Molecular Microbial Ecology

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The Challenge for Microbial Ecology Habitat Culturability (%) Seawater 0.001-0.1 Freshwater 0.25 Sediments Soil 0.3 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

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

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

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

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)

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.

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)

BLAST Basic 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

16S RNA sequences Santelli et al. 2008

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)

MPS Approaches Schematic courtesy of B. Crump

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

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? > 36,000 eubacterial species! ~3,000 archea species

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

Head et al. 1998 38

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

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

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)! 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

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

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! 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!