1. Molecular Microbial Ecology

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4. The Challenge for Microbial Ecology
Habitat
Culturability (%)
Seawater
Freshwater
0.25
Sediments
Soil
0.3
How do you study something you can’t grow in the lab?
From Amann et al Microbiological Reviews

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

7. A more classical approach
Head et al. 1998 7

8. 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

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

10. Usual procedure in molecular microbial ecology:
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
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

11. 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)

12. TRFLP: The first one is called Terminal Restriction Fragment Length Polymorphism or TRFLP. The first part of the method is the same as Clone Library sequencing, but one of the primers is labeled with a fluorescent molecule or with P-32. The mix of PCR products are then cut up with a restriction enzyme. Restriction enzymes seek out very specific short sequences on a piece of DNA and they cut the DNA only at those sequences. In this example I am using a restriction enzyme that cuts at the sequence “ggcc”. It cuts these pieces of DNA in different places, because there are differences in the sequences. Only the terminal piece of each different gene is labeled, and they are all different lengths. You then separate these pieces of DNA using electrophoresis. This is done in a gel matrix of agarose or acrylamide. An electric field is set up across the gel and DNA migrates towards the positive pole. Smaller pieces of DNA move faster, and so the fragments are separated by length. The banding pattern is used as a community fingerprint, with each band representing some subset of the organisms in the original sample.

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)

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

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

22. BLAST Basic Local Alignment Search Tool

23. Alignments of sequences

26. 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)

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

29. 16S RNA sequences
Santelli et al. 2008

30. Community Overlap
Santelli et al. 2008

31. 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)

32. MPS Approaches
Schematic courtesy of B. Crump

33. 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
Illumina sequencing (next gen.)
2,000
0.0007 35
From Hugenholtz and Tyson 2008

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

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

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

38. Head et al. 1998 38

39. Fluorescent In-Situ Hybridization (FISH)
FISH: Another use of probes is quite different. It is called fluorescent in situ hybridization, which unfortunately abbreviates to FISH. With this method you actually use probes to label whole organisms. In one approach cells are immobilized on a microscope slide. The Cell walls of the organisms are made permeable and probes are introduced. The slides are then looked at under a microscope and the fluorescent cells are counted. Another approach is to count the fluorescently labeled cells with a flow cytometer.
Schleper et al. 2005

40. 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

41. Quantitative PCR

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

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

47. Metagenomics a.k.a., Community Genomics, Environmental Genomics Does not rely on Primers or Probes (apriori knowledge)!
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS
Image courtesy of John Heidelberg

48. Access genomes of uncultured microbes:
Metagenomics
Access genomes of uncultured microbes:
Functional Potential
Metabolic Pathways
Horizontal Gene Transfer …
Keep sequencing genomes of isolates to ground truth and intepert metagenomics; in particular relate it to primary productio nand other key trains in the SS

49. 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

50. … 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!