| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 102 === Vibrio vulnificus is a marine bacterial species causing fulminant septicemia and severe wound infections in humans. Although a number of virulence factors have been identified, how the virulence of this organism is regulated remains mostly unknown. Our laboratory has fortuitously isolated a spontaneous, noncytotoxic and avirulent V. vulnificus mutant, NY303, from a vplvvhA mutant of the wild-type strain, YJ016. This mutant was also defective in motility/chemotaxis, and showed altered outer membrane protein profile. In this study, the genome sequence of mutant NY303 determined by next-generation sequencing was compared with the published whole genome sequence of strain YJ016, and four single nucleotide polymorphisms were identified in NY303. The mutation leading to the phenotype of NY303 was mapped to the gene encoding leucine-responsive regulatory protein (lrp) by characterizing the specific gene-knockout mutants. To identify the genes regulated by Lrp, we compared the transcriptomes of YJ016 and a lrp mutant that were analyzed by RNA-seq with the 4 h bacterial culture, and we identified 416 and 212 genes that were apparently (〉 2-fold change) down- and up-regulated, respectively, in the lrp mutant. Among the 21 regulator genes whose mRNA levels were shown to be altered by RNA-seq, 10 were confirmed by real-time RT-PCR. None of the mutants disrupted in these genes were shown to be different from strain YJ016 in cytotoxicity and virulence in mice. To perform genome footprinting by high-throughput sequencing (GeF-seq) for identification of the promoters directly bound by Lrp, I have also constructed a V. vulnificus strain expressing an Lrp with a His6 tag to be used in this experiment.
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