Ethidium bromide accumulation assay

Efflux activity was measured with ethidium bromide as previously described (97). Briefly, cultures were grown to an OD₆₀₀ of 0.4. Cells were washed with 20-mM potassium phosphate buffer supplemented with 1-mM MgCl₂ and diluted to an OD₆₀₀ of 0.2. Ethidium bromide and CCCP (Sigma-Aldrich) were added to final concentrations of 50 µg/mL and 100 µM, respectively. Cells were incubated at 23°C for 1 h with aeration. Afterward, cells were harvested by centrifugation for 10 min at 23°C at 3,000 × g and resuspended in 20-mM potassium phosphate buffer with 1-mM MgCl₂. Cells were energized with a final concentration of 1% glucose, and ethidium bromide efflux was measured for over 40 min at excitation and emission wavelengths of 530 and 600 nm, respectively, in a FLUOstar OMEGA plate reader (BMG Labtech, Germany).