# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
###### Abstract
The type I to VI TA modules, which are classified according to the nature of the antitoxin and to their mode of toxin inhibition, are implicated in the fine tuning of multiple cellular functions and are associated with cell survival under different stress conditions [20; 21]. The largest group is formed by type II TAs, which were first described as loci that enforce the maintenance of plasmids via post-segregational killing [22], and their ability to stabilize their mobile elements is considered as a selfish property [21; 23]. In a type II TA system, which comprises a pair of genes coding for two proteins, the antitoxin forms a tight complex with the toxin to avoid its action [18; 19; 20; 21; 23]. Under certain stress conditions, the unstable antitoxin is rapidly degraded by host ATP-dependent proteases, freeing the proteolytically stable toxin [18; 20]. The free toxin, which targets essential cellular processes such as RNA, DNA and protein synthesis, cell division, general stress response, etc., triggers a biphasic cell inactivation curve [24]. A variant of the HipAB type II TA system, rather than deletion of _hipBA_ operon, has been shown to contribute to the formation of antibiotic persistence in _E. coli_ cells. The _hipA7_ mutation, which maps in the HipA toxin, produces a high fraction of persisters [7; 25]. HipA activates the stringent response, and by increasing (p)ppGpp levels, leads to persister formation [26; 27; 28]. However, a strong link between the induction of TA systems and antibiotic persistence is missing, and further work is needed to establish whether physiological levels of a toxin contribute to antibiotic persistence [23; 29; 30; 31]. Deletion of _E. coli_- and _B. subtilis_-encoded chromosomal type II TA systems have no effect on antibiotic persistence [12; 31]. The type II TAs of the \(\zeta\)-\(\varepsilon\) superfamily, which is among one of the most broadly distributed TA modules in nature, are found in major human and plant pathogens. TAs of this superfamily are composed of two toxin monomers (with \(\zeta\) and PezT as representatives), that interact with their cognate dimeric \(\varepsilon_{2}\)/PezA\({}_{2}\) antitoxin forming an inactive \(\zeta\varepsilon_{2}\zeta\) or PezT-PezA\({}_{2}\)-PezT complex [32; 33]. \(\zeta\)/PezT interacts with its activator/substrate, the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) [34]. UNAG is an essential precursor of bacterial cell wall biosynthesis [34]. The structures of the inactive \(\zeta\varepsilon_{2}\zeta\) or PezT-PezA\({}_{2}\)-PezT complex, alone or UNAG bound, have been reported [32; 33; 34; 35]. The interaction of the antitoxin with the toxin sterically blocks ATP-Mg\({}^{2+}\) binding by the \(\zeta\)/PezT toxin, but not with UNAG [32; 33; 34; 35]. Therefore, the interactions with ATP and \(\varepsilon_{2}\)/PezA\({}_{2}\) are mutually exclusive [32; 33]. The structure of the inactive \(\zeta\varepsilon_{2}\zeta\) or PezT-PezA\({}_{2}\)-PezT complex, both alone or UNAG bound, has been reported [32; 33; 34; 35]. Toxin \(\zeta\) hydrolyses ATP in the presence of UNAG and phosphorylates a small fraction of UNAG, rendering it irreversibly inactive [15; 34; 36; 37]. Transient toxin \(\zeta\) expression, at or near physiological concentrations, leads to several cellular responses in _B. subtilis_ cells growing in S7 minimal medium, at 37 \({}^{\circ}\)C with aeration. At early stages of transient toxin \(\zeta\) expression (up to 15 min), the \(\zeta\) UNAG-dependent ATPase decreases the ATPpool, and indirectly it alters the intracellular ATP/GTP ratios [37; 38]. Such perturbation affects the concentration of the transcription initiation nucleoside triphosphates (ATP, GTP), and indirectly regulates RNA polymerase transcription initiation [39]. This mechanism, which is crucial for rapid adjustment of gene expression in response to environmental changes [40], alters the expression of \(\sim\)2% of total _B. subtilis_ genes (among those, it induces _relA_ and _comGA_ gene expression) [38].