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Diversity of uncultured candidate division SR1 in anaerobic habitats James P. Davis Microbial & Molecular Genetics Oklahoma State University Overview • • • • • Background Materials and Methods Results Summary & Conclusion Future Work 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 – Hydrocarboncontaminated Soil – Kessler Farm Field Soil – Anaerobic Fresh-Water Pond Sediments – Waste Water Treatment Plant Results – Division Specific Primers SR1 Primers DNA Zodletone Sed. Bovine Rumen Bovine Feces WWTP Theta Pond Sed. Duck Pond Sed. Kessler Soil HC Contam soil SR1-445F / SR1-1075R [+] + [+] – [+] [+] – [+] Uni-8F / SR1-1075R – + [+] – – – – – Table 1: [+] Indentified only by Nested PCR Uni-8F / SR1-914R + – [+] – – – – – Uni-27F / SR1-914R – + – – – [+] – – 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