Pre-Methylation of Foreign DNA Improves Conjugation Efficiency in the Fish Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a known fish pathogen causing bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS). The diseases cause tissue damage and tail rot in young and adult fish. The issue is prevalent in fisheries in the Pacific Northwest and the diseases generally affect salmonids. Genetic manipulations in F. psychrophilum CSF 259-93, the most problematic strain in rainbow trout fisheries in U.S., is scarce due to strain’s ability to use restriction modification systems to destroy foreign DNA. Pre-methylation of foreign DNA has been shown to improve the efficiency of DNA transfer via transformation in other bacteria. The goal of this research project is to pre-methylate foreign DNA (plasmids) by insertion of three methyltransferase encoding genes to the plasmid pACYC184 individually and use of the constructed plasmids (pSS01, pSS02, and pSS03) to methylate the tested plasmid pCP11 in E. coli, followed by conjugation to transfer pCP11 to F. psychrophilum CSF 259-93. Our first conjugation trial showed that two of the constructed plasmids (pSS01 and pSS02) were able to protect pCP11, with 33 and 5 F. psychrophilum colonies containing pCP11 obtained, respectively. The success to improve the conjugation efficiency in F. psychrophilum CSF 259-93 would allow us to further construct deletion mutants with decreased virulence, which can be potentially used in creating live attenuated vaccines to prevent the BCWD and RTFS diseases, and thus reduce the economic losses in aquiculture