## 4 Discussion  
Proteomic profiling is a valuable tool that can assist in predicting the physiological characteristics of bacterial cells under specific conditions. In this study, we examined the differential proteins in cultures of _M. smegmatis_ with overexpression of VapC compared to the wild-type strain. We then compared these proteins with the known features of bacterial stress responses. Specifically, we observed a decrease in the abundance of carbohydrate metabolism proteins, such as pyruvate kinase, pyruvate dehydrogenase, aconitate hydratase, and fumarate hydratase, in addition to an increase in citrate synthase representation. Some enzymes were also substituted with their isoforms, such as isocitrate lyase and succinate-semialdehyde dehydrogenase. These changes suggest the activation of the glyoxylate shunt and 4-aminobutyric acid shunt, (which are tricarboxylic acid cycle bypass pathways), indicating that the cells are preparing for a stationary phase and potentially entering a  
Figure 3: The results of qRT-PCR analysis performed in M. smegmatis samples treated with 5 \(\upmu\)g/mL tetracycline (Tet+) in comparison with untreated control cultures (Tet\(-\)). The mRNA expression levels of vapC (**a**) and mazF (**b**) were calculated relative to 16S mRNA transcripts and presented as fold change in mean \(\pm\) SD format. An asterisk (*) indicates statistically significant differences with the point of 0 h.  
-dormant state [31; 32]. Furthermore, we observed an increase in the abundance of proteins involved in fatty acid biosynthesis (at least eight over-represented proteins) and a decrease in enzymes involved in fatty acid catabolism (six under-represented proteins). This suggests that the bacteria may be accumulating fatty acids in the form of triacyl glycerides to counteract stressful conditions such as hypoxia and nutrient starvation, which are often encountered during stationary phase and dormancy development [33; 34]. Additionally, we observed an increase in the abundance of HemL protein, which participates in porphyrin biosynthesis. Porphyrin accumulation is a characteristic of dormant cells in the case of _M. smegmatis_[35], further supporting the assumption that the cells are transitioning to a stationary phase and developing dormancy. On the other hand, the transition to the stationary phase and resting state development is usually associated with the activation of a general stress response [36]. Interestingly, overexpression of the VapC toxin led to similar changes in the proteomic data. We observed an increase in the abundance of the ClpP protease subunit, accompanied by the detection of the ClpX subunit as a unique protein. This suggests the activation of proteolysis, the breakdown of unfolded and misfolded proteins, and polypeptides with aborted synthesis [37]. However, microbial cells typically try to refold injured and denatured proteins before resorting to proteolysis. Therefore, changes in chaperone abundance would be expected under VapC overexpression. Indeed, we identified the molecular chaperone DnaJ, which can effectively restore aggregated and misfolded proteins independently of ATP availability [38], potentially replacing the decreased levels of DnaK and GroEL chaperones [39; 40] under VapC overexpression. Furthermore, it is commonly observed that stress conditions and the transition to the stationary phase result in a reduction in metabolic activity and energy production. This notion is supported by our findings, as we observed a downregulation of carbohydrate metabolism enzymes in response to these conditions. This possible decrease in metabolic activity may also be connected with a reduced respiration rate and decreased electron flow in the respiratory chain. Since the respiratory chain is a major source of reactive oxygen species in cells [41], the endogenous oxidative stress is reduced under these conditions [42].