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Expansion of the Tetracycline-Dependent Regulation Toolbox for Helicobacter pylori

机译：扩展幽门螺杆菌的四环素依赖性调控工具箱

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In an effort to gain greater understanding of the biology and infection processes of Helicobacter pylori , we have expanded the functionality of the tetracycline-dependent gene regulation ( tet ) system to provide more improved and versatile genetic control and facilitate the generation of conditional mutants to study essential genes. Second-generation tetracycline-responsive H. pylori uPtetO 5 promoters were based on the mutated core ureA promoter. Single point mutations at either the ribosomal binding site or the start codon were introduced to shift the regulatory range of three uPtetO 5 derivatives. All promoters were tested for regulation by TetR and revTetR using dapD , a gene essential to peptidoglycan biosynthesis, as a reporter. All tet promoters were effectively regulated by both TetR and revTetR, and their regulation windows overlapped so as to cover a broad range of expression levels. tet promoters uPtetO 5m1 and uPtetO 5m2 could be sufficiently silenced by both TetR and revTetR so that the conditional mutants could not grow in the absence of diaminopimelic acid (DAP). Furthermore, through the use of these inducible promoters, we reveal that insufficient DAP biosynthesis results in viable cells with altered morphology. Overall, the development and optimization of tet regulation for H. pylori will not only permit the study of essential genes but also facilitate investigations into gene dosage effects on H. pylori physiology.
机译：为了进一步了解幽门螺杆菌的生物学和感染过程，我们扩展了四环素依赖性基因调控（tet）系统的功能，以提供更完善和通用的遗传控制，并促进条件突变体的研究必需基因。第二代四环素反应性幽门螺杆菌uPtetO 5启动子是基于突变的核心ureA启动子。在核糖体结合位点或起始密码子处引入单点突变，以改变三种uPtetO 5衍生物的调节范围。使用肽聚糖生物合成必不可少的基因dapD作为报告基因，测试了所有启动子的TetR和revTetR调控。 TetR和revTetR均有效地调节了所有tet启动子，并且它们的调节窗口重叠，从而涵盖了广泛的表达水平。 tet启动子uPtetO 5m1和uPtetO 5m2可以同时被TetR和revTetR沉默，以使条件突变体在没有二氨基庚二酸（DAP）的情况下无法生长。此外，通过使用这些诱导型启动子，我们发现不足的DAP生物合成导致形态改变的活细胞。总体而言，针对幽门螺杆菌的tet调控的发展和优化不仅将允许研究必需基因，而且还将有助于研究基因剂量对幽门螺杆菌生理的影响。

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