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首页> 外文期刊>Journal of bacteriology >Identification and Mutagenesis by Allelic Exchange of choE, Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi
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Identification and Mutagenesis by Allelic Exchange of choE, Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi

机译：通过等位基因交换choE的鉴定和诱变，编码来自细胞内病原体红球菌的胆固醇氧化酶

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The virulence mechanisms of the facultative intracellular parasiteRhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, thechoE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified inBrevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis andMycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10?2 and 10?3. Twelve percent of the recombinants were double-crossover choE mutants. The choEmutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choEisogenic mutants will be a valuable tool for the molecular analysis ofR. equi virulence.
机译：兼性细胞内寄生虫 Rhodococcus equi 的毒力机制仍不清楚。在这种致病性放线菌的候选毒力因子中，有一种分泌的胆固醇氧化酶，一种假定的破坏膜的毒素。我们鉴定并鉴定了编码该酶的基因 choE 单顺反子。它的蛋白质产物ChoE与 sterevister sterolicum 和 Streptomyces spp中鉴定出的其他分泌的胆固醇氧化酶同源。 ChoE还与结核分枝杆菌和麻风分枝杆菌编码的假定的胆固醇氧化酶具有显着相似性。与 R一起使用的遗传工具。 Equi 发展不佳。在这里，我们描述了可用于该细菌的第一个靶向诱变系统。它基于自杀质粒，选择性标记（来自 Salmonella 的 aacC4 阿普霉素抗性基因）和同源重组。通过插入 aacC4 基因破坏 choE 等位基因，将其克隆到pUC19中，并通过电穿孔引入 R中。当量在10 ？2 和10 ？3 之间的频率分离出choE 重组子。百分之十二的重组体是双杂交的 choE 突变体。 choE 突变与产生鞘磷脂酶的细菌（ ivanovii ）的合作性（CAMP样）溶血作用丧失有关。通过从pVK173-T（赋予潮霉素抗性的pAL5000衍生物）表达 choE 实现功能互补。我们的数据表明，ChoE是 R的重要细胞溶解因子。等。我们用于生成 choE 同基因突变体的高效靶向诱变程序将是 R分子分析的有价值的工具。等毒力。

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