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

Abstract

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. 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.

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

“…The TetR/Pip‐OFF repressible promoter system has been successfully used to construct several conditional mutants in Mycobacterium tuberculosis (Boldrin et al ., 2010). However, the high strength of the P ptr promoter, on which the system is based, may represent a problem when the target gene is physiologically expressed from a weak promoter.…”

“…1). Mutated and wt promoters were cloned upstream of a promotorless egfp gene in the integrative plasmid pFRA61, containing the TetR/Pip‐OFF system (Boldrin et al ., 2010). The resulting plasmids were transformed in both Mycobacterium smegmatis mc 2 155 and in M. tuberculosis H37Rv (Table S1).…”

“…The main aim of this work was to mutagenize the repressible promoter P ptr to increase the tunable range of the TetR/Pip‐OFF repressible system (Boldrin et al ., 2010). The pip promoter encloses three Pip operators each containing two directly repeated 4 bp palindromes separated by a 3 bp spacer.…”

“…In previous work, we reported the development of a repressible promoter system (TetR/Pip‐OFF) based on two chromosomally encoded repressors and the tunable promoter P ptr (Boldrin et al ., 2010). In this system, entirely integrated in single copy in the mycobacterial genome, the gene encoding the tetracycline‐responsive repressor TetR is constitutively expressed, while the gene coding for the pristinamycin‐responsive repressor Pip is expressed from a TetR‐dependent promoter, and the gene of interest is expressed from P ptr , a strong promoter repressible by Pip.…”

“…In the absence of ATc, TetR represses pip expression and the target gene is expressed; in the presence of ATc, TetR releases the promoter of the Pip gene resulting in repression of P ptr . The system can be further modulated by the addition of pristinamycin I, which can alleviate P ptr repression by Pip (Boldrin et al ., 2010). …”