Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli

Abstract

The cytoplasmic and outer membranes of gram-negative bacteria can be isolated from spheroplasts, and separated on sucrose density gradients. Lysis of spheroplasts causes extensive membrane fragmentation and since the characteristics of the fragments obtained by different lysis procedures need not be identical, the influence of the disruption method on membrane composition has been examined. Spheroplasts of Escherichia coli J5 were lysed by osmotic shock, which did not significantly separate the cytoplasmic and outer membranes, but resulted in mixed membrane vesicles. Lysis in the French press and by sonication caused extensive membrane fragmentation and separation. Sonication, however, also caused some fusion between fragments of the outer and the cytoplasmic membranes; this intermembrane fusion increased with sonication time. When the cytoplasmic and outer membranes were well separated and intermembrane fusion was minimal or absent, the cytoplasmic and outer membrane fragments were heterogeneous with respect to density and overall phospholipid, protein and lipopolysaccharide composition. In addition, cytoplasmic, but not outer, membrane fragments were also heterogeneous with respect to protein composition. It is concluded, therefore, that membrane fragments obtained from the cytoplasmic and outer membranes are heterogeneous independently of the lysis procedures used to obtain these fragments. Possible reasons for this heterogeneity are discussed.