# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
###### Abstract
However, an increased expression of SOS response genes or proteases that selectively induce cell-cycle arrest is not observed [38]. At 15-30 min times, the concerted action of _relA_ and _comGA_ increases the (p)ppGpp pool and indirectly the levels of the essential c-di-AMP second messenger [15; 38]. Toxin \(\zeta\) expression induces a cellular dormant state, but a reduced subpopulation, which shows no genetic modification, survives toxin action [10; 15; 38]. At 30-90 min, the synthesis of macromolecules (DNA, RNA, proteins) is inhibited and the membrane potential is impaired, with \(\sim\)35% of total cells transiently stained with propidium iodide (an indicator of membrane compromised cells) [38]. At 90-120 min, the cell wall biosynthesis is reduced by \(\zeta\)-mediated phosphorylation of a small UNAG fraction, leading to the accumulation of unreactive UNAG-3P [34; 38]. In addition, (p)ppGpp mediates the inhibition of peptidoglycan metabolism [41]. Antitoxin \(\varepsilon\) expression blocks ATP binding by toxin \(\zeta\), thereby inhibiting its ATPase and phosphotransferase activities. Antitoxin \(\varepsilon\) reverses the toxic effect exerted by \(\zeta\) in cell proliferation, and only \(\sim\)10% of total cells remained stained with propidium iodide [38; 42; 43]. This is consistent with the fact that the toxin \(\zeta\) action is reversible by nature even in distantly related bacteria, such as _E. coli_ and _B. subtilis_ cells [38; 43]. Transient toxin \(\zeta\), expression sensitizes cells to Amp treatment, and antitoxin \(\varepsilon\) expression reverses the \(\zeta\)-induced dormant state, and the steady state condition is re-established [15; 38; 43]. The reversibility of \(\zeta\) action upon longer periods of time in the presence of Amp is unknown. Therefore, we have re-examined the mode of action of toxin \(\zeta\), at or near physiological concentrations in _B. subtilis_ cells growing in the minimal S7 medium, to understand how toxin \(\zeta\) contributes to reduce Amp persistence. In this study, we show that toxin \(\zeta\) induces a dormant although still metabolically active state. The study of SPP1 in cells where toxin \(\zeta\) is expressed reveals that \(\zeta\) induces an active slow-growth rather than a growth-arrested condition and abortive infection. Amp addition at twice the minimum inhibitory concentration (2\(\times\) MIC) halts _B. subtilis_ proliferation, leading to a biphasic time-inactivation curve, which is maintained by a long period of time. Transient toxin \(\zeta\) expression sensitizes cells to Amp action during a long time period. Antitoxin \(\varepsilon\) expression switches back \(\zeta\)-induced dormancy, and promotes the exit of toxin-facilitated Amp dormants without observing a point of no return. Inactivation of _recA_, which suppresses the SOS response, marginally reduced the relative rate of antibiotic persistence. Transient \(\varepsilon\) expression reverses the negative effect of the toxin and indirectly awakes Amp dormants to levels observed in the _rec\({}^{+}\)_ control. This toxin-mediated sensitization of bacterial cells to Amp and the understanding of the conditions that inhibit antitoxin action will facilitate targeted engineering of their activity towards the development of anti-persistence agents.