Single Cell Microbiology

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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 onstudies at population level.

Information obtained mostly from population level data.

Development of new tools & techniques for studyingindividual microbial cell.

Single-Cell Techniques reveal:-

i) Environmental distribution

ii) Invisible processes

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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.

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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 successfullife style strategy.

Types of Heterogenity:-Genetic.Biochemical/MetabolicPhysiologicalBehavioral

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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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Individual cellular differences in macromolecular

composition

Phenotypic expression of genetic phenomena ofmutants.

Random transcription events and "noise.

Asymmetric distribution of proteins between motherand daughter cells.

Retention of oxidatively damaged proteins withinmother cell.

Quantity of certain macromolecular components.

Biochemical / Metabolic Heterogenity

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Physiological Heterogenity

Stems primarily through cell cycle. Driven by microenvironmental factors.

Describe morphological differences b/w individualcells:-

a) Difference in size & shape.b) Internal characteristics.

e.g. -

YeastSize differences

Bud scarringSurface wrinklingVariation in vacuole size

BacteriaDifferences in cell volume

Cell shapeBuoyant densityNucleoid morphology

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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.pd fMachineA pdf writer that produces quality PDF files with ease!




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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

orb/w pathogens & their hosts.

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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

regulatedistinct biochemical or genetic processes

Certain proteins change their "subcellular address.

Actin polymerization in Listeria monocytogenes.

Protease secretion in Vibrio cholerae.

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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 thesemesoscopic or macroscopic phenomena and theirmicroscopic, cellular origins.

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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 ofHeisenberg's "uncertainty principle".

Bridson and Gould "quantal microbiology"

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Tools and Technologies

Fluorescence

Rapid & more sensitive than colorimetric techniques.

Facilitate staining of microbial cells according to theirindividual properties.

Compatible with living cells.

Principle:- e-

Photon

e-

Fluorophore

fluorescence

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Fluorescence Staining techniques

Allow observation of:-

Protein expression & behavior

Substrate uptake

Binding and release of individualchemoattractant molecules to cell surfacereceptors

Selective degradation of uniparental DNA

within newly formed algal zygotes

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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 placepractical constraints on the fluorescent dye.

Advantages:-Including large extinction coefficientsReduced 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.

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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-specificfluorescence of target cells.

Recent FISH-based methods:-Detect low-copy-number targets on plasmid orchromosomal 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.pd fMachine





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Fluorescently labeled antibodies:-

Detection of diagnostic molecular binding events.

Can be directed against surface antigens or againstinternal targets.

Used for discrete localization of specific proteinswithin 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 dynamicevents occurring in living cells.

In Escherichia coliregular pole-to-pole oscillation for GFP-MinD wasobserved Raskin and de Boer theroized that cell may use this protein as a"measuring device" to continuously probe the location of the center of thecell.

Construction of whole-cell sensors for in situ monitoring

of:-Cytoplasmic viscosity & internal pH of bacterial cellsProtein diffusion rates in living cells.Investigation of quorum-based interspecies communication.

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Introduction of fluorescence-shifted spectral variants:-

Chimeric "nanosensor proteins based on the fusion ofECFP a bacterial maltose binding periplasmic protein, andEYFP. (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 thismethod.

Cells in a liquid sample Passed individually

Intense light sourceData collected & saved

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Facilitates valuableinsights into connectionsbetween single-cell and

population-level.

Elucidating thatapoptosis is not limitedto eukaryotes but may

also be active inprokaryotic systems.

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Laser Scanning Cytometry

Solid-phase cytometric technology for collecting laser-induced fluorescence from cell samples on membrane filter.

Well suited for the observation of cellular properties asa function of time.

e.g. *monitoring the kinetics of fluorescence staining inliving cells.

*observing interactions between neighboring cells.

Ability to concentrate cells prior to analysis givesdefinite practical advantages over flow cytometry.

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LSC instruments provide:-

Rapid means of counting, quantifying, andrecording the distribution of fluorescent events ona filter.

Visual information on both cell morphology andthe spatial distribution of fluorescence within eachcell.

Exposure to excitation source for long periods cancreate 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 MicroscopiesCan yield information on both the topography & physcio-chemical properties of a sample surface.

The SPMfamily 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. Usedto investigate charge development on biological surfaces.

Tapping-mode imaging

Alternative method for measurements of "soft" biologicalsurfaces.Chemically functionalized tips oscillates over surface thusminimizing frictional forces.pd fMachine




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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 electrochemicalcharecterization of single living microbial cell.

Scanning tip is an ultramicroelectrode designed formeasuring charge transfer reactions.

Redox maps are generated as tip is scanned in x-yplane above an electrochemically active cell.

e.g.Electrochemical visualization of production in singlealgal protoplasts on exposure to light.

O2

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Microspectroscopic Methods

Single cell resolution methods, enable observation oftarget properties within specific cells.

Provide biochemical information on overall macromolecularcomposition of cells.

Minimal sample preparation requirement enable analysisfor living cells.

Raman microspectroscopyMicrobeam analysis

Electrorotation

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Raman microspectroscopy

Raman effect:-

Induced emission of light resulting from the inelasticscattering of a small number of photons.

Raman spectra provide information on molecular vibrational

states.

Energy range:-UV (e.g., 257 nm), visible, and infrared excitationfrequencies.

Common visible sourcesArgon-ion lasers ( 514 nm) and helium-neon lasers ( 632 nm).

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Applications:-To investigate biochemical differences b/w cells in morphologically

heterogenous cultures ofClostridia.

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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MicromanipulationUsed to address a cell to a specific position in a liquid medium.

Allow stable positioning of cells for observations during Single cellassay.

Used to measure forces exerted by microorganisms on its

environment.

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Microcapillary Electrophoresis


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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-techmethods form recurrent themes in Single-Cell

Microbiology.

Availability of high-throughput sequencing methods andincreased computing power has fueled a rapid pace ofdiscovery 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 witheconomy, speed & flexibility.

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Still this is only the scratching the surface regarding

the complexity of microbial cells, there is much more toexplore in future of Single-Cell Microbiology.

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Single Cell Microbiology