## 2 Results and Discussion
### Toxin \(\zeta\)Y83C Leaves Significant Amounts of UNAG In Vivo
The level of toxin in non-induced BG689 cells is too low (\(<\)10 \(\zeta\)Y83C monomers/colony forming units, CFUs) to measurably alter the growth rate in minimal medium S7 (MMS7) [12]. Previously it was observed that induction of the _xyIR_\(P_{\chi\mathrm{yl}A}\zeta\)Y83C cassette (BG689 cells), by addition of 0.5% Xyl, increased \(\zeta\)Y83C levels to a plateau with a toxin concentration of \(\sim\)300 toxin monomers/CFUs at \(\sim\)10 min, and the steady-state level of the toxin remained for at least 240 min [12]. When exponentially growing BG689 cells, growing in MMS7, reached moderate-density (\(\sim\)5 \(\times\) 10\({}^{7}\) cells/mL) xylose (Xyl) 0.5% was added to express \(\zeta\)Y83C toxin as described Materials and Methods [14,15]. As already observed, transient exposure to sub-physiological concentrations of free \(\zeta\)Y83C induced dormancy and produced a typical biphasic survival curve, with a sub-fraction of cells (1-\(5\times 10^{-5}\) cells) non-inheritable tolerant to toxin action upon 120 min of exposure (Figure 3) [14,15]. When exponentially growing BG689 cells (\(\sim\)5 \(\times\) 10\({}^{7}\) cells/mL in MMS7) were transiently exposed to Fos or Amp (at twice the minimal inhibitory concentration [MIC]) or to Van (at 4 times the MIC) for 120 min, these bactericidal antimicrobials caused a cessation of cell proliferation, and rendered a fraction of 0.8 to 5 \(\times\) 10\({}^{-3}\) phenotypic tolerants (Figure 3). This multidrug tolerance of the isogenic population of antimicrobial sensitive cells is termed bacterial persistence [28, 29, 30]. To test whether toxin action depletes the UNAG pool and this leads to enhanced antimicrobial persistence when cells are treated with cell wall inhibitors moderate-density exponentially growing BG689 cells were treated with Xyl (to induce \(\zeta\)Y83C toxin expression) and transiently exposed to the antimicrobial. Experiments are showed when the two compounds (_i.e._, Xyl and the antimicrobial) were added simultaneously, but the same results were obtained when first cells were exposed to toxin action for 30 min, and later the antimicrobial was added. Toxin \(\zeta\)Y83C expression and Fos, Van or Amp treatment additively decreased the rate of cell survivals to 0.9 to 2 \(\times\) 10\({}^{-6}\) (Figure 3). Since the three antimicrobial show a similar outcome in the presence of free \(\zeta\)Y83C toxin, it is unlike that the enhanced sensitivity could be attributed to a perturbation of cell envelop stress (see above). It is likely that: (I) \(\zeta\) toxin expression only decreases the UNAG pool, and Fos, Van or Amp use the remaining fraction to further inhibit the plating of toxin tolerant cells; and (II) \(\zeta\) toxin expression additively enhances the efficacy of the antimicrobials rather than making cells insensitive to Fos, Van or Amp treatment. Alternatively, transient expression of \(\zeta\) toxin, by inactivating the UNAG substrate, inhibits the action of the MurA-MurF enzymes, and this inhibition leads to loss of cell shape and integrity, with cells becoming more prone to autolysis, when they are additionally treated with any of these antimicrobial. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strains** & **Relevant genotype** & **Reference** \\ \hline BG687 1  & [12] \\ BL21(DE3) 2  & [12] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Bacterial strains used.