| Summary: | Toxin–antitoxin (TA) systems are widely present in bacterial genomes. <i>Mycolicibacterium smegmatis</i>, a common model organism for studying <i>Mycobacterium tuberculosis</i> physiology, has eight TA loci, including <i>mazEF</i> and <i>vapBC</i>. This study aims to investigate the physiological significance of these TA systems. Proteomic profiling was conducted on a culture overexpressing the VapC toxin, and the involvement of VapC in <i>M. smegmatis</i> stress responses to heat shock and antibiotic treatment was examined. While deciphering the underlying mechanisms of the altered stress resistance, we assessed the antibiotic susceptibility of <i>vapBC</i>, <i>mazEF</i>, and double <i>vapBC-mazEF</i> deletion mutants. Additionally, the mRNA levels of <i>vapC</i> and <i>mazF</i> were measured following tetracycline supplementation. The results reveal changes in the abundance of metabolic enzymes and stress response proteins associated with VapC overexpression. This activation of the general stress response leads to reduced thermosensitivity in <i>M. smegmatis</i>, but does not affect susceptibility to ciprofloxacin and isoniazid. Under tetracycline treatment, both <i>vapC</i> and <i>mazF</i> expression levels are increased, and the fate of the cell depends on the interaction between the corresponding TA systems.
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