Single Cell Microbiology

Welcome

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Single-Cell Microbiology: Tools, Technologies, and Applications Dr. Rajesh Kumar

Ph.D (Dairy Microbiology) Molecular Biology Unit Dairy Microbiology Division N.D.R.I

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Introduction Traditionally the field of microbiology has been focused on studies at population level.

 Information obtained mostly from population level data.  Development of new tools & techniques for studying individual microbial cell.  Single-Cell Techniques reveal:i) Environmental distribution ii) Invisible processes pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Cont…

Single-Cell Methods: Direct micro or nanoscale measurement.  Microphysiological studies of metabolite.  Protein elemental localization.  Intracellular water dynamics.  Offers discrete microbial observation.  Microbial viability phenomenon. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Microbial Heterogenity  Variability is hallmark of biological systems.  Important practical consequences: Antibiotic & biocide resistance.  Productivity & Stability of fermentation.  Potential of pathogen. Phenotypic plasticity forms the basis of successful ‘life style strategy’.

 Types of Heterogenity:Genetic. Biochemical/Metabolic Physiological Behavioral

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Genetic Heterogenity  Phage-related phenomena.  Copy number of mobile genetic elements.  Intracellular genetic heterogeneity.  Asymmetric distribution of genetic material.  Accumulation of DNA damage due to cell aging.  Loss of gene silencing.

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Biochemical / Metabolic Heterogenity ‘‘Individual cellular differences in macromolecular composition’’

 Phenotypic expression of genetic phenomena of mutants.  Random transcription events and "noise“.  Asymmetric distribution of proteins between mother and daughter cells.  Retention of oxidatively damaged proteins within mother cell.  Quantity of certain macromolecular components. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Physiological Heterogenity  Stems primarily through cell cycle.  Driven by microenvironmental factors.  Describe morphological differences b/w individual cells:a) Difference in size & shape. b) Internal characteristics. e.g. Yeast Bacteria •Size differences •Differences in cell volume •Bud scarring •Cell shape •Surface wrinkling •Buoyant density •Variation in vacuole size •Nucleoid morphology pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Behavioral Heterogenity  Observable consequence of cell-to-cell variation.  Stem from:*Genetic mutation. *Stochastic processes.  Responses to chemotactic or phototactic stimuli.  Measurement of swimming speed or direction.

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ADVANTAGES OF SINGLE-CELL APPROACHES  Plate counting and light microscopy.  Recent technological and methodological innovations: Advances in computing or imaging technologies  Development of culture-independent methods  Use of Single-Cell Approaches:Revealing Cryptic Processes. Observing Discrete and Dynamic Events within Living Cells. Relating Microscopic, Mesoscopic, and Macroscopic Observations. Caveat: the "Uncertainty Principle“.

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Revealing Cryptic Processes Microorganisms  carryout a no. of processes 

have substantial impact on human life  Urgent need of proper set of tools to acess:Gene transfer & environmental distribution. Biochemical interactions b/w microbial cells or b/w pathogens & their hosts. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Observing Discrete & Dynamic evnts within living cells Previously bacterial cell "...amorphous vessel…’’ 

Now much more complex than previously imagined.  Discrete subcellular domains 

regulate distinct biochemical or genetic processes  Certain proteins change their "subcellular address“.  Actin polymerization in Listeria monocytogenes.  Protease secretion in Vibrio cholerae. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Relating Microscopic, Mesoscopic, and Macroscopic Observations Coordinated multicellular activities:Aggregation. Development of specialized structures. Colony pattern formation. :• fruiting-body development in myxobacteria. • mound and slug formation in Dictyostelium discoideum. • chiral colony morphology in Bacillus subtilis. Single-cell resolution enable connections b/w these mesoscopic or macroscopic phenomena and their microscopic, cellular origins. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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A Caveat: the "Uncertainty Principle" Single-Cell methods enable the observation of living cells.  However, this expose cells to:-

*Potentially toxic fluorescent dyes * intense light *electric or magnetic energies *physical manipulation. *experience of increased metabolic load.  This, in effect, is the biological equivalent of Heisenberg's "uncertainty principle".  Bridson and Gould … …"quantal microbiology" pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Tools and Technologies Fluorescence  Rapid & more sensitive than colorimetric techniques.  Facilitate staining of microbial cells according to their individual properties.  Compatible with living cells. e-

Principle:Photon

fluorescence

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Fluorescence Staining techniques Allow observation of: Protein expression & behavior  Substrate uptake  Binding and release of individual chemoattractant molecules to cell surface receptors  Selective degradation of uniparental DNA within newly formed algal zygotes  Bacterivory and drug efflux pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Fluorescence ratio Imaging Microscopy Provide insights into dynamic cellular events: Physiological responses of spoilage organisms to chemical stress. or  Cellular inactivation b/c of antimicrobial treatment. CFSE  Probe.

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Fluorescent nanocrystals or Quantum dots Incompatible spectral or chemical properties can place practical constraints on the fluorescent dye. Advantages:Including large extinction coefficients Reduced susceptibility to photobleaching. Most intriguing properties:Narrow, size-dependent emission spectra May be excited with a single UV light source. Can be directed to specific tissues or cell types. Long-term labeling of live cells

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Targeting of Quantum dots into specific tissues of mouse

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Fluorescence insitu hybridization & Immuno fluorescence Fluorescently labeled nucleic acid probes are hybridized to complementary rRNA targets. The aggregate signal leads to the sequence-specific fluorescence of target cells. Recent FISH-based methods:Detect low-copy-number targets on plasmid or chromosomal DNA. Different from rRNA-targeted FISH b/c it utilizes polynucleotide probes. The resulting signal characterized by the formation of a fluorescent "halo" around the periphery of target cells.Thus named RING-FISH. 20

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Fluorescently labeled antibodies: Detection of diagnostic molecular binding events.  Can be directed against surface antigens or against internal targets.  Used for discrete localization of specific proteins within individual cells.  Unlike FISH, it does not require cell permeabilization so can be used on living cells

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Green fluorescent protein and related reporters Versatile tool for in vivo visualization of protein expression, localization, and functionality. The true power of GFP is as a visual reporter of dynamic events occurring in living cells. In Escherichia coli regular pole-to-pole oscillation for GFP-MinD was observed Raskin and de Boer theroized that cell may use this protein as a "measuring device" to continuously probe the location of the center of the cell.

 Construction of whole-cell sensors for in situ monitoring of:•Cytoplasmic viscosity & internal pH of bacterial cells •Protein diffusion rates in living cells. •Investigation of quorum-based interspecies communication. 22 pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

Cont…

Introduction of fluorescence-shifted spectral variants:Chimeric "nanosensor“ proteins based on the fusion of ECFP a bacterial maltose binding periplasmic protein, and EYFP. (Fehr et al) 

Nanosensors  bind to maltose  confermational change More efficient FRET from ECFP to EYFP

In S. cerevisiae, changes in ECFP/EYFP FRET ratios enabled maltose uptake and compartmentation.

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Flow cytometry  A powerful fluorescence-based diagnostic tool.  Enables rapid analysis of entire cell populations.  Specific Flow cytometers add to the versatility of this method. Passed individually

Intense light source







Cells in a liquid sample

Data collected & saved

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 Facilitates valuable insights into connections between single-cell and population-level.

 Elucidating that apoptosis is not limited to eukaryotes but may also be active in prokaryotic systems.

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Laser Scanning Cytometry  Solid-phase cytometric technology for collecting laserinduced fluorescence from cell samples on membrane filter.  Well suited for the observation of cellular properties as a function of time. e.g. *monitoring the kinetics of fluorescence staining in living cells. *observing interactions between neighboring cells.  Ability to concentrate cells prior to analysis gives definite practical advantages over flow cytometry. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Cont…

LSC instruments provide: Rapid means of counting, quantifying, and recording the distribution of fluorescent events on a filter.  Visual information on both cell morphology and the spatial distribution of fluorescence within each cell. Exposure to excitation source for long periods can create photobleaching of fluorescent labels

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Single-cell determination of yeast glycogen content by image cytometry.

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Single-molecule analysis of cyclic AMP receptor occupancy on the surface of a Dictyostelium discoideum cell during chemotaxis.

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Scanning Probe Microscopies Can yield information on both the topography & physciochemical properties of a sample surface. The SPM family of tools:1) STM 2) AFM 3) SECM 4) MFM

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Atomic force microscopy Modes of imaging:- contact, noncontact, and tapping . Contact imaging  "dragging" the tip across the sample. Noncontact imaging  Based on electrostatic deflection of the probe tip. Used to investigate charge development on biological surfaces. Tapping-mode imaging  Alternative method for measurements of "soft" biological surfaces. Chemically functionalized tips oscillates over surface thus 31 minimizing frictional forces.

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Applications:Measure discrete interaction forces in the piconewton range. Ultrastructural studies of surfaces of living microbial cells. AFM cantilevers with functionalized tips can be used as "nanobiosensors"

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Scanning Electrochemical Microscopy  SPM based tool for mapping redox activity in living cells.  Well established technique for electrochemical charecterization of single living microbial cell.  Scanning tip is an ultramicroelectrode designed for measuring charge transfer reactions.  Redox maps are generated as tip is scanned in x-y plane above an electrochemically active cell. e.g. Electrochemical visualization of O2 production in single algal protoplasts on exposure to light. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Microspectroscopic Methods  Single cell resolution methods, enable observation of target properties within specific cells.  Provide biochemical information on overall macromolecular composition of cells.  Minimal sample preparation requirement enable analysis for living cells. Raman microspectroscopy Microbeam analysis Electrorotation

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Raman microspectroscopy Raman effect:Induced emission of light resulting from the inelastic scattering of a small number of photons. Raman spectra provide information on molecular vibrational states. Energy range:UV (e.g., 257 frequencies.

nm),

visible,

and

infrared

excitation

Common visible sources Argon-ion lasers ( 514 nm) and helium-neon lasers ( 632 nm). pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Applications:To investigate biochemical differences b/w cells in morphologically heterogenous cultures of Clostridia.

Reagentless identification of individual bacterial spores.

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Microbeam analysis Include sensitive methods for characterizing single cell elemental composition. Allow measurement of concentration, chemical state & cellular location of biologically relevant inorganic nutrients. Elemental map of an individual cell can generate in a single pass.

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Micromanipulation Used to address a cell to a specific position in a liquid medium. Allow stable positioning of cells for observations during Single cell assay. Used to measure forces exerted by microorganisms on its environment.

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Microcapillary Electrophoresis  Isoelectric focusing of whole cells.  Electrophoretic separation of intracellular analytes from a single cell after lysis. Single cell  loading into capillary  lysed  analyzed

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Conclusion AND Future Perspectives  “high-tech”methods form recurrent themes in Single-Cell Microbiology.  Availability of high-throughput sequencing methods and increased computing power has fueled a rapid pace of discovery in genomics, proteomics and related fields.  Use of Mathematical modeling in Single-Cell Microbiology:How to control & improve microbial fermentations. Represent an additional source to test hypothesis with economy, speed & flexibility. pdfMachine A pdf writer that produces quality PDF files with ease! Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across nearly all Windows platforms, simply open the document you want to convert, click “print”, select the “Broadgun pdfMachine printer” and that’s it! Get yours now!

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Cont… “Still this is only the scratching the surface regarding the complexity of microbial cells, there is much more to explore in future of Single-Cell Microbiology.”

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