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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?

Head et al. 1998

The grand picture, from environment to identification

Head et al. 1998

A more classical approach

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

EUKARYA

ARCHAEABACTERIA

EUKARYA

You Are Here

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)

BLASTBasic 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

Santelli et al. 2008

16S RNA sequences

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)

Schematic courtesy of B. Crump

MPS Approaches

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

V3, V6 and V6 hypervariable regions in 16S rRNA genes, and taxon specific conserved primer sites

for PCR (%coverage = % species amplified)

~3,000 archea species

> 36,000 eubacterial

species!

How many species in 1 L of vent fluid?

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

• 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

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

Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways

Horizontal Gene Transfer…

Metagenomics

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!

•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