Abstract:The phenotypic characteristics of Vibrio anguillarum are related to the pathogenicity of the bacteria, such as swimming motion, ability of membrane formation, and extracellular protease production. To identify the genes affecting phenotypic changes in V. anguillarum, this study used transposon mini-Tn10 (pLOF/Kana) to construct a library of V. anguillarum M3 mutant strains and to screen the strains and related genes that affect phenotypic changes. It is proved that there is a certain correlation between mutants causing these phenotypic changes and the virulence. Mutations of 1152 strains of M3 mutant library were screened, and mutant strains with significant changes in swimming ability (strain 6G_1), casein enzyme activity (strains 5A_11, 7B_12, and 7E_12), gelatin enzyme activity (strain 7H_1), and biofilm formation ability (strains 5E_2, 6A_2, and 6E_12) were noted. Further analysis revealed that a phosphodiesterase-related gene mutation caused increased swimming capacity (P<0.05), leuD, rseB, and thiQ mutations caused a significant decrease in caseinase activity (P<0.05), and potD mutations caused a significant decrease in gelatinase activity (P<0.05). Moreover, mutations in leuO, ilvH and grpB resulted in a significant decrease in the ability to form bacterial membranes (P<0.05). Moreover, we observed a virulent infection in these mutant strains, which showed that LD50 of wild type M3 was 2.04 times higher than that of 6G_1 and the virulence was relatively increased. Additionally, 5A_11, 7B_12, and 7E_12 LD50 were 2.96 times, 3.25 times, and 3.36 times higher than that of wild-type M3, respectively. The LD50 with the strain 7H_1 was 1.25 times higher than that of wild M3, and the LD50 with the strains 5E_2, 6A_2, and 6E_12 were 3.34, 4.08, and 1.84 times higher than that of wild M3, respectively. These results lay a foundation for further study on the pathogenic mechanism of V. anguillarum.
