Biosensor Assay for Antimicrobial Activity

CWBIs and negative control compounds were either from Sigma–Aldrich or the sources listed in Table S2. Susceptibility testing²⁹ was used to define appropriate concentrations of control compounds for assay. Biosensor strains were cultured in tryptone soya broth (TSB; Oxoid) at 37°C with vigorous aeration to an OD₆₀₀ of 0.2 and challenged with antimicrobial agents for 60 min. In the case of biosensor constructs in Ts mutants, strains were grown at 30°C to an OD₆₀₀ of 0.2, before shifting the temperature to 42°C for 60 min. Post-challenge, OD₆₀₀ was measured to allow changes in cell density to be accounted for in calculating biosensor induction. An aliquot of culture (typically 200 µL) was centrifuged, and the washed cells resuspended in 0.5 volumes of AB buffer^{30 (link)} containing lysostaphin (15 mg/L) and the fluorogenic β-galactosidase (β-gal) substrate, 4-methylumbelliferyl β-D-galactopyranoside (MUG, 500 mg/L; Sigma–Aldrich), and incubated at 25°C with shaking for 90 min. Production of β-gal was determined as described.^{30 (link)}