Escherichelin activity assay on P. aeruginosa.

PW5011 (pvdA-E02::ISlacZ/hah) (55, 56) was grown in LB broth overnight at 37°C under shaking conditions and then back-diluted into succinate medium (25) at 10⁸ CFU/ml. The growth experiments were performed in a final volume of 100 μl in 96-well plates. Escherichelin, solubilized in ethanol, was added to the cultures at increasing concentrations. The final ethanol (vehicle) concentration in all cultures was 5% (v/v). The cultures were grown at 37°C under shaking conditions for 20 hours, at which point they were serially diluted in PBS and plated on LB agar for CFU quantification.