Rv1255c, a dormancy-related transcriptional regulator of TetR family in Mycobacterium tuberculosis, enhances isoniazid tolerance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is exposed to diverse stresses inside the host during dormancy. Meanwhile, many metabolic and transcriptional regulatory changes occur, resulting in physiological modifications that help M. tuberculosis to adapt to these stresses. The same physiological changes also cause antibiotic tolerance in dormant M. tuberculosis. However, the transcriptional regulatory mechanism of antibiotic tolerance during dormancy remains unclear. Here, we showed that the expression of Rv1255c, an uncharacterised member of the tetracycline repressor family of transcriptional regulators, is upregulated during different stresses and hypoxia-induced dormancy. Antibiotic tolerance and efflux activities of Mycobacterium smegmatis constitutively expressing Rv1255c were analysed, and interestingly, it showed increased isoniazid tolerance and efflux activity. The intrabacterial isoniazid concentrations were found to be low in M. smegmatis expressing Rv1255c. Moreover, orthologs of the M. tuberculosis katG, gene of the enzyme which activates the first-line prodrug isoniazid, are overexpressed in this strain. Structural analysis of isoforms of KatG enzymes in M. smegmatis identified major amino acid substitutions associated with isoniazid resistance. Thus, we showed that Rv1255c helps M. smegmatis tolerate isoniazid by orchestrating drug efflux machinery. In addition, we showed that Rv1255c also causes overexpression of katG isoform in M. smegmatis which has amino acid substitutions as found in isoniazid-resistant katG in M. tuberculosis.

Mycobacterium tuberculosis TetR family transcriptional regulator Rv1019 is a negative regulator of the mfd-mazG operon encoding DNA repair proteins.

Rv1019, a member of an uncharacterized tetracycline resistance regulator family of transcriptional regulators of Mycobacterium tuberculosis H37Rv, was found to be differentially expressed during dormancy and reactivation in vitro. In this study, we show that this protein binds to its own promoter and acts as a negative regulator of its own expression. It forms dimers in vitro which is essential for the DNA binding activity. We also show that Rv1019 and downstream genes Rv1020 (mfd) and Rv1021 (mazG) are cotranscribed. Constitutive expression of Rv1019 in M. smegmatis downregulated MSMEG_5423 (mfd) and MSMEG_5422 (mazG), suggesting that Rv1019 negatively regulates these downstream genes which encode key proteins involved in DNA repair. M. smegmatis expressing Rv1019 was found to be sensitive to DNA-damaging environments, suggesting its role in regulating the DNA damage response in mycobacterium.