Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that the TCA-wall consisted of mostly peptidoglycan. By digesting the TCA-wall with lysozyme, the circular structures were greatly disturbed, and they disappeared after 60 min of treatment. From these observations it can be expected that the peptidoglycan is arranged in a concentric circular manner in the newly generated cell wall of Staphylococcus.     

Surface arrays on the cell wall of Spirillum metamorphum.

A complex and easily disrupted arrangement of macromolecules was present on the outer (lipopolysaccharide) membrane of the cell wall of Spirillum metamorphum. Separation of the arrays from the cell and spontaneous reassembly into regularly structured complexes usually occurred during preparation for electron microscopy. Freeze etchings, thin sections, and optical diffraction analysis of negatively stained fragments indicated that they consisted of two sets of a thin layer which was studied with 3-nm particles arranged in a loose (OL). The OSL consisted of a hexagonal arrangement of 8-nm disks and the OL of a thin layer which was studied with 3-nm particles arranged in a loose rectangular manner. The OSL of reassembled fragments displayed numerous broken delta-linkers between units and a center-to-center spacing of half the expected distance, which suggests that an interdigitation of two OSL arrays had occurred. The observations combined with freeze etchings and thin sections of whole cells suggested a possible reassembly mechanism. The normal surface arrangement of these layers on cells was thought to consist of the OL overlying one set of OSL which was loosely adherent to a thin amorphous backing layer.     

The demonstration of the existence of an interlayer between the cytoplasmic membrane and the cell wall proper of staphylococci

By disintegration of the cell wall of staphylococci a definite interlayer located between the cytoplasmic membrane and the cell wall proper could be demonstrated for the first time (=MW-interlayer). This MW-interlayer contains a sort of cloddy material in which clusters of embedded ring-like disks are hexagonally arranged in a crystal-like manner. The ring-like disks, approximately 40 Å in diameter and with center-to-center spacings of approximately 75 Å, lie in direct contact either with a rhombically arranged fibrillar network of the outer parts of the cytoplasmic membrane or they themselves are part of (or interconnected by) such an apparently rhombical network. The crystal-like arranged ring-like disks of the interlayer between the cytoplasmic membrane and the cell wall shall be called MW-particles in order to differentiate them from intramembrane particles and particles on the outer surface of the cell wall. At present, nothing more than speculation on the function of the MW-particles located within the space where final processes of the cell wall polymerization are taking place is possible.Abbreviations MW membrane-wall - EF external face - PF protoplasmic face - PS protoplasmic surface - IM intramembrane     

Ultrastructure of the cell wall of a Synechocystis strain

The ultrastructure of the cell wall of a Synechocystis strain, isolated from the Gulf of Finland, was studied using several electron microscopic techniques. This cyanobacterium has numerous projections which were observed to penetrate the cell wall complex. An additional layer (AL) was associated with the outer membrane. An additional external wall layer (EL) was connected to the outer membrane complex by thin fibers as revealed by ruthenium red staining. A hexagonal arrangement of the subunits in the additional external wall layer with a lattice constant of 15.5 nm was found.     

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.     

Mode of cell separation and arrangement of Staphylococcus

The process of cell separation and arrangement of Staphylococcus was investigated using a scanning electron microscope. After two cycles of cell division, the Staphylococcal cells cultured on an agar medium were generally observed to be arranged in three morphological types: linear, square, and crooked arrangements. Results of the examination of cell surface structure revealed that separations had occurred in these clustered cells following two patterns. One type of second separation occurred parallel to the transversal axis of the preceding pair of the parental cells (X-type) and the other occurred tangential to it (Y-type). In the former type, the four daughter cells were usually arranged tetragonally after the separations, and in the latter type they were arranged either linearly or crookedly depending on the direction of the second separation. The final pattern of the cell arrangement was thus determined by the type of septal wall formation and the direction of cell separation. After several cycles of cell divisions, the cells were finally arranged in an irregular grape-like cluster, even though the cross walls were formed regularly at the rectangular face of the preceding cross walls.     

Regular arrangement of wall polymers in staphylococci

Concentric circular structures were observed on the newly exposed surface of the wall of Staphylococcus epidermidis and on its isolated cross-wall. These structures were removed by treatment with trichloroacetic acid. Chemical analysis revealed that after treatment with trichloroacetic acid most of the wall phosphorus was extracted but more than half of the N-acetylglucosamine remained associated with the wall. These observations suggest that polysaccharides are likely to be arranged circularly on the surface of the wall.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.     

Surface architecture of the plant cell: Biogenesis of the cell wall,with special emphasis on the role of the plasma membrane in cellulose biosynthesis

Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amourphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi-derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi-derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross-linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze-etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed “ridges.” The ridges interlink with definitive rows of particles associated with the PF fracture face of the inner leaflet of the plasma membrane. These particles are termed “granule bands,” and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.     

Ultrastructure and partial characterization of a regular array in the cell wall of Lactobacillus brevis.

The cell wall of Lactobacillus brevis was revealed by electron microscopy to have an outer layer composed of a regular array. The morphological unit of the regular array appeared to consist of four spherical subunits, each about 2 nm in diameter, which were arranged in a tetragonal pattern about 4.5 by 7.0 nm in dimension. The regular array was composed of the tetragonal units in rows in two directions at an angle of about 75 degrees to each other. The average spacing between the rows was about 10 nm in one direction and about 7 nm in the other. The tetragonally arranged subunits were removed from the cell wall by treatment with guanidine hydrochloride, urea, or sodium dodecyl sulfate (SDS) but not by the action of ethylenediaminetetraacetate, nonionic detergents, or proteolytic enzymes except pepsin. The regular subunits were shown to be composed of a protein with a molecular weight of about 51,000 by SDS-polyacrylamide gel electrophoresis.     

Distribution of protein A on the surface of Staphylococcus aureus

Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Characterization and reassembly of a regular array in the cell wall of Clostridium difficile GAI 4131

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mM CaCl2. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins and freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.     

The fine structure of smooth muscle cells and their relationship to connective tissue in the rabbit portal vein

Summary The smooth muscle of rabbit portal vein was studied by electron microscopy with particular emphasis on the mechanical linkage between the muscle cells and on the distribution of connective tissue.The media of this vein is composed of inner circular and outer longitudinal muscle layers which are orientated almost perpendicularly to each other. The muscle of the inner circular layer shows very irregular contours with much branching and anastomosing of the cytoplasmic processes, which often make membrane contacts with neighbouring cells to form an extensive network of cytoplasmic processes. The muscle cells of the outer longitudinal layer are arranged in densely packed bundles and are spindle-shaped, with no branching processes. Opposing dense areas from neighbouring cells, with variable gap distances (30–100 nm) and close membrane contacts (intermediate junctions) with a gap of 11 nm were observed in both circular and longitudinal muscle layers.In the terminal regions of muscle cells in both circular and longitudinal layers a specialized anchoring structure was present which was closely related to extracellular elastic tissue. Muscle cells in the longitudinal layer showed the most elaborate structure, the tapering end of the muscle cell showing a honeycomb-like structure penetrated by columns of connective tissue compounds. The functional implications of these structures are discussed.     

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.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

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.     

Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.