Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.     

Direct proof of a "more-than-single-layered" peptidoglycan architecture of Escherichia coli W7: a neutron small-angle scattering study

A neutron small-angle scattering study was performed to determine the thickness and the scattering density profile of isolated peptidoglycan sacculi of Escherichia coli W7 in aqueous suspension (D2O). The maximum thickness (7 +/- 0.5 nm) of the sacculus from the exponential-phase cells was large enough to suggest the existence of a more-than-single-layered architecture. The experimental density profile across the thickness of the sacculus did not allow an unambiguous differentiation between a single-layered architecture characterized by completely extended peptide side chains projecting from the sugar strands or, alternatively, a partially triple layered structure. To resolve this ambiguity, sacculi were labeled with deuterated wall peptides. Comparison of the two experimental profiles indicated that the sacculus is more than single layered across its surface, with about 75 to 80% of its surface single layered and 20 to 25% triple layered.     

Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10–15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala: Glu: GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M 1) alanine is replaced by threonine, however. Neutral sugars and-in some strains-additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or d-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three l-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min.The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.Non Standard Abbreviations SDS sodium dodecylsulfate - EDTA ethylenediaminetetra acetic acid - DNP dinitrophenyl Dedicated to Prof. Dr. Adolf Butenandt on the occasion of his 75th birthday     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

Bacterial growth does require peptidoglycan hydrolases

Most bacteria surround their cytoplasmic membrane with a net‐like, elastic heteropolymer, the peptidoglycan sacculus, to protect themselves from bursting due to the turgor and to maintain cell shape. It has been assumed that growing bacteria require peptidoglycan hydrolases to open meshes in the peptidoglycan net allowing the insertion of the newly synthesized material for surface expansion. However, peptidoglycan hydrolases essential for bacterial growth have long remained elusive. In this issue of Molecular Microbiology Singh et al. ( 2012 ) report the identification in Escherichia coli of three new DD‐endopeptidases (Spr, YdhO and YebA) which are collectively required for peptidoglycan growth. Cells depleted of the three enzymes fail to incorporate new peptidoglycan, indicating that the cleavage of cross‐links by the new endopeptidases is needed for surface growth of the sacculus. These results are corroborated by recent data showing that Bacillus subtilis cells require the DL‐endopeptidase activity of CwlO or LytE for growth.     

Amount of peptidoglycan in cell walls of gram-negative bacteria.

The amount of diaminopimelic acid (Dap) in the cell wall of Escherichia coli was measured in two ways. A radiochemical method first described by us in 1985 (F. B. Wientjes, E. Pas, P. E. M. Taschner, and C. L. Woldringh, J. Bacteriol. 164:331-337, 1985) is based on the steady-state incorporation of [3H]Dap during several generations. Knowing the cell concentration and the specific activity of the [3H]Dap, one can calculate the number of Dap molecules per sacculus. The second method measures the Dap content chemically in sacculi isolated from a known number of cells. With both methods, a value of 3.5 x 10(6) Dap molecules per sacculus was obtained. Combined with electron microscopic measurements of the surface area of the cells, the data indicate an average surface area per disaccharide unit of ca. 2.5 nm2. This finding suggests that the peptidoglycan is basically a monolayered structure.     

Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 10(7) N/m(2) was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intact E. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927-953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301-366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.     

Novel peptidoglycans in Caulobacter and Asticcacaulis spp.

Peptidoglycan sacculi free of poly-beta-hydroxybutyric acid were prepared from whole cells of four species of Caulobacter and two species of Asticcacaluis and from morphological mutants of Caulobacter crescentus and Caulobacter leidyi. Acid hydrolysates of the sacculi were analyzed quantitatively, and each of the hydrolysates was found to contain significant amounts of only five ninhydrin-reactive compounds: alanine, glutamic acid, alpha , omega-diaminopimelic acid, muramic acid, and glucosamine. Four types of peptidoglycans were distinguishable on the basis of the molar ratios among these five compounds. The respective ratios were as follows: in C. leidyi, 2:1:1:1:0.8; in Asticcacaulis biprosthecum, 1.7:1.6:1.1:0.7; in the cells of the remaining species, 2:1:1:1.2:0.8; and in stalks shed by the abscission mutant 2NY66, 2:1:1:1:1.67. Thus, in addition to some species differences among these caulobacters, it was found that the peptidoglycan sacculus of the stalked C. crescentus cell is chemically differentiated; the cellular peptidoglycan is richer in muramic acid than is the peptidoglycan of typical gram-negative bacteria, and the peptidoglycan of the stalk is correspondingly rich in glucosamine. Empirical formulas for the repeating units of the peptidoglycans have been inferred on the basis of the molar ratios of their amino components.     

Specific interaction of the tetragonally arrayed protein layer of Bacillus sphaericus with its peptidoglycan sacculus.

Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.     

Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.     

Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.     

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.     

Process of cellular division in Escherichia coli growth pattern of E. coli murein

The growth pattern of the murein-sacculus which determines the shape of the Escherichia coli cell was studied by the use of high-resolution autoradiography with the electron microscope. The murein was pulse labelled with ³H-labelled diaminopimelic acid as a specific murein precursor and sacculi were prepared immediately. The radioactivity of the nascent murein appeared on the auto- radiographs at a well-defined growth zone in the central area of the sacculus. This was true regardless of the size of the cells. Pulse chase experimenta show rapid mixing of labelled murein with pre-existing murein and its even distribution over the whole surface of the sacculus.     

Murein (peptidoglycan) binding property of the essential cell division protein FtsN from Escherichia coli

The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.     

The permeability of the wall fabric of Escherichia coli and Bacillus subtilis.

To study the overall structure of the peptidoglycan fabric of the sacculi of gram-negative and gram-positive walls, actively growing cultures of Escherichia coli and Bacillus subtilis were treated with boiling sodium dodecyl sulfate solutions. The sacculi were then treated with enzymes to eliminate proteins and nucleic acids. These intact saccoli were probed with fluorescein-labeled dextrans with a range of known molecular weights. The penetration of the probes could be monitored by the negative-staining appearance in the fluorescence microscope. At several chosen times, the molecular weight fraction that allowed barely observable entry of the fluorescein-labeled probe and the molecular weight fraction that penetrated to achieve almost, but not quite, the concentration of probe in the solution external to the sacculi were determined. From three pairs of times and molecular weights that met one or the other of these two criteria, the effective pore size could be calculated. The minimum size of protein molecule that could diffuse through the pores was also calculated. Two mathematical models, which gave essentially the same results, were used to interpret the experimental data: one for the permeation of random coils through a surface containing holes and the other for rigid spheres diffusing through water-filled cylindrical pores. The mean estimate of the effective hole radius in walls from E. coli is 2.06 nm, and that of the effective hole size in walls from B. subtilis is 2.12 nm. These results are supported by experiments in which the loss of preloaded cells was monitored. Various fluorescein-labeled dextran samples were mixed with samples of intact cell walls, held for a long time, and then diluted. The efflux of the dextrans was monitored. Neither large nor small dextrans stained under these conditions. Only with dextran samples of a sufficiently small size were the sacculi filled during the preincubation period, and only with the largest of these could the probe not escape quickly. From the pore (or mesh) size, it can be concluded that the wall fabric of both organisms has few imperfections and that the major passageway is through the smallest possible pore, or "tessera," formed by the maximal cross-linking of the peptides from glycan chain to glycan chain compatible with the degree of rotational flexibility of the chains of repeating disaccharides of N-acetyl muramic acid and N-acetyl glucosamine. A tessera is composed of two chains of eight saccharides cross-linked by two octapeptides. The size of a globular hydrophilic molecule, if it did not bind to wall components, that could pass freely through the meshwork of an unstretched sacculus of either organism is roughly 25 kDa. We stress that this is only a rough estimate, and it may be possible for proteins of less than 50 kDa to pass through the native wall during normal growth conditions.     

Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.     

Structure of the rigid-layer of Rhizobium cell wall. III. Electron microscopic evidence for the cellulose microfibrils association with peptidoglycan sacculi

The morphology of peptidoglycan layer of Rhizobium cell wall was examined by transmission electron microscopy. Peptidoglycans were isolated from intact cells after treatment with sodium dodecyl sulfate, extraction with aqueous 45% phenol and then with a mixture of chloroform-methanol. Finally rigid layers were digested with trypsin and chymotrypsin. The results indicate the presence of lump or bar-like structures on the surface of the cell shaped peptidoglycan sacculi. Evidence is provided suggesting that the cellulose microfibrils arise directly from these excrescences found on the peptidoglycan surface. Digestion with cellulase removed all cellulose microfibrils whereas the lumps and bars remained as an integral part of the Rhizobium peptidoglycan.     

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.