Location of the Fracture Faces Within the Cell Envelope of Acinetobacter Species Strain MJT/F5/5

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.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp

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.     

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

Cell envelope associations of Aquaspirillum serpens flagella.

Specific regions of the cell envelope associated with the flagellar basal complex of the gram-negative bacterium Aquaspirillum (Spirillum) serpens were identified by studying each of the envelope layers: outer membrane, mucopeptide, and plasma membrane. The outer membrane around the flagella insertion site was differentiated by concentric membrane rings and central perforations surrounded by a closely set collar. The perforations in both the outer membrane and the isolated mucopeptide layer were of a size accomodating the central rod of the basal complex but smaller than either the L or the P disks. The P disk of the complex may lie between the mucopeptide and the outer membrane. Electron microscopy of intact, spheroplasted, or autolyzed preparations did not adequately resolve the location of the inner pair of disks of the basal complex. Freeze-etching, however, revealed differentiation within the plasma membrane that appeared to be related to the basal complex. The convex fracture face showed depressions which are interpreted as impressions of a disk surrounded by a set of evenly spaced macromolecular studs and containing a central "plug" interpreted as the central rod. In thin sections, blebs, which appear to be associated with the flagellar apparatus, were seen on the cytoplasmic side of the plasma membrane. Superimposing the dimensions of the flagellar basal complex and the spacings of the cell envelope layers and using the position of the L disk within the outer membrane for reference, showed that the S disk might be within and the M disk beneath the plasma membrane. A tentative model was developed for comparison with that based on the structure of the Escherichia coli basal complex.     

Localization of tetramethylphenylenediamine-oxidase in the outer cell wall layer of Neisseria meningitidis.

A highly active tetramethylphenylenediamine-oxidase has been found in association with the cell wall blebs, evaginations of the outer wall membrane, of Neisseria meningitidis. Isolated wall blebs consumed oxygen in the presence of ascorbate-tetramethylphenylenediamine but not in the presence of succinate, whereas cell envelope preparations are active in both substrates. The ratio of succinate dehydrogenase/tetramethylphenylenediamine-oxidase activities in preparations of envelopes was approximately 100 times that in isolated wall blebs, indicating that the outer membrane preparations were highly purified.     

Protein Excretion through Bleb Formation by PE4LA,a Mutant of Escherichia coli

A mutant of E. coli (PE4LA) excreted approximately 15% of total cellular protein without cell lysis. The materials in the culture supernatant of the mutant were precipitated with 5% cold TCA. Protein, lipopolysaccharide (LPS), and phospholipid were found in a ratio of approximately 5:6:1. In electrophoretical analyses, exoproteins appeared to contain both periplasmic and outer membrane proteins.

An electron microscopic study showed that PE4LA cells had many blebs around the cell surface and that these blebs were surrounded by double track layers. Some vesicles were also observed as free forms of blebs, while the parent cells had neither blebs nor vesicles. The vesicles appeared to be rich in LPS and lacked phosphatidylglycerol, compared to the outer membrane.

The physiological and morphological data suggested alterations in the PE4LA cell surface, but what was altered remains obscure. It was concluded that PE4LA cells do not have a substantial increase in permeability, but rather have some defect in the cell envelope organization, which causes the formation of blebs with periplasmic proteins.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

Effect of Ethylenediaminetetraacetate upon the Surface of Escherichia coli

The effect of ethylenediaminetetraacetate (EDTA) on the envelope of two strains of Escherichia coli (B and Cla) was studied with freeze-fracturing methods. Untreated cells showed the outer membrane's outer surface with a fine texture of randomly spaced depressions of about 4.5-nm diameter; small areas with symmetrical arrangements of structural surface elements were also observed. The outer membrane's fracture plane revealed a random distribution of particles on its "concave" plane, only occasionally interrupted by particle-free areas. The "convex" aspect of the outer membrane's fracture plane showed only a few scattered particles. The cleavage plane of the inner membrane was often interrupted by many localized elevated plateaus, at which the cleaving process had, for short distances, switched to the outer membrane. The effects of EDTA treatment were mainly seen in the structure of the freeze-etched outer membrane: (i) the pits as well as the symmetrical surface elements of the outer membrane's outer surface had disappeared; (ii) a number of plateaus (about 20 to 50/cell) were seen at which a cleavage plane within the inner membrane had switched to the hydrophobic portion of the outer membrane (outer membrane's fracture plane). These plateaus were also visible in untreated cells; however, EDTA treatment apparently caused an increased exposure of plateaus. Surface areas, exposed by freeze-etching, revealed the underlying plateaus as elevations in the surface contour of the cell, suggesting a slower etching rate in the zones of the plateaus relative to the rest of the outer membrane. Well-defined, particle-free patches in the outer membrane's fracture plane, concave, were more frequent and larger in size after EDTA treatment than in the controls. In the presence of glycerol, the cells often cleaved in the outer membrane's fracture plane, but isolated plateaus were rarely observed. After metabolic poisoning of cells for 15 to 25 min at 37 degrees C, the plateaus had widened. These data suggest that the material of the plateaus has a slow rate of lateral diffusion. Placement of EDTA-treated cells in fresh medium at 37 degrees C caused, after 3 to 5 min, the reoccurrence of the pitted surface structure. We propose that the plateaus represent localized zones, at which newly synthesized lipopolysaccharide has been inserted.     

Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed "prospheroplast." The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast.     

Immunocytochemical Identification and Localization of Active and Inactive alpha-Amylase and Pullulanase in Cells of Clostridium thermosulfurogenes EM1

Clostridium thermosulfurogenes EM1 formed blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by phosphate limitation or high dilution rates. Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded. Bleb and vesicle formation was also observed in the absence of substrates that induce alpha-amylase and pullulanase synthesis. No correlations existed between bleb formation and the presence of active enzyme. Similar blebs could also be observed in a number of other gram-positive bacteria not producing these enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of alpha-amylase and pullulanase in C. thermosulfurogenes EM1, two different antisera were applied. One was raised against the enzymes isolated from the culture fluid; the other was produced against a peptide synthesized, as a defined epitope, in analogy to the N-terminal amino acid sequence (21 amino acids) of the native extracellular alpha-amylase. By using these antisera, alpha-amylase and pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning, blebs could be seen, and the immunolabel pinpointing alpha-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the blebs. Cells exhibiting high or virtually no enzyme activity were labeled similarly with both antisera. This finding strongly suggests that alpha-amylase and pullulanase may occur in both active and inactive forms, depending on growth conditions.     

Physiology and ultrastructure of Leptothrix discophora SS-1

Leptothrix discophora strain SS-1 (ATCC 43182) is a Gram-negative, Mn²⁺-oxidizing, aerobic heterotroph which lost its sheath-forming ability after 18 months of cultivation on laboratory media. SS-1 possesses high 6-phosphogluconate dehydratase and KDPG aldolase activities, and a very low level of phosphofructokinase, indicating carbohydrate catabolism by the Entner-Doudoroff pathway. The strain is polarly flagellated, accumulates PHB up to 67% of its dry weight when grown in pyruvate-containing medium, and has a G+C content of 69.8 mol%. These properties indicate that L. discophora is essentially a pseudomonad which can form a sheath and oxidize Mn²⁺. Ultrastructural observations made before SS-1 lost its sheath-forming ability indicated two cell types. Short, flagellated, non-sheathed cells seen under the electron microscope probably corresponded to swarmer cells observed under phase-contrast microscopy. These cells contained plate organelles and PHB granules, and produced extracellular blebs approx. 25–50 nm in diameter. Larger sheathed cells also contained plate organelles, PHB granules, and blebs that were often sandwiched between the outer membrane and the sheath. Cells grown in the presence of added Mn²⁺ were surrounded by an extensive fibrillar matrix, rendered electron dense by precipitation of manganic oxide. The matrix was connected to various points of the cell by outer membrane evaginations or electron dense threads. We propose that the outer membrane blebs represent vehicles for excretion of unorganized sheath material and/or Mn²⁺-oxidizing protein produced by L. discophora.Parts of this work were presented previously (W.C. Ghiorse, Abst. Annu. Meet. Am. Soc. Microbiol., 1981, N 64, p 183)     

Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli.

Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Defeat of colicin tolerance in Escherichia coli ompA mutants: evidence for interaction between colicin L-JF246 and the cytoplasmic membrane.

Escherichia coli ompA mutants are tolerant to colicin L-JF246. This tolerance can be overcome by a variety of treatments that have as their target the outer membrane or the peptidoglycan layers of the cell envelope. Thus, increasing the concentration of colicin L, releasing lipopolysaccharide from the outer membrane by treatment of intact cells with ethylenediaminetetracetic acid (EDTA), converting cells to spheroplasts by treatment with lysozyme-EDTA or penicillin, or trypsin, treatment of intact cells will result in an increased colicin sensitivity. These treatments alter the outer membrane of ompA mutants and suggest that the altered outer membrane may allow the penetration of at least a portion of the colicin L molecule to a site of action located within this barrier. To substantiate this, we have demonstrated that membrane vesicles prepared from ompA mutants are sensitive to colicin L and that 14C-labeled colicin L binds rapidly to both the outer and inner membrane fractions of the cell.     

Effect of the local anesthetic dibucaine on the surface membrane in sterol-manipulated Tetrahymena pyriformis studied by freeze-fracture electron microscopy

The effect of the local anesthetic dibucaine on the membrane ultrastructure of sterol-manipulated Tetrahymena pyriformis (NT-1 strain) was studied by freeze-fracture electron microscopy. Dibucaine-treated, ergosterol-replaced Tetrahymena cells had marked alterations in their plasma membranes. IMP-free small depressions (exoplasmic fracture face) and protrusions (protoplasmic fracture face) were formed on the plasma membranes which was in contact with the outer alveolar membrane. In addition, large IMP-free surface "blebs" covered with hexagonally-arranged depressions and protrusions appeared on both the plasma and outer alveolar membranes. These "blebs" were pinched off when the membranes were severely affected. Our previous study (28) demonstrated that the plasma membrane of dibucaine-treated native Tetrahymena cells that contain tetrahymanol showed vertical displacement of its intramembranous particles and that subsequently a smooth, flat surface appeared. Therefore, the structural changes in ergosterol-replaced membranes produced by dibucaine differ strikingly from changes in the native membranes. The remarkable difference in the ultrastructural deformation of the plasma membrane probably is due to a difference in the membrane lipid composition induced by sterol-manipulation.     

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation.Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.     

Fracture Faces in the Cell Envelope of Escherichia coli

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.