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首页> 外文期刊>Applied Microbiology >Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens
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Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens

机译：产气荚膜梭菌中可控制基因表达的乳糖诱导型启动子系统的构建和表征

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Clostridium perfringens is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with C. perfringens , a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an Escherichia coli-C. perfringens shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene bgaL ( bgaR -P _(bgaL) ). To measure transcription at the bgaL promoter in pKRAH1, the E. coli reporter gene gusA , encoding β-glucuronidase, was placed downstream of the P _(bgaL) promoter to make plasmid pAH2. When transformed into three strains of C. perfringens , pAH2 exhibited lactose-inducible expression. C. perfringens strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene bglR , encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the yfp - pilB gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in C. perfringens . Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a pilC mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.
机译：产气荚膜梭状芽胞杆菌是革兰氏阳性厌氧性病原体，可导致人类和动物的许多疾病。尽管存在一些与产气荚膜梭菌一起工作的遗传工具，但目前缺乏严格调控的诱导型启动子系统。因此，我们构建了一个基于质粒的启动子系统，当添加乳糖时，该系统提供了调控的表达。该质粒（pKRAH1）是大肠杆菌-C。产气荚膜梭菌载体，其包含编码转录调节子BgaR的基因，以及基因bgaL（bgaR -P _（bgaL））上游的启动子。为了测量pKRAH1中bgaL启动子的转录，将编码β-葡萄糖醛酸苷酶的大肠杆菌报告基因gusA置于P_（bgaL）启动子的下游，以制备质粒pAH2。当将其转化为三种产气荚膜梭菌菌株时，pAH2表现出乳糖诱导的表达。产气荚膜梭状芽孢杆菌菌株13，是一种常用菌株，具有内源性β-葡萄糖醛酸苷酶活性。我们突变了基因bglR，该基因编码假定的β-葡萄糖醛酸苷酶，并观察到内源性活性降低了89％，而没有乳糖。用于调节基因表达的系统和具有低β-葡萄糖醛酸苷酶活性的13菌株突变体的这种组合是研究重要致病细菌中的基因调节和蛋白质表达的有用工具。我们使用该系统表达yfp-pilB基因，该基因由黄色荧光蛋白（YFP）编码基因与组装的ATPase基因融合而成，该基因参与了产气荚膜梭菌IV型菌毛依赖性滑行运动。在野生型菌株中的表达表明，YFP-PilB主要定位于细胞的两极，但是在pilC突变体中，YFP-PilB定位在整个细胞中，这表明PilB的极性定位需要膜蛋白PilC。

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