Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis

Objective: Most non-tuberculous mycobacteria exhibit intrinsic resistance against the anti-tuberculosis drug isoniazid (INH). We previously found that a pyrazinamidase/nicotinamidase of Mycobacterium smegmatis , named PzaA, has an enzymatic activity to hydrolyze INH, which may contribute to intrinsic resistance. Furthermore, PzaA expression is strongly induced by INH under nitrogen-depleted conditions, although the precise mechanism of this phenomenon remains unclear. Here, we aimed to reveal the mechanism underlying the INH-dependent induction of PzaA using a transcriptomic approach. Methods: RNA sequencing was performed to identify INH-inducible genes other than pzaA. 5 ' rapid amplification of cDNA ends analysis was employed to identify the transcription start sites of INH-induced transcription units. The function of a LuxR-like regulator gene (MSMEI_1050) found within the gene cluster containing pzaA was confirmed by gene deletion and complementation experiments involving INH hydrolysis assay and quantitative reverse transcription PCR. Results: RNA sequencing revealed 23 genes that INH strongly induced under conditions of nitrogen depletion, 17 of which were in a gene cluster containing pzaA. This cluster comprised at least three transcription units, including a non-INH-inducible monocistronic unit containing MSMEI_1050. Deletion of this gene deprived M. smegmatis of the ability to respond to INH, and complementation restored this ability. Conclusions: MSMEI_1050 plays a key role in INH-dependent gene regulation. The precise mechanism of action is to be determined in future studies.