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Chemical Composition and Ultrastructure of Native and Reaggregated Membranes from Protoplasts of Bacillus cereus

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of Bacillus cereus T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C₁₅ and C₁₇ and of normal C₁₆ acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl₂. The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl₂ concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl₂ concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments.