Department of Molecular Medicine, University of Padova, 35121, Padova, Italy.
Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Station 19, 1015, Lausanne, Switzerland.
Microb Biotechnol. 2018 Jan;11(1):238-247. doi: 10.1111/1751-7915.12875. Epub 2017 Oct 27.
A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was P , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected P to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme.
在过去的几年中，已经开发出一系列用于分枝杆菌的调控基因表达系统，以促进对必需基因的研究、验证新的药物靶标并评估其脆弱性。其中，TetR/Pip-OFF 可诱导的启动子系统已成功应用于多种分枝杆菌，包括在体外和体内。在该系统的第一个版本中，可诱导的启动子是 P ，这是来自吸水链霉菌的强 Pip 可诱导启动子，这可能会阻碍具有低基础表达水平的基因的有效下调。在这里，我们报告了一种增强型系统，可更有效地控制低水平表达的基因。为此，我们对 P 进行了靶向诱变，产生了 16 种不同强度的启动子。其中三种弱于野生型启动子，选择并表征了这三种启动子，结果表明它们确实可以提高 TetR/Pip-OFF 可诱导的系统在体外和体内的性能，增加其严格性。最后，我们使用这些启动子构建了一系列对 DprE1 抑制剂具有不同敏感性的细菌生物传感器，并开发了一种全细胞筛选测定法来鉴定该酶的抑制剂。
