Ferrero R L, Cussac V, Courcoux P, Labigne A
Unité des Entérobactéries, Institut National de la Santé et de la Recherche Médicale U199, Institut Pasteur, Paris, France.
J Bacteriol. 1992 Jul;174(13):4212-7. doi: 10.1128/jb.174.13.4212-4217.1992.
Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.
通过等位基因置换构建了幽门螺杆菌的同基因脲酶阴性突变体。含有脲酶结构基因（ureA和ureB）的幽门螺杆菌DNA克隆区域被mini-Tn3-Km转座子插入破坏。用卡那霉素-ureB破坏的衍生质粒对幽门螺杆菌细胞进行电转化，导致分离出卡那霉素抗性的幽门螺杆菌转化体。电转化能力似乎仅限于某些野生型幽门螺杆菌分离株；（10个测试分离株中）只有1个分离株能持续被转化。对两个卡那霉素抗性的幽门螺杆菌转化体进行了进一步研究，结果显示它们脲酶阴性。Southern杂交分析表明，脲酶阴性突变体是通过等位基因交换构建的，包括用卡那霉素-ureB破坏的拷贝同时替换ureB基因以及载体的丢失。用抗幽门螺杆菌血清对同基因ureB突变体的全细胞提取物进行免疫印迹研究，结果表明不存在表观分子量为61 kDa的多肽；因此，突变体不再合成UreB产物。产生稳定的、基因工程改造的幽门螺杆菌脲酶突变体将有助于研究脲酶在幽门螺杆菌感染发病机制中的作用。
