# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
###### Abstract  
Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive \(\zeta\)-\(\varepsilon_{2}\)-\(\zeta\) TA complex is composed of one labile \(\varepsilon_{2}\) antitoxin dimer flanked by two stable \(\zeta\) toxin monomers. Free toxin \(\zeta\) reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-\(N\)-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of \(\zeta\) and \(\varepsilon\) levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin \(\zeta\) induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin \(\zeta\)-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin \(\zeta\) expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin \(\varepsilon\) expression, by reversing \(\zeta\) activities, facilitates the exit of Amp-induced dormancy both in \(rec^{+}\) and \(recA\) cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions. toxins-antitoxin system; cell wall inhibition; persistence; nucleotide hydrolysis; uridine diphosphate-\(N\)-acetylglucosamine  
The understanding of bacterial persistence might enable us to devise treatments against such threat. Toxin \(\zeta\), which is a UNAG-dependent ATPase, interferes with purine nucleotide homeostasis, and this physiological heterogeneity induces reversible dormancy and a subpopulation of survivors. Transient \(\zeta\) expression indirectly reduces Amp persistence without massive autolysis. Antitoxin \(\varepsilon\) expression reverses toxin action and induces the exit of cells from an Amp dormant state. A deeper understanding of the metabolic state to inhibit toxin action, which sensitiizes bacterial cells to different antibiotics, and disruption of antitoxin-mediated reversion of toxic action, are crucial to overcome antibiotic persistence. (_Escherichia coli_ being the best-characterized representative) starvation-induced (p)ppGpp contributes to antibiotic persistence, whereas in bacteria of the Firmicutes phylum, the persistence effectors are more complex [7; 8; 9; 10]. It has been proposed that in _S. aureus_ the _bona fide_ persistence effector is ATP depletion [11], but treatment with ATP synthesis inhibitors also induces (p)ppGpp accumulation and decreases GTP levels [12]. In _Bacillus subtilis_, which is the best-characterized representative of the Firmicutes phylum, the synthase-hydrolase ReIA, and the two small alarmones synthases SasA (also termed RelP or YwaC) and SasB (RelQ or YjbM) downregulate ATP and GTP levels, increase (p)ppGpp, and indirectly the cyclic 3,5-diadenosine monophosphate (c-di-AMP) pools [13; 14]. Antibiotic persistence might be controlled by deregulating (p)ppGpp and c-di-AMP pools [10; 15]. In response to antibiotics that target cell wall biosynthesis, as the bactericidal \(\beta\)-lactam ampicillin (Amp), (p)ppGpp and c-di-AMP synthesis are induced in _B. subtilis_ cells [16]. Furthermore, the _sasA_ transcripts are increased by certain classes of cell-wall-active antibiotics (Amp among them) that trigger a membrane stress, and increase (p)ppGpp and c-di-AMP levels [13; 17]. Bacterial toxin-antitoxin (TA) modules, which are ubiquitous across a broad range of extrachromosomal elements and are present in bacterial and archaeal chromosomes, are operons encoding a toxin that interferes with vital cell processes and an antitoxin that counteracts toxin [18; 19; 20; 21].