Construction And Character Analysis Of Streptococcus Iniae CpsJ Knock-out Mutant

Streptococcus iniae (used name Streptococcus shiloi) is a major pathogen of aquaculture fishes. Recent years, outbreaks of fish diseases resulted in S.iniae infection were frequently reported, which caused numerous financial loss. The capsule is an essential virulence factor, which is consist of many polysaccharides. The operon conduct the synthesis of S.iniae capsule are very large, which contains 21 open reading frames at all. cpsJ is an important gene of the capsule synthesis operon, it encodes one UDP-Glc epimerase, which catalyse the composing of precursors of heteropolysaccharide assembling the capsule.This study knocked out the cpsJ gene induced by temperature sensitive plasmid pJRS233, replacing the cpsJ with chloramphenicol resistant gene and then constructed knockout mutant ΔcpsJ. Compared the mutant and wild type strains in gram stain observation, colonies observation, grow rate determination, adherence and invasion test, whole blood killing test and challenge test to preliminarily investigate the fuction of cpsJ. Results showed compared with wild type, the mutant formed longer chain length and smaller colonies at the same culture conditions; In the grow rate test, OD600 value of mutant broth when the grow curve run up to plateau was 0.42, while the wild type broth was 1.28; in the adherence and invasion test, the adherence rate of mutant to epithelioma papulosum cyprini was 25.72%, the wild type’s was 1.35%; the invasion rate of mutant is 1.72%, the wild type’s was 0.34%; in whole blood killing test, the survival rate of mutant was 45.94%, wild type’s was 137.39%; in challenge test, survival rate of fishes infected with mutant broth by intraperitoneal injection was 75%, survival rate of fishes infected with wild type broth was 50%.The result illustrated that the AcpsJ mutant has weaken virulence than the wild type strain. The cpsJ gene play a essential role in capsule synthesis.