Transcript Cottrell et al., 2005 discussion

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 reamplified 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 & Discussion
ARCHAEA
LUCA
• Cytophaga-like
Bacteria?
• Beta-Proteobacteria?
• Actinobacteria?
EUCARYA
Cytophaga-like
bacteria?
• Polysaccharide
degradation!
• Flavobacteriales
• Chitinophaga
• Flectobacillus
β-Proteobacteria?
• Broad freshwater
distribution.
• Chemoautotrophic
bacteria are well
represented.
• Rhodoferax
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?