Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens
Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens
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DOI：
10.1128/aem.01572-10
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发表时间：
2011-02-01
影响因子：
4.4
通讯作者：
Okabe, Akinobu
中科院分区：
文献类型：
--
作者：
Nariya, Hirofumi;Miyata, Shigeru;Okabe, Akinobu
许多致病梭状芽胞杆菌会产生毒素和酶。为了便于毒力因子的全基因组鉴定及其有用产物的生物技术应用，我们开发了一种针对产气荚膜梭菌的无标记框内缺失方法，该方法允许高效的反选择和多基因干扰。该系统包括GalKT基因干扰物和自杀Galk质粒，其中克隆了用于框架内缺失的目的基因的两个片段。该系统被用来干扰生物体的阿尔法毒素基因，证明了该系统的准确和简单。它还被用来构建两个不同的毒力减毒株HN1303和HN1314：前者是调节毒力因子表达的VIRRS操纵子的干扰物，后者是编码α、β和kappa毒素的六个基因的干扰物；梭状芽胞杆菌痛样蛋白酶；190 kDa的分泌蛋白；以及可能的细胞壁裂解内肽酶。比较两种干扰剂的生长能力和分泌蛋白的本底水平，表明HN1314比HN1303更适合作为重组蛋白大规模生产的宿主。
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, HN1303 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 alpha, theta, and kappa 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 HN1303 as a host for the large-scale production of recombinant proteins.