Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis. Issue 1 (27th October 2017) - Boldrin, Francesca
Summary: 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 ptr, 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 ptr 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. Abstract : We report an enhanced TetR/Pip‐OFF repressible promoter system that allows more effective control of genes expressed at low level. Subjecting the Pptr promoter to targeted mutagenesis, we produced several promoters with different strength, able to improve the performances of TetR/Pip‐OFF repressible system both in vitro and in vivo increasing its stringency. Such promoters were used to construct bacterial biosensors with different sensitivity to DprE1 inhibitors and to develop a whole cell screening assay to identify inhibitors of this enzyme.