創傷弧菌為生長於海洋之革蘭氏陰性菌，會感染人類和鰻魚並造成嚴重的全身性感染症。創傷弧菌依其感染的天然宿主不同，可區分為三種生物型，其中只有生物二型的菌株會感染鰻魚。實驗室先前的研究已證實創傷弧菌生物二型菌株具有一共有的質體，而此質體的存在會促進生物二型菌株在鰻魚血清中的存活，進而對鰻魚造成毒力。我們進一步發現，生物二型野生株CECT4999之毒力質體pR99上的基因vep07為此菌株在鰻魚血清中存活所必需。然而，當此基因單獨送入無pR99的菌株時，雖然可以正常合成出蛋白質，卻不能使該菌株在鰻魚血清中存活，表示在此毒力質體pR99上可能尚有其他的基因參與在其中。因此，在本研究中我以兩種方法來尋找pR99上可以與vep07共同作用以回復細菌在鰻魚血清中存活能力的基因。首先，我將目前已知其他兩個會影響生物二型菌株在鰻魚血清中存活能力的基因vep20與vep71，或是pR99上不同的DNA片段送入已帶有vep07的無pR99的菌株中，發現它們都不能幫助vep07回復細菌在鰻魚血清中的存活能力。之後，我進行pR99上大規模之基因剔除以尋找其上參與對鰻魚血清之抗性的基因。先前已知在pR99中具有一與細菌抵抗鰻魚血清殺菌作用沒有關聯之rtx基因組，因此我在保留rtx基因組與已知的毒力基因vep07、vep20、vep71的情況下，對pR99進行大規模的基因剔除。過程中，我確認pR99上的mazEF毒素─抗毒素基因組與兩處可能之質體複製起始點為pR99穩定維持在菌體中所需。最後，在只保留vep07、vep20、vep71、rtx基因組、mazEF與其中一個質體複製起始點(位在vep27序列上)所在的vep21-vep31 DNA片段之情況下，該突變株仍然可以在鰻魚血清中存活。另外，將vep21-vep31 DNA片段從pR99剔除後，此突變株也能在鰻魚血清中存活。這些結果顯示pR99上大多數基因對生物二型菌株在鰻魚血清中存活是不必要的。

Vibrio vulnificus, a gram-negative bacterium inhabiting the estuarine water, may cause severe systemic infectious diseases in humans and eels. Strains of this bacterial species are classified into three biotypes (BTs) based on their host range and phenotypic traits, and the eel-pathogenic strains are classified as BT2. We have demonstrated that all the BT2 strains carry a common plasmid that is essential for bacterial survival in eel serum and virulence for the eel. We further found that vep07 in the virulence plasmid, pR99, of a BT2 strain, CECT4999, was an important virulence gene associated with the bacterial resistance to eel serum killing effect. However, vep07 alone is not sufficient for conveying bacterial survival in eel serum, suggesting that there may be other gene(s) in pR99 that are associated with the function of Vep07. Two approaches were used to identify the genes complementing with vep07 for survival of BT2 strains in eel serum in this study. In one approach, vep07 was coexpressed with vep20 and vep71, two other genes of pR99 previously shown to be associated with resistance to eel serum, or genes in various DNA fragment(s) from pR99. None was found to enable the plasmid-cured strain to grow in the eel serum, however. In another approach, a large-scale deletion of pR99 was conducted. Plasmid pR99 contains an rtx gene cluster of about 20 kb that is known to be dispensable for resistance to eel serum and two putative replication origins, one between vep03 and vep04 and the other within vep27. This plasmid also contains a TA (toxin-antitoxin) module, mazEF, required for plasmid stability. Deletion of DNA fragments except for vep07, vep20, vep71, the rtx gene cluster, mazEF and vep21-vep31 from pR99 did not impair the bacterial resistance to eel serum. On the other hand, the mutant deleted of vep21-vep31 from pR99 was still able to grow in the eel serum. These results together suggest that most of the genes of pR99 were not essential for the survival of BT2 strains in eel serum.

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