1. Diversity of uncultured candidate division SR1 in anaerobic habitats James P. Davis Microbial & Molecular Genetics Oklahoma State University

2. Overview Background Materials and Methods Results Summary & Conclusion Future Work

3. 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

4. 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

5. 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

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

7. Materials & Methods Primer Design Environmental Sampling DNA Extraction Polymerase Chain Reaction (PCR) Cloned PCR Products Sequencing Preliminary Phylogenetic Analysis

8. 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

9. Results – Division Specific Primers

10. 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

11. 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

12. 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

13. Acknowledgements Miller, Fathepure, Shaw Labs DeSilva Lab Dr. Duncan Cody Shiek