3.5. Real-Time Efflux Assays

Real-time efflux assays were conducted with Nile red, 1,2′-DNA, and BM-27 according to protocols published earlier [20,21,22] with slight modifications. Briefly, 20 mL MH2 broth was inoculated with a colony from a freshly grown MH2 agar plate and cultivated overnight at 37 °C with shaking (200 rpm). Bacterial cells were harvested by centrifugation (3220 g, 10 min) and washed twice with PBS. The pellets were suspended in PBS containing 1 mM MgCl₂ and adjusted to an OD₆₀₀ of 1. Efflux arrest was induced by incubating with the proton gradient uncoupling agent CCCP (carbonyl cyanide 3-chlorophenylhydrazone, final concentration 5 µM) at 37 °C for 20 min followed by dye loading with Nile red or BM-27 to final concentrations of 10 µM at 37 °C for 2 h. 1,2′-DNA was added to a final concentration of 32 µM (4 h incubation, 37 °C). To monitor the real-time efflux, 180 µL aliquots of cells were washed by centrifugation (5800 g, 2 min) and resuspended in the same volume of PBS containing 1 mM MgCl₂. After 40 s of fluorescence recording with the TECAN plate reader M200 Pro, cells were re-energized by the addition of glucose to a final concentration of 1 mM and the measurement was continued for 360 s (at 37 °C). For Nile red the excitation and emission wavelengths were 544 and 650 nm, respectively, for BM-27 400 and 457 nm, and for 1,2′-DNA 370 and 420 nm.