DM)andseparatedbyblue-nativePAGE(BN-PAGE)and subsequentSDS-urea-PAGE(16-22%acrylamide).Smallmembrane polypeptidegenepsbTc wasinactivatedbyreplacementwitha chloramphenicolcassetteandcompletesegregationwasconfirmed. Results: Whenthethylakoidmembranesweresolubilizedwithlow concentrationsofDM,therecoveryofthePSIImonomerwashigher thanthedimerinBN-PAGE,whilesolubilizationwithhigh concentrationsofDMgaverisetolowerrecoveryofthePSIImonomer thanthedimer.ThechlorophyllcontentinBN-PAGEestimatedby monochromaticscanningat670nmconfirmedtheinversecorrelation betweenthedimericandmonomericbandsdependingontheDM concentrations,despitethatmaximalrecoveryofthetotalPSIIwas achievedabove0.6%DM.Thiscorrelationwasverifiedasspot intensityinthetwo-dimensionalPAGE.Namely,spotsforthePSII integralsubunitsreflectedthechlorophylldistributioninBN-PAGE, beingexclusivelyfoundinthemonomerandthedimerbands.These resultssuggestthatsolubilizationwithhighconcentrationofDM artificiallyfacilitatesthedimerizationofthemonomer.Wealsostudied sequentialtreatmentofPSIIwith0.5%and4.5%DM.After solubilizationofthethylakoidswith0.5%DM,thesolubilizedPSIIwas recoveredbycentrifugationandfurthertreatedwithorwithout additionof4.5%DM(final5.0%).Resultsthusobtainedwerevery similartothoseintheone-stepsolubilization.Thisindicatesthata majorpartofthedimerisgeneratedfromthemonomerafter solubilizationbyactionoftheadditionalDMtreatment.Inthecrystal structure,therearefourDMmoleculesatthemonomer-monomer interfaceofPSII.ThismaysuggestthatthedimericPSIIinthecrystalis artificiallystabilizedbyinsertionoftheDMmolecules.Therearealso PsbTcpolypeptidesatthemonomer-monomerinterfaceinthePSII crystal.WestudiedthePsbTc-depletedPSIIbyBN-PAGEafter solubilizationwithDM.AtlowconcentrationsofDM,verylittleamount ofthePSIIdimerwasrecovered,whiletherecoveryofthedimerwas increasedathighconcentrations.ThisresultsuggeststhatPsbTcis essentialforthestabilityofthedimerinlowconcentrationsofDM,but isnotessentialforthedimerizationdrivenbyhighconcentrationsof DM.
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Conclusion: WeconcludethatthePSIIcomplexexistsinthethylakoid asamonomeroralooselyassociateddimerwhichissurroundedby lipids.PsbTcisessentialforstabilizationofthedimerbutnotessential forthedimerizationdrivenbyhighconcentrationofDM.
P.134 THREE INDIVIDUAL POINT MUTATIONS IN SYNECHOCOCCUS PCC6301 RUBISCO CONFER INCREASED OXYGEN TOLERANCE IN VIVO UNDER AEROBIC CHEMOAUTOTROPHIC GROWTH CONDITIONS IN RHODOBACTER CAPSULATUS SBI/II-. BrianWitte,StephanieScott,SriramSatagopan,F.RobertTabita. OhioStateUniversity,Columbus,OH,USA. Introduction: Ribulosebisphosphatecarboxylase/oxygenase (RubisCO)isthekeyenzymeoftheCalvin-Benson-Bassham(CBB) pathway,andisdirectlyorindirectlyresponsibleformostofthe organiccarbononEarth.Despiteitsimportance,theenzyme possessesconfusedsubstratespecificityandoftenwastescellular carboninincorporatingO2 insteadofCO2.Thisstudyusesrandom mutagenesisoftheRubisCOgenefromSynechococcus PCC6301and apowerfulbiologicalselectionsystemthatwaspreviouslydeveloped inRhodobacter capsulatus toselectformutantenzymesthatconfer increasedoxygentolerancein vivo.Further,themutantRubisCOs havebeenpurifiedandcharacterizedin vitro.Wehypothesizethata specificregionoftheRubisCOlargesubunitisespeciallyimportantfor interactionswithO2. Methods: Thegenesencodingthelarge(rbcL)andsmall(rbcS) subunitsofRubisCOfromSynechococcus PCC6301wereclonedin tandemintopUC19.Thevectorwithinsertwasusedasthetemplate inrepeatedroundsofmutagenicpcr.ThepoolofmutagenizedrbcLS wassubclonedintothebroadhostrangeplasmidpRPS-MCS3and subsequentlytransferredintotheRubisCO-deletionstrainofR. capsulatus SBI/II-.Individualcolonieswereusedforcreatingreplica platesforincubationunderphotoheterotrophic(minimalsaltsplus malate,CO2/H2 atmosphere),photoautotrophic(minimalsalts,CO2/H2 atmosphere)andchemoautotrophic(minimalsaltsplates,CO2/H2/O2 atmosphere).LevelsofintracellularRubisCOweremonitoredusing Westernblotstoascertaintheapproximatefractionoftotalprotein comprisedofRubisCO.Mutanted6301genesthatcomplemented growthunderallautotrophicconditionsweresubclonedinto Escherichia coli expressionvectorpSKB3andtheproductspurified andcharacterizedin vitro. Results: Usingthe6301numberingscheme,mutationsatresidues 259,269and375enabled6301RubisCOtocomplementgrowthin SBI/II- underaerobicchemoautotrophicconditions.Growthcurvesof SBI/II- complementedwitheachmutant,aswellasWT6301,are provided.Thekineticdatawithrecombinantproteinsindicatedthat eachmutationconfersasubtlydifferenteffectonRubisCO mechanism,althoughKm(CO2), Km(O2) andkcat wereaffectedto differentdegrees. Conclusions: ItisdifficulttosaythatanyoneofthemutantRubisCOs istruly“better”althoughtheydodefinitelyconferanincreased toleranceforoxygenunderchemoautotrophicgrowthconditionsand enablegrowthunderconditionswherethewild–typeenzymedoes not.Interestingly,noneoftheresiduesidentifiedinthisstudyare locateddirectlywithintheactivesiteoftheenzyme.Previousstudies haveshownthatalterationstoactivesiteresiduesalwaysresultsina lossofenzymefunction.Rather,thisstudyseemstoindicatethe importanceofresiduesneighboringtheactivesitethatcouldaffect theaccessofgaseoussubstratestotheenediolofribulose bisphosphateinthereactioncenter.Itisalsopossiblethatoneor August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes moreoftheseresiduesalterinteractionswithR. capsulatus intracellular chaperonessuchthat theobservedgrowthunderaerobicconditions maybedueinparttogreaterstabilityofthemutantenzymes. Nevertheless,thecombinationofinvivocomplementationandgrowth underselectiveconditionscombinedwithin vitro enzymological studies,projectsanintriguingnewdirectionforresearchintothebasis forRubisCO’sinteractionwithoxygen.
P.135 THE REDOX-SENSING MECHANISM OF REGB IN RHODOBACTER CAPSULATUS. JiangWu,CarlBauer. IndianaUniversity,Bloomington,Indiana,USA. RegB/RegAisaglobalredox-respondingtwo-componentsystemthat regulatesmanyenergy-generatingandenergy-utilizingprocessesin Rhodobacter capsulatus. Previousstudieshaveshownthat thesensor kinaseRegBmodulatesitskinaseactivityandsenseredoxsignals throughtwodifferentsignal-inputsites:aubiquinone-bindingsite locatedinaperiplasmicloopwhichcanmonitorstheoxidationstates ofubiquinonepoolandaredox-activecysteineinthecytosolic domainthatrespondstocellularredoxstate.Theubiquinone-binding siteandredox-activecysteineappeartoworkindependentlyandboth controlapartofRegBactivity. TofurtherelucidatethemechanismbywhichRegBsensestheredox signal,therolesoftwohighlyconservedresidues inthe transmembranedomain,R31andQ38,havebeenanalyzedby mutagenesisstudy.Mutationsatbothoftheseresiduescompromised theinhibitoryeffectofoxidizingconditiononRegBactivity,indicating thatR31andQ38areinvolvedinredoxsensing.Toinvestigatehow exactlyRegBinteractswithitsredoxsignalubiquinone,thebinding affinitiesofoxidizedubiquinoneanalogQ0andreducedQ0 toRegB havebeendeterminedbyIsothermalTitrationCalorimetry(ITC).ITC determinedtheKaofoxidizedQ0 toRegBwhilethereisno detectablebindingaffinityforreducedQ0.ConsistentwithITCdata,in vitro phosphorylationassayshowedthattwomutationsinthe ubiquinone-bindingsite,N110QandF112Y,willgiveRegBhigher activityinthepresenceofoxidizedQ0.In vivo, bothmutantshad elevatedphotosystemexpressionlevelsunderaerobicconditions. Thus,weconcludethatoxidizedubiquinoneistheformthatbindto RegBandinhibitRegBactivity,whenubiquinoneisconvertedto reducedform,itwillbedissociatedfromRegBandRegBactivitywill bereleased.
P.136 DISCOVERY OF NOVEL O2-TOLERANT NIFE-HYDROGENASES FROM ENVIRONMENTAL MICROBES TO CONSTRUCT A CYANOBACTERIAL RECOMBINANT FOR SOLAR H2 PRODUCTION. QingXu,GergelyMaroti,WalterVargas,YingkaiTong,ShibuYooseph. J.CraigVenterInstitute,Rockville,MD,USA. Hydrogenisacleanalternativetogasolineandotherfossilfuels. Photosyntheticcyanobacteriahaveattractedconsiderableattentionin recentyearssincetheycanphotolyticallysplitwatertosupportH2 productionthroughhydrogenasecatalysis.However,onemajor drawbackofthebiophotolysisprocessisthattheirH2-evolving hydrogenasesareextremelysensitivetoO2.TodevelopanO2tolerantbiophotolyticsystem,wearesearchingforO2-tolerant NiFe-hydrogenasesthatcanbetransferredintocyanobacteria.The oceansharboranabundanceofmicroorganismswithH2-production capabilityandtheyaregoodresourcesforidentifyingnovelO2August 9 to 14, 2009 • Montréal, QC, Canada
Final Program and Abstracts toleranthydrogenases.TheVenterInstituterecentlyconducteda metagenomicstudyofthemarineplanktonicmicrobesintheglobal ocean.Wesearchedthemetagenomicdataofthisglobalocean samplingprojectforputativenovelNiFe-hydrogenasesbyusing probabilisticmodelingmethods.Oneofthenovelhydrogenases identifiedfromtheglobaloceanwasclonedfromenvironmentalDNA samples,whichshowsstronghomologytoaknownO2-tolerant hydrogenasefromaphototropicbacteriumThiocapsa roseopersicina. TheclonedenvironmentalDNAcontainsthestructuralgenes(hynS andhynL)ofthisnovelhydrogenase.Locatedimmediatelyupstream ofhynS/hynL,twoaccessorygenes(hupH andhynD)werealso identified.ThroughheterologousexpressioninT. roseopersicina,we convertedtheenvironmentalDNAintoafunctionalNiFehydrogenase.TransferringthenovelNiFe-hydrogenaseinto cyanobacteriumSynechococcus elongates PCC7942isinprogress.
P.137 RHODOBACTER CAPSULATUS HBRL AND IRR: TWO MORE TRANSCRIPTION FACTORS INVOLVED IN THE REGULATION OF THE HEME BIOSYNTHESIS. SébastienZappa,CarlE.Bauer. DepartmentofBiology,IndianaUniversity,Bloomington,IN,USA. ThepurplephotosyntheticbacteriumRhodobacter capsulatus presentsaversatilemetabolism,capableofgrowingfromdarkaerobic tolightanaerobicconditions.Inordertoachievesuchmetabolic adaptations,thesynthesisofpigmentshastobetightlyregulatedand thebalancebetweenthemajortetrapyrrolesheme, bacteriochlorophyllandcobalaminmaintainedatadequatelevels.It hasbeenshowninthelabthatseveraltranscriptionfactorsare involvedintheregulationoftetrapyrrolesynthesis, i.e. RegA,CrtJ, AerR.Morerecently,studieswereundertakenabouttwonew transcriptionfactors,namelyHbrLandIrr.Whilethefirstoneisa memberoftheLysRfamily,thesecondisaFurrepresentative.Both werestudiedwithregardtotheireffectonhemebiosynthesis,asa functionoftheexogenoushemecontentortheavailabilityofironin thegrowthmedium.Theirrespectiveroleswereinvestigatedby geneticandbiochemicalapproaches.Indeed,genedisruptedstrains wereproducedandtheirphenotypes,regardingtheexpressionof hemesynthesisgenes,wereanalyzed.Inaddition,bothproteinwere heterologouslyexpressedinEscherichia coli TUNER(DE3),usingthe pSUMOvector.Purificationoftherecombinantproteinbyaffinity chromatographyenabledtoundertakestudiesregardingDNAand/or co-factorbinding.ItappearedthattheHbrLdeletedstrainisvery sensitivetoiron-chelatedmedia,whiletheIrrmutantshowsacellular hemecontentapproximately25%higherthanthewild-typeandthe HbrL-deletedstrains.Regardinggeneexpressionoftheheme biosynthesispathway,whileHbrLshowsacontrolonhemA,hemBand hemZ,itlookslikeIrrhasaneffectonhemCandhemE.Finally,the biochemicalcharacterizationgavesomeinsightsoftheDNAbinding mechanism,suchtheasthebindingofHbrLtopromoterssuchas hemAandpuf,asshownbygelmobilityshiftanalysis,andpreliminary DNAseIfootprintingassay.Asaconclusion,thisworkconfirmsthat HbrLandIrrareinvolvedintheregulationofthecomplextetrapyrrole synthesispathway,inadditiontopreviouslycharacterizedtranscription factors.
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P.138 METABOLIC ENGINEERING OF SYNECHOCYSTIS SP. PCC 6803 A SYSTEMS BIOLOGY APPROACH. YvonneZilliges,Jan-ChristophKehr,WolfgangLockau,Thomas Börner. Humboldt-UniversitätzuBerlin,InstitutfürBiologie, Berlin,Germany. Introduction: Cyanobacteria,alsoknownasblue-greenalgae, arethe onlyknownoxygenicphotoautotrophicprokaryotes.Theyplayamajor roleinglobalecologyandtheassimilationofinorganiccarbonfrom theatmosphere.Thegenomeoftheunicellularcyanobacterium Synechocystis sp.StrainPCC6803wasthefirsttobefullysequenced ofanyphototrophicorganism.Thisstrainthusrepresentsanideal candidateforasystems-leveldescriptionofprimarymetabolismand itsregulation–ultimatelyaimingataniterativeconstructionofa computationalmodelofthecoremetabolicprocesses.The combinationofametabolicnetworkmodelwith biochemicaland physiologicaldataallowforadeepunderstandingofphotosynthetic andmetabolicprocessesandtheirregulationinasystemsbiology approach. Methods and Results: Inourstudies,weanalyzedseveralmutants whereenzymesofglycolysisandespeciallyofpyruvatemetabolism,of metabolismofstoragecompoundsandoftheCalvincyclewere manipulated.Thesemutantswerecharacterizedwithrespectto growthparameters,pigmentationandintra-aswellasextracellular metabolitesbyionchromatographyandopticenzymatictestsfor metabolicfingerprinting.Thesedataareusedforasystems-level descriptionofprimarymetabolismanditsregulation.Thisapproachto amodelisalsoassistedbytranscriptomicdataandwillbefurther refinedbytheincorporationofexperimentallydeterminedkinetic parametersofselectedenzymes. Conclusion: Theestablishmentofanaccuratemodelcombines differentdatafromtranscriptomics,metabolomicsandkinetics.Using theestablishedmethodologyofflux-balanceanalysis,weevaluatethe mainmodesofmetabolism,identifymetabolicbottlenecksand correlatedreactionsets. Basedonthecomputationalmodel,mutationscouldbeintroduced andtheresultingphenotypescomparedtothetheoreticalpredictions. Thefinalmodelisexpectedtopermittheselectionofcandidate genesfortheoptimizationofbiotechnologicalprocesses.
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Exhibitors
ENVIRONMENTAL PROTEOMICS
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Satlanticdevelopsactivefluorometersforthestudyofalgaland bacterialphotosynthesis.Wealsoofferreal-timein-situnutrient sensorsandsophisticatedinstrumentintegrationandwaterquality systemsthatenablereal-timeoperationaldecision-making.Our customdesigncapabilitiesenableustoprovideourclientswith theidealsolutionpackage.
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Qubit’sPhotobioreactorsareusedforprecisephototrophic cultivationofalgaeandcyanobacteria.Theyfeatureaunique combinationofcultivatorandmonitoringdevice.Temperature, aerationgascomposition,Lightintensityandspectralcomposition canbesetwithahighaccuracy.Inaddition,cultivationconditions canbedynamicallyvariedaccordingtoauserdefinedprotocol.
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Final Program and Abstracts
13th International Symposium on Phototrophic Prokaryotes
Notes
148
August 9 to 14, 2009 • Montréal, QC, Canada
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Index of Presenters Abed,Raeid
P.090
Dragnea,Vladimira
P.009
Iwamori,Shunsuke
Adams,David
OC-11.6,P.035
Dragomani,Tierna
P.098
Jackson,Owen
Ahn,Chi-Yong
P.066
Druga,Bogdan
P.042
Jacobsen,Jacob
P.046
Akinwole,Philips
P.057
Duggan,Paula
P.099
Johansen,HenningKristian
P.064
P.036
Eaton-Rye,JulianJ
Al-Shehri,Abdulrahman Alexova,Ralitza
OC-12.5
Allison,L.F.
P.037
Eddie,BrianJ. Eisenhut,Marion
OC-6.1 P.100 OC-6.6
Elhai,Jeff
OC-18.3
Espie,George
P.101
Karp,Peter
Ferreira,Daniela
P.043
Katayama,Mitsunori
Kaplan,Aaron
Attia,Magdy
P.038
Autenrieth,Caroline
P.001
Fewer,David
OC-13.2
Kawabata,Tadashi
Azai,Chihiro
P.002
Florencio,FranciscoJ.
OC-10.4
Keren,Nir
Bagheri,Homayoun
P.078
Flores,Enrique
OC-1.6 P.003
Bauer,Carl Beatty,J.Thomas
PL-6.1 PL-7.2,P.004
Bennette,Nicholas
P.091
Bergman,Brigitta
PL-2.1
Bibby,ThomasS.
OC-1.2
Bird,David Biswas,Avijit Blankenship,Robert
Forchhammer,Karl
PL-3.3
KhalfaouiHassani,Bahia
Fortin,Nathalie
OC-15.5
Kirilovsky,Diana
Frigaard,Niels-Ulrik
OC-13.1
Kitashima,Masaharu Knoop,Henning
P.114
Galmozzi,CarlaV.
P.102
Kobayashi,Masayuki
P.015
P.103
Komada,Jun
P.070
GarciaCostas,Amaya
P.061
Korn,Anja
P.016
P.092
Garcia-Pichel,Ferran Ghosh,Robin
OC-16.5,P.104 P.105
Gomolla,Dorothea
P.011
Bryan,Samantha
P.094
Gorlenko,Vladimir
PL-1.3,P.062
Grammel,Hartmut
OC-15.4
OC-9.2 P.006,P.007
Grossman,Arthur
Burroughs,Nigel
P.039
Gugger,Muriel
Campbell,Douglas
P.095
Haande,Sigrid
Cardona,Tanai
P.032
Hagemann,Martin
Kurmayer,Rainer Kushige,Hiroko
OC-11.1,P.017
PL-4.1
Laguna,Rick
P.047
Le,ThiAnhTuyet
P.048
Lechno-Yossef,Sigal
P.018
PL-2.2
Lee,Young-Ki Lindblad,Peter Liu,Jinjie
OC-5.4
P.063
Liu,Zhenfeng
OC-4.3
PL-3.2
Ludwig,Frank
P.081 P.019
Ludwig,Marcus
Christman,Harry
OC-5.1
Haimovich,Maya
OC-11.5
Luther,Amanda
Cox,RaymondP.
P.060
Hanson,ThomasE.
OC-4.1
Csotonyi,JuliusT.
P.040
Harwood,Caroline
PL-9.1
PL-5.1
Hervás,Manuel
P.012
OC-7.1
Hess,Wolfgang
OC-11.3
OC-14.2 P.079
P.071 OC-18.1
P.080
P.106
P.097
P.115
Kyndt,John
Hahn,Alexander
OC-17.3,P.041
OC-15.6
OC-3.3,P.044
P.008,P.096
August 9 to 14, 2009 • Montréal, QC, Canada
OC-8.1
Gao,Qunjie
Golden,Susan
Dorador,Cristina
P.113
Knaff,David
P.059
Doná,Clelia
P.014 PL-6.2
P.010
P.005
Dey,Swati
OC-9.4
P.077
Brown,IgorI.
DePhilippis,Roberto
P.013 OC-16.6
Gaertner,Katrin
Boulay,Clémence
Dangel,Andrew
P.112
P.058 PL-1.1
Daldal,Fevzi
OC-2.4
Fuhrmann,Eva
Giani,Alessandra
Chen,Min
OC-6.5,OC-10.1
Kerfeld,Cheryl
P.093
Burnap,Robert
OC-1.5
PL-3.1
Bonsu,ErnestO.
Bryant,DonaldA.
P.111
Jungblut,AnneD.
OC-10.5
Batyrova,Horcheska
OC-3.2
Joshi,Gauri
Appel,Jens
Ballot,Andreas
OC-5.2
Johnson,Zackary
Alvey,Richard
OC-14.1
P.110
Maeda,Isamu
P.116 OC-17.1,P.117
Magnuson,Ann
OC-12.3
Maness,Pin-Ching
OC-18.2
Masepohl,Bernd
PL-6.3
Hirose,Yuu
P.107
Masukawa,Hajime
Hosokawa,Norimune
P.108
Mazard,Sophie
Inoue-Sakamoto,Kaori
P.045
McNeely,Kelsey
OC-12.2
Ishizuka,Takami
P.109
Meeks,Jack
OC-13.3
P.049 P.065
149
13th International Symposium on Phototrophic Prokaryotes
Final Program and Abstracts
Index of Presenters and Chairs Mella-Herrera,Rodrigo Meyer,Terry Midorikawa,Takafumi Miller,Mette Mulkidjanian,ArmenY. Mullineaux,Conrad Mundt,Sabine Narikawa,Rei
P.118 P.020,P.021
Shastik,Evgeny
P.073
Shen,Gaozhong
OC-9.6
Wu,Jiang
P.135
Xu,Qing
P.136
P.119
Sherman,Louis
PL-5.2,P.129
Xu,Yu
P.056
OC-14.3
Shimura,Yohei
P.074
Yamamoto,Haruki
P.030
PL-1.2,P.022
Sicora,Cosmin
P.033
Yeager,Chris
OC-15.2
OC-5.3
Sivonen,Kaarina
PL-9.3
P.050
Song,Hong-Gyu
P.053
Yurkov,Vladimir
OC-2.2
Zannoni,Davide
OC-17.2
Yin,Liang
OC-7.2,P.031
OC-16.3
Spence,Edward
P.067
Nenninger,Anja
P.082
Stadnichuk,Igor
OC-11.2
Zappa,Sébastien
P.137
Nicolaisen,Kerstin
P.120
Stal,Lucas
OC-10.2
Zilliges,Yvonne
P.138
Steglich,Claudia
OC-11.4
Núñez-Cardona,M.T. Oh-oka,Hirozo Ohki,Kaori Ohta,Hisataka
P.072,P.083 P.121 OC-3.1 P.122
Stensjö,Karin
P.131
Summerfield,Tina
OC-12.1
Summers,Michael
OC-5.5
Otaki,Hiroyo
OC-2.6
Suzuki,Eiji
Ouchane,Soufian
OC-7.3
Suzuki,Iwane
Overmann,Jörg
OC-2.1
Tabita,F.Robert
KN-2.1
P.023
Takaichi,Shinichi
OC-8.2,P.054
Owttrim,GeorgeW. Palinska,KatarzynaA. Park,Jeong-Jin
OC-1.4 P.123
Partensky,Frederic
PL-5.3
Pedersen,MarieØ.
OC-9.1
Pereira,Sara
P.051
Pitt,FrancesD.
OC-10.3
Preuflel,Karina Razi,Saiqa Redding,Kevin
OC-12.4 PL-7.1
Tamagnini,PaulaM.
PL-9.2
Tang,Kuo-Hsiang
OC-4.2,P.026
Tank,Marcus
OC-1.1
Tekucheva,Darya
P.055
Thiel,Teresa
OC-5.6
Thiel,Vera
P.086
P.124
Tomitani,Akiko
P.087
P.052
Tse,JessicaLi
P.132
Tsukatani,Yusuke
P.027
OC-6.4
Rosgaard,Lisa
P.125
Tsygankov,Anatoly
Saha,SushantaKumar
P.126
Vasquez,Yasmin
P.034
Sakamoto,Toshio
OC-10.6
Ventura,Stefano
OC-1.3,P.068
Sakurai,Hidehiro
OC-15.1,P.127
Salerno,GracielaL.
P.128
Sandmann,Gerhard
OC-15.3
Vermaas,Wim
OC-6.2,OC-9.3
Vioque,Agustin
P.075
OC-8.3
Vogl,Kajetan
P.076
Schaefer,Steffani
OC-6.3
Walsby,A.E.
Schluchter,Wendy
OC-16.2
Watanabe,Mai
Schmetterer,Georg
OC-16.1
Watson,SusanB.
Schumann,Susanne Schwarz,Christoph Schwarz,Rakefet Setterdahl,Aaron Severin,Ina Shabeb,Mohamed
150
P.084 OC-2.5 OC-16.4 P.025 OC-2.3 P.085
KN-1.1 P.133 OC-12.6
Wegener,KimberlyM. Wilmotte,Annick Wilson,Adjélé Witte,Brian Wolk,PeterC. Woronowicz,Kamil
P.028
P.069,P.088,P.089 P.029 P.134 PL-4.2 OC-9.5
August 9 to 14, 2009 • Montréal, QC, Canada
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