Detection of lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis

Abstract

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.