Boneca Ivo G, Ecobichon Chantal, Chaput Catherine, Mathieu Aurélie, Guadagnini Stéphanie, Prévost Marie-Christine, Colland Frédéric, Labigne Agnès, de Reuse Hilde
Groupe Biologie et Génétique de la Paroi Bactérienne, Unité de Pathogénie Bactérienne des Muqueuses, Department of Microbiology, Institut Pasteur, 75724 Paris, France.
Appl Environ Microbiol. 2008 Apr;74(7):2095-102. doi: 10.1128/AEM.01348-07. Epub 2008 Feb 1.
The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.
大肠杆菌-幽门螺杆菌穿梭载体pHeL2被改造，以引入大肠杆菌的可诱导LacI(q)-pTac系统，其中启动子经改造后受幽门螺杆菌RNA聚合酶的控制。采用幽门螺杆菌的amiE基因启动子组成型表达LacI(q)阻遏物。报告基因lacZ的表达由pTac（pILL2150）或ureI基因启动子的修饰版本驱动，在ureI基因启动子的修饰版本中，在核糖体结合位点与ATG起始密码子之间引入了一个或两个LacI结合位点和/或突变核苷酸（pILL2153和pILL2157）。通过测量β-半乳糖苷酶活性来评估启动子活性。pILL2150是一个严格调控的表达系统，适用于分析低水平表达的基因，而pILL2157非常适合在幽门螺杆菌中受控表达编码重组蛋白的基因。为了证明这些工具的实用性，我们构建了分别编码幽门螺杆菌青霉素结合蛋白1和3的推定必需基因pbp1和ftsI的条件突变体。将这两个基因克隆到pILL2150中，并导入亲本幽门螺杆菌菌株N6。然后，通过用非极性卡那霉素盒替换染色体上携带的pbp1和ftsI基因，使其失活。失活严格依赖于添加异丙基-β-D-硫代半乳糖苷。因此，我们能够构建出第一批幽门螺杆菌条件突变体。最后，我们证明，重组质粒在体外甲基化后，可以导入具有不同遗传背景的多种幽门螺杆菌分离株中。
