在大腸桿菌DH5α中由吲哚引起生物膜形成所必需的蛋白質及其功能

Abstract

ABSTRACT 之前我們已經以二維膠片的分析技術發現當大腸桿菌的重要病原菌O157被處以抗生素枯草桿菌素時會經由EvgS/EvgA雙蛋白質系統引起Tryptophanase基因的表現，並進而分解Tryptophan引起Indole的產生，接著引起生物膜的形成以及一系列相關蛋白質的表達。 在這篇論文中我們以被跳躍子基因破壞的大腸桿菌DH5α突變株作為研究材料，進一步研究哪些基因為由Indole引起大腸桿菌形成生物膜所必須，結果發現當yhiP、cyaA、trkA、phr、fimE突變時大腸桿菌無法為Indole引起生物膜的形成。經由互補實驗我們也進一步證實這些基因確實為Indole引起大腸桿菌形成生物膜所必須。我們也利用報導基因分析再進一步證實Indole確實能引起這些基因的表現，其中又以yhiP、cyaA活性增加至200% 以上最為明顯﹔另外經由互補實驗和報導基因分析我們也部分的闡明了Indole與這些基因上游的轉錄調控子之間的關聯﹔以及各種常見抗生素對這些基因表現的影響。除此之外，在這篇論文中也以butanol等實驗材料分析了這些基因對大腸桿菌形成生物膜以對抗逆境的貢獻。 另外由於我們之前發現了在大腸桿菌中蛋白質B0363能為autoinducer (indole)引發表現且為金黃色葡萄球菌IcaA的類似物，因為它被預測是被分泌至細胞膜外側的部分水溶性蛋白質較易於分析，所以在這篇論文中我們也表達純化了這個蛋白質並做了相關的活性分析並且得到了高純度且具有活性的蛋白質。

ABSTRACT By using the two-dimensional gel method, we already discovered that when E. coli O157 was exposed to bacitracin, the expression of tryptophanase gene was induced through the EvgS/EvgA two-component system. Tryptophanase then converted tryptophan to indole,which caused biofilm formation as well as a series of related protein expression. In this study, we screened the E. coli DH5α transposon library mutants to find out which genes are crucial for indole induced biofilm formation. These results show that when yhiP, cyaA, trkA, phr, and fimE genes are disrupted by transposon, indole can not induce these mutant strains to form biofilm. By using complementary assays, we confirmed that these genes are truly necessary for Indole induced biofilm formation in E. coli DH5α. Besides, we also used the reporter gene analysis method to further confirm that indole can cause these genes expressions with the promoter activity of yhiP, cyaA increased up to 200%. Moreover, we also clarified the relationship between indole and expressions of some transcription factor genes upstream to these genes, as well as the influences of each kind of common antibiotic to the expressions of these genes. In addition, we also used other experiment material such as butanol to test if expressions of these genes also contribute to the resistence to the adverse circumstance when E. coli formed biofilm. Besides, the expression of B0363 can be induced byindole in E.coli. B0363 is an analogous protein of IcaA of staphylococcus, which is secreted to outside of cell after transcription. I also expressed and purified this protein successfully and made sure it has catalytic activity.