Membrane surface charge measurement

To evaluate bacterial surface charge, we performed a fluorescein isothiocyanate-labeled poly-L-lysine (FITC-PLL) (Sigma) binding assay. In brief, FITC-PLL is a polycationic molecule that binds to anionic lipid membrane in a charge-dependent manner and is used to investigate the interaction between cationic peptides and charged lipid bilayer membranes⁷⁰. The assay was performed as previously described^25,71. Briefly, bacterial cells were grown overnight in MS medium, centrifuged and washed twice with 1X PBS buffer (pH 7.4). The washed bacterial cells were re-suspended in 1× PBS buffer to a final OD₆₀₀ of 0.1. A freshly prepared FITC-PLL solution was added to the bacterial suspension at a final concentration of 6.5 µg/ml. The suspension was incubated at room temperature for 10 min, and pelleted by centrifugation. The remaining amount of FITC-PLL in the supernatant was determined fluorometrically (excitation at 500 nm and emission at 530 nm) with or without bacterial exposure. The quantity of bound molecules was calculated from the difference between these values. A lower binding of FITC-PLL indicates a less net negative surface charge of the outer bacterial membrane.