Development of Genetic Manipulation Techniques for the Fish Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a known fish pathogen, causing bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), predominantly in salmonids. The disease causes tissue damage and tail rot in young and adult fish. The issue is prevalent in fisheries in the Pacific Northwest and the treatments often entail the use of antibiotics. Genetic manipulations in F. psychrophilum are scarce, due to most of the strains’ ability to destroy foreign DNA via restriction enzymes. The virulence mechanisms of F. psychrophilum are not well understood. Lack of genetic manipulation tools in F. psychrophilum has also hampered the development of live attenuated vaccines to prevent BCWD and RTFS in aquaculture. In this study, we identified two methyltransferases, HpaIIM and ScrFIM, in F. psychrophilum strain CSF259-93, the most problematic strain in rainbow trout fisheries in the United States. A helper plasmid pSS05 carrying both HpaIIM and ScrFIM encoding genes was constructed and used to pre-methylate the target DNA to improve the efficiency of DNA transfer by conjugation. By using pSS05 in combination with a previously developed markerless deletion system, we constructed a mutant lacking gldN, a core component of the type IX secretion system. The gldN deletion mutant was deficient in secreted proteolytic activity, colony spreading, single cell motility, and virulence on rainbow trout. The pre-methylation system developed in this study also functions in other F. psychrophilum strains. It may assist identification of virulence factors and development of live attenuated vaccines in multiple F. psychrophilum strains