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

Flavobacterium psychrophilum is a Gram-negative bacteria that produce proteins that cause tissue degradation leading to diseases like bacterial cold-water diseases (BCWD) and rainbow trout fry syndrome (RTFS), among salmonids. The manifestation of these infections includes skin ulcers, necrotic myositis, septicemia, and exophthalmia. Bacterial cold-water diseases are prominent when water is 16oC and below. Among salmonids, F. psychrophilum mainly targets Coho salmon, Rainbow trout, Atlantic salmon, and Ayu. Adult fish are highly susceptible to necrotic lesion caused by BCWD whereas young fish is highly susceptible to hemorrhagic septicemia caused by RTFS. Global economic losses in aquaculture are caused due to BCWD and RTFS. Therefore, it is necessary to address the damages caused by infections caused by F. psychrophilum. The goal of this study is to develop genetic manipulation tools that can be used to understand the virulence mechanisms of F. psychrophilum. Specifically, we will construct helper plasmids carrying methyltransferase genes that can methylate and protect foreign DNA during conjugation between E. coli and F. psychrophilum. We have been cloning two methyltransferase genes from F. psychrophilum (acuIM and its downstream gene) into the pACYC184 plasmid. We plan to test if the helper plasmids carrying acuIM and its downstream methyltransferase would protect foreign DNA and increase the conjugation efficiency of F. psychrophilum. These tools will further allow us to generate avirulent or less virulent mutants that can be developed as live attenuated vaccines