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Molecular Microbial Ecology

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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  1. Molecular Microbial Ecology

  2. ??? ??? ??? ???

  3. ??? ??? ??? ???

  4. The Challenge for Microbial Ecology How do you study something you can’t grow in the lab? From Amann et al. 1995 Microbiological Reviews

  5. The grand picture, from environment to identification Head et al. 1998

  6. A more classical approach Head et al. 1998

  7. 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

  8. BACTERIA Universal Tree of Life BACTERIA ARCHAEA ARCHAEA You Are Here EUKARYA EUKARYA

  9. 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

  10. 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)

  11. 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)

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

  13. What do you do with the sequences? • Perform a similarity search (database) • Align the sequences (common ancestry) • Build a tree (phylogeny and taxonomy)

  14. BLASTBasic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/Blast.cgi

  15. Alignments of sequences

  16. Build a Tree (Phylogeny) Reconstructing evolutionary history and studying the patterns of relationships among organisms

  17. 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)

  18. Built clone libraries from deep-sea rocks • Compared them to one another and other habitats

  19. 16S RNA sequences Santelli et al. 2008

  20. Community Overlap Santelli et al. 2008

  21. 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)

  22. MPS Approaches Schematic courtesy of B. Crump

  23. The next generation sequencing methods From Hugenholtz and Tyson 2008

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

  25. How many species in 1 L of vent fluid? > 36,000 eubacterial species! ~3,000 archea species

  26. Now we know who is there:What next? • Quantify particular groups: FISH or qPCR

  27. Head et al. 1998

  28. Fluorescent In-Situ Hybridization (FISH) Schleper et al. 2005

  29. 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

  30. Quantitative PCR

  31. Primers used to amplify mcrA, an important gene for adaptation to anoxic sediments (note different primers are used to detect different groups)

  32. Now we know who and how many:What next? • Metagenomics • RNA-based methods • Many many more…

  33. Metagenomicsa.k.a., Community Genomics, Environmental GenomicsDoes not rely on Primers or Probes (apriori knowledge)! Image courtesy of John Heidelberg

  34. Metagenomics Access genomes of uncultured microbes: Functional Potential Metabolic Pathways Horizontal Gene Transfer …

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