Wuenscher M D, Köhler S, Goebel W, Chakraborty T
Institut für Genetik und Mikrobiologie, Universität Würzburg, Federal Republic of Germany.
Mol Gen Genet. 1991 Aug;228(1-2):177-82. doi: 10.1007/BF00282463.
A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts). An internal fragment of the listeriolysin gene (lisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 x 10(-3) via lisA homology and these cells could be selected at 42 degrees C using a plasmid-encoded erythromycin resistance. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species.
开发了一种用于插入失活单核细胞增生李斯特菌染色体基因的质粒整合技术。构建了一种李斯特菌-大肠杆菌穿梭载体（pLSV1），其携带来自质粒pTV32(Ts)的温度敏感型革兰氏阳性复制起点。将李斯特菌溶血素基因（lisA）的一个内部片段克隆到pLSV1中，构建成pLSV2。在单核细胞增生李斯特菌pLSV2转化体中，质粒pLSV2通过lisA同源性以2×10(-3)的频率整合到单核细胞增生李斯特菌染色体中，并且可以在42℃下使用质粒编码的红霉素抗性筛选这些细胞。质粒整合导致lisA基因破坏，产生截短的、具有免疫交叉反应性的李斯特菌溶血素蛋白，并丧失溶血表型。还描述了一种改进的李斯特菌原生质体转化方法，该方法有助于对李斯特菌属进行基因操作。
