The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy

The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37°C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroylnaphthalene (Laurdan) as fluorescent probes. No phase transition of bulk lipids was observed in these preparations at the range of temperature studied. The anisotropy values (r_s) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress. Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface. Membrane preparations showed r_s higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter. This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.