# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### Toxin \(\zeta\) Induces a Slow-Growth Active State  
In \(\gamma\)-Proteobacteria, deletion of chromosomally-encoded type II TA loci neither confers a fitness benefit nor influences stress tolerance [48; 49]. Similarly, deletion of \(B\). _subtilis_-encoded type II TA systems has no effect on antibiotic persistence [12]. To gain insight on the molecular and physiological bases of toxin-induced dormancy, dormant cells were infected with a lytic bacteriophage (phage) and the infective cycle was followed. Except TA systems that abort infection of a specific phage in selected hosts [50], and those phages that encode _bona fide_ TA systems [23], it has been observed that overexpression of type II toxins contributes to abortive infection and causes exclusion of lytic phages from the bacterial population [26; 51]. It has been also shown that in the arms race of a bacterium and its phage, a \(\lambda\) lytic variant was able to infect and kill survival bacteria, despite their dormancy state [52], and that T4 infection inactivates MazF toxin activity [53]. In contrast, it has been also shown that phage \(\lambda\) cannot overcome a toxin treat, since toxin overexpression, acting as a defense mechanism, protects the bacterial culture by increasing (p)ppGpp levels, and the alarmone might antagonize \(\lambda\) phage development [54]. Moreover, toxin RnIA overexpression cleaves phage T4 mRNAs to antagonize T4 amplification [55]. Therefore, phage infection of toxin-induced dormant cells can be a powerful tool to understand the physiology of toxin-induced dormancy and to address how the toxin \(\zeta\) expression sensitizes cells to toxin \(\zeta\) expression. A set of isogenic \(B\). _subtilis_ strains, lacking probhages (SP\(\beta\) and PBSX), conjugative genetic elements, CRISPR-Cas and restriction systems, and having a deficiency in the _rsbV_ gene, whose function is crucial for the activation of RNA polymerase \(\alpha^{\text{B}}\)-mediated general stress response [56], were used to characterize the impact of toxin \(\zeta\) expression on cell dormancy (Table 1). The BG1127 (pCB799) strain (in the presence or absence of IPTG, doubling time \(59\pm 3\) min) and BG1125 (pCB799) strain (in the absence of IPTG, doubling time \(58\pm 2\) min) grown in the minimal S7 medium supplemented with traces of Xyl (0.005%) at 37 \({}^{\circ}\)C with shaking had a similar doubling time (Figure S2). Both cell cultures reached a plateau at \(\sim\)\(3.5\times 10^{9}\) cells mL\({}^{-1}\) (Figure S2). By contrast, if IPTG (2 mM) was added at OD\({}_{560}\) = 0.2, the growth curve of the BG1125 (pCB799) strain was halted and after 720 min the OD\({}_{560}\) was not significantly decreased (Figure 1 and Figure S2), but the plating efficiency was significantly reduced (Figure 2A) [38]. 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. At this time (-20 min), the culture was divided, 2 mM IPTG (time zero) was added to a half, and both cultures were incubated for 20 min to allow toxin-induced dormancy. The total number of cells were measured as colony-forming units (CFUs) by plating them on LB plates lacking IPTG (Figure 1A and Figure S3). At -5 min, both cell cultures, containing or not IPTG, were infected with phage SPP1 at a multiplicity of infection (_moi_) of 1 (to minimize the noise of free phages), and incubated for further 5 min. Then, free phages were removed by centrifugation, and this time was considered as 0 min in our experiment. At 0 min, the number of cells was measured by plating on LB agar plates and plaque-forming units (PFUs), to determine the number of infected centers, cells were measured by plating appropriate dilutions of the culture in a lawn of exponentially growing BG214 cells (indicator strain) onto LB-Mg (supplemented with 10 mM MgCl\({}_{2}\)) plates (Figure 1A,B).