## 2 Results and Discussion
### Prolonged Action of Toxin \(\zeta\)Y83C Does not Induce Massive Cell Lysis  
To test the latter hypothesis were analyzed if prolonged expression of sub-physiological concentrations of \(\zeta\)Y83C (360 or 480 min) might exhaust the metabolite pool and BG689 cells might reach the limits of their \"capacity\" to exit the dormant state, and they cannot be longer rescued from the \"point of no return\". As already observed, after 16 h of \(\zeta\)Y83C toxin expression the survival rate drops to 2-\(8\times 10^{-7}\)[16]. BG689 cells were grown up to optical density (OD\({}_{600}\)) of 0.2 in MMS7 and divided into two aliquots. To one aliquot inducer (0.5% Xyl) was added (time zero) and samples were collected at different times (120, 240, 360 and 480 min after induction) (Figure 4). In the presence of Xyl, the increment in OD\({}_{600}\) halted after one doubling time, and remained constant during the 480 min interval (Figure 4A). At these time points, samples were withdrawn, and the cells were stained with membrane-permeant SYTO 9 (green fluorescence) and membrane-impermeant PI (red fluorescence), appropriate dilutions were plated in LB plates and the survival rate was measured (Figure 4B). Transient expression of \(\zeta\)Y83C toxin for 120 min or longer periods of time induced dormancy, and a similar sub-fraction of 2-6 \(\times\) 10\({}^{-5}\) survivals became tolerant of toxin action (Figure 4B). Within the first 360 min of \(\zeta\)Y83C toxin expression, the proportion of PI permeable cells slightly increased (\(\sim\)1.2-fold). At later times (480 min) the proportion of PI stained cells increased \(\sim\)2-fold (\(\sim\)65% of total  
Figure 3: Cell wall inhibitors and \(\zeta\) toxin expression show an additive effect. BG689 cells containing the short living \(\zeta\) variant (\(\zeta\)Y83C) gene were grown to \(\sim\)5 \(\times\) 10\({}^{7}\) cell/mL in MMS7. Then Xyl (0.5% to induce expression of the \(\zeta\)Y83C toxin) or an antimicrobial or both was added and the culture was incubated for 120 min. Cells were washed and appropriate dilutions were plated to count the survivals. The results are the average of at least four independent experiments and are within a 10% standard error. cells stained with PI) with respect to the proportion of cells stained with PI after 120 min of toxin expression (Figure 4B). It is likely that prolonged \(\zeta\)Y83C expression might not induce massive cell lysis. **Figure 4.** Effect of prolonged action of toxin \(\zeta\)Y83C on membrane permeability and cell survival. (**A**) Growth curve of BG689 cells containing \(\zeta\)Y83C gene. Cells were grown to \(\sim\)\(2.5\times 10^{7}\) cell/mL in MMS7 (OD\({}_{600}\) = 0.2), to half of the culture Xyl (0.5%) was added to induce \(\zeta\)Y83C expression (time zero) and OD\({}_{600}\) was followed over time in the two cultures. The arrow denotes the time of Xyl (0.5%) addition; (**B**) Aliquots of the cultures were taken at time zero, 120, 360 and 480 min after Xyl addition (denoted as \(+\)). The cells were fixed, stained with SYTO 9 and PI, and analyzed by fluorescence microscopy (black lane), at the same time, cells were washed and appropriate dilutions were plated in LB plates to count the survivals. The numbers of relative CFUs (black bars) at the indicated times after \(\zeta\)Y83C toxin expression are relative to the non-induced control at the same time (denoted as -) taken as 1 (grey bar). Error bars show 95% confidence intervals of more than three independent experiments. When similar experiments were performed in _E.