# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion  
### Experimental Rationale  
Resistance, tolerance, and persistence are independent solutions used by bacteria to survive an antibiotic action (Supplementary Figure S1) [1]. A bacterial population resistant to antimicrobial, which grows under drug pressure, uses mutation-associated defense mechanisms (such as antibiotic inactivation/modification, increased efflux, or target modification) to confer a resistant phenotype with or without a fitness cost (Figure S1) [19; 44; 45]. Persistence is a special case of tolerance, and often the two terms are interchangeable [45]. Tolerance is the general ability of a population to survive a longer antibiotic treatment without a significant change in the MIC (Figure S1, dotted line), whereas persistence represents a subpopulation of cells that can survive the antibiotic treatment (Figure S1, dotted vs. orange broken dotted line) [1]. The growth of susceptible bacteria is challenged by the addition of an antibiotic (e.g., Amp), but a subpopulation of stochastic variants, by undergoing a period of slow- or non-growth, escapes the antibiotic action, leading to a bimodal time-inactivation curve of cell persistence (Figure S1, orange broken dotted line) [1]. In _B. subtilis_, the mechanisms responsible for antibiotic persistence have not been delineated, and the fate of persistents after a prolonged exposure to the antibiotic before returning to permissive conditions is poorly understood. Here, Amp-induced cell-wall stress, by inducing _sasA_ expression (one of the two small alarmone synthases), reduces the GTP pool and increases c-di-AMP and ppGpp levels [10; 13; 17]. This state might be transient and all cells might \"awake\" after removal of the antibiotic. In previous studies, the number of toxin \(\zeta\) and antitoxin \(\varepsilon\) molecules from a _S. pyogenes_ plasmid-borne \(\varepsilon\)-\(\zeta\) TA cassette was determined in the _B. subtilis_ host grown in the minimal S7 medium [43; 46]. An orthogonal control of \(\zeta\) and \(\varepsilon\) was constructed by placing the \(\zeta\) gene under the control of an isopropyl-\(\beta\)-D-thiogalactoside (IPTG) inducible promoter (_lacI_, _P\({}_{hsp}\zeta\), _spc_ cassette), was ectopically integrated into the _B. subtilis amty_ locus (Table 1) [38]. The antitoxin \(\varepsilon\) gene, which reads from a xylose (Xyl)-inducible promoter (_xylR_, _P\({}_{xylA}\varepsilon\), cat_ cassette), was cloned into the middle-low-copy number pCB799 plasmid (~8 copies/cell) [38]. In _B. subtilis_, BG1125 pCB799 cells grown in S7 are prone to genome rearrangements, by transcriptional escape of the \(\zeta\) gene from the _P\({}_{hsp}\)_ promoter. The Xyl concentration required to reduce genome rearrangements was estimated to be 0.005% [10; 38]. BG1125 (pCB799) grows in the S7 medium with 0.005% Xyl at 37 \({}^{\circ}\)C with a doubling time of (58 \(\pm\) 2 min) (Figure S2) [38]. Then, the IPTG concentration necessary to express similar levels of toxin from the native system, and the Xyl concentration necessary to express sufficient \(\varepsilon_{2}\) to inactivate the toxin \(\zeta\) action was estimated [38]. Transient toxin \(\zeta\) expression, at or near physiological concentrations, induces reversible cell dormancy. Here, a large cell fraction enters into a toxin-induced dormant state that cannot form colonies since they minimize their metabolic activity. By contrast, a small subpopulation escapes the toxin \(\zeta\) action without diminishing their metabolic activity. When the inducer of toxin expression was removed, the cells exited the transient dormant state and formed colonies, leading to bimodal toxin survivors (Figure S1, blue broken dotted line) rather than toxin tolerants (Figure S1) [15]. These cells that escape the toxin \(\zeta\) action show no genetic changes [38]. Transient toxin \(\zeta\) expression sensitizes cells to the Amp treatment [10; 15]. In the BG1125 [pCB799] strain, the presence of IPTG or Xyl does not affect the MIC for Amp [10].