許多對於細菌生長而言是必須的蛋白質，往往在過量表現時反而對宿主有害，而可能必須利用誘導式表現載體來選殖此類基因。創傷弧菌為一嗜鹽性弧菌，屬於人類伺機性病原菌。我們實驗室曾研究一個廣泛存在於革蘭氏陰性菌外膜上的蛋白質TolC，其參與運送許多分子如抗生素、細胞毒素等。若破壞其基因，會降低創傷弧菌對膽鹽的耐受性、紅黴素的抗性、HEp-2細胞的毒性以及老鼠的致病性。但當我們使用一個broad-host-range的質體pJRD215攜帶tolCvv進行補償實驗時，發現無法於大腸桿菌中成功的選殖到在tolCvv沒有突變的重組質體，因而無法回復突變株所喪失的功能。我們推測，以此質體選殖tolCvv時，可能由於過量表現TolCvv而對宿主有害，因此決定建立一個可在大腸桿菌與創傷弧菌中複製的誘導式表現載體，來構築tolCvv補償性質體。在本研究中，我們將可受L-arabinose誘導的DNA片段（araC-PBAD）插入pJRD215的衍生質體中，最先得到了一個在araC基因上有三個點突變的質體pMH01，隨後在縮短了選殖的DNA片段後，得到了一個在araC不具突變的質體pMH02。當我們使用pMH01選殖創傷弧菌vvn與lacZvv基因，發現兩者皆可以成功的送入創傷弧菌中，且可透過arabinose的添加而增加基因的表現量。故我們進一步使用此載體建構tolCvv補償性質體，第一次成功得到了正確的重組質體，但於大腸桿菌中發現，TolCvv在未誘導時即有基礎表現量，使大腸桿菌對膽鹽有耐受性；然而，當以arabinose誘導TolCvv表現時，大腸桿菌的生長便會受到抑制，也減低了此菌株對膽鹽的耐受能力。將此質體送入tolC突變株後發現，TolC仍有基礎表現量，也使得在未添加arabinose時，TolC補償株完全回復了突變株所喪失的功能，確定了TolC在創傷弧菌對小鼠的毒力所扮演的角色。另一方面，我們測試了在araC沒有突變的質體pMH02誘導基因表現的能力，發現其誘導能力的確較pMH01佳，但當使用此質體表現TolCvv時，基礎表現量仍能回復突變株對膽鹽的耐受性。研究結果顯示pMH01與pMH02兩個誘導性表現載體，將可選擇用以在創傷弧菌大量表現所轉殖的蛋白質，或使用於進行補償實驗。

Many genes that encode essential products for bacterial growth may exert toxicity to the host cells when overexpressed. To study this kind of genes, the inducible systems are more desirable. In our laboratory, we have been focusing on the identification of virulence factors in Vibrio vulnificus, which is a gram-negative halophilic marine bacterium causing severe wound infection and septicemia. One of our targets was the tolC gene, which encodes an outer membrane protein for transport of a variety of molecules. We have previously isolated a TolC-deficient mutant strain, MW021, which is attenuated in resistance to bile and erythromycin, non-cytotoxic to the HEp-2 cells, and much less virulent in mice. However, when we tried to clone the entire tolC gene into E. coli to further complement the mutant, we were not able to obtain a clone without mutation in tolC. This suggests that overexpression of TolCvv might be toxic for the host cells. Therefore, we intended to establish an inducible system in V. vulnificus to solve this problem. In this study, I have cloned the araC-PBAD DNA fragment, contained the PBAD promoter together with the regulator AraC whose actively is controlled by L-arabinose, into a derivative of the broad-host-range plasmid, pJRD215. The first plasmid we obtained, designated pMH01, was found to harbor three mutations in araC. We then cloned a short DNA fragment and finally obstained a construct, pMH02, that contained no mutation in araC. pMH01 was shown to express vvn (V. vulnificus nuclease gene) and lacZvv cloned downstream of the pBAD promoter in the presence of arabinose in a dose-dependent manner. I then cloned tolCvv into pMH01, and for the first time that this gene can be readily cloned into a plasmid without any mutation. We found that TolCvv was expressed at a basal level in E. coli DH5α without induction by arabinose and the basal level expression could make DH5α resistant to bile. However, addition of arabinose to induce TolCvv resulted in growth retardation and reduced resistance to bile of the host cell. When pMH01-tolCvv was transferred to a ΔtolC V. vulnificus mutant, MW021 by conjugation. The expression of TolCvv in the outer membrane, and the defects of the mutant in resistance to bile salt and erythromycin, cytotoxicity to the HEp-2 cells, and the virulence to mice was restored without arabinose induction. On the other hand, we found that compared to pMH02, the expression of gene cloned downstream of PBAD in pMH02 was induced to a much higher level by arabinose. However, the expression of tolCvv from pMH02 also restored the ability of growth on TCBS agar plate without arabinose induction. These results suggest that pMH01 and pMH02 can be selectively used for overexpression of the cloned gene or performing the complementation experiments.

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