Fig. 5. Tetracyclines delocalize membrane proteins.

Effects of tetracycline, anhydrotetracycline, and gramicidin (positive control) on protein localization. B. subtilis LB318 (168 amyE::spc mgfp-minD aprE::cat mcherry-minC) a and TNVS205 (168 aprE::cat mcherry-mreB) b were treated with 2 µg/ml tetracycline, 2 µg/ml anhydrotetracycline, or 1 µg/ml gramicidin, respectively. Arrows indicate abnormalities in protein localization patterns. Scale bars 2 µm. c Quantification of microscopy images pertaining to (a) and (b). MinC localization depends on MinD resulting in MinC always being affected when MinD is delocalized. Hence, the numbers of affected cells are the same for both proteins and only one graph is shown. A MinCD-expressing cell was counted as affected, when it lost its typical septal/polar localization pattern by displacement of the fluorescence signal into the cytosol and/or membrane patches. MreB-expressing cells were counted as affected, when the regular MreB localization was disturbed by gaps (typical for tetracycline), patches (typical for anhydrotetracycline), or displacement of the fluorescence signal into the cytosol (typical for gramicidin and partially anhydrotetracycline). Images were quantified manually. Sample size was ≥100 individual cells per condition. Error bars show standard deviation of the mean of three replicate experiments. Circles indicate individual datapoints. d Effects of tetracycline, anhydrotetracycline, and gramicidin on the membrane potential of B. subtilis 168 measured with DiSC(3)5. An exemplary graph out of three biological replicates is shown. e Effects of tetracycline, anhydrotetracycline, and gramicidin on fluid membrane microdomains of B. subtilis 168 cells stained with the fluid lipid domain dye DiIC12. Arrows indicate abnormal membrane domain stains. Scale bar 2 µm (f) Quantification of microscopy images pertaining to (e). Images were quantified manually. Cells were counted as affected, when the regular DiIC12 staining pattern deviated from the control (regular distribution, regular size, typically 6–15 spots per cell) by accumulation of the dye in irregular, large, and/or less than 6 foci). Sample size was ≥100 individual cells per condition. Error bars show standard deviation of the mean from three replicate experiments. Circles indicate individual datapoints.