Construction Of Gene Knockout Strains And Phenotypic Analysis Of Vibrio Alginolyticusnce Virulence Strain

Vibrio alginolyticus, one of the normal flora of the marine environment, is a serious bacterial pathogen to aquatic animals and humans. However, the virulence gene system and the pathogenic mechanism are still unknown. Recently, compared genomics analysis and genetic mutation technique were introduced to find out the virulence genesof V. alginolyticus. In this paper, four full-length genes, obtained by SON-PCR and included five genes (motB、prs2、vasD、vasC、ggdef), were regarded as the target genes for exploring their function. motB encodes flagellar motor protein MotB; prs2encodes phosphoribosyl pyrophosphate synthetase (PRS2); vasD encodes type VI secretion lipoprotein VasD; vasC encodes type VI secretion forkhead associated (FHA) domain protein; ggdef encodes GGDEF domain protein. In order to determinate the role of these five genes in V. alginolyticus, we obtained above the five genes’ deletion fragments by SOE PCR and respectively connected to suicide vectors to construct five recombinant plasmids (pDM4-△wotB、pDM4-Aprs2、pDM4-AvassD、pDM4-AvasC and pDM4-Aggdef). Then, the five recombinant plasmids were transformed into the donor bacterium and then transformed into the V. alginolyticus by conjugal transfer to obtain the gene deletion mutants strains of motB, prs2, vasD, vasC and ggdef, which were AmotB、Aprs2、AvasD、AvasC and Aggdef.The growth curve, swimming ability, flagellation ability, biofilm formation ability and the virulence of each gene deletion mutant strain was determined by a strandard method. The results show that the growth curve of each gene knockout mutant strain is consistent with that of the wild type strain, being late logarithmic phase after12h cultured; on the0.3%(w/v) ager plate after16h cultured in the30℃, the diameters of swimming circle of gene deletion mutant strains were6.26mm-16.24mm and exhibited significant difference (P<0.01) to the that (17.61mm) of the wild strain;△prs2,△vasD,△ggdef and the wild strain could form a flagellum but△motB and△vasC not;△vasD and Aggdef had not formed biofilm and the biofilm formation ability of△vasC was obviously decreased (P<0.01). The result of the infection experiment showed that the virulence of wild strain, Aprs2,△vasD, and△vasC to Litopenaeus vannamei was stronger than those of△motB and△ggdef. Compared with the wild strain, the death time and mortality of△motB and△ggdef of is later and lower.In summary, motB involved in the flagellum assembly and associated with the swimming ability; prs2associated with the swimming ability; vasC and vasD related to biofilm formation and swimming ability, and vasC may also involved in the flagellum formation; ggdef associated with the swimming ability, flagellum formation and biofilm formation. We inferred that the five genes mentioned above were involved in assembly of flagellum, swimming ability, and/or biofilm formation, which means that they are important virulence genes.