# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### RecA Inactivation Does not Alter the Antitoxin Awake of Toxin and Amp Dormants  
Toxin-induced dormants are metabolically active slow-growing cells, and this may enhance the development of heritable genetic changes, since many of the stress-response programs involved in the survival of persisters can also accelerate genome-wide mutagenesis [58]. In Proteobacteria, both stochastic and stress-induced slow-growth persisters have been shown to depend on the SOSresponses [58; 62]. Furthermore, the SOS response induces the expression of certain type I and II TA modules in a subpopulation of _E. coli_[5; 63]. In Proteobacteria and Firmicutes, bactericidal antibiotics (e.g., Amp), regardless of the drug-target interaction, induce changes in cellular metabolism that promote the production of highly deleterious hydroxyl radicals, leading to cell killing [64]. Amp-induced hydroxyl radicals, may damage template bases and introduce single strand nicks. These nicks, through DNA replication, can be converted into one-ended double-strand breaks (DSBs), that cause a significant induction of the SOS response [64; 65] and maintenance of Amp persistence in _E. coli_ cells [66]. However, DNA damage caused by sublethal doses of a fluoroquinolone in _E. coli_ promotes persistence, but not Amp [5]. The recombinase _recA_, which is a central player in the SOS response, contributes to the repair of the damaged template bases that escape specialized repair, and of the nicks that are converted in one-ended DSBs during DNA replication [67; 68]. The contribution of the SOS response to the DSB repair, however, differs substantially between _E. coli_ and _B. subtilis_ cells. In _B. subtilis_, the inability to induce the SOS response does not compromise the DSB repair [69]. In _E. coli_ cells, _recA_ plays a critical role in the appearance of apoptotic phenotypes and in pushing the cell towards its death [65], and inactivation of _recA_ potentiates killing by different antibiotics, Amp among them [64]. In _B. subtilis_ cells, transient toxin \(\zeta\) expression [38; 43] or extended cell cycle arrest [70] does not induce the SOS response. To test whether _recA_ inactivation sensitizes cells to toxin or Amp, a _ArecA_ mutation was mobilized onto the _B. subtilis_ BG1125 (pCB1226) strain by SPP1-mediated generalized transduction, rendering the BG1889 (pCB1226) strain (Table 1). Plasmid pCB1226 is a derivative of pCB799, but the _ermC_ gene was selectively inactivated so that the _ArecA_ mutation could be introduced in _B. subtilis_ BG1125 (pCB1226) cells by \(\mathrm{Erm^{R}}\) selection (Table 1). BG1889 (pCB1226) cells were grown in S7 medium, supplemented with 0.005% Xyl, to \(\mathrm{OD}_{560}=0.2\) at 37 \({}^{\circ}\)C with shaking. CFUs at this \(\mathrm{OD}_{560}\) are 3-6 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\), in agreement with previous reports showing that in _B. subtilis_ only 10-20% of _ArecA_ cells can form colonies in plates in the absence of DNA damage [71]. Then, IPTG (2 mM), Amp (3 \(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\)), or both IPTG and Amp were added (0 \(\mathrm{\SIUnitSymbolMicro m}\)), and the culture was maintained for 900 min (Figure 3). In the absence of ITPG and presence of traces of Xyl (0.005%), the BG1889 (pCB1226) cells had a poor fitness, formed small-size colonies, and the plating efficiency was reduced \(\sim\)100-fold after 900 min of incubation (Figure 3, filled grey triangles). This result indicates that a significant proportion of _ArecA_ cells may have a prolonged defect in DNA repair and chromosomal segregation, which reduces cell viability during prolonged growth. The number of Amp persisters and toxin survivors during the first 120 min was lower \(\sim\)\(2\times 10^{-3}\) and \(\sim\)\(4\times 10^{-6}\), respectively, but persistence to both Amp and toxin (\(\sim\)\(4\times 10^{-7}\) frequency of persisters) in the _ArecA_ context (Figure 3, orange filled squares and blue empty rhomb vs. grey filled triangles) was similar to that of the _rec\({}^{+}\)_ control (see Figure 2A).