## 2 Results and Discussion
### Toxin \(\zeta\)Y83C Leaves Significant Amounts of UNAG In Vivo  
_In vitro_ assays showed that \(\zeta\) is a poor kinase, phosphorylating only a fraction of the UNAG present. However, these assays might not extrapolate to the _in vivo_ situation. It could be that _in vivo_ there is(are) some unknown factor(s) that may stimulate the phosphotransfer reaction. To test whether \(\zeta\) toxin reduces or depletes the UNAG pool _in vivo_, and by this way it reduces or blocks the biosynthetic pathway of peptidoglycans different types of antimicrobials, which act at different levels of the two stage process of cell wall biosynthesis, were used. It has been established that the first step of peptidoglycan (murein) biosynthesis is a cytosolic stage that consists in the transfer of an enolpyruvate moiety from phosphoenolpyruvate (PEP) to the C3 position of \(N\)-acetylglucosamine moiety of UNAG, a reaction catalyzed by two MurA enzymes in Firmicutes (the ubiquitous MurA [MurA or MurA1] and MurAB [or MurA2] [18]), to yield enolpyruvateyl-UDP-\(N\)-acetylglucosamine (EP-UNAG). The synthesis of these murein precursors can be inhibited by: (I) \(\zeta\)-mediated phosphorylation of UNAG to form unreactive UNAG-3P; and (II) a naturally occurring PEP analogue, Fosfomycin (Fos), which binds covalently to the active site of MurAA (or MurAB). Fos, in the presence of the UNAG substrate inhibits cell wall biosynthesis by blocking the accumulation of EP-UNAG [19,20]. EP-UNAG is transformed by a series of cytosolic steps, which results in the formation of the UDP-\(N\)-acetyl-muramic acid-pentapeptide that is translocated to the membrane acceptor and through different steps the \(N\)-acetylglucosamine/\(N\)-acetylmuramic acid-pentapeptide is covalently linked to the lipid carrier molecule undecaprenol via a pyrophosphate ester bridge (Lipid II). Vancomycin (Van) prevents incorporation of \(N\)-acetylglucosamine and \(N\)-acetylmuramic acid-peptapeptide subunits into the peptidoglycan matrix, and interferes with the Lipid II cycle [21]. Then, the Lipid II building block is used as the substrate for the polymerization reaction, consisting of the assembly into glycan chains by transglycosylation and peptide cross-linking of the pentapeptide moiety by transpeptidation, reactions catalyzed by Classes A or B penicillin-binding proteins [22, 23, 24]. Amp acts as an irreversible inhibitor of these transeptidases [25,26]. We reasoned that if \(\zeta\) toxin depletes the UNAG pool, cells will become tolerant to antimicrobials that act downstream of toxin action, leading to non-inheritable drug tolerance (persistence). To test this hypothesis, \(\zeta\) toxin induced cells were exposed to Fos, Van or Amp and the antimicrobial drug tolerance was analyzed. These antimicrobials, in addition of inhibiting cell wall synthesis as explained above, induce two regulatory systems to cope with stress in Firmicutes: the extractoplasmic function (ECF) \(\sigma\) factors and/or cell envelope stress-sensing two component systems, which are linked to other stress responses [27]. Van, which is the strongest cell envelope-perturbing agent, induces the synthesis of a large number of genes controlled by specific \(\sigma\) factors (\(\sigma^{\mathrm{V}}\), \(\sigma^{\mathrm{M}}\), \(\sigma^{\mathrm{W}}\) and \(\sigma^{\mathrm{Y}}\)) and two components systems (e.g., LiaRS), Fos alters the expression of genes controlled by \(\sigma^{\mathrm{M}}\) and \(\sigma^{\mathrm{W}}\); and Amp alters the expression of genes through cell envelope stress-sensing two component systems (e.g., BlaRI, MecRI) [27]. If the three antimicrobials show a similar behavior upon \(\zeta\) toxin we can omit any specific contribution of cell envelop stress. To perform these experiments we took advantage of the short-lived toxin variant, \(\zeta\)Y83C (half-life \(\sim\)28 min) under control of a Xyl inducible promoter (_xylR_\(P_{\mathrm{XylA}}\zeta\)Y83C expression cassette) (Table 1).