Debowski Aleksandra W, Sehnal Miriam, Liao Tingting, Stubbs Keith A, Marshall Barry J, Benghezal Mohammed
Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia
Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia.
Appl Environ Microbiol. 2015 Dec;81(23):7969-80. doi: 10.1128/AEM.02191-15. Epub 2015 Sep 11.
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 uPtetO5 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 uPtetO5 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 uPtetO5m1 and uPtetO5m2 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.
为了更深入地了解幽门螺杆菌的生物学特性和感染过程，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、更通用的基因控制，并便于生成条件性突变体来研究必需基因。第二代四环素响应性幽门螺杆菌uPtetO5启动子基于突变的核心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uPtetO5衍生物的调控范围。使用肽聚糖生物合成必需基因dapD作为报告基因，测试了所有启动子受TetR和revTetR的调控情况。所有tet启动子均受TetR和revTetR有效调控，且它们的调控窗口重叠，从而覆盖广泛的表达水平范围。tet启动子uPtetO5m1和uPtetO5m2可被TetR和revTetR充分沉默，使得条件性突变体在缺乏二氨基庚二酸（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DAP生物合成不足会导致形态改变的活细胞。总体而言，幽门螺杆菌tet调控的开发和优化不仅将允许对必需基因进行研究，还将促进对基因剂量对幽门螺杆菌生理学影响的研究。
