Molecular microbial ecology
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Molecular Microbial Ecology. ???. ???. ???. ???. ???. ???. ???. ???. The Challenge for Microbial Ecology. 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.

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Molecular Microbial Ecology

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Molecular microbial ecology

Molecular Microbial Ecology


Molecular microbial ecology

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Molecular microbial ecology

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The challenge for microbial ecology

The Challenge for Microbial Ecology

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

The grand picture, from environment to identification

Head et al. 1998


Molecular microbial ecology

A more classical approach

Head et al. 1998


Molecular microbial ecology

  • 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


Molecular microbial ecology

BACTERIA

Universal Tree of Life

BACTERIA

ARCHAEA

ARCHAEA

You Are Here

EUKARYA

EUKARYA


Primers can be designed to amplify hypervariable regions but are specific to eubacteria vs archae

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

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 microbiology1

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)


Molecular microbial ecology

Denaturing gradient gel electrophosis (DGGE): DNA melts at a certain point


What do you do with the sequences

What do you do with the sequences?

  • Perform a similarity search (database)

  • Align the sequences (common ancestry)

  • Build a tree (phylogeny and taxonomy)


Molecular microbial ecology

BLASTBasic Local Alignment Search Tool

http://blast.ncbi.nlm.nih.gov/Blast.cgi


Alignments of sequences

Alignments of sequences


Build a tree phylogeny

Build a Tree (Phylogeny)

Reconstructing evolutionary history and studying the patterns of relationships among organisms


Alternative routes for molecular ecological studies in microbiology2

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)


Molecular microbial ecology

  • Built clone libraries from deep-sea rocks

  • Compared them to one another and other habitats


Molecular microbial ecology

16S RNA sequences

Santelli et al. 2008


Community overlap

Community Overlap

Santelli et al. 2008


Alternative routes for molecular ecological studies in microbiology3

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)


Molecular microbial ecology

MPS Approaches

Schematic courtesy of B. Crump


The next generation sequencing methods

The next generation sequencing methods

From Hugenholtz and Tyson 2008


Molecular microbial ecology

V3, V6 and V6 hypervariable regions in 16S rRNA genes, and taxon specific conserved primer sites for PCR (%coverage = % species amplified)


Molecular microbial ecology

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

Now we know who is there:What next?

  • Quantify particular groups: FISH or qPCR


Molecular microbial ecology

Head et al. 1998


Molecular microbial ecology

Fluorescent In-Situ Hybridization (FISH)

Schleper et al. 2005


Molecular microbial ecology

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

Quantitative PCR


Molecular microbial ecology

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

Now we know who and how many:What next?

  • Metagenomics

  • RNA-based methods

  • Many many more…


Molecular microbial ecology

Metagenomicsa.k.a., Community Genomics, Environmental GenomicsDoes not rely on Primers or Probes (apriori knowledge)!

Image courtesy of John Heidelberg


Metagenomics

Metagenomics

Access genomes of uncultured microbes:

Functional Potential

Metabolic Pathways

Horizontal Gene Transfer


From the most simple microbial communities

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

… 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!


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