Genetic Variation of Renibacterium salmoninarum genes in infected salmonids Jeffrey Burnett HHMI Summer Investigator Dr. Dan Rockey Laboratory Biomedical Sciences Jeffrey Burnett HHMI Summer Investigator Dr. Dan Rockey Laboratory Biomedical Sciences

13 Oct 2007Jeffrey Burnett - HHMI1 Renibacterium salmoninarum Causes bacterial kidney disease (BKD) Wild and farmed salmonid species Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI2 Relevance Why is this a problem? We eat salmonids We depend on salmonids to keep an ecosystemic balance in our local rivers and streams R. salmoninarum devastates whole populations; endangered fish stocks Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI3 Relevance All salmonids are susceptible to BKD coho salmon (Oncorhynchus kisutch) brook trout (Salvelinus fontinalis) brown trout (Salmo trutta) chinook salmon (Oncorhynchus tshawytscha) rainbow trout (Oncorhynchus mykiss) Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI4 Renibacterium salmoninarum Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI5 Relevance Prevalence Found in majority of countries Economic impact felt worldwide Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI6 Relevance Economics close to home Local: Oregon Hatcheries $143,000 Largest Global Impact: Chile,S.A. and Europe Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI7 Big Picture Drug / Vaccine to eliminate bacteria Difficult to treat Current treatments ineffective Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI8 Goal of My Project Genome analysis American Tissue Culture Collection (ATCC) Accurate representation ERGO by Integrated Genomics  Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI9 Specific Goal Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI10 Hypothesis Due to extended laboratory culture, the genome of strain ATCC33209 has extensive mutations not representative of what is found in nature Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI11 Genetic analysis Introduction Hypothesis Methods Results Discussion Conclusion Dipeptide Permease Protein Citrate Synthase Protein Tetracycline Resistance Protein P Fibronectin Binding Protein

13 Oct 2007Jeffrey Burnett - HHMI12 Genomic DNA wt - 2 fish kidneys (A,B) Mt239 ATCC Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI13 Primer Design Flank apparent frameshifts identified by ERGO Introduction Hypothesis Methods Results Discussion Conclusion Tetracycline Resistance Protein P

13 Oct 2007Jeffrey Burnett - HHMI14 Experiment PCR products inserted into expression vectors Plasmids transformed into Escherichia coli Plasmids purified from bacteria Sent for sequencing Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI15 Results Center for Genome Research and Biocomputing (CGRB) - OSU 20 sequences in both directions Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI16 Results Introduction Hypothesis Methods Results Discussion Conclusion Y = yes, the sequence is identical to the ATCC sequence N = no, the sequence received is different from the ATCC sequence

13 Oct 2007Jeffrey Burnett - HHMI17 Results Verification of first round of results Reconstruct plasmids from different samples of DNA strains Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI18 Results Introduction Hypothesis Methods Results Discussion Conclusion All of the samples marked “N” ran in duplicate, returned the same results

13 Oct 2007Jeffrey Burnett - HHMI19 Discussion Findings are contrary to what we had originally hypothesized Genes are actually more mutated in the other strain isolate DNA that we tested Introduction Hypothesis Methods Results Discussion Conclusion

13 Oct 2007Jeffrey Burnett - HHMI20 Conclusion Introduction Hypothesis Methods Results Discussion Conclusion My research suggests that the ATCC sequence is representative of what is found in nature The bacteria is acquiring more mutations in its genome than the original ATCC strain

13 Oct 2007Jeffrey Burnett - HHMI21 Acknowledgements Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Dan Rockey Laboratory Sara Weeks Gina Capri Integrated Genomics 