# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### Discrete Subpopulations of Toxin \(\zeta\) Survivors and Amp Persisters  
The subpopulation of cells that survive an antibiotic treatment, are termed persisters [1; 45]. The rate of killing by Amp is strictly proportional to the rate of bacterial growth, and Amp persistence is associated with a pre-existing phenotypic switching to a stochastic non-growing state (or leftover cells from high-stress stationary-phase inoculums) [57; 58]. To analyze the interconnection between toxin survivors and Amp persisters and whether a prolonged growth arrest causes an irreversible cell cycle arrest (point of no return), we have used BG1125 (pCB799) cells (Table 1). 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, and then 2 mM IPTG (0 min) was added to induce toxin expression, and the reaction incubated for 900 min (Figure 2A). Toxin \(\zeta\) expression quickly reduced the cell plating efficiency showing a biphasic time-inactivation curve (Figure 2A, blue empty rhombs), suggesting that toxin \(\zeta\) expression reaches a steady-state in a short time and it remains for at least 900 min. It is likely that toxin \(\zeta\) expression induces a slow-growth active state, but a rare fraction (\(3\)-\(6\times 10^{-5}\)) exits this dormant state and survives, or a preexisting population of non-growing cells are insensitive to toxin action (type I-like persisters) (see [59]). Alternatively, expression of the \(\zeta\) gene triggers the expression of any of the type I and/or type II TA modules present in the background, and the loss of cell viability and the observed surviving cells arise via the overlapping activation of multiple toxins, even with different activities. To test whether the frequency of survivors may increase in response to the spontaneous induction of other chromosomally encoded toxins present in the background, IPTG was removed, by washing the culture with the pre-warmed S7 medium, containing 0.5% Xyl (to induce antitoxin \(\varepsilon\) expression), and cells were preincubated for 15 min before plating on LB plates containing 0.5% Xyl, but lacking IPTG. Antitoxin \(\varepsilon\) expression reversed \(\zeta\)-induced dormancy, by significantly increasing cell viability nearly to full values almost similar to the time of IPTG addition (Figure 2A, blue filled rhombs). Similar results are observed when IPTG is not removed from the medium [15]. It is likely that: (i) Toxin \(\zeta\)-induced dormancy rather than cell lysis, as judged by the non-significant variation in the OD\({}_{560}\) = 0.2 during a 720-min interval (Figure S2); (ii) toxin \(\zeta\) is not triggering a spontaneous induction of toxins from TA systems present in the background; and (iii) toxin \(\zeta\) transiently halts DNA replication and triggers a phenotypic heterogeneity [38]. In addition, the expression of the antitoxin \(\varepsilon\) expression reversed \(\zeta\)-induced growth arrest even after a prolonged dormancy (equivalent to \(\sim\)15 mass doubling periods) (Figure 2A, blue filled rhombs). To address whether antibiotic persisters are produced due to a stochastic entrance into the stationary phase, the overnight culture was extensively diluted and 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. It is expected that \(<\)0.3% of the total cells in the actively growing inoculum are old cells from the stationary-phase inoculum. Amp (at 2\(\times\) MIC, 3 \(\mathrm{\SIUnitSymbolMicro\text{$\mu$g mL}^{-1}}\)) was added, and the culture was maintained up to 900 min. At indicated times, the cells were washed to remove the antibiotic, and plated in LB lacking Amp. The removal of Amp was sufficient to reveal that a large fraction of cells (\(>\)99% of total cells) cannot form colonies upon exposure to bacteriolytic Amp (Figure 2A, filled orange squares).