Diversity and quantification of candidate division SR1 in various anaerobic environments James P. Davis and Mostafa Elshahed Microbiology and Molecular Genetics Oklahoma State University Presented at: Oklahoma Academy of Science ANNUAL TECHNICAL MEETING TULSA COMMUNITY COLLEGE – SOUTHEAST CAMPUS Friday, November 2, 2007

Introduction Bacterial 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

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 EnvironmentMSD Zodletone30.52% Rumen4.24% Feces4.17% Theta9.34% Duck24.90% Existing SR1 Sequences36.22% All Sequences40.79%

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 EnvironmentCoverage Zodletone Spring88.04% Rumen/Feces100.00% Theta Pond50.00% Duck Pond93.33%

SR1 Abundance - Quantitative PCR Gene copy number per gram of sediment Standard Deviation Zodletone Spring Sediment7.96x10+04±1.81x10+04 Bovine Rumen1.24x10+06±1.44x10+05 Duck Pond Sediment6.28x10+04±5.56x10+04 Theta Pond Sediment4.20x10+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