## 2 Results and Discussion
### Toxin \(\zeta\) Reversibly Induces a Halt in Cell Proliferation  
Previously, it was shown that transient exposure (120 min) to physiological concentrations of free wt \(\zeta\) reversibly induces dormancy, produces a sub-fraction of membrane-compromised cells to be stained with PI (25%-35% of total cells), and a minor subpopulation of _B. subtilis_ cells become tolerant of toxin action. Subsequent expression of the \(\varepsilon_{2}\) antitoxin facilitates the exit from the dormant state and a fraction of membrane compromised cells recover their alteration of the membrane potential [14]. Toxin \(\zeta\) expression inactivated a fraction of UNAG and Fos used the remaining fraction of unphosphorylated UNAG to further inhibit cell proliferation (see Figure 3). If \(\zeta\) toxin expression only reduces the UNAG pool and/or UNAG-P poisons the exit from the dormant state, expression of \(\varepsilon_{2}\) antitoxin might lead to different outcomes in the presence of Fos: First, if \(\zeta\) toxin expression and Fos addition deplete the UNAG pool, cells should not be recovered by antitoxin expression and cell wall biosynthesis can be rescued only after _de novo_ synthesis of UNAG or UNAG-3P has to be re-activated by an unknown pathway. Second, the concert action of both \(\zeta\) toxin expression and Fos addition triggers cell lysis by unbalancing the control of peptidoglycan biosynthesis. Finally, if \(\zeta\) toxin expression decreases a fraction of the UNAG pool, without depleting it, \(\varepsilon_{2}\) antitoxin expression may reverse toxin action and the remaining UNAG is used to rescue cell proliferation. To discriminate between all these possible outcomes, cell survival and the proportion of PI stained cells were analyzed after \(\varepsilon_{2}\) antitoxin expression. When exponentially growing BG1125 cells (Table 1) reached a moderate-density (\(\sim\)5 \(\times\) 10\({}^{7}\) cells/mL in MMS7) 1 mM IPTG was added to induce \(\zeta\) toxin expression. As already observed, expression of nearly physiological wt \(\zeta\) toxin concentrations for 120 min induced dormancy, a fraction of the cell population (\(\sim\)30%) was stained with PI, and \(\sim\)1 \(\times\) 10\({}^{-5}\) survivals tolerant to toxin action were obtained (Table 2). Subsequent expression of the \(\varepsilon_{2}\) antitoxin, after 120 min of IPTG addition, by Xyl (0.5%) addition, led to exit of the dormant state with CFUs increasing \(\sim\)5000-fold, and only a small fraction (\(\sim\)10%) of cells were stained with PI (Table 2) [14,15]. Alternatively, resumption of cell growth might dilute the relative proportion of PI stained cells up to 10% of total cells. In the absence of IPTG, addition of Fos to moderate-density exponentially growing BG1125 cells for 120 min reduced plating efficiency of the cells to levels similar to the ones observed in the BG689 background (\(\sim\)3 \(\times\) 10\({}^{-3}\) survivals), and as expected for a bactericidal antimicrobial the proportion of cells permeable to PI increased (\(\sim\)70% of total cells) (Table 2). Expression of the \(\varepsilon_{2}\) antitoxin under these conditions did nor alter the observed outcome (\(\sim\)70% of total cells stained with PI, and \(\sim\)3 \(\times\) 10\({}^{-3}\) survivals). When moderate-density exponentially growing BG1125 cells were exposed to toxin (by addition of 1 mM IPTG) and Fos action, the toxin markedly enhanced the efficacy of Fos treatment, but no additive effect on membrane permeation to PI was observed (Table 2). Then, transient expression of the \(\varepsilon_{2}\) antitoxin led to partial exit of the dormant state, which increased \(>\)2000-fold CFUs, but it failed to decrease the proportion of PI permeable cells (Table 2).