E6376

Fresh antibiotic solutions were prepared for each experiment: ampicillin (dissolved in sterile MilliQ water; Sigma–Aldrich A9518), erythromycin (dissolved in 96% ethanol; Sigma–Aldrich E6376), gentamicin (dissolved in sterile MilliQ water; Sigma–Aldrich G3632), norfloxacin (dissolved in sterile MilliQ with 1% glacial acetic acid; Fluka N9890) vancomycin (dissolved in sterile MilliQ; Sigma–Aldrich V2002) and co-trimoxazole, which is comprised of one part trimethoprim (dissolved in sterile MilliQ with 1% glacial acetic acid; Sigma–Aldrich 92131) and five parts sulphamethoxazole (dissolved in acetone; Fluka S7507). H₂O₂ (30% in water; Sigma–Aldrich 216763) was diluted to 20 mM in sterile MilliQ water.

Bacterial strains used in this study are described in Supplementary Table 1 and can be obtained upon request from the referenced lab. All strains were grown at 37 °C on plates of Luria-Bertani broth (LB, Becton Dickinson (BD) #244610) with 1% agar (w/v, Sigma #400400010). Overnight cultures and subcultures were grown in LB at 37 °C with shaking. For the Δpyk mutant, the LB agar plates and LB medium were supplemented with 5% sodium pyruvate (w/v, Sigma #P5280). For the Tn::icaA, Tn::icaB, and Tn::icaC transposon library mutants, the LB agar plates and LB medium contained 5 μg/mL erythromycin (Sigma-Aldrich #E6376). For the experiments that used LB supplemented with itaconate (Aldrich #129204), the pH of the medium was corrected to 7.0 with 10N sodium hydroxide (Fisher Scientific #SS-2661). Bacterial concentrations were quantified by optical density at 600 nm (OD₆₀₀) and confirmed by serial dilution and plating on LB agar plates.