中文摘要
　　創傷弧菌為棲息在海水環境中之細菌，會造成人類嚴重的傷口感染和致命性的敗血症。本實驗室先前的研究發現在創傷弧菌內有一個調控子SmcR，具有調控蛋白酶基因vvp以及細胞溶解毒素的功能，而進一步利用DNase I footprint analysis分析發現SmcR與vvp 啟動子區域結合的位置涵蓋了49個鹼基對 (-35~-83)，且在此區域有一不完美之inverted repeats序列存在而當中有14個鹼基對 (spacer) 將它們隔開。為了了解這些inverted repeats序列與SmcR結合的關聯性，我們利用site-directed mutagenesis的方式，在vvp啟動子此區域，置入七個不同的點突變，並在其下游與lacZ融合，再將含此構造的DNA片段插入一自殺質體，然後藉由創傷弧菌染色體上之lacZ與質體上的lacZ部分，進行homologous recombination使質體插入染色體中，而產生一系列的突變菌株。從測試B-galactosidase活性的結果顯示，四個發生在vvp啟動子區域之inverted repeats上的點突變，造成vvp的表現活性下降；相反的，其餘三個點突變，其中一個在inverted repeats區域，兩個在spacer中之菌株，其vvp的表現不受影響。然而，從EMSA實驗觀察這些具有點突變的vvp啟動子區域與SmcR作用發現，不論vvp啟動子區域攜帶那些點突變，都具有與SmcR結合的能力。因此，我們推測在創傷弧菌vvp啟動子上之inverted repeats序列與SmcR之間的結合以及基因的表現，可能需要透過其它因子的參與來達成。

　　另一方面，我們發現在SmcR上第39至59個之胺基酸與LuxR家族中的之helix-turn-helix (HTH) DNA-binding domain 具有很高的相似性。為了了解SmcR HTH區域在與vvp啟動子區域之間的結合上所扮演的角色，我們建構了攜帶不同smcR片段的質體。很意外的，當我們在帶有vvp啟動子區域與lacZ 融合片段且smcR缺失的菌株中由質體表現smcR63-205以及 smcR101-205時，雖然它們在HTH區域有缺失，但仍具有活化vvp啟動子的功能；然而在表現smcR32-205以及smcR1-99時，即使它們HTH區域還存在，但卻喪失活化vvp啟動子的能力。另外，我們也將不同的smcR片段在vvp啟動子區域上含有不同點突變的菌株中表現。結果發現，在表現smcR63-205時，lacZ在不同突變株內的表現模式與表現完整SmcR的菌株相同；而在表現smcR101-205時，LacZ的活性在不同的點突變間並無明顯的差異。我們進一步純化出不含HTH區域之蛋白質SmcR63-205，利用EMSA試驗此蛋白質是否能與帶有不同點突變之vvp啟動子區域之結合，結果發現SmcR63-205與SmcR一樣，皆能與帶有不同點突變之vvp啟動子結合。從以上的結果我們推測，SmcR C端的區域可能較HTH區域在與DNA間的結合以及調控基因的表現上扮演更重要的角色。

Abstract

　We have found previously that SmcR, a transcriptional activator, is involved in the regulation of the metalloprotease (vvp) and cytolysin genes in Vibrio vulnificus, a marine bacterium causing wound infections and fatal septicemia. The putative SmcR-binding site in the vvp promoter region has been identified by DNase I footprint analysis, in which an inverted repeat sequence with a spacer of 14 bp was found. To determine the involvement of this inverted repeat region in binding with SmcR and transcriptional activation of vvp, seven point mutations at different positions in this region were created. The promoter mutations together with a downstream lacZ gene (Pvvp-lacZ) that served as the reporter were cloned individually into a suicide plasmid and subsequently introduced into V. vulnificus chromosome via homologous recombination between the lacZ gene in the plasmid and that in the chromosome. By assaying the B-galactosidase activity in these strains, we found that four mutations that were located in the inverted repeats resulted in significant decreases in vvp promoter activity. However, another mutation in the inverted repeat and two mutations in the spacer did not affect the vvp promoter activity. We further examined the binding of SmcR to vvp promoter with the various mutations in this region by electrophoresis motive shift assay (EMSA). There was little difference between the wild type vvp promoter and the mutated ones, even those showing reduced vvp promoter activity, in binding with SmcR. One explanation for this inconsistency is that other factors may be involved in binding with vvp promoter region and regulation of vvp expression.

　On the other hand, the amino acid sequence from residues 39 to 59 of SmcR showed significant homology to the helix turn helix (HTH) DNA-binding domains of LuxR family. To determine the role of this putative HTH domain in binding with vvp promoter, five SmcR mutants were generated. Surprisingly, when expressed from a plasmid in the strain with a Pvvp-lacZ fusion, SmcR devoid of the HTH domain, namely, SmcR63-205 and SmcR101-205, retained the ability to activate the vvp promoter. On the contrary, those containing the HTH domain, SmcR29-205 and SmcR1-99, did not activate the vvp promoter. Furthermore, when combined with the various vvp promoter mutations, the B-galactosidase expression patterns of SmcR63-205 were similar to those of the intact SmcR. However, for some reason not clear at this moment, the B-galactosidase expression levels of the various vvp promoter mutants were similar to one another in the presence of SmcR101-205. We further examined the binding of SmcR63-205 to the vvp promoter with the various mutations by EMSA, and found that just like the intact SmcR, it bound to all the mutated promoter sequences. These results suggest that the SmcR C-terminal domain may be more important than the HTH domain for binding with and activation of vvp promoter.

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