The outer membrane of Proteus mirabilis I. Isolation and characterization of the outer and cytoplasmic membrane fractions

Abstract

1. 1. The crude envelope preperation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was seperated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a singel trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped strutures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. 3. The outer membrane fraction consisted of protien (∼40%), lipopolysaccharide (∼36%) and lipid (∼18%) and had a density of about 1.22 g/cm 3 . The cytoplasmic membranse fraction consisted mostly of protein (∼56%) and lipid (∼38%), had a density of about 1.16 g/cm 3, and contained almost all the NADH oxidase. succinate and d-lactate dehydrogenase activities of the crude envelope preparation. 4. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6–7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.