Determination of membrane charge

Determination of bacterial membrane charge

The net electrostatic charge of the OM of whole bacteria and the CM of spheroplasts was measured by a well-established approach using FITC-labelled Poly-L-Lysine (PLL; Sigma-Aldrich). Samples (300 µl) of whole cells or spheroplasts produced as described above were washed and incubated at room temperature for 10 minutes in Tris buffer (0.03 M, pH 8.0, with 20% sucrose added for spheroplasts) containing FITC-PLL at a final concentration of 20 µg ml^-1. To remove any FITC-PLL that had not bound to the cellular surface, the samples were washed thoroughly by three rounds of centrifugation (4,000 x g, 5 minutes) and resuspension in Tris buffer. Washed cells (200 µl) were seeded into the wells of a black-walled microtitre plate, and the amount of FITC-PLL attached to the samples was quantified by measuring the fluorescence in a Tecan Infinite 200 Pro multiwell plate reader (excitation at 490 nm, emission at 525 nm). The less FITC-PLL binding to the surface of whole cells/spheroplasts, the more positively-charged the membrane was – with the cationic FITC fluorophore having reduced affinity for the membrane bilayer.