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

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

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

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

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.

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

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

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

MAUVE output : courtesy Dr. Greg Wiens, NWFSC

Sequencing results Introduction Hypothesis Methods Results Conclusion

VS.

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

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

Genes selected for analysis Introduction Hypothesis Methods Results Conclusion

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

Chemically competent E.coli cell

X 60 samples

Center for Genome Research and OSU

Results Introduction Hypothesis Methods Results ConclusiontetPdppD/F c. syn FBPA+++ B++++ mt ATCC++++

Sequencing results Introduction Hypothesis Methods Results Conclusion

Sequencing results Introduction Hypothesis Methods Results Conclusion

Sequencing results Introduction Hypothesis Methods Results Conclusion

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

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

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