Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.
Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli.
复制标题
DOI：
10.1016/j.gene.2011.01.006
复制
发表时间：
2011-06
期刊：
影响因子：
3.5
通讯作者：
Takeshi Shimizu;Y. Ohta;Hiroyasu Tsutsuki;M. Noda
中科院分区：
文献类型：
--
作者：
Takeshi Shimizu;Y. Ohta;Hiroyasu Tsutsuki;M. Noda
利用发光杆菌（Photorhabdus luminescens）luxCDABE基因构建了一种新的染色体-质粒杂交生物发光报告系统（C-P报告系统），用于真实的实时监测肠出血性大肠杆菌（EHEC）中滋贺毒素1（Stx 1）和滋贺毒素2（Stx 2）的表达。已克隆发光假单胞菌的luxCDABE基因并将其分成luxCDAB盒和luxE基因。将无启动子的luxE基因以单拷贝形式从肠出血性大肠杆菌染色体上的stx 1和stx 2的下游引入，并将其他luxCDAB基因在多拷贝数表达质粒上表达到相同的细胞中。这些Stx 1-和Stx 2-生物发光报告菌株表达生物发光到细菌细胞中时，无启动子的luxE基因的表达分别响应于stx 1和stx 2的启动子活性而表达。生物发光的表达水平与Stx 1和Stx 2生物发光报告菌株中Stx 1和Stx 2的产生水平相同，并且这些菌株以与亲本EHEC菌株相同的相应水平产生两种Stx。使用这些报告菌株，我们研究了在EHEC中Stx 1和Stx 2的表达谱。我们发现，在肠出血性大肠杆菌中，Stx 1和Stx 2的产生在与肠上皮细胞和巨噬细胞接触后增强。然而，在这些条件下，Stx 1和Stx 2在EHEC中的表达谱彼此不同。因此，这些结果表明，该C-P报告系统是有用的，用于确定细菌的基因表达谱。
A novel chromosome–plasmid hybrid bioluminescent reporter system (C–P reporter system) utilizing Photorhabdus luminescens luxCDABE genes has been constructed to monitor the expression of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) in enterohemorrhagic Escherichia coli (EHEC) in real time. The luxCDABE genes of P. luminescens have been cloned and divided into a luxCDAB cassette and a luxE gene. A promoter-less luxE gene introduced downstream from stx1 and from stx2 on EHEC chromosomes in single copies, and other luxCDAB genes were expressed on a multicopy number expression plasmid into the same cells. These Stx1- and Stx2-bioluminescent reporter strains expressed bioluminescence into bacteria cells when the expression of the promoter-less luxE gene was expressed in response to the promoter activity of stx1 and stx2, respectively. The expression levels of bioluminescence were identical to the production levels of Stx1 and Stx2 in the Stx1- and Stx2-bioluminescent reporter strains, and these strains produced both Stxs at the same respective levels as those of the parent EHEC strains. Using these reporter strains, we examined the profiles of Stx1 and Stx2 expression in EHEC. We found that production of both Stx1 and Stx2 in EHEC was enhanced upon contact with intestinal epithelial cells and within macrophages. However, the expression profiles between Stx1 and Stx2 in EHEC were different from each other under these conditions. Thus, these results suggested that this C–P reporter system is useful for determining the gene expression profile of bacteria.