We characterized physical and chemical properties of cell-membrane fragments from Bacillus subtilis 168 (trpC2) grown at pH 5.0, 7.0 and 8.5. Effects of long-term bacterial adaptation reflected in growth rates and in changes of the membrane lipid composition were correlated with lipid order and dynamics using time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. We demonstrate that the pH adaptation results in a modification of a fatty acid content of cellular membranes that significantly influences both the lipid-chain order and dynamics. For cultivation at acidic conditions, the lipid order increases and membrane dynamics decreases compared to pH 7.0. This results in rigid and ordered membranes. Cultivation at pH 8.5 causes slight membrane disordering. Instant pH changes induce qualitatively similar but smaller effects. Proton flux measurements performed on intact cells adapted to both pH 5.0 and 8.5 revealed lower cell-membrane permeability compared to bacteria cultivated at pH optimum. Our results indicate that both acidic and alkalic pH stress represent a permanent challenge for B. subtilis to keep a functional membrane state. The documented adaptation-induced adjustments of membrane properties could be an important part of mechanisms maintaining an optimal intracellular pH at a wide range of extracellular proton concentrations.

• MeSH
• Bacillus subtilis growth & development   metabolism   physiology   MeSH
• Cell Membrane metabolism   physiology   MeSH
• Diphenylhexatriene metabolism   MeSH
• Fluorescence Polarization MeSH
• Hydrogen-Ion Concentration MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Diphenylhexatriene MeSH

Lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis was found by using density gradient centrifugation. Crude membranes (CM) present in the whole cell lysate were separated into three fractions of increasing density (F, CI, CII). Substantial difference exists in the amount of protein recovered from these fractions, the relative ratio being 15 : 35 : 50. The qualitative protein composition (by SDS-PAGE) of the fractions varies markedly as well. The lipid components extracted from the fractions are also distributed in different proportions, viz. 40 : 40 : 20. The spectrum of fatty acids (FA), detected in lipids of F fraction and analyzed by GC-MS exhibits the same profile as that found in CM; in contrast, fractions CI and CII undergo extensive FA reconstruction. Thermotropic behavior of fractions measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicates significant variations of microviscosity (r(s)) within the F, CI and CII fractions. The protein-to-lipid ratio plays evidently a key role in affecting the physical state of the cytoplasmic membrane. Microdomains of different density coexist in the membrane and exhibit heterogeneity in both chemical composition and "physical state"; the increased de novo synthesis of FA induced by the cold exclusively in fractions CI and CII indicates correlation with an altered physiological state of bacterial metabolism.

• MeSH
• Bacillus subtilis chemistry   physiology   ultrastructure   MeSH
• Cell Membrane chemistry   physiology   ultrastructure   MeSH
• Centrifugation, Density Gradient MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescence Polarization MeSH
• Fatty Acids analysis   MeSH
• Membrane Lipids chemistry   physiology   MeSH
• Membrane Proteins chemistry   physiology   ultrastructure   MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fatty Acids MeSH
• Membrane Lipids MeSH
• Membrane Proteins MeSH

The temperature dependence of fluorescence anisotropy, lifetime and differential tangent of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its polar trimethylammonium derivative (TMA-DPH) were investigated in cytoplasmic membranes of Bacillus subtilis. The fluorescence parameters were compared in the two types of membranes prepared from bacteria cultivated at 20 and 40 degrees C. Steady-state anisotropy measurements showed that within a broad range of temperatures, membranes cultivated at 20 degrees C exhibit significantly lower values than those prepared from cells cultivated at 40 degrees C. The temperature dependence of lifetime and differential tangent measurements (differential polarized phase fluorimetry) were fully consistent with steady-state anisotropy data of both DPH and TMA-DPH. The low anisotropy values in the case of TMA-DPH could be explained by a shorter lifetime and higher temperature-induced decrease as compared with DPH. Surprisingly, the temperature dependence of rotational rate R calculated according to the model of hindered rotations (Lakowicz 1983) gave misleading results. When increasing the temperature from 5 to 25 degrees C, a marked drop of rotational relaxation rate was observed. The minimum R values were measured between 25 and 30 degrees C and further increase of temperature (up to 60 degrees C) was reflected as increase of the R values. Therefore, a new model of "heterogeneous rotations" was developed. This model assumes that even at low temperatures (approaching 0 degrees C) where the differential tangent reaches zero, a fraction of fast rotating molecules exists. The ratio between fast and slowly rotating molecules may be expressed by this model, the newly calculated rotational rates are fully consistent with anisotropy, lifetime and differential tangent measurements and represent the monotonically increasing function of temperature.

• MeSH
• Bacillus subtilis metabolism   ultrastructure   MeSH
• Cell Membrane metabolism   MeSH
• Diphenylhexatriene analogs & derivatives   metabolism   MeSH
• Fluorescence Polarization MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Diphenylhexatriene MeSH