Growth, stability and colony morphology of MqsR variants. A. Growth curves in LB medium with 1 mM IPTG induction at 37°C for BW25113 ΔmqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐mqsR (MqsR), pBS(Kan)‐mqsR 2‐1 (MqsR 2‐1) and pBS(Kan)‐mqsR 20‐14 (MqsR 20‐14). Error bars indicate standard deviation (n = 3). B. Structure of MqsR in ribbon representation with MqsA in surface representation (based on Protein Data Bank accession code 3HI2). Cyan indicates MqsR active site residues (K56, Q68, Y81 and K96), yellow indicates the residues (K3 and N31) where the substitutions occur in MqsR 2‐1 (K3N and N31Y), and orange indicates the residues (R9, L35 and V70) where the substitutions occur in MqsR 20‐14 (R9C, L35F and V70I). C. Protein stability of native MqsR and the MqsR variants (MqsR K3N, MqsR N31Y and MqsR 2‐1). Western blot (upper panel) and SDS‐PAGE show the protein levels of His‐MqsR and variants detected by a His‐tagged antibody. MqsR and the variants were induced from pET28a‐based plasmids in E. coli BL21 (DE3) via 1 mM IPTG. Arrows indicate the MqsR proteins. D. Colony morphology of BW25113 ΔmqsR strains containing pBS(Kan) (empty plasmid), pBS(Kan)‐mqsR and pBS(Kan)‐mqsR 2‐1 grown on LB agar plates at 37°C after 24 h. Scale bars indicate 1 cm. Representative images are shown. E. Observation of BW25113 ΔmqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐mqsR and pBS(Kan)‐mqsR 2‐1. IPTG was added to LB for 2 h to produce native MqsR and MqsR 2‐1 (middle row), while glucose (0.2%) was added to LB to repress MqsR production (upper row). Lower row shows the result of Live/Dead staining after producing MqsR and MqsR 2‐1. Live cells are stained in green, and dead cells are stained in red. Scale bars indicate 10 µm. Representative images are shown.

Persister cell formation of MqsR variants. BW25113 ΔmqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐mqsR, pBS(Kan)‐mqsR 2‐1 and pBS(Kan)‐mqsR 20‐14 were grown to a turbidity of 0.5 at 600 nm in LB with 1 mM IPTG at 37°C, adjusted to a turbidity of 1, and exposed to 26 µg ml⁻¹ ampicillin with 1 mM IPTG for 24 h. Error bars indicate the standard deviation (n = 3).

Effect of isogenic mutations on persister cell formation. BW25113 and its isogenic mutants were grown to a turbidity of 1 in LB at 37°C and exposed to 20 µg ml⁻¹ ampicillin for 5 h (A). Time‐course of persister formation of BW25113 ΔrpoS exposed to 20 µg ml⁻¹ ampicillin for 6 h (B). Persister formation of BW25113 ΔrpoS exposed to 1 µg ml⁻¹ ciprofloxacin (Cipro) for 5 h (C). Persister formation of PA14 ΔrpoS exposed to 1 µg ml⁻¹ Cipro for 5 h (D). Error bars indicate the standard deviation (n = 3).

Persister formation after oxidative and acid stresses. Cell survival (%) with oxidative stress (20 mM H₂O₂) for 10 min (A) and with acid stress (pH 2.5) for 2 min (B). BW25113 and its isogenic mutants were grown to a turbidity of 1 in LB at 37°C and exposed to H₂O₂ or pH 2.5. C. Persister cell formation of BW25113 and its isogenic mutants exposed to 20 µg ml⁻¹ ampicillin for 5 h after oxidative or acid stress. Error bars indicate the standard deviation (n = 3).

Schematic of persistence and resistance with rpoS. Upon stresses such as UV exposure, acid shock, heat shock, oxidative stress and starvation, most cells induce resistance by RpoS (left panel). For a small percentage of cells, stresses also increase persister formation (right panel). MqsA is degraded by proteases (ClpXP and Lon), and MqsR 2‐1 with enhanced toxicity degrades mRNA transcripts including stress responsive mRNAs. The degradation of rpoS mRNA by the toxin leads to downregulation of the acid (gadB, gadX), osmotic (osmY), and multidrug (mdtF) resistance systems. Then, upon antibiotic treatment, cells enter a dormant state. ‘→’ indicates induction, and ‘⊥’ indicates repression or differential mRNA decay.