## 3 Results  
VapC Overexpression Lands to Proteomic Changes in Abundance of Proteins Involved in Primary Metabolism and Stress Response  
In a previous study, we demonstrated the 23S rRNA cleavage when VapC was overexpressed in _M. smegmatis_. Additionally, we observed significant alterations in the components of the protein-synthesis machinery under these conditions [11]. In the current work, we analyzed the substantial modifications in the proteins involved in metabolic processes and stress responses according to the proteomic profiling data obtained earlier in _M. smegmatis_ cultures overexpressing VapC (please refer to the data availability statement to find the deposited data). During the process of quantitative proteome analysis, we specifically chose distinct polypeptides that were detected in all the samples examined. Only those polypeptides that exhibited an increase in at least two-fold or a decrease in more than two-fold under VapC overexpression, in comparison to the control wild-type samples, were considered for further investigation. A detailed analysis of proteomic data allowed us to observe certain changes in primary metabolic and regulatory processes under VapC overexpression (see Figure 11). Specifically, a decrease in abundance was detected for a number of enzymes involved in glycolysis (pyruvate kinase MSMEG_3227, pyruvate dehydrogenase subunits MSMEG_2280, MSMEG_4323, MSMEG_0903), tricarboxylic acid cycle (e.g., aconitate hydratase MSMEG_3143, fumarate hydratase MSMEG_5240), and pentose-phosphate shunt (transketolase MSMEG_3103) of carbohydrate metabolism. At the same time, the abundance of citrate synthase MSMEG_5672, which is involved in glyoxylate shunt, was increased, whereas isocitrate lyase MSMEG_0911 was replaced by isoenzyme MSMEG_3706. A similar phenomenon was observed for gamma-aminobutyric acid shunt, i.e., succinate-semialdehyde dehydrogenase MSMEG_6452 was substituted by isoenzyme MSMEG_0889. The data on the abundance of proteins involved in amino acid metabolism under VapC overexpression demonstrated the decrease amidst synthase enzymes, such as tryptophan synthase MSMEG_3220, asparagine synthase MSMEG_2594, methionine synthase MSMEG_4185, carbamoyll phosphate synthase MSMEG_3047, etc. (nine enzymes overall). However, the abundance ratio of glutamine synthetase MSMEG_4290 was 94-fold higher when compared to the control. Moreover, three enzymes of amino acid catabolism wereidentified as under-represented proteins, and two more proteins were over-represented in this category (refer to Figure 11). VapC overexpression also affected the abundance of a number of enzymes participating in fatty acid biosynthesis as most of them were over-represented, whereas certain enzymes attributed to fatty acid degradation pathways were under-represented to a large extent. Acetyl-CoA acetyltransferase MSMEG_5273 was substituted by isoenzyme MSMEG_0373 following VapC overexpression, and its abundance ratio was nearly 85-fold higher in comparison with the control strain (Figure 11). Furthermore, we detected three enzymes encoded by _pur_ genes to be under-represented according to our proteomic data, which implied that nucleotide metabolism could have been affected by VapC overexpression as well. At the same time, three isoforms of inosine 5-monophosphate dehydrogenase were identified as over-represented. A notable find was the increased abundance of HemL protein (MSMEG_0969) which is involved in tetrapyrrole biosynthesis and thus participates in porphyrin production (Figure 11). Simultaneously, significant changes in the abundance of stress response proteins were observed under VapC overexpression. In this group, carbon starvation protein A (MSMEG_2259) was the most over-represented (a 29-fold increase in abundance in comparison with the control), it was followed by GTP pyrophosphokinase RelA (MSMEG_2965) with a fourfold increase in abundance relative to the control.