molecular microbial ecology.?? habitatculturability (%) seawater 0.001-0.1 freshwater 0.25 sediments...
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Molecular Microbial Ecology
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Habitat Culturability (%)Seawater 0.001-0.1
Freshwater 0.25Sediments 0.25
Soil 0.3
From Amann et al. 1995 Microbiological Reviews
The Challenge for Microbial Ecology
How do you study something you can’t grow in the lab?
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Head et al. 1998
The grand picture, from environment to identification
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Head et al. 1998
A more classical approach
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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
EUKARYA
ARCHAEABACTERIA
EUKARYA
You Are Here
ARCHAEA
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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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Santelli et al. 2008
16S RNA sequences
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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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Schematic courtesy of B. Crump
MPS Approaches
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From Hugenholtz and Tyson 2008
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
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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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~3,000 archea species
> 36,000 eubacterial
species!
How many species in 1 L of vent fluid?
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Now we know who is there:What 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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• 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
Quantitative (Real Time) PCR
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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 many:What next?
• Metagenomics
• RNA-based methods
• Many many more…
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Metagenomics a.k.a., Community Genomics, Environmental Genomics
Does not rely on Primers or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
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Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways
Horizontal Gene Transfer…
Metagenomics
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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!
•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!
Venter et al. 2004