Abstract:[Objective] To study the regulatory effect of TetR family transcription factor Ms0606 on the drug resistance of Mycobacterium smegmatis. [Methods] Firstly, the growth curve was measured to detect the regulatory role of Ms0606 in mycobacterial resistance; electrophoretic mobility shift assay and DNase I footprinting assay were used to identify the conservative sequence recognized by the transcription factor Ms0606, and then explore its potential target genes. Secondly, reverse transcription-qPCR and β-galactosidase activity experiments were used to detect the regulatory effect of Ms0606 on the target gene Ms0608. The potential roles of Ms0606 in the regulation of drug resistance are further discussed. [Results] Overexpressing Ms0606 made mycobacteria more sensitive than the wild-type strain in response to isoniazid, whereas disrupting Ms0606 is resistant to isoniazid; Ms0606 could recognize a conserved 22 bp palindromic motif within the upstream region of its self-operon, using the palindrome sequence to search M. smegmatis genome, it is found that Ms0606 may regulate 5 potential target genes; Reverse transcription-qPCR and β-galactosidase activity experiments revealed Ms0606 acts as a repressor and negatively regulates Ms0608 expression, which may affect mycobacterial antibiotic resistance. [Conclusion] I have identified a new TetR family transcriptional regulator of Mycobacterium smegmatis encoded by Ms0606, which regulates mycobacterial sensitivity to isoniazid, and further characterized its target genes and its regulatory mechanism for mycobacterial resistance.
