## 4 Discussion
Consistent with this, we found that oxidative stress-related proteins, such as thioredoxin reductase, which participates in antioxidant defense through the conversion of thiol and disulfide bonds [43], and SuffBCD complex proteins, which are the components of the Suf machinery that is responsible for de novo iron-sulfur (Fe-S) cluster biogenesis [44], were less abundant under VapC overexpression. In contrast, the Mn-superoxide dismutase SodA was replaced by the copper/zinc superoxide dismutase SodC, which is an enzyme induced during the stationary phase and dependent on the SigB factor. This enzyme provides protection against reactive oxygen intermediates at the surface of mycobacteria [45]. Additionally, the catalase KatG was substituted with an alternative isoenzyme, possibly similar to the stationary phase KatE of _E. coli_ which is known to be less efficient though better fitting for stressful environments [46; 47]. These two substitutions (SodA and KatG) may indicate a shift in the oxidative defense system of mycobacteria from internal to external challenges, similar to the oxidative stress experienced by cells within macrophages in the case of MTB or BCG infection [45; 48; 49]. Although the (p)ppGpp-synthase ReIA was found to be highly expressed under VapC overexpression, the observed features of the stringent response were atypical. Contrary to the presumable increase in amino acid synthesis [50], we actually observed a decrease in the abundance of amino acid synthetase enzymes. Nonetheless, this decrease aligns with the overall reduction in synthetic processes during the transition to stationary phase and bacterial dormancy [36; 51]. Additionally, we found that the abundance of proteins involved in purine nucleotide metabolism was altered, which also happens under enhanced (p)ppGpp production [52]. Surprisingly, we also observed an increase in the abundance of glutamine synthetase MSMEG_4290, which catalyzes the conversion of glutamate to glutamine. This contradicted our initial hypothesis that it would be repressed along with other amino acid synthetases. The physiological significance of this increase may be related to ammonium fixation [53] or,more probably, the utilization of amino groups following protein degradation [54] due to the premature release of incompletely synthesized polypeptide molecules out of the ribosome under aminoacyl-tRNA deficiency [55]. The synthesized glutamine may also play a role in the conversion of 2-ketoglutarate to glutamate, a key step in the 4-aminobutyric acid pathway (we also detected the increased abundance of succinate-semialdehyde dehydrogenase MSMEG_0889 that is the component of this pathway) [56]. Additionally, the upregulation of glutamate decarboxylase MSMEG_1574 suggests a potential role in acid stress resistance [57]. Overall, our proteomic profiling revealed changes indicative of a transition to alternative energy production processes, activation of emergency-associated biochemical pathways, and a general stress response. These proteomic features likely alter the physiology of mycobacterial cells. The conducted investigation showed that VapC indeed participates in the regulation of bacterial sensitivity to high temperatures (Figure 1a). Activation of proteases and changes in the representation of chaperones observed under conditions of VapC overexpression are well consistent with increased heat resistance. Accordingly, the AvapBC strain demonstrates an increasing sensitivity to this adverse factor. Although the proteomic alterations observed in _M. smegmatis_ cells upon VapC overexpression did not confer any protective advantage against the effects of tetracycline, the role of VapC was clearly pronounced upon deletion of the _vapBC_ locus (Figure 1b). At the same time, the VapBC TA system did not affect the ability of mycobacterial cells to adapt to the action of ciprofloxacin and isoniazid. This is most likely related to the mechanism of action of antibiotics. Ciprofloxacin belongs to fluoroquinolones and is aimed at inhibiting DNA gyrase [58].