Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, 49076, Osnabrück, Germany.
Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, 49076, Osnabrück, Germany.
Int J Med Microbiol. 2019 Jan;309(1):54-65. doi: 10.1016/j.ijmm.2018.11.001. Epub 2018 Nov 16.
The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of P can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.
细菌毒力因子的表达受宿主或环境因素的控制，大多数毒力基因在实验室条件下不表达。对细菌毒力因子的分子结构和细胞功能的研究需要实验控制表达的系统。我们描述了一种简单而强大的系统，该系统基于 tetA 启动子和同源的 TetR 阻遏物。在 P 的控制下表达可以通过非抗生素类四环素衍生物如脱羟四环素（AHT）诱导。Tet-on 表达盒可用于替换感兴趣的染色体基因或操纵子的天然启动子。Tet-on 质粒允许在同源或异源宿主生物中进行游离表达。我们展示了 Tet-on 系统在控制鞭毛组装和运动、肠致病性大肠杆菌伴侣/usher 家族表面黏附素和耶尔森氏菌肠侵袭性大肠杆菌自转运黏附素的表面表达方面的应用。由于诱导剂 AHT 可以轻易穿透细菌包膜和哺乳动物细胞膜，该系统也可用于控制细胞内细菌的毒力基因。我们展示了细胞内沙门氏菌和大肠杆菌 III 型分泌系统效应蛋白的受控合成、易位和功能。
