Molecular Microbial Ecology
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The Challenge for Microbial Ecology
Habitat
Culturability (%)
Seawater
0.001-0.1
0.25
0.25
0.3
Freshwater
Sediments
Soil
How do you study something you can’t
grow in the lab?
From Amann et al. 1995 Microbiological Reviews
The grand picture, from environment to identification
Head et al. 1998
A more classical approach
Head et al. 1998
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
Universal
Tree of Life
BACTERIA
BACTERIA
You Are
Here
EUKARYA
EUKARYA
ARCHAEA
ARCHAEA
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
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)
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)
Denaturing gradient gel electrophosis (DGGE): DNA melts at a
certain point
What do you do with the sequences?
• Perform a similarity search (database)
• Align the sequences (common ancestry)
• Build a tree (phylogeny and taxonomy)
BLAST
Basic Local Alignment Search Tool
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Alignments of sequences
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)
• Built clone libraries from deep-sea
rocks
• Compared them to one another and
other habitats
16S RNA sequences
Santelli et al. 2008
Community Overlap
Santelli et al. 2008
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)
MPS Approaches
Schematic courtesy of B. Crump
The next generation sequencing methods
Platform
Million base
Cost per
Average read
pairs per run base (cents) length (base
pairs)
Dye-terminator
(ABI 3730xl)
(classic method)
0.07
0.1
700
454-Roche
pyrosequencing
(next gen.)
400
0.003
400
2,000
0.0007
35
Illumina
sequencing
(next gen.)
From Hugenholtz and Tyson 2008
V3, V6 and V6 hypervariable regions in 16S rRNA
genes, and taxon specific conserved primer sites
for PCR (%coverage = % species amplified)
How many species in 1 L of vent fluid?
> 36,000
eubacterial
species!
~3,000
archea
species
Now we know who is there:
What next?
• Quantify particular groups: FISH or qPCR
Head et al. 1998
Fluorescent In-Situ Hybridization (FISH)
Schleper et al. 2005
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
Quantitative PCR
Primers used to amplify mcrA, an
important gene for adaptation to
anoxic sediments (note different primers are
used to detect different groups)
Now we know who and how many:
What next?
• Metagenomics
• RNA-based methods
• Many many more…
Metagenomics
a.k.a., Community Genomics, Environmental Genomics
Does not rely on Primers or Probes (apriori knowledge)!
Image courtesy of John Heidelberg
Metagenomics
Access genomes of uncultured microbes:
Functional Potential
Metabolic Pathways
Horizontal Gene Transfer
…
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
… 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!