Techniques in microbial ecology I

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Techniques in microbial ecology (I)
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Questions

• Who is there?
• How much of it?
• What is its function?
• How can we obtain it in pure culture?

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I. Culture-independent analysis The use of
Biomarkers

• What is a biomarker?
• How could it be used in microbial ecology?
• - DNA
• - RNA
• - Proteins
• - Fatty acids
• DNA and Fatty acids are more suitable as
  biomarkers than RNA and Proteins. Why?

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Overview of 16S rRNA gene based analysis
What is PCR ?
What is cloning ?
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DNA genes in ecological and evolutionary studies

• DNA biomarkers
• Universal
• Ribosomal RNA (rRNA) genes (5S, 16S, 23S)
• Transfer RNA (tRNAs) aminoacyl transferase.
• RecA gene (codes for RecA Protein, Required for
  DNA recombination)
• Elongation factors Efg, Eftu, required for
  peptide elongation during translation.
• 16S is the most widely used universal biomarker
  in microbial ecology.

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Group specific primers

• Targeting 16S rRNA gene.
• Utilizing signature sequences of a specific group
  (Division, class, family, genus, species or
  strain) of microorganisms.
• Targeting functional genes. Genes present only in
  the group of interest
• Sulfate-reducing bacteria DSR
  (Dissimilatory sulfite reductase), and APS
  adenosine-5'-phosphosulfate reductase. Genes in
  the sulfate-reduction pathway.
• Nitrate reducing bacteria narG (nitrate
  reductase), nirK and nirS (nitrite reductase),
  nosZ (nitrous oxide reductase). Genes in the
  nitrate-reduction pathway.
• Methanogenic bacteria mcrA methyl-Coenzyme M
  reductase. Genes in the methanogenic pathway.
• Photosynthetic purple bacteria pufM, coding a
  protein in the photosynthetic reaction center of
  purple bacteria.

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II. DNA typing methods

• What is the difference between typing methods and
  cloning and sequencing approaches ?
• DGGE-TGGE.
• 16S-23S intergenic spacer (ITS) analysis
• RFLP, T-RFLP

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DGGE and TGGE (Denaturing / temperature gradient
gel electrophoresis)

• Separate DNA fragments based on difference in
  sequence (GC) content.
• Double stranded DNA fragments denatures according
  to their GC content.
• If mixed PCR products are subjected to a gradient
  of denaturing agents (Urea, Formamide) or a
  temperature gradient, a mixture of DNA fragments
  with different sequence could be separated.
• Advantages Allows a quick overview of the
  microbial diversity on one gel.
• Bands could be cut, sequenced.
• Disadvantages Inaccurate, incomplete separation,
  especially in complex communities.

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Extract DNA
Sediment
Amplify using 16S primers
GC 30
GC 50
GC 70
Low conc. of denaturing agent, Low temperature
High conc. of denaturing, high temperature
Less Gs and Cs, less bonds Between the DNA
strands, Earlier denaturation.
PAGE gel
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DGGE is useful in determining The level of
microbial diversity The similarities and
differences between 2 ecosystems
Thermophilic Vs Mesophilic water treatment
reactors Source Timothy M. LaPara, James
Alleman, Purdue University
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Restriction fragment length polymorphismRFLP

• RFLP. Use of restriction enzyme(s) to cut DNA
  fragments, comparing the banding patterns
  produced.
• Procedure amplify the 16S gene, clone, perform
  restriction digest on the library of clones,
  determine restriction patterns.
• Advantages Quick overview of the level of
  diversity.
• Useful as a pre sequencing screening method

RFLP of 16S genes of bacteria isolated from lake
Erie http//www.fredonia.edu/biology/molgen.html
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Intergenic (ITS) spacer analysis

• Organization of the 16S-23S operon.
• Spacer regions have different lengths.
• Procedure Amplify the region, run on a gel.
  Separate according to length.
• A piece of the 16S rRNA gene could be included to
  aid in identification by sequencing.
• Very similar to DGGE, TGGE in concept

16S
spacer
23S
1054f-16S
21r-23S
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III. DNA probes in quantitative microbial ecology

• Probe a DNA (or RNA) strand that can be labeled
  and hybridizes to a complementary sequence in a
  mixture of DNA.
• Useful for quantification, visualization of cells
  in-situ
• Phylogenetic 16S probes.
• - 16S rRNA gene has universally
  conserved, domain conserved, phylum conserved
  sequences.
• - Different Labeled probes with
  various levels of specificity could be designed.

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Membrane Hybridizations (Slot blot hybridization)

• The relative abundance of a specific group of
  interest could be quantified.
• Design, label a 16S group specific, universal
  primers.
• Probe labeling Either with radioactive Phosphate
  or with non radioactive dyes (Cy3 labeled dCTP).
• Extract DNA, fix on a membrane.
• Hybridize to group-specific, universal probe.
• Wash off extra probe.
• Measure radioactivity left on the membrane.
• Appropriate controls are needed.

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Fluorescent in situ hybridization(FISH)

• Probe hybridizes to the rRNA in the cell
  ribosomes, cells become uniformly Probe labeled
  to a dye (Biotin, Cy3 labeled dCTP, digoxigenin)
• Cells are permiabelized to allow the penetration
  of the dye-labeled probe.
• Cells having the target sequence will be
  fluorescent, could be observed with under a
  fluorescent microscope,

Growth of type 1851 in sludge AEM 70 158-1588
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FISH applications

• Quantification of a specific group (counting
  cells Vs Universal primer or stain)
• Visualization of the cells of interest (usually
  uncultured microorganisms), examination of
  microbial consortia, physical association between
  two groups of microorganisms (Syntrophy).

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IV. Quantification of active community

• RNA extraction, quantification.
• RNA is a better indicator for active community
  than DNA.
• RNA extraction coupled to membrane hybridization
• RNA extraction coupled to qPCR (RT-PCR)
• Use of 13C labeled isotopes
• Add 13C labeled substrate to your environmental
  sample,
• labeled will be assimilated, form to 13C labeled
  DNA.
• 13C labeled DNA is heavier, could be separated by
  ultracentrifugation.
• 13C labeled DNA could be amplified, cloned, and
  sequenced.
• Great tool to determine the fate of a single,
  specific substrate, especially in enrichments.

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Phospholipid fatty acid (PLFA) analysis

• Phospholipids (ester linked in bacteria, ether
  linked in Archaea) provides a good measure of the
  microbial biomass containing intact cellular
  membranes (living cells).
• Various groups of microorganisms have signature
  phospholipids.
• Procedure. Extract Phospholipids from
  environmental samples, run on Gas Chromatography
  -Mass spectroscopy (GC-MS), Observe
  characteristic lipids, their relative quantities.
• Differentiates down to the genus level is
  sometimes possible.
• Changes in PLFA profiles could sometimes be
  telling of the environmental condition e.g.
  Starvation, entry to the stationary phase, etc.

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Other biomarkers

• Ergosterol for fungi.
• Photosynthetic pigments for photosynthetic
  bacteria, algae.
• Biodegradation Intermediates as indicators of
  biodegradation.

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V. Activity measurements Use of Radioisotopes

• Activity, process oriented rather than species
  oriented
• Measures, quantifies a specific process e.g
  methanogenesis from 14C02, methanogenesis from
  14C acetate, Sulfate reduction fro 35SO42-.
• Also useful for determining the group of
  microorganisms responsible for a specific process
  e.g. fate of 14C acetate in freshwater
  environments (methanogenesis, sulfate-reduction,
  and photoheterotrophy)

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Activity measurements sensor analysis

• Extremely small electrodes that can measure
  various parameters Inserted in various depths in
  the environment and records readings in less than
  0.1 mm intervals.
• Useful in studying activities of various
  microbial groups in microbial mats.
• Revealed high levels of sulfate-reduction in oxic
  layers of microbial mats, their close association
  with oxygenic cyanobacteria.