Genetic Variation of Renibacterium salmoninarum genes in infected salmonids

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

2. Renibacterium salmoninarum • Causes bacterial kidney disease (BKD) • Wild and farmed salmonid species Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

3. 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 Jeffrey Burnett - HHMI

4. 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 Jeffrey Burnett - HHMI

5. Renibacterium salmoninarum http://oregonstate.edu/dept/salmon/projects/images/4BKD.jpg Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

6. Relevance • Prevalence • Found in majority of countries • Economic impact felt worldwide Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

7. Relevance • Economics close to home • Local: Oregon Hatcheries • 2004 - $143,000 • Largest Global Impact: • Chile,S.A. and Europe Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

8. Big Picture • Drug / Vaccine to eliminate bacteria • Difficult to treat • Current treatments ineffective Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

9. Goal of My Project • Genome analysis • American Tissue Culture Collection (ATCC) 33209 • Accurate representation • ERGO by Integrated Genomics Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

10. Specific Goal Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

11. 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 Jeffrey Burnett - HHMI

12. Genetic analysis • Fibronectin Binding Protein • Dipeptide Permease Protein Introduction Hypothesis Methods Results Discussion Conclusion • Citrate Synthase Protein • Tetracycline Resistance Protein P Jeffrey Burnett - HHMI

13. Genomic DNA • wt - 2 fish kidneys (A,B) • Mt239 • ATCC33209 • 684 Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

14. Primer Design • Flank apparent frameshifts identified by ERGO Introduction Hypothesis Methods Results Discussion Conclusion • Tetracycline Resistance Protein P Jeffrey Burnett - HHMI

15. 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 Jeffrey Burnett - HHMI

16. Results • Center for Genome Research and Biocomputing (CGRB) - OSU • 20 sequences in both directions Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

17. 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 Jeffrey Burnett - HHMI

18. Results • Verification of first round of results • Reconstruct plasmids from different samples of DNA strains Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

19. Results Introduction Hypothesis Methods Results Discussion Conclusion • All of the samples marked “N” ran in duplicate, returned the same results Jeffrey Burnett - HHMI

20. 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 Jeffrey Burnett - HHMI

21. 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 Introduction Hypothesis Methods Results Discussion Conclusion Jeffrey Burnett - HHMI

22. Acknowledgements • Howard Hughes Medical Institute • Dr. Kevin Ahern • Dr. Dan Rockey Laboratory • Sara Weeks • Gina Capri • Integrated Genomics Jeffrey Burnett - HHMI