A GntR family transcription factor positively regulates mycobacterial isoniazid resistance by controlling the expression of a putative permease

Jialing Hu, Lei Zhao, Min Yang

Bacteria use transcriptional regulation to respond to environmental stresses. Specifically, exposure to antibacterial drugs is deemed to be an atypical stress, and altering transcriptional regulation in response to such stress can increase bacterial drug resistance. However, only a few transcription factors that regulate drug resistance have been reported. In the present study, a GntR family transcription factor, encoded by the MSMEG_0535 (Ms0535) gene, was shown to be an isoniazid (INH) resistance regulator in Mycobacterium smegmatis. When the Ms0535 gene was overexpressed, cells showed a significant increase in INH resistance. First, the interaction between Ms0535 and its own promoter was determined, and a conserved 26-bp palindromic DNA binding motif was identified using electrophoretic mobility shift and DNaseI footprinting assays. Second, quantitative reverse transcription-PCR assays showed that Ms0535 acted as a transcriptional activator, and positively regulated its own expression, as well as that of a permease encoded by the MSMEG_0534 (Ms0534) gene. Similar to the case for the Ms0535 gene, a recombinant Ms0534-overexpressing strain also exhibited increased INH resistance compared with the wild-type strain. Furthermore, we showed that Ms0535 and Ms0534 deletion strains were more sensitive to INH than the wild-type strain. Interestingly, overexpressing Ms0534 in the Ms0535 deletion strain enhanced its INH resistance. In contrast, the Ms0534 deletion strain was still sensitive to INH even when Ms0535 was overexpressed. These findings suggest that Ms0534 is an effector protein that affects INH resistance in M. smegmatis. In summary, the GntR transcriptional regulator Ms0535 positively regulates INH resistance by transcriptionally regulating the expression of the Ms0534 permease in M. smegmatis. These results improve our understanding of the role of transcriptional regulation in INH drug resistance in mycobacteria.