Isolation of Outer Membranes with an Ordered Array of Surface Subunits from Acinetobacter

Abstract

A method has been developed for the isolation of outer membranes from Acinetobacter sp. strain MJT/F5/199A. Washed cells were broken in a French press and, after deoxyribonuclease and ribonuclease treatment, removal of intact cells, and four washes in 20 mosmol phosphate buffer, pH 7.4, with centrifugation at 25,000 x g for 10 min, preparations of cell wall fragments from which almost all pieces of plasma membrane had been removed resulted. Treatment of the cell walls with lysozyme and further washing, in the presence of 20 mM MgCl2, yielded preparations of outer membranes. Electron microscopy of freeze-etched preparations shows that a regular pattern of subunits is present on the outer surfaces of intact cells. After negative staining, these subunits are visible on isolated walls and outer membranes; they can be removed by brief treatment with papain. In section, the cell wall structure is that typical of gram-negative bacteria, but the subunits are not detectable on the surface of the outer membrane. The outer membrane retains the appearance of a "unit membrane" in the cell wall, isolated outer membrane, and papain-treated outer membrane fractions. Both cell walls and outer membranes contain a high percentage of protein (76 and 84%, respectively) and not more than 5% carbohydrate, of which glucose and galactose are constitutents. The outer membranes of this Acinetobacter thus differ in structure and composition from those of bacteria in the Enterobacteriaceae.

Freeze-Etching of the Cell Envelope of an Acinetobacter Species Which Carries a Regular Array of Surface Subunits

“…The appearance of the envelope in thin section is shown in Fig. 14; evidence of the composition and appearance by negative staining of the various layers has been obtained in earlier studies (26,27). Outside the plasma membrane (pm), the dense, peptidoglycan-containing layer (d) is adjacent to an intermediate, protein-containing layer (i) which appears wrinkled by negative staining.…”

“…Preparations of isolated cell walls and outer membranes which had received at least four washes in 20 mosmolal phosphate buffer (27) were also examined by freeze-etching, and show the same array of subunits as that seen on intact cells. However, the spaces between the subunits seem to penetrate deeper into the cell wall and outer membrane preparations ( Fig.…”

“…Effect of lysozyme on layers of the cell envelope. To help in identification of the layers, lysozyme treatment was used, since this is known from earlier work (27) to remove the dense, peptidoglycan-containing layer, leaving the outer membrane still carrying the regular array of subunits, as shown by negative stain- ing. The appearance in section is seen in Fig.…”

“…strain MJT/F5/199A is a gram-negative bacterium which has already been extensively studied by electron microscopy, using negative staining and thin-sectioning techniques (26). (The problems involved in the taxonomic position of this organism have been discussed by Thornley, Glauert and Sleytr [27].) A method of isolating cell walls and outer membranes from a preparation of cell envelopes has been described (27).…”

“…(The problems involved in the taxonomic position of this organism have been discussed by Thornley, Glauert and Sleytr [27].) A method of isolating cell walls and outer membranes from a preparation of cell envelopes has been described (27). An array of regularly arranged subunits is present on the isolated cell walls and outer membranes and is thought to be located on the outer surface of the intact cell.…”