Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

Abstract

A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance

“…Alanyl tRNAs tolerate mutation of their anticodons because recognition by their cognate synthetase is mediated by a highly conserved motif in the acceptor stem (16) and mutant tRNAs are still charged normally with alanine (16). In the absence of inducer, low-level expression of abnormal tRNAs in EMED from the tetracycline-inducible promoter (17) resulted in higher mistranslation rates (Fig. 1B) with limited growth inhibition (Fig.…”

Inhibition of the Sole Type I Signal Peptidase of Mycobacterium tuberculosis Is Bactericidal under Replicating and Nonreplicating Conditions

“…One transformant (transformant 2) showed slightly reduced growth at low ATc concentrations, suggesting that the ATc-dependent regulation of lepB expression was lost in this strain. Escape mutants are often seen with all of the available tetracycline-regulated promoters used in M. tuberculosis, where accumulation of mutation(s) in the promoter/operator region leads to loss of ATc-dependent growth phenotypes (9). However, the clear ATc dependence of two of the three transformants confirms that downregulation of lepB expression does reduce the growth of M. tuberculosis.…”

Identification of Essential Operons with a Rhamnose-Inducible Promoter in Burkholderia cenocepacia

“…Another general strategy for identifying essential genes is functional suppression either by antisense mRNA induction (15,49) or by the transposon-based delivery of inducible promoters such as the arabinose-regulated promoter (P BAD ) (25) and the tetracycline-inducible promoter (5,13). When a conditionally lethal phenotype is obtained, the identified gene downstream of the inserted promoter is usually defined operationally as essential.…”