Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

Abstract

ABSTRACT Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (P ompA ) was fused to sacB to construct a counterselectable marker for F. columnare . F. columnare carrying P ompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying P ompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB , were deleted. The Δ cslA and Δ cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (Δ cslA Δ cslB ) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G 4 and of the chondroitin lyase-deficient Δ cslA Δ cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes .