Molecular Microbial Ecology

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Molecular Microbial Ecology
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The Challenge for Microbial Ecology
Habitat Culturability ()
Seawater 0.001-0.1
Freshwater 0.25
Sediments 0.25
Soil 0.3
How do you study something you cant grow in the
lab?
From Amann et al. 1995 Microbiological Reviews
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The grand picture, from environment to
identification
Head et al. 1998
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A more classical approach
Head et al. 1998
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• Ribosomal RNA (rRNA)
• Everybody has it
• Contains both highly conserved and variable
  regions
• -allows making comparisons between different
  organisms
• over long periods of time (evolutionary history)
• Not laterally transferred between organisms
• Huge and growing database

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Universal Tree of Life
BACTERIA
BACTERIA
ARCHAEA
ARCHAEA
You Are Here
EUKARYA
EUKARYA
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Primers can be designed to amplify hypervariable
regions, but are specific to Eubacteria vs. Archae

• 16S rRNA Bacteria primer pairs
• Several hypervariable regions
• 16S rRNA Archaea primer pairs
• Several hypervariable regions

• Usual procedure in molecular microbial ecology
• Obtain environmental sample (soil, seawater,
  fresh water, organism such as human gut)
• Extract total DNA
• PCR amplify and obtain amplicons
• Or clone DNA, and grow up clones
• Genotype/sequence DNA
• Characterize microbial diversity

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Alternative routes for molecular ecological
studies in microbiology

• Get community fingerprint via T-RFLP
  fingerprint profiles
• Get community fingerprint via DGGE and sequence
  bands
• Get species identification by
• Clone and sequence clones
• Skip cloning, go straight into sequencing
  (massively parallel sequencing, MPS)

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Alternative routes for molecular ecological
studies in microbiology

• Get community fingerprint via T-RFLP
• Get community fingerprint via DGGE and sequence
  bands
• Get species identification by
• Clone and sequence clones
• Skip cloning, go straight into sequencing
  (massively parallel sequencing, MPS)

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Denaturing gradient gel electrophosis (DGGE) DNA
melts at a certain point
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What do you do with the sequences?

• Perform a similarity search (database)
• Align the sequences (common ancestry)
• Build a tree (phylogeny and taxonomy)

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BLASTBasic Local Alignment Search Tool
http//blast.ncbi.nlm.nih.gov/Blast.cgi
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Alignments of sequences
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Alternative routes for molecular ecological
studies in microbiology

• Get community fingerprint via T-RFLP
• Get community fingerprint via DGGE and sequence
  bands
• Get species identification by
• Clone and sequence clones
• Skip cloning, go straight into sequencing
  (massively parallel sequencing, MPS)

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• Built clone libraries from deep-sea rocks
• Compared them to one another and other habitats

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16S RNA sequences
Santelli et al. 2008
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Community Overlap
Santelli et al. 2008
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Alternative routes for molecular ecological
studies in microbiology

• Get community fingerprint via T-RFLP
• Get community fingerprint via DGGE and sequence
  bands
• Get species identification by
• Clone and sequence clones
• Skip cloning, go straight into sequencing
  (massively parallel sequencing, MPS)

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MPS Approaches
Schematic courtesy of B. Crump
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The next generation sequencing methods
Platform Million base pairs per run Cost per base (cents) Average read length (base pairs)
Dye-terminator (ABI 3730xl) (classic method) 0.07 0.1 700
454-Roche pyrosequencing (next gen.) 400 0.003 400
Illumina sequencing (next gen.) 2,000 0.0007 35
From Hugenholtz and Tyson 2008
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V3, V6 and V6 hypervariable regions in 16S rRNA
genes, and taxon specific conserved primer sites
for PCR (coverage species amplified)
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How many species in 1 L of vent fluid?
gt 36,000 eubacterial species!
3,000 archea species
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Now we know who is thereWhat next?

• Quantify particular groups FISH or qPCR

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Head et al. 1998
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Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
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Quantitative (Real Time) PCR

• Detection of amplification-associated
  fluorescence at each cycle during PCR
• No gel-based analysis
• Computer-based analysis
• Compare to internal standards
• Must insure specific binding of probes/dye

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Quantitative PCR
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Primers used to amplify mcrA, an important gene
for adaptation to anoxic sediments (note
different primers are used to detect different
groups)
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Now we know who and how manyWhat next?

• Metagenomics
• RNA-based methods
• Many many more

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Metagenomics a.k.a., Community Genomics,
Environmental GenomicsDoes not rely on Primers
or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
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Metagenomics
Access genomes of uncultured microbes Functional
Potential Metabolic Pathways Horizontal Gene
Transfer
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From the Most Simple Microbial Communities

• Acid Mine Drainage (pH 0!)
• Jillian Banfield (UC Berkeley)
• Well-studied, defined environment with 4
  dominant members
• Were able to reconstruct almost entire community
  metagenome
• Tyson et al. 2004

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to the potentially most diverse!
Venter et al. 2004

• The Sorcerer II Global Ocean Sampling Expedition
• J. Craig Venter Institute Sequence now, ask
  questions later
• Very few genomes reconstructed
• Sequenced 6.3 billion DNA base pairs (Human
  genome is 3.2) from top 5 m of ocean
• Discovered more than 6 million genes and they
  are only halfway done!