創傷弧菌屬於生活於海洋的細菌，感染人類往往造成猛爆型敗血症與嚴重傷口感染。儘管先前的研究已鑑定出各種毒力因子，包括細胞毒殺能力、細胞貼附能力、莢膜等，然而這些毒力因子是如何被調控大部分還未知。我們實驗室之前意外地在構築野生株YJ016 的vplvvhA 突變株時分離到一株沒有細胞毒性及小鼠毒力的自發性突變株NY303，其運動性/趨化性也有缺陷，並且顯示出外膜蛋白質的表現發生了變化，這些現象顯示此自發性突變可能發生在某個全面性調控子的基因上。本研究使用次世代定序技術(NGS) 將此突變株進行全基因體定序，再將之與已發表之YJ016 全基因體序列進行比對。最終確認了四個單一核苷酸變異(SNPs)，它們都位在分別被註解為leucine-responsive transcriptional regulator (lrp), ABC transporter permease, UDP-glucose pyrophosphorylase 以及hypothetical protein 的編碼序列中。我們進一步分離lrp 突變株，發現它與當初的自發性無毒力之突變株有諸多相似，包括細胞毒殺力、運動性/趨化性與對小鼠的毒力都有缺陷，推測lrp 可能涉及創傷弧菌毒力之調控。我們以RNA-seq 分析野生株與lrp 突變株在LB 培養基生長4 小時候的全轉錄體，發現 lrp突變株與野生株相比，有416 和212 個基因的表現量分別有明顯 (≧ 2 倍改變) 的下降以及上升的情形。在這些基因之中，為了縮小被Lrp 所調控之基因的範圍，我選擇了調控基因，並且利用real-time RT-PCR 確認了10 個調控基因的表現有受到明顯的影響。然而，進一步分離這些基因的突變株並分析其細胞毒性和對小鼠的毒力，並沒有任何一株突變株與野生株有明顯的不同。為了鑑定Lrp 直接結合上的啟動子區域，我在創傷弧菌中構築了Lrp 標定His6-tag 的菌株，準備用以進行基因體足跡結合高通量定序(GeF-seq) 之實驗。透過RNA-seq 與Gef-seq 這兩種方法，希望未來可以了解創傷弧菌的毒力調控網絡。

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 vplvvhA 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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