# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### Toxin \(\zeta\) Expression Induces Amp Dormancy in Non-Growing Cells  
To re-evaluate whether Amp persisters are produced due to a stochastic entrance into the stationary phase or upon transient toxin \(\zeta\) expression, and if toxin expression or Amp addition to stationary phase cells affects the persistence level and its subsequent exit, high-density slow- or non-growing stationary phase _B. subtilis_ BG1125 (pCB799) cells, which grew overnight in the S7 medium supplemented with 0.005% Xyl, were used. The overnight culture was normalized to \(\sim\)\(1\times 10^{9}\) cells mL\({}^{-1}\) with the pre-warmed S7 medium, supplemented with 0.005% Xyl and split. Amp (3 \(\upmu\)g mL\({}^{-1}\)), IPTG (2 mM), or both Amp and IPTG were added and the cells incubated up to 240 min. At the indicated times, IPTG, Amp, or both were removed from the medium and the culture was plated on LB plates. Since in the absence of both, Amp and IPTG, the number of cells did not significantly increase at 240 min (Figure 2B, filled grey triangles) and BG1125 (pCB799) cells grown in S7 supplemented with 0.005% Xyl usually had an usual lag-time of 30-45 min (Figure S2), we assume that the high-density cells remain in the stationary phase during the experimental period.  
The MIC for Amp was estimated using low-density cells (1-\(3\times 10^{6}\) cells mL\({}^{-1}\) for 16 h, at 37 \({}^{\circ}\)C), and no correction by per-cell in the Amp concentration was performed. At a variable time, and upon removal of the antibiotic, a significant fraction could not form colonies, but a subpopulation of cells persisted to Amp action (3-\(5\times 10^{-2}\) survivals). As expected, the proportion of persisters was significantly higher than in exponentially growing cells (Figure 2A vs. Figure 2B, filled orange squares). This is consistent with the observation that the rate of killing of Amp is strictly proportional to the rate of bacterial growth [57]. Similar results were observed in the presence of 6 \(\upmu\)g mL\({}^{-1}\) Amp (data not shown). It is likely that the lower proportion of cell death might correlate with the less proportion of metabolically active cells in the stationary phase, but in the only Xyl condition the cells are not proliferating.  
In the presence of IPTG, the subpopulation of toxin \(\zeta\) (3-\(6\times 10^{-5}\) survivals) did not significantly increase during time and was similar in both, exponential growth and stationary phase (Figure 2A,B, empty blue rhombs). Similar results were observed when a low-lived \(\zeta\) variant (\(\zeta\)Y83C) is used [10], suggesting that when toxin \(\zeta\) is expressed, it triggers dormancy in high-density stationary phase cells. When Amp and IPTG were added, a low proportion of high-density stationary phase cells survive toxin \(\zeta\) and Amp action (4-\(7\times 10^{-8}\) survivals) (Figure 2B, empty purple circles).  
To test whether the expression of \(\varepsilon\) antitoxin also reverses the effect of \(\zeta\)-mediated dormancy in high-density non-growing cells, Xyl was added at the indicated times. The expression of \(\varepsilon\) antitoxin reversed the effect of the \(\zeta\) toxin (3-\(5\times 10^{-1}\) survivals) and partially reversed toxin and Amp (4-\(5\times 10^{-2}\) survivals) (Figure 2B, filled blue rhombs and purple circles). It is likely that: (i) Toxin \(\zeta\) induces a reversible dormant state in high-density, non-growing cells rather than entering in a stochastic viable but not-culturable state; (ii) there are two different subpopulations of Amp persisters: Stochastic (3-\(5\times 10^{-2}\) survivals) and toxin-induced (4-\(7\times 10^{-8}\) survivals); and iii) antitoxin expression reverses the apparent sensitization of toxin dormants to Amp action.