# Antitoxin \(\varepsilon\) Reverses Toxin \(\zeta\)-Facilitated Ampicillin Dormants
## 4 Materials and Methods
### Growth Conditions  
BG1125 (pCB799) and BG1125 (pCB1226) cells were grown to a mid-exponential phase (OD\({}_{560}\) = 0.2) in S7 medium supplemented with methionine, tryptophan, and 0.005% Xyl at 37 \({}^{\circ}\)C with shaking [43]. Under this condition, cells grew in S7 medium, with a doubling time of 50-60 min. Transient toxin and/or antitoxin expression was induced by IPTG (2 mM) and/or Xyl (0.5%) addition. Before plating, cells were centrifuged and resuspended in the fresh LB medium to remove the inductor or the antibiotic, and dilutions were plated on LB agar plates. The survival rate was derived from the number of CFUs in a given condition relative to the CFU of the non-induced/non-antibiotic-treated control. All plates were incubated for 20 h at 37 \({}^{\circ}\)C.  
The lytic SPP1 phage was used to test the toxin effect. BG1125 bearing _lacl-P\({}_{\mathrm{hsp}}\) wt\(\zeta\)_, and pCB799-borne _xylR-P\({}_{\mathrm{xylA}}\) wt\(\varepsilon\)_ gene were grown to a mid-exponential phase at 37 \({}^{\circ}\)C in S7 medium, supplemented with methionine, tryptophan, 0.005% Xyl, and 10 mM MgCl\({}_{2}\). Toxin expression was induced by adding IPTG 2 mM and after 15 min cells were infected with phage SPP1 at a multiplicity of infection (_moi_) of 1 in the presence or absence of IPTG. After 5 min, cells were centrifuged to remove unabsorbed phages and resuspended in S7 medium with 10 mM MgCl2 (0 min) and the culture was incubated for 60 or 120 min. The inducers were removed by washing, and the phage titer (PFUs) was calculated by plating the appropriate dilution deposited in a lawn of exponentially growing BG214 cells onto LB plates supplemented with 10 mM MgCl\({}_{2}\) (LB-Mg) to measure PFUs as described [72]. The plates were then incubated for 18-20 h at 37 \({}^{\circ}\)C.  
The MIC for Amp (Sigma-Aldrich, St. Louis, MO, USA) was estimated by exposing 1-3 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\) (16 h, 37 \({}^{\circ}\)C) in the minimal S7 medium, with an increasing Amp concentration under shaking (240 rpm). The minimal concentration that gave no growth overnight (1.5 ug mL\({}^{-1}\)) was defined as MIC. The Amp concentration used was twice the MIC (2\(\times\) MIC) or 3 ug mL\({}^{-1}\).  
The following data are available online at [[http://www.mdpi.com/2072-6651/12/12/801/s1](http://www.mdpi.com/2072-6651/12/12/801/s1)]([http://www.mdpi.com/2072-6651/12/12/801/s1](http://www.mdpi.com/2072-6651/12/12/801/s1)), Figure S1: Graphic illustration of the distinct responses that may occur after Amp and toxin \(\zeta\) action, Figure S2. Growth curves of BG1127 (pCB799) and BG1125 (pCB799) strains in the presence or absence of IPTG, Figure S3. BG1125 (pCB799) in the presence or the absence of IPTG.  
[PERSON]-d.A. and [PERSON] designed the experiments; [PERSON]-d.A. and [PERSON] performed the experiments, [PERSON] coordinated the research; [PERSON]-d.A. and [PERSON] interpreted the data; [PERSON]-d.A., [PERSON] and [PERSON] drafted the manuscript; and [PERSON] wrote the manuscript. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the Spanish Ministerio de Ciencia e Innovacion/Agencia Estatal de Investigacion (MCI/AEI)/FEDER, EU) PGC2018-097054-B-100 to J.C.A.  
We thank [PERSON] and [PERSON] for critical reading and comments on the manuscript.  
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.