Rhodomyrtone causes highly fluid membrane domains.

(A) Membrane fluidity of B. subtilis 168 treated with different concentrations of rhodomyrtone measured with laurdan generalized polarization (laurdan GP, on a scale from -1 (very fluid) to 1 (very rigid)). Benzyl alcohol served as control for fluidization. (B) Laurdan GP microscopy of B. subtilis 168. Cells were treated with 1x MIC of rhodomyrtone for 10 min. Arrows indicate fluid membrane patches. Scale bar 2 μm. Boxes represent areas magnified in (C). (C) Rigidification of the rest membrane. Arrows indicate position of the cell membrane. Left panels show magnified cutout from (B) with the same color scale. Right panels show a false-colored image depicting the laurdan GP picture in grey and the laurdan fluorescence stain (460 nm) in red to visualize the position of the plasma membrane (see Material and methods for details of GP calculation from images). The GP shifts from green (GP of ~0.3) to white (GP of ~0.5) showing rigidification of the membrane. (D) Quantification of membrane fluidity in individual membrane patches and in the rest membrane of cells stained with laurdan. 100 μM CCCP was used as positive control. (E) Fatty acid adaptation of B. subtilis after one doubling time under exposure to sub-inhibitory concentrations (0.25 μg/ml, 0.5x MIC) of rhodomyrtone. This concentration was chosen based on the growth effect of the rhodomyrtone-treated cultures in large culture flasks (see S15 Fig). Error bars represent standard error of the mean of two biologically independent replicate experiments.