Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Appl Environ Microbiol. 2011 Feb;77(4):1375-82. doi: 10.1128/AEM.01572-10. Epub 2010 Dec 23.
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.
许多致病性梭菌会产生毒素和酶。为了促进病原菌毒力因子的全基因组鉴定和其有用产物的生物技术应用，我们开发了一种用于产气荚膜梭菌的无标记框内缺失方法，该方法可实现有效的反向选择和多基因缺失。该系统包括一个 galKT 基因缺陷体和一个自杀型 galK 质粒，该质粒中克隆了用于框内缺失的目标基因的两个片段。通过使用该系统来缺失该生物体的α-毒素基因，证明了该系统的准确性和简单性。该系统还用于构建了两种不同的减毒菌株 HN1303 和 HN1314：前者是 virRS 操纵子的缺失体，该操纵子调节毒力因子的表达，后者是编码α、θ 和 κ 毒素的六个基因、一种类凝乳蛋白酶、一种 190kDa 分泌蛋白和一种假定的细胞壁裂解内肽酶的缺失体。从生长能力和分泌蛋白的背景水平两方面比较这两种缺失体，结果表明 HN1314 比 ΗΝ1303 更适合作为大规模生产重组蛋白的宿主。
