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Freeze-etching was applied to preparations, with and without glycerol, of Acinetobacter sp. strain MJT/F5/199A, consisting of intact cells after normal growth or after incubation with chloramphenicol, spheroplasts, and isolated cell walls and outer membranes. Etched preparations show that a regular array of subunits forms the surface of normal cells. Near the zones of constriction in dividing cells, blebs and irregularities are seen, and some blebs, consisting of both surface subunits and outer membrane, are released from the cells. The cross-fractured cell envelope shows four layers which are related to the structures seen in section as follows: cw1, which is not visible in section, contains the surface subunits; cw2 consists of all or part of the outer membrane; cw3 includes the intermediate and dense, peptidoglycan-containing layers; within these cell wall layers is the plasma membrane. Internal fracture of the plasma membrane occurs under all conditions tested, but the fracture plane in the cell wall is only revealed in chloramphenicol-treated cells or normal cells freeze-fractured with glycerol present; the characteristic fracture faces are not seen in spheroplasts or isolated outer membranes. The concave fracture face cw2 consists of densely packed granules, while the convex face cw3 is fibrillar. The probable location of this fracture plane is discussed. After incubation with chloramphenicol, the outer surface of the cells is obscured by extracellular material, the dense peptidoglycan-containing layer is increased in thickness, and the cytoplasm contains rounded bodies bounded by one or more unit membranes.

The cell wall of the gram-negative bacterium Acinetobacter species strain MJT/F5/5 shows in thin section an external "additional" layer, an outer membrane, an intermediate layer, and a dense layer. Negatively stained preparations showed that the additional layer is composed of hexagonally arranged subunits. In glycerol-treated preparations, freeze-etching revealed that the cell walls consist of four layers, with the main plane of fracture between layers cw 2 and cw 3. The surface of [Formula: see text] 2 consisted of densely packed particles, whereas [Formula: see text] 3 appeared to be fibrillar. In cell envelopes treated with lysozyme by various methods, the removal of the dense layer has detached the outer membrane and additional layer from the underlying layers, as shown in thin sections. When freeze-etched in the absence of glycerol, these detached outer membranes with additional layers fractured to reveal both the faces [Formula: see text] 2 and [Formula: see text] 3 with their characteristic surface structures, and, in addition, both the external and internal etched surfaces were revealed. This experiment provided conclusive evidence that the main fracture plane in the cell wall lies within the interior of the outer membrane. This and other evidence showed that the corresponding layers in thin sections and freeze-etched preparations are: the additional layer, cw 1; the outer membrane, cw (2 + 3); and the intermediate and dense layers together from cw 4. Because of similarities in structure between this Acinetobacter and other gram-negative bacteria, it seemed probable that the interior of the outer membrane is the plane most liable to fracture in the cell walls of most gram-negative bacteria.

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.

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 MgCl(2), 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.

Chemical analysis of fractions of the cell envelope of Acinetobacter sp. strain MJT/F5/199A, prepared by breakage in the French press and removal of plasma membranes, followed by sequential treatment with lysozyme and with papain, confirmed the existence of layers previously identified by electron microscopy. Outside the plasma membrane and periplasmic space, the envelope is composed of (i) a peptidoglycan-containing dense layer, (ii) an intermediate layer, (iii) a lipopolysaccharide-containing outer membrane, and (iv) an ordered array of protein subunits. A small amount of carbohydrate (3%) is found associated with protein in the fraction containing both the surface subunits and the intermediate layer. The papain-treated outer membranes contain 67% protein, 24% lipid, together with 11% lipopolysaccharide, and about 6% of non-lipopolysaccharide hexosamine. Lipid is located only in the papain-treated outer-membrane and is mainly phospholipid: 29% phosphatidyl glycerol, 30% phosphatidyl ethanolamine, and 40% cardiolipin. The principal fatty acid is C(18:1). Significant amounts of alcohols(16:1) and alcohols(18:1), which are found in Acinetobacter waxes, were recovered from the outer membrane.