Construction and characterization of the angR mutant of Vibrio anguillarum via insertional inactivation

Vibrio anguillarum strain M3 was isolated from cultured diseased flounder Paralichthys olivaceus. In order to determine whether chromosomal angR gene plays an important role in infecting flounder host, part fragment of a virulence regulatory gene angR from V anguillarum M3 genome was cloned and ligated with suicide plasmids pNQ705 in the present study. The conserved fragment of angR gene was amplified from M3 genome and inserted into suicide plasmid pNQ705, a kind of conditional replicon. The recombinant plasmid was transferred into Vibrio anguillarum M3 strain genomic DNA through bacterial conjugation and homologous recombination. A mutant V anguillarum strain with angR gene mutation was constructed and screened successfully using TCBS medium containing chloramphenicol. PCR identification and sequencing showed that the recombinant plasmid inserted into Vibrio anguillarum genome DNA, as expected design, resulted in insertional inactivation of angR gene. Results of artificial infection experiment showed that virulence of mutant Vibrio anguillarum strain reduced dramatically as compared with the wild strain M3.