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首页> 外文期刊>Applied Microbiology >Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens
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Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens

机译：产气荚膜梭状芽孢杆菌反向选择框内缺失方法的开发与应用

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摘要

Many pathogenic clostridial species produce toxins and enzymes. To facilitate genome-wide identification of virulence factors and biotechnological application of their useful products, we have developed a markerless in-frame deletion method for Clostridium perfringens which allows efficient counterselection and multiple-gene disruption. The system comprises a galKT gene disruptant and a suicide galK plasmid into which two fragments of a target gene for in-frame deletion are cloned. The system was shown to be accurate and simple by using it to disrupt the alpha-toxin gene of the organism. It was also used to construct of two different virulence-attenuated strains, ΗΝ1303 and HN1314: the former is a disruptant of the virRS operon, which regulates the expression of virulence factors, and the latter is a disruptant of the six genes encoding the α, θ, and κ toxins; a clostripain-like protease; a 190-kDa secretory protein; and a putative cell wall lytic endopeptidase. Comparison of the two disruptants in terms of growth ability and the background levels of secreted proteins showed that HN1314 is more useful than ΗΝ1303 as a host for the large-scale production of recombinant proteins.
机译：许多致病梭菌产生毒素和酶。为了促进全基因组毒力因子的鉴定及其有用产品的生物技术应用，我们开发了产气荚膜梭状芽胞杆菌的无标记框内缺失方法，该方法可进行有效的反选择和多基因破坏。该系统包括galKT基因破坏剂和自杀galK质粒，其中克隆了用于框内缺失的靶基因的两个片段。通过使用该系统破坏生物体的α-毒素基因，该系统被证明是准确而简单的。它还被用于构建两种不同的毒力减毒株，HN1303和HN1314：前者是virRS操纵子的破坏物，后者调节毒力因子的表达，后者是编码α的六个基因的破坏物， θ和κ毒素;类梭菌蛋白酶190 kDa的分泌蛋白;和推定的细胞壁溶解性内肽酶。在生长能力和分泌蛋白的本底水平方面比较两种破坏剂表明，HN1314比HN1303作为宿主大规模生产重组蛋白更有用。

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