## 3 Results
### vapBC and _mazEF_ TA Systems of M. smegmatis Show Different Effects under Tetracycline Treatment  
After 24 h of exposure to tetracycline, no effect of the _mazEF_ deletion on the viability of the _M. smegmatis_ was observed (Figure 2). At the same time, the double deletion strain (\(\Delta\)vapBC\(\Delta\)mazEF) developed a restoration of the wild-type phenotype as the culture death observed with _vapBC_ deletion was abolished. After 48 h of tetracycline exposure, the significance of the MazEF system was pronounced even more, i.e., \(\Delta\)vapBC\(\Delta\)mazEF cultures showed not only a reversion of the \(\Delta\)vapBC phenotype to the wild type but also a higher survival rate of bacterial cultures in the presence of this antibiotic (Figure 2, Table 2). A similar level of CFU value was observed in the \(\Delta\)mazEF strain. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Strain**} & \multicolumn{3}{c}{**Minimum Inhibitory Concentration, \(\mu\)g/mL**} \\  & **Tetracycline** & **Ciprofloxacinacin** & **Isoniazid** \\ \hline wt & 0.313 & 0.250 & 0.940 \\ \(\Delta\)vapBC & 0.313 & 0.250 & 0.940 \\ \(\Delta\)mazEF & 0.313 & 0.250 & 0.940 \\ \(\Delta\)vapBC\(\Delta\)mazEF & 0.313 & 0.250 & 0.940 \\ \(\mathrm{wt}+\Delta\)find & 0.313 & 0.250 & 0.940 \\ \(\mathrm{vapC}\) over & 0.313 & 0.250 & 0.940 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Minimum inhibitory concentrations of three antibiotics for all _M. smegmatis_ strains included in this research. Figure 1: VapC toxin is involved in _M. smegmatis_ stress susceptibility. Suspensions of 48 h _M. smegmatis_ cells of the wild type, \(\Delta\)vapBC, and wild-type cells transformed with the empty pMind vector (\(\mathrm{wt}+\mathrm{pMind}\)), and VapC overexpressing (vapC over) cells were subjected to a 10 min heat treatment at 60 and 70 \({}^{\circ}\)C (**a**) or treated with 5 \(\mu\)g/mL tetracycline for 24 h (**b**). Following antibiotic treatment, bacterial cells were washed with saline and plated into Petri dishes. Data are represented in relative CFU/mL units (%), which were calculated with respect to CFU/mL numbers in the untreated cultures at the starting time point. Mean values were calculated out of at least 3 independent experiments and are represented with standard deviation margins. An asterisk (*) indicates statistically significant differences with the corresponding control culture (see bacterial strains, media, and growth conditions subsection in the Materials and Methods section). Myobacteria are known to develop a dormant state that is characterized by the ability of cells to remain alive but not to form colonies on solid media, so the growth of colonies on Petri dishes does not always reflect the true survival rate of the bacteria. The ratio of the dormant cells in the population can be measured by comparing CFU values with the results of the most probable number (MPN) test [26]. After conducting such an analysis, we found that the CFU decrease in \(\Delta\)vapBC strain under tetracycline treatment was partially associated with cell transition to an unculturable dormant state, although the survival rate recession was still caused mostly by cell death (Table 2). It is important to note that in the \(\Delta\)vapBC\(\Delta\)mazEF strain, the percentage of dormant cells was reversed to the level of the wild-type strain, although the absolute values of viability were almost threefold higher than in the control. In many cases, complementation of a deleted locus is important for confirming the restoration of the wild-type phenotype. However, in the case of toxin-antitoxin loci, this is not a trivial task, as in the wild type, the expression of toxins and antiotoxins is driven by their own promoter, and the adjustment of expression may occur stochastically and be coupled with post-translational regulation, particularly with proteolysis of antitoxin molecules.