# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 1 Introduction  
Bacteria sense and respond to environmental stress with responses that can require dramatic cellular reprogramming. Protein toxins, of the toxin-antitoxin (TA) modules, are implicated in multiple cellular functions and are associated with cell survival under different stress conditions. Toxins and their cognate antitoxins have been detected in the chromosomes of bacteria, their phages and low-copy number plasmids, as well as in archaea, with more than 6000 putative modules identified so far [1]. The nature and activity of the antitoxins was used to classify the TA systems into six different types (I to VI). The largest group is formed by type II TAs [2]. In the type II TAs, which comprise a pair ofgene-coding proteins, the antitoxin forms a tight complex avoiding the action of the toxin [2; 3; 4; 5]. In the presence of stress, the unstable antitoxin is rapidly degraded, freeing the more stable toxin. Then, the free active type II toxin targets essential cellular processes such as RNA, DNA and protein synthesis, cell division, etc., and reversibly induces inhibition of cell proliferation (dormant state) [2; 3; 4; 5; 6; 7; 8; 9]. These TAs may also contribute to antibiotics persistence [2; 6; 10; 11], although a direct link between induction of TA systems and persistence to antibiotics has recently been challenged [12]. Type II toxins of the \(\zeta\) superfamily, which are among the most broadly distributed in nature, sensitize bacterial cells to different antibiotics [13; 14; 15; 16]. To understand how the toxin \(\zeta\) might contribute to reducing recalcitrant and recurring infections, we have examined its molecular mechanism of action. The TA cassette of the \(\zeta\)-\(\varepsilon\) superfamily, which has been found in major human and plant pathogens, is phylogenetically well conserved [17; 18]. In Firmicutes, toxin \(\zeta\) from _Streptococcus pyogenes_, _S. agalacatine_, _Enterococcus faecalis_, _Clostridium perfingens_ or _Staphylococcus aureus_ (\(\sim\)285 amino acids) share -43% sequence identity with the shorter _S. pneumoniae_ PezT or _S. suis_ SezT toxin (\(\sim\)255 amino acids) [19; 20]. In solution, two \(\zeta\) or PezT monomers interact with their cognate antitoxin dimers (\(\varepsilon_{2}/\)PezA\({}_{2}\)), with ATP-Mg\({}^{2+}\) (denoted as ATP) and with its target uridine diphosphate-N-acetylglucosamine (UNAG), which is an essential precursor of bacterial cell wall biosynthesis [19; 21; 22; 23]. The interaction of toxin and antitoxin leads to the formation of a biological non-toxic hetero-tetrameric (\(\zeta\varepsilon_{2}\zeta/\)PezT-PezA\({}_{2}\)-PezT) complex. The structure of the inactive \(\zeta\varepsilon_{2}\zeta\) complex, both alone or UNAG bound, has been reported [19; 21; 22; 23]. In these structures, the ATP binding site has been putatively assigned based on similarity to chloramphenicol phosphodiesterase [19; 22]. The crystal structure of the \(\zeta\varepsilon_{2}\zeta\) TA complex and the complex bound to UNAG showed that the ATP binding pocket of the \(\zeta/\)PezT toxin is sterically blocked by the binding of the \(\varepsilon_{2}/\)PezA\({}_{2}\) antitoxin, but the antitoxin cannot interfere with UNAG binding [19; 21; 22; 23]. Therefore, the interactions with ATP and \(\varepsilon_{2}/\)PezA\({}_{2}\) are mutually exclusive [19; 22]. In the presence of UNAG, toxin \(\zeta\) primarily hydrolyses ATP rather than another nucleotide cofactors [24; 25]. Toxin \(\zeta\), which fails to undergo auto-phosphorylation [24], transfers the ATP \(\gamma\)-phosphate (P\(\gamma\)) to a fraction of the C3\({}^{\prime}\)-OH group of the amino sugar of the N-acetylglucosamine moiety of the peptidoglycan precursor UNAG amino sugar, producing irreversibly unreactive UNAG-P [23; 24]. UNAG-P cannot be utilized by the MurA (MurAA and MurAB) enzyme(s) that catalyze the transfer of enolpyruvate from phosphoenolpyruvate to the C3\({}^{\prime}\)-OH group of the N-acetylglucosamine moiety of UNAG [23].