# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 2 Results and Discussion
### Toxin \(\zeta\) Induces a Slow-Growth Active State
In parallel, free phages from the supernatant of centrifugation were determined, to test whether centrifugation was sufficient to remove them or polyclonal anti-SPP1 antibodies were necessary to the inactive free phages. Since in the minus IPTG condition the number of total cells minus free phages was not significantly different from the number of infected centers, we have omitted the use of polyclonal anti-SPP1 antibodies. After 20 min of toxin induction IPTG, CFUs were reduced by \(\sim\)1000-fold at a time when compared to the parallel culture in the absence of IPTG (Figure 1A and Figure S3, black vs. grey bar). After infection with SPP1, the proportion of infected centers measured as PFUs was similar in the minus and plus IPTG conditions (Figure 1B, black and grey bars), suggesting that a large fraction of dormant cells were infected by SPP1. In the absence (grey bar) and presence of IPTG (black bar), the phage titer, which correlates to the cell population infected by SPP1, was similar and in good agreement with the expected for a one-hit kinetic. From the input phages, \(\sim\)71% and \(\sim\)67% infected the cells, respectively, defining the infected centers (Figure 1B) and \(\sim\)30% were recovered from the supernatant after centrifugation. In parallel, the total number of cells at -20, 0, 60, and 120 min in the absence of phage SPP1 was also measured as CFUs by plating them on LB plates lacking IPTG (Figure S3). The SPP1 infection cycle takes 35-45 min in cells growing in the minimal medium, thus the infected cells were incubated for 60 min, and then the relative number of progeny phage per infected cell (burst size) was deduced for the minus and plus IPTG conditions. At 60 min and in the presence of IPTG, cells remained in the dormant state (Figure 1A vs. Figure S3), suggesting that SPP1 does not encode (a) gene(s) whose function can neutralize toxin \(\zeta\), action. At 60 min, the phage SPP1 titer from cultures where the toxin had been induced was similar to the number of infected cells at 0 min. However, in the absence of IPTG, the PFUs greatly increased. The SPP1 relative burst size (the ratio of the total number of SPP1 progeny to the number of input phage) from three independent experiments was quantified by counting cells and phages taking in to account the supernatant of the cultures (Figure 1A,B). The SPP1 burst size significantly increased (\(\sim\)80 SPP1 phages/infected cell). These results suggest that SPP1, upon infecting toxin-dormant cells neither enters in the amplification cycle nor promotes the exit of the dormant state. To test whether dormant cells antagonize or cannot support SPP1 amplification or if dormant cells can support SPP1 amplification upon toxin neutralization at 60 min post-infection, Xyl (0.5%) was added to induce antitoxin \(\varepsilon\) expression, and the cultures were incubated for further 60 min. At 120 min, the number of total cells and the SPP1 burst-size were quantified as described above. In the  
Figure 1: Toxin dormants infected with the lytic phage SPP1 are metabolically active. Survival rate of BG1125 (pCB799) cells infected by the SPP1 phage after \(\zeta\) toxin induction. BG1125 (pCB799) cells were grown in the minimal S7 medium, containing traces of Xyl (0.005%) and 10 mM MgCl\({}_{2}\) to OD\({}_{560}\) = 0.2 at 37 \({}^{\circ}\)C. At this time (-20 min) the culture was divided in two. To one half, IPTG (2 mM) was added (black bars) to induce \(\zeta\) expression (-20 min) and incubated for 15 min. After 15 min, phage SPP1 at \(moi=1\) was added to both cultures and cells were incubated for further 5 min. At 0 min, cells were centrifuged to remove unadsorbed phages and resuspended in S7 medium, containing 0.005% Xyl and 10 mM MgCl\({}_{2}\). Colony-forming units (CFUs) were counted plating on a LB agar (time 0) and both infected cultures were incubated for further 60 min (60 min). At 60 min, 0.5% Xyl was added to induce antitoxin \(\varepsilon\) expression to neutralize toxin action, and both infected cultures were incubated for further 60 min (120 min).