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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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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



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


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)



What do you do with the sequences
What do you do with the sequences? certain point

  • Perform a similarity search (database)

  • Align the sequences (common ancestry)

  • Build a tree (phylogeny and taxonomy)


BLAST certain pointBasic Local Alignment Search Tool

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



Build a tree phylogeny
Build a Tree (Phylogeny) certain point

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)



16S RNA sequences microbiology

Santelli et al. 2008


Community overlap
Community Overlap microbiology

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)


MPS Approaches microbiology

Schematic courtesy of B. Crump


The next generation sequencing methods
The next generation sequencing methods microbiology

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? taxon specific conserved primer sites for PCR (%coverage = % species amplified)

> 36,000 eubacterial species!

~3,000 archea species


Now we know who is there what next
Now we know who is there: taxon specific conserved primer sites for PCR (%coverage = % species amplified)What next?

  • Quantify particular groups: FISH or qPCR


Head taxon specific conserved primer sites for PCR (%coverage = % species amplified)et al. 1998


Fluorescent In-Situ Hybridization (FISH) taxon specific conserved primer sites for PCR (%coverage = % species amplified)

Schleper et al. 2005


Quantitative (Real Time) PCR taxon specific conserved primer sites for PCR (%coverage = % species amplified)

  • 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 taxon specific conserved primer sites for PCR (%coverage = % species amplified)


Primers used to amplify taxon specific conserved primer sites for PCR (%coverage = % species amplified)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: taxon specific conserved primer sites for PCR (%coverage = % species amplified)What next?

  • Metagenomics

  • RNA-based methods

  • Many many more…


Metagenomics taxon specific conserved primer sites for PCR (%coverage = % species amplified)a.k.a., Community Genomics, Environmental GenomicsDoes not rely on Primers or Probes (apriori knowledge)!

Image courtesy of John Heidelberg


Metagenomics
Metagenomics taxon specific conserved primer sites for PCR (%coverage = % species amplified)

Access genomes of uncultured microbes:

Functional Potential

Metabolic Pathways

Horizontal Gene Transfer


From the most simple microbial communities
From the Most taxon specific conserved primer sites for PCR (%coverage = % species amplified)“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
taxon specific conserved primer sites for PCR (%coverage = % species amplified) 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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