Development of Techniques for the Genetic Manipulation of the Fish Pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the phylum Bacteroidetes, is the causative agent to infect salmonid fish with the bacterial cold-water disease (BCWD) resulting substantial economic losses in aquaculture. Infected fish with BCWD appeared to have tissue eruptions damaging their gills and fins. Efficient way to wipe out possible infections caused by F. psychrophilum was not still discovered. Current control of the outbreak relies on standard antibiotic administrations. The aim of this research is to advance current genetic manipulation techniques including DNA transfer in different F. psychrophilum strains. This will allow us to identify virulence genes, understand the pathogenicity of F. psychrophilum, and eventually develop live attenuated vaccines that can be used in aquaculture to effectively prevent diseases caused by this bacterium. Twenty-seven of F. psychrophilum strains (#1 to #27) were obtained from the U.S. Department of Agriculture. Conjugation was used as a primary DNA transfer method to transfer pCP11 plasmid (Replicative Plasmid) from Escherichia coli S17-1 -pir to F. psychrophilum cells. pCP11 contains an erythromycin resistance gene as the selection marker, which allows us to evaluate conjugation efficiency. We have optimized conjugation and growth conditions over the course of the research for 11 strains (1-5,12,14,15,22,25,26). pCP11 was successfully transferred to strains #15 and #26 confirmed by Polymerase Chain Reactions using primers spanning the erythromycin resistance gene ermF. We are applying a sacB-mediated strategy by using repetitive counterselection and conjugation to isolate spontaneous mutants of THCO2-90 (#26) that are more efficient in conjugation. Two spontaneous mutants FP107, FP108 were isolated from #26 strain by using sacB -mediated strategy