Methylation of foreign DNA overcomes the restriction barrier of Flavobacterium psychrophilum and allows efficient genetic manipulation

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms F. psychrophilum uses to cause disease are poorly understood. Despite considerable effort, most strains of F. psychrophilum have resisted attempts at genetic manipulation. F. psychrophilumrestriction-modification (R-M) systems may contribute to this resistance. Restriction endonucleases (REases) rapidly degrade nonself DNA if it is not properly methylated by their cognate DNA methyltransferases (MTases). We used comparative genomics to show that R-M systems are abundant in F. psychrophilum and that strain-specific variations partially align with phylogeny. We identified two critical type II R-M systems, HpaII-like (FpsJI) and ScrFI-like (FpsJVI), that are conserved in most of the sequenced strains. Protection of foreign DNA against HpaII and ScrFI was achieved by expression of the MTases M.FpsJI and M.FpsJVI in Escherichia coli. Furthermore, deleting the two REase genes from F. psychrophilum resulted in efficient conjugative DNA transfer from E. coli into the otherwise genetically intractable F. psychrophilum strain CSF259-93. This allowed us to construct a CSF259-93 mutant lacking gldN, a core component of the type IX protein secretion system. The pre-methylation system developed in this study functions in all tested F. psychrophilum strains harboring HpaII-like and ScrFI-like REases. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines or other measures to control BCWD