Effects of rhodomyrtone on the cell envelope of B. subtilis.

(A) Impact of rhodomyrtone on growth of B. subtilis. Arrow marks time point of antibiotic addition. (B) Influence of rhodomyrtone on cell wall synthesis. B. subtilis was treated with 0.5 μg/ml (1x MIC) or 1 μg/ml (2x MIC) rhodomyrtone (Rho), 1 μg/ml gramicidin S (GraS), 2 μg/ml daptomycin (Dap), or 10 μg/ml MP196 for 15 min and subsequently fixed in a 1:3 mixture of acetic acid and methanol. Impairment of lipid II synthesis results in extrusion of the cell membrane through holes in the cell wall. Daptomycin, gramicidin S, and MP196, all of which inhibit lipid II synthesis by interfering with cell wall synthesis proteins at the membrane, served as positive controls. Scale bar 2 μm. (C) Membrane potential measured with the fluorescent dye DiSC(3)5. Arrow indicates time point of antibiotic addition. (D) Staining with propidium iodide. The fluorescent dye is a reporter for the presence of large membrane pores or severe membrane disruption. The detergent SDS, which causes pores by forming membrane micelles, was used as positive control. Error bars represent the standard error of the mean (SEM) of three biological replicates. (E) Efflux of potassium ions measured with the intracellular potassium-selective fluorescent dye APG-2. B. subtilis 168 was stained with APG-2 for 1 h prior to antibiotic addition (arrow). The K⁺/Na⁺ channel ionophore gramicidin served as positive control. (F) Activity of the respiratory chain measured by reduction of resazurin to resofurin. CCCP and NaN₃ were used as positive controls. CCCP uncouples the respiratory chain from the proton gradient and NaN₃ is an inhibitor of the cytochrome c oxidase (complex IV of the respiratory chain). Error bars represent standard deviation of the mean of three biological replicates. Note the different time scales in (A), (C), and (E).