Diversity and quantification of candidate division SR1 in various anaerobic

environments

James P. Davis and Mostafa ElshahedMicrobiology and Molecular Genetics

Oklahoma State University

Presented at:Oklahoma Academy of Science

ANNUAL TECHNICAL MEETINGTULSA COMMUNITY COLLEGE – SOUTHEAST CAMPUS

Friday, November 2, 2007

IntroductionBacterial Diversity

Culture-independent surveys, based on 16S gene analysis, indicates that there are many novel, yet-uncultured, bacteria in the environment

An “Unculturable” Majority Apart from 16S gene

based analysis, little is known regarding: metabolic pathways, physiological activities, and community interactions

Rappe and Giovannoni Annual Rev. Micr. 2003 369-394

Candidate Division SR1

Few 16s rRNA sequences are present in databases (<40)

Found mainly in anaerobic habitats• Deep-sea hydrothermal

vents and sediments• Sulfur-rich sediments• Termite gut

Why SR1?• Interest was prompted

because SR1 was detected in Zodletone Spring using general bacterial primers

Purpose• Survey different

environments for SR1 and to determine the diversity and abundance in each environment

Culture-Independent Research Conducted

Sampling and DNA Extraction

Primer Design

Polymerase Chain Reaction (PCR)

Cloning and Sequencing

Phylogenetic and Statistical Analysis

Abundance - Quantitative PCR

Environments Surveyed

Anaerobic

Zodletone Spring – sulfur/sulfide-rich, anaerobic spring in S.W. Oklahoma

Bovine Rumen/Feces

Duck/Theta Ponds – fresh water ponds (low sulfur/sulfide)

Aerobic

Kessler Farm Soil

Hydrocarbon contaminated soil

Primer Design and Confirmation

Primers• General bacterial primers and

SR1-sequence specific primers (based on known 16S rRNA gene sequences)

• 9 primer pair combinations tested

• Best primer pair was a combination of forward universal primer and a reverse SR1 specific primer

Initial Sequence Analysis

• Operational Taxonomic Units – representative sequence

• 159 sequences in 30 OTUs• SR1 not detected in any aerobic

environment• SR1 detected in environments

with high and low sulfur concentrations

Phylogenetic Tree for Candidate Division SR1

•Zodletone Spring sequences have the highest diversity•Bovine Rumen/Feces sequences have the lowest diversity•5 Novel Groups•Interesting Animal Group

Maximum Sequence Divergence of SR1

Maximum Sequence Divergence•The highest percent difference between two sequences in a given set of sequences•The higher the MSD, the higher the diversity of the sequences

Known sequences found in GenBank

Known sequences + sequences from this study

Environment MSDZodletone 30.52%

Rumen 4.24%Feces 4.17%Theta 9.34%Duck 24.90%

Existing SR1 Sequences 36.22%

All Sequences 40.79%

Comparative Diversity of SR1 in different environments

Rarefaction Curve•Plots number of OTUs against number of sequences•A plateau indicates sampling has reached saturation at the species level•Zodletone Spring has highest species richness•Rumen has lowest species richness

Species Richness Estimates

• Coverage is an estimate of the percentage of new species already encountered

• Coverage = 1 - (n/N); where, n = number of OTUs with only 1 sequence and N = total number of sequences in the environment

Environment CoverageZodletone Spring 88.04%

Rumen/Feces 100.00%Theta Pond 50.00%Duck Pond 93.33%

SR1 Abundance - Quantitative PCR

Gene copy number per gram of

sediment

Standard Deviation

Zodletone Spring Sediment 7.96x10+04 ±1.81x10+04Bovine Rumen 1.24x10+06 ±1.44x10+05

Duck Pond Sediment 6.28x10+04 ±5.56x10+04Theta Pond Sediment 4.20x10+06 ±1.30x10+06

Summary and Conclusions

SR1 specific primers were designed to target the 16S rRNA gene These primers identified SR1 in most anaerobic environments tested (found

only in anaerobic environments) SR1 was detected in 2 non-sulfur rich environments (Duck & Theta Pond). This

suggests the SR1 is not restricted to sulfur-rich sites like Zodletone Springs and Sulfur River, KY

Phylogenetic analysis indicates the highest level of diversity of SR1 community in Zodletone Spring, while there was the least amount of diversity in the Bovine Rumen and species richness estimates confirmed the diversity levels

Quantification shows very high copy number for Theta and Rumen, but low numbers for Zodletone Spring indicating that diversity is indirectly proportional to abundance