# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### Transient Toxin \(\zeta\) Expression and Amp Addition Reduce the Subpopulation of Persisters  
Transient toxin \(\zeta\) expression sensitizes cells to the presence of different antibiotics [10; 37]. At least two mechanisms can be envisioned: (i) Toxin dominants are sensitive to antibiotic action and stochastically enter in a viable but not-culturable state with resuscitation independent of antitoxin expression; or (ii) antibiotic persistence is the sum of stochastic and transient antibiotic-induced dormants with resuscitation dependent of antitoxin expression. To test the above hypotheses, BG1125 (pCB799) cells were grown in S7 medium, supplemented with 0.005% Xyl, to OD\({}_{560}\) = 0.2 at 37 \({}^{\circ}\)C with shaking. Then, 2 mM IPTG and 3 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g mL}^{-1}}\) Amp were added, and at indicated times cells were washed and plated in the absence of both IPTG and Amp. A typical biphasic decline curve was observed (Figure 2A, purple empty circles). Amp and toxin action significantly decreased the proportion of survivals (1-2 \(\times\) 10\({}^{-7}\)) when compared to only \(\zeta\) (3-6 \(\times\) 10\({}^{-5}\) survivors) or only Amp (1-0.5 \(\times\) 10\({}^{-2}\) persisters) (Figure 2A, purple empty circles vs. blue empty rhombs and orange filled squares). The persistent cells stochastically switch back to a growing state, upon plating in the absence of IPTG and Amp, with the fraction of persisters remaining nearly constant for \(\sim\)900 min (Figure 2A, purple empty circles). It is likely that: (i) Amp persisters are in an active state that allows toxin expression; (ii) toxin \(\zeta\), expression does not increase antibiotic persistence; (iii) toxin expression triggers a network of intracellular stress responses that significantly increases cell dormancy; and (iv) toxin-induced dormancy facilitates Amp dormants that resuscitate upon antitoxin expression.  
Transient Antitoxin \(\varepsilon\) Expression Reverses \(\zeta\)-Facilitated Exit of Amp Dormants  
To test whether transient \(\zeta\) expression enhances Amp efficacy with the cells entering in a viable but not-culturable state or if \(\zeta\) expression induces dormancy of Amp persisters, BG1125 (pCB799) cells were grown in the minimal S7 medium, supplemented with 0.005% Xyl, to OD\({}_{560}\) = 0.2 at 37 \({}^{\circ}\)C with shaking. Then, 2 mM IPTG and Amp (3 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g mL}^{-1}}\)) were added and the cells incubated up to 900 min (Figure 2A, purple empty circles). At indicated times, IPTG and Amp were removed by washing the culture with the pre-warmed S7 medium, containing 0.5% Xyl to induce antitoxin \(\varepsilon\) expression, and cells were preincubated (15 min) before plating (Figure 2A, purple filled circles). The antitoxin \(\varepsilon\) recovered cell proliferation to levels equivalent to that of Amp persisters, even after 900 min of toxin \(\zeta\) and Amp action (Figure 2A, purple filled circles). These data altogether suggest that: (i) Toxin \(\zeta\) expression and Amp addition induce prolonged dormancy (at least for 15 h) rather than programmed-cell death, as reportedfor the _E. coli_MazEF and RelBE locus (reviewed in [61]); (ii) \(\zeta\)-induced dormancy is insensitive to Amp action; (iii) toxin-induced dormant cells awake and form colonies even after 15 h of toxin and Amp action, rather than triggering cell lysis or inducing a viable but not-culturable state (Figure 2A, purple filled circles); and (iv) the orthogonal control of \(\zeta\) and \(\varepsilon_{2}\) levels revealed that a TA system can be an important element in coping with Amp stress; and (v) toxin survivors and Amp persistence are not epistatic.