Discovering Dual Cyanobacteria Metabolism

biology + medicine

Ancient Survivors in a Hostile Environment Evolve

Two Radically Different

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ctopus Spring in Yellowstone National Park is one of the most inhospitable places on the planet. Yet, life flourishes there at temperatures that reach nearly 90°C (194° F), close to the temperature of the boiling water that emanates from the source of the spring. Living in effluent channels of the hot springs at temperatures ranging from under 50°C to just above 70°C are Synechococcus. These singlecelled cyanobacteria are part of a complex and diverse microbial ecosystem forming mat-like structures on the surface of the springs. This past summer, Stanford Scientists Devaki Bhaya, Arthur Grossman, and Anne Soisig Steunou from the Carnegie Institution’s Department of Plant Biology collaborated with

Arthur Grossman and Anne Soisig Steunou

Metabolic Processes

Cross section of gelatinous microbial mat shows the complex structure of the ecosystem. At the top centimeter lives Synechococcus cyanobacteria, one of the many producers; below this level resides other heterotrophs, including Acidobacterium and several green non-sulfur bacteria, such as Roseiflexus and Chloroflexus (photoheterotrophs).

by Benjamin Tran

genomicists, population biologists, evolutionary biologists and physiologists from Maryland, Montana, Connecticut and Denmark to investigate the question: What are the interactions of different species that are required for survival in microbial communities?

Residents of the Mat The microbial mats of Octopus Spring are highly organized ecosystems where different organisms perform different functions in the community. Synechococcus live in the top 1 mm layer of the microbial mat and are the primary producers, using sunlight to convert carbon dioxide and water into oxygen and energy-rich sugar. Cyanobacteria are believed to have evolved about 3 billion years ago, making them the oldest known microorganisms on the earth to perform photosynthesis. Other residents of the mat include heterotrophs—organisms that cannot produce their own food, and several types of green non-sulfur bacteria known as photoheterotrophs – organisms that use light as an energy source but can not convert carbon dioxide into energy. As a result of Synechococcus photosynthesis, the other organisms of the mat supplied Octopus Springs in Yellowstone National Park is seemingly unwelcoming to life, even in winter.

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Nitrogen Fix Nitrogen is one of the nutrients required by all organisms for the building blocks of nucleic acids and proteins. Animals (and carnivorous plants) can directly consume nitrogen by eating foods that contain it, whereas plants and certain bacteria must reduce or “fix” atmospheric N2 into larger carbon–containing compounds in order to assimilate it into their systems. The microbial mat receives some nitrogen and other required nutrients from organic material that falls into and decomposes in the steamy waters. However, it needs far more nitrogen than what is supplied by the springs. N2 fixation is a problem for photosynthetic cells, since oxygen produced during photosynthesis inhibits the function of the enzyme complex that fixes N2. Because Synechococcus performs photosynthesis, it was widely dismissed as a candidate for N2 fixation.

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When Night Falls

John Heidelberg of The Institute for Genomic Research (TIGR) sequenced genomes of two different Synechococcus strains, OS-A which can live up to 65°C and OS-B’ which can live up to 60°C. While these two genomes had similar gene

“It was as if at least one of the genomes shattered, and the genes were put back together in an almost random order.” content, the arrangement of the individual genes appeared to be “scrambled.” According to Steunou, “Analysis of orthologous genes revealed that the cyanobacteria were very similar in terms of gene content.” However, as coauthor Grossman states, “It was as if at least one of the genomes shattered, and the genes were put back together in an almost random order.” This startling difference in genome architecture raises questions about the evolution of Synechococcus ecotypes and the relationships of these ecotypes to other microorganisms in the mat community.

layout design:Loren Alegria

nifD Arthur Grossman and Anne Soisig Steunou

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A Question of Genetics

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light intensity (μmol photon m-2 sec-1)

While investigating the extent of diversity within microbial mats and the interactions between the various members of the mat environment, the group discovered a pronounced duality in the metabolic nature of the cyanobacteria that populate these mats. During the day, Synechococcus proceeds normally with photosynthesis, fixing inorganic carbon and releasing eight times the amount of oxygen needed to fully saturate the mat. However, as the intensity of light decreases from 1000 to around 50 –100 µmol photons per square meter per second, the cyanobacteria stop photosynthesis and the organisms in the mat

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This cartoon of the nitrogenase machine depicts the process for nitrogen fixation. To produce two molecules of NH3 and one H2 molecule, the machine takes in, among other things, 16 units of ATP energy currency.

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with carbon energy in the form of sugar, and oxygen which they need for respiration.

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The plot of the light intensity as time progresses shows that as intensity falls around 50 –100 µmol·m-2·s-1, the nif genes for nitrogen fixation “turn on.”

consume the oxygen more quickly than it can be produced. The mat quickly becomes depleted of oxygen. So what does Synechococcus do to survive? The group looked at its gene expression to find out. Surprisingly, they discovered that since Synechococcus cannot spatially separate photosynthesis and N2 fixation, it solves the mat’s photosynthetic and nitrogenfixing needs instead by temporally separating these tasks. The researchers found that the Synechococcus genome contains a nif gene cluster - nifH, nifK, nifJ, nifF, and nifD – which encodes for nitrogenase, the enzyme needed for N2 fixation. Previous work suggested that Synechococcus growing at the high temperatures of the hot spring mats were not able to fix N2. However, Steunou’s group found that while there is no transcription of nif genes during the day, the absence of oxygen at night allows the nif transcripts to accumulate and nitrogenase activity can be readily measured. Under these conditions, the cyanobacteria are able to fix atmospheric nitrogen (N2) into ammonia (NH3), the form of nitrogen they require for cell growth.

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Fermentation – it’s not just for beer!

The energy cost of running what Steunou calls the “Nitrogenase Machine” is high: to produce two molecules of ammonia and one molecule of hydrogen gas requires 16 ATP molecules (adenosine triphosphate, the cellular energy currency). Where does all this energy come from? When photosynthesis shuts down at night, the mat becomes oxygen starved, reducing the expression of the respiration genes coxA and cydA. Respiration is an efficient energygenerating pathway that requires oxygen to release the energy stored in sugars. With respiration turned down, the cells must rely on fermentation, a pathway that can proceed without oxygen. However, fermentation produces little energy relative to photosynthesis and respiration, and so nitrogenase activity during the evening is generally low. During the first hour and a half of sunrise, the mats remain anaerobic at the same time that photosynthesis just begins to function. It is at this time that ATP availability increases, making more energy available for N2 fixation. Thus, as Grossman states, “Much of the N2 fixation seems to be driven by the generation of energy by photosynthetic electron transport.” Even so, once photosynthetically generated O2 accumulates in the mat, nitrogenase activity is inhibited. To sum it up, during the early morning, the rate of respiration

Testing the Hypothesis The group’s method of testing for the presence and activity of the nitrogenase enzyme was based on a common procedure that has been used by many laboratories to measure nitrogenase. Nitrogenase can be assayed for its ability to reduce acetylene (C2H2) to ethylene (C2H4), which is readily detectable by gas chromatography (GC). “We took a cork bore sample of the mat, placed it in a test tube with acetylene, sealed it, and kept in it the effluent channel,” Steunou describes. “After a couple of hours we stopped the reaction by the addition of formaldehyde and subsequently measured the amount of ethylene and acetylene in the vial using GC.” This provided a quantitative measure of nitrogenase activity in the mat.

in the mat is fast enough to prevent O2 accumulation and the denaturation of the nitrogenase complex. As the morning wears on, the light levels increase and O2 accumulates, which causes destruction of nitrogenase activity and the switching off of the nitrogenase genes.

The Future of Microbial Mat Research These findings provide insight into the assimilation and utilization of nitrogen in the hot spring environment and help to define the metabolic processes and their interactions that shape this community of organisms. Grossman and Steunou agree that much work still needs to be done in order to fully understand these complex ecosystems. Future research projects will investigate the genetic relationships between cyanobacteria and other microbes in the mats as well as the environmental parameters that trigger metabolic switching. S Benjamin Tran is currently a freshman who is looking to major in mechanical engineering or chemical engineering. He is an avid sudoku enthusiast and enjoys playing tennis and working on the Stanford Solar Car Project in his spare time. Special thanks to Arthur Grossman and Anne Soisig Steunou for their help with this article.

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Testing the microbial mats

The Metabolic Switch Gene expression of Synechococcus during the day and night are summarized here: Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes decline in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fall into two categories. Some (ldh, pdhB, ald, and ackA) decrease in the evening, whereas others (pflB, pflA, adhE, and acs) increase at the end of the day and remain high throughout the night. Transcripts of nif genes are expressed only at the end of the day when the mat becomes anoxic.

NPS Photo Bob Lindstrom

“…future research projects will investigate the genetic relationships between cyanobacteria and other microbes in the mats and the environmental parameters that trigger metabolic switching”