1. Genetic Variation of Renibacterium salmoninarum Genes Jeffrey Burnett Dr. Dan Rockey Laboratory Biomedical Sciences College of Veterinary Medicine

2. Acknowledgements Dr. Dan Rockey Dr. Katharine G. Field Dr. Ling Jin Dr. Damir Alzhanov Dr. Kevin Ahern Wanda Crannell Rockey Lab – Brendan, Kelsey, Sara Introduction Hypothesis Methods Results Conclusion

3. Acknowledgements Dr. Greg Wiens Whole Genome Sequencing of Renibacterium salmoninarum – USDA-CREES 2004-35600-14173 Howard Hughes Medical Institute Introduction Hypothesis Methods Results Conclusion

4. Who should care… Anyone who enjoys salmon – fishing / recreation – gastronomy Ecosystem Balance Economics – Communities depend on salmon for subsistence Introduction Hypothesis Methods Results Conclusion

5. Who should care… Introduction Hypothesis Methods Results Conclusion Smith, Pedro et al. Principales enfermedades de los peces salmonídeos cultivados en chile. Monografías de Medicina Veterinaria, Vol.21(2), diciembre 2001.

6. R. salmoninarum vs. Arthrobacter spp. Arthrobacter spp. – Diverse group of soil-dwelling microbes – 5.9 Mb genome – Used in bioremediation, extracellular polysaccharide synthesis Introduction Hypothesis Methods Results Conclusion

7. R. Salmoninarum vs. Arthrobacter spp. R. salmoninarum – 3.1 Mb genome – Fastidious – Facultative intracellular pathogen – Farmed and wild salmon species Worldwide – Vertical and horizontal transmission Introduction Hypothesis Methods Results Conclusion

8. Genome Sequencing Project R. salmoninarum ATCC33209 – Completed in December 2007 – Inactivated open reading frames – Supports reductive evolution hypothesis Introduction Hypothesis Methods Results Conclusion

9. MAUVE output : courtesy Dr. Greg Wiens, NWFSC

10. Sequencing results Introduction Hypothesis Methods Results Conclusion

13. VS.

14. Hypothesis The highly frameshifted genome sequence of Renibacterium salmoninarum ATCC 33209 is an artifact of extensive culture in vitro. Introduction Hypothesis Methods Results Conclusion

15. Acquired R. salmoninarum samples ATCC33209 – Chinook Salmon Leaburg, Oregon, USA (1974) mt239 – Atlantic Salmon (Salmo salar), Scotland 684 – Brown Trout (Salmo Trutta), Norway Clinical samples (A and B) – Chinook Salmon (Onchorhyncus tschawytscha) Bonneville Fish Hatchery, Oregon Introduction Hypothesis Methods Results Conclusion

16. Genes selected for analysis Introduction Hypothesis Methods Results Conclusion

17. Sequencing results dppD/F (F) – 5’ GGC.CGC.GAT.GTG.TCG.ATG.GTT.TT dppD/F (R) – 5’ GCC.CAA.GTG.CGG.CAC.TGC.AGC C. Syn. (F) – 5’ GCG.CGG.AGA.GAA.GTT.CTT.GTG C. Syn. (R) – 5’ CGA.TGC.GGT.GCG.ACG.TTT.T tetP (F) – 5’ GCC.TAG.CGA.CGC.AAA.AG tetP (R) – 5’ ATA.GTG.ACT.AAG.CAA.TCG.GTG FBP (F) – 5’ CTG.ACG.CCA.ACG.GTA.AAT.ACA.CC FBP (R) – 5’ GGC.GGA.TTC.TCA.ACA.CTC.ACG Introduction Hypothesis Methods Results Conclusion

18. Chemically competent E.coli cell

19. X 60 samples

20. Center for Genome Research and Biocomputing @ OSU

21. Results Introduction Hypothesis Methods Results ConclusiontetPdppD/F c. syn FBPA+++ B++++ mt239++++ 684+++ ATCC++++

22. Sequencing results Introduction Hypothesis Methods Results Conclusion

23. Sequencing results Introduction Hypothesis Methods Results Conclusion

24. Sequencing results Introduction Hypothesis Methods Results Conclusion

25. Conclusions Sequence suggests significant reductive evolution Transition to pathogenic lifestyle No evidence that the ATCC 33209 strain is irrepresentative of what is found in nature Introduction Hypothesis Methods Results Conclusion

26. Conclusions Mutations represented in the sequence genome are not a function of culture in vitro Introduction Hypothesis Methods Results Conclusion

27. Questions?