Diversity of uncultured candidate division SR1 in

anaerobic habitatsJames P. Davis

Microbial & Molecular Genetics

Oklahoma State University

Overview

• Background

• Materials and Methods

• Results

• Summary & Conclusion

• Future Work

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

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Candidate Division SR1

• Few 16S sequences are present in databases

• Found mainly in anaerobic habitats– Deep-sea hydrothermal vents and

sediments– Sulfur-rich sediments– Termite gut– Human oral cavity

SR1 Relevance

• Why SR1?– Interest was prompted because SR1 was

detected in Zodletone Springs using general bacterial primers

• Purpose– Survey different environments for SR1 and

to determine the diversity in each environment

Hypothesis

• Candidate division SR1 will be detected in a variety of anaerobic environments, including those with high and low sulfur contents.

Materials & Methods

• Primer Design

• Environmental Sampling

• DNA Extraction

• Polymerase Chain Reaction (PCR)

• Cloned PCR Products

• Sequencing

• Preliminary Phylogenetic Analysis

Survey of SR1 in a variety of anaerobic environments

• Primer Design– 4 different primers

targeting 16S rRNA genes of SR1 sequences were designed

– These division specific primers were used together and in conjunction with general bacterial primers

• Sampling– Bovine rumen and feces– Zodletone Springs

Sediment– Hydrocarbon-

contaminated Soil– Kessler Farm Field Soil– Anaerobic Fresh-Water

Pond Sediments– Waste Water Treatment

Plant

Results – Division Specific Primers

DNA

SR1-445F / SR1-1075R

Uni-8F / SR1-1075R

Uni-8F / SR1-914R

Uni-27F / SR1-914R

Zodletone Sed. [+] – + – Bovine Rumen + + – + Bovine Feces [+] [+] [+] –

WWTP – – – – Theta Pond Sed. [+] – – – Duck Pond Sed. [+] – – [+]

Kessler Soil – – – – HC Contam soil [+] – – –

Table 1: [+] Indentified only by Nested PCR

SR1 Primers

Preliminary evidence that distinct SR1 members are present in different environments

•High level of diversity among the Zodletone clones – 11 sequences in 5 operational taxonomic units• The 5 Rumen clones are all the same OTU•The data shows that the primer design was effective because all the sequences belonged to SR1

Summary & Conclusion

• SR1 specific primers were designed to target the 16S rRNA gene and were validated

• These primers identified SR1 in most environments tested

• Preliminary phylogenetic analysis indicates a high level of diversity among the SR1 community in Zodletone, while there is less diversity in the Bovine Rumen

• 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

Future Work

• Continue sequencing more clones of SR1 in all environments

• Investigate other uncultured divisions that are prevalent in anaerobic environments such as, TM7, WS5, SC3, OP11, OD1, and OD2

Acknowledgements

• Miller, Fathepure, Shaw Labs

• DeSilva Lab

• Dr. Duncan

• Cody Shiek