Cottrell, M. T., L. A. Waldner, L. Yu, and D. L. Kirchman. 2005. Bacterial diversity of metagenomic and PCR libraries from the Delaware River. Environmental Microbiology. 2005. 7(12): 1883-1895.

• What is a metagenomic clone library?

• What is the utility of metagenomic analysis?

• What is a 16SrDNA clone library?

• What is FISH?

• Do metagenomic libraries (key to link function to taxa for non-culturables) represent similar diversity as PCR library and FISH?

• Why study bacterial diversity in rivers?

Metagenomic clone fosmid library

• What’s a fosmid?• Gentle DNA extraction to avoid

excessive shearing.• Ligate 40 kb fragments, package

with phage proteins, infect host bacterium, and select for clones.

• Screen clones for 16SrDNA by PCR and DGGE, bands are re-amplified and sequenced (160 bp).

16SrDNA PCR Library

• Take same DNA extract as above and PCR amplify 16SrDNA.

• Clone 16SrDNA amplicons in plasmid vector and transform host bacterium.

• Screen clones by amplified ribosomal DNA restriction analysis (ARDRA); same as RFLP on 16SrDNA amplicons.

• Representative clone for each unique fingerprint was sequenced (~1400 bp).

FISH• Filter paraformaldehyde fixed river bacteria onto

polycarbonate filter.• Treat cells on filter with hybridization buffer with

Cy3 labeled oligonucleotide probe; then wash, dehydrate, and mount on slide with DAPI stain.

Data Analysis• Identify 16SrDNA clone sequences:

– BLASTN database similarity search– ARB software Tool

• Alignment and phylograms with ARB tools.

• Library coverage comparison (LIBSHUFF).

• Checked for heteroduplex artifacts in the PCR library via reconditioning.

Results & Discussion

• How did coverage over evolutionary distance compare between the two library types (Fig 4)?

• Were heteroduplexes a large problem for the PCR library?

• Which was method gave more diversity?

• What were the differences in composition of major taxa between methods (Table 1)?

Results & DiscussionLUCA

ARCHAEA

EUCARYA

• Cytophaga-like Bacteria?

• Beta-Proteobacteria?• Actinobacteria?

• Polysaccharide degradation!

• Flavobacteriales

• Chitinophaga

• Flectobacillus

Cytophaga-like bacteria?

• Broad freshwater distribution.

• Chemoautotrophic bacteria are well represented.

• Rhodoferax

β-Proteobacteria?

Actinobacteria?

• High G+C Content Gram Positive

• Mostly Sporichtya

• DNA extraction problems with Gram+?

• No strong terrestrial “signal” (e.g. no low Firmicutes).

Conclusions

• What was learned about bacterial diversity in large rivers?

• How well did the metagenomic library represent the bacterial community?

• Any weaknesses in sampling or experimental design?