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

UNLABELLED

causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms uses to cause disease are poorly understood. Despite considerable effort, most strains of have resisted attempts at genetic manipulation. restriction-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 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 . Furthermore, deleting the two REase genes from resulted in efficient conjugative DNA transfer from into the otherwise genetically intractable strain CSF259-93. This allowed us to construct a CSF259-93 mutant lacking , a core component of the type IX protein secretion system. The pre-methylation system developed in this study functions in all tested 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.

IMPORTANCE

Bacterial cold-water disease (BCWD) caused by is a problem for salmonid aquaculture worldwide, and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most strains resist DNA transfer, thus limiting genetic studies of their virulence mechanisms. restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here, we used DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into nine diverse strains that had previously resisted DNA transfer. Using this approach, we constructed a gene deletion mutant that failed to cause disease in rainbow trout. Further genetic studies could help determine the molecular factors involved in pathogenesis and may aid development of innovative BCWD control strategies.