Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites

Abstract

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. The isolation of these sites from cell membrane fractions of Salmonella anatum is described. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate oxidase and .beta.-NADH oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), S. anatum was infected with phage .epsilon.15, which causes a rapid change (conversion) in the cell''s O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage .epsilon.34. The fractions of intermediate density (Int. M) from briefly converted cells bound both phage .epsilon.34 and E2-specific antibody. EM investigations showed that .epsilon.34 was absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. The Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. Int. M material also exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. This membrane fractionation allows isolation of a variety of activities associated with adhesion sites from Int. M regions.