Cell membrane permeability assays

Membrane permeability was measured through N-phenyl-1-naphthylamine (NPN) uptake as previously described (48). Briefly, K. pneumoniae MS6671 was grown to OD₆₀₀ of 0.5 in CA-MHB. Cells were pelleted, washed, and resuspended in an equal volume of 5 mM Hepes buffer (pH 7.2). The bacterial suspension was then allowed to equilibrate at room temperature for 30 min. Following equilibration, bacterial suspensions were treated with combinations of either PBT2 + NPN or EDTA + NPN. PBT2 was added to a final concentration of 0 to 64 μM, EDTA was added to a final concentration of 2 mM, and NPN was added to a final concentration of 10 μM. Immediately after the addition of PBT2 + NPN or EDTA + NPN, NPN fluorescence was measured every 15 s for up to 3 hours at 37°C using a FLUOstar Omega microplate reader (BMG Labtech, Germany) with the excitation and emission wavelengths set at 355 and 460 nm, respectively. Response curves were determined by subtracting the background fluorescence of each respective treatment in the absence of bacteria. All experiments were done in biological triplicate and measured in technical triplicates.