Measuring Bacterial Membrane Surface Charge

To measure bacterial membrane surface charge, we carried out a fluorescein isothiocyanate-labelled poly-L-lysine- (FITC-PLL) (Sigma) binding assay (Kintses et al., 2019 (link); Rossetti et al., 2004 (link)). FITC-PLL is a polycationic molecule that binds to an anionic lipid membrane in a charge-dependent manner and is used to investigate the interaction between cationic peptides and charged lipid bilayer membranes. The assay was performed as previously described (Kintses et al., 2019 (link)). Briefly, bacterial cells were grown overnight in LB medium, and then centrifuged, and washed twice with 1× phosphate-buffered saline (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.