Establishment of an inducible system in Vibrio vulnificus

• Main Authors: Ming-Hsiang Chen, 陳銘祥
• Other Authors: Lien-I Hor
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/18481873348482127141

碩士 === 國立成功大學 === 微生物及免疫學研究所 === 94 === 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.