Development of a repressible mycobacterial promoter system based on two transcriptional repressors.

Mycobacterium tuberculosis is still one of the most deadly microorganisms as it is responsible for ∼2-million human deaths every year. The lack of an efficient vaccine makes antimicrobial treatment the principal strategy to fight tuberculosis, but the emergence of multidrug-resistant (MDR), and, more recently, extensively drug-resistant (XDR) M. tuberculosis strains, is endangering this strategy and makes the development of new antitubercular drugs a global urgency. Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. Moreover, they are required to characterize the function of essential genes, which cannot be deleted without obtaining a lethal phenotype.While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth.