title: Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.
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 (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB 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.
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Xiao Lin
authors.initials: XL
authors.lastname: Zhang
authors.affiliation: College of Fisheries, Jimei University, Xiamen, Fujian Province, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Zhi Xin
authors.initials: ZX
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Hai Xia
authors.initials: HX
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McBride
authors.affiliation: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, China pinnie@ihb.ac.cn.
authors.forename: Pin
authors.initials: P
authors.lastname: Nie
mesh_terms: D000818:Animals; D002347:Carps; D002808:Chondroitin Lyases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D045826:Flavobacteriaceae Infections / Q000382:microbiology / Q000473:pathology / Q000662:veterinary*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D014774:Virulence; D037521:Virulence Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D037521:Virulence Factors; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases