# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 3 Conclusions  
This study provides new insights that allow us to unravel the mechanisms underlying the mode of action of the toxin \(\zeta\) dormancy and indirectly of toxin and Amp dormants. Transient toxin \(\zeta\) expression, at or near physiological concentrations, induces a slow-growth dormant state, and a small fraction of cells (persisters) is insensitive to the toxin action in _rec\({}^{+}\)_ and _recA_ backgrounds. Toxin \(\zeta\)-induced dormant cells can be infected with the lytic phage SPP1 with a similar efficiency as exponentially growing cells, but the amplification cycle is halted. Neutralization of the toxin, by inducing antitoxin expression, allows dormant cells to exit this state and this allows phage amplification. It is likely that toxin dormants are metabolically active rather than in a growth-arrested state.  
Figure 3: Inactivation of _recA_ does not affect the frequency of \(\zeta\) survivors and Amp persisters. BG1889 (pCB799) cells were grown in the minimal S7 medium, containing traces of xylose (Xyl; 0.005%) to OD\({}_{560}\) = 0.2 (with CFUs of 1–3 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\)) at 37 \({}^{\circ}\)C. Then, the cultures were divided. To the indicated cultures, Xyl (0.5%) to induce \(\varepsilon\) expression as the control, IPTG (2 mM) to induce \(\zeta\) expression, Amp (3 \(\upmu\)g mL\({}^{-1}\)) or both IPTG and Amp were added (0 min), and the cultures were incubated for 900 min. At various times, samples were withdrawn and plated in LB agar plates. At various times, aliquots were taken and 0.5% Xyl was added to induce antitoxin expression, and the cultures were incubated for 15 min before being plated in LB agar plates containing Xyl, but lacking IPTG (filled blue rhomb), or both IPTG and Amp (filled purple circle). Data are shown as mean \(\pm\) standard error of the mean (SEM), from >4 independent experiments.  
Amp action kills \(\sim\)99% of susceptible cells, but stochastic Amp persisters (frequency of 1-0.5 \(\times\) 10\({}^{-2}\)) can form colonies upon removal of the antibiotic. Toxin survivors are significantly rare (\(\sim\)3 \(\times\) 10\({}^{-5}\)), and in the presence of Amp the proportion of bacterial Amp and toxin survivors was further reduced (by 100-200-fold), suggesting a non-epistatic effect. To further evaluate the viable but non-culturable state hypothesis, we inactivated _recA_. In _E. coli_ cells, _recA_ contributes to apoptotic cell death in Amp treated cells [64], but the proportion of Amp persisters was not affected in the absence of _recA_[5]. In _B. subtilis_ cells, toxin \(\zeta\) and Amp persistence are not significantly affected in _recA_ cells when compared to _rec\({}^{+}\)_ control, and transient antitoxin \(\varepsilon\) expression is necessary and sufficient to switch off toxin-induced dormancy, and to awake the fraction of toxin-facilitated Amp-induced persisters both in the _rec\({}^{+}\)_ and _recA_ cells. We propose that in exponentially growing cells, two different subpopulations of Amp persisters are found, stochastic and toxin-induced dormancy, that also induce long term Amp dormancy.