Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.     

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.     

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.     

Cadmium replaces calcium in the cell wall ofUlva lactuca

Electron microscopy, in conjunction with X-ray microanalysis, was used to investigate the effects of exposure to cadmium on the elemental composition of the macroalgaUlva lactuca. The cell wall was the only region of the cell to show any marked change in chemical composition as a result of exposure to cadmium, with less calcium evident in cadmium-treated thallus compared with untreated thalli. The cell wall ofU. lactuca is a complex structure made up of polysaccharides consisting of many-branched chains composed mostly of rhamnose and galactose subunits. Some of the hydroxyl groups on the subunits are substituted by sulphate groups. Borate is associated with the rhamnose subunits, which contain no sulphate groups, and calcium binds to borate, cross-linking the rhamnose groups. The borate-calcium complex adds rigidity to the cell wall; the replacement of calcium by cadmium will, therefore, influence the rigidity of the thallus. The ecological significance of this work is discussed with respect to the ability of the alga to withstand grazing or emersion.     

Cell wall assembly in vitro from Chlamydomonas reinhardi

Summary The conditions for in vitro dissociation and reassembly of the cell wall of Chlamydomonas reinhardi have been investigated. The cell wall dissociates in aqueous 8 M lithium chloride to two homogeneous components (6.8 S₂₀w and 9.3 S₂₀w)and dialysis of the cell wall subunits against water causes reassembly of a product having the same overall morphology and chemically similar to the original cell walls. Cell wall subunits in 8 M lithium chloride alone do not reassemble on dialysis against water to form cell walls but a nucleation centre has to be provided.     

Three-dimensional structure of a cell wall glycoprotein

Electron microscopy and computer image analysis have been used to determine the three-dimensional structure of the crystalline glycoprotein cell wall layer of the alga Lobomonas piriformis. Images of negatively stained specimens, tilted through a range of angles up to 70 °, were combined to give a map of the molecular envelope to a resolution of 2.0 nm. The cell wall layer consists of crystalline plates the centres and edges of which display distinctly different but isomorphous structures. A comparison of three-dimensional reconstructions of the two areas shows the difference probably to be due to a conformational change of one of the glycoprotein subunits. The structure consists of two sets of dimers composed of rod-shaped subunits which lie with their long axes approximately in the plane of the crystal. The centre-edge transition may have significance in the pathway of accretion of new subunits during cell wall growth.     

Electron Microscopy of the Cell Wall of Rickettsia prowazeki

Purified Rickettsia prowazeki were found to undergo morphological changes resembling plasmolysis when stained with uranyl acetate, resulting in rod-like forms. Sequential electron micrographs of disintegrating organisms provide evidence for the cell wall origin of these rod-like forms. The substructure of the cell wall was discerned by using negative-contrast electron microscopy. The wall was found to be composed of repetitive subunits with a periodicity of 13 nm and was surrounded by a thin membrane.     

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits.

Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.     

Regular Array in the Cell Wall of Lactobacillus fermenti as Revealed by Freeze-Etching and Negative Staining1

Freeze-etching of Lactobacillus fermenti F-4 (NCTC 7230) revealed that the outer layer of the cell wall was composed of a regular array in which parallel lines ran obliquely to the longitudinal axis of the cell with an average distance between the centers of about 9.6 nm and were intersected by thinner lines with an average periodicity of approximately 6.2 nm at an angle of about 75°. Occasionally the direction of the striation was discontinuously shifted near one end of the cell. Beneath the regular array the middle cell wall layer packed with granules and the smooth inner cell wall layer were discernible and the mesosomes were also visible in the cytoplasm. When the ultrastructure of isolated outer cell wall fragments was examined by negative staining, the regular array appeared to be composed of subunits, about 3.6 nm in diameter, which were arranged in a tetragonal pattern. The tetragonal array consisted of the subunits in rows in two directions at an angle of about 75° to each other. The average spacing between the rows was about 9.3 nm in one direction and 5.5 nm in the other direction.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

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.     

Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.     

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.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Detachment and Chemical Characterization of the Regularly Arranged Subunits from the Surface of an Acinetobacter

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.     

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

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.     

Cell Wall of Mycobacterium lepraemurium Strain Hawaii

The chemical properties of the cell wall of Mycobacterium lepraemurium strain Hawaii were investigated. Five subunits of the cell wall, arabinose mycolate, mycolic acids, tetrapeptide (Ala-Gln-diaminopimelic acid-Ala), disaccharide (N-acetylglucosaminyl-beta-1,4-N-glycolylmuramic acid), and arabinogalactan, were obtained, and their chemical structures were identified.     

Ultrastructure of Clostridium tetani. I. Structural features of the surface structures of vegetative cells]

Crest-like structures formed by internal layer of cell wall and cytoplasmic membrane were revealed in G1.tetani 471 by electron microscopy with the use of negative contrasting, ultrathin sections and freezing-etching. The transverse section of these crest-like structures was 56.3 nm and they were localized 4 to 6 in one row girdling the protoplast in different directions. Ring-like subunits located in rows with the periodicity of 5.9 nm, perpendicularly to the long axis of the cell, were revealed on the surface of the cell wall.     

The invertebrate myosin filament: subfilament arrangement of the solid filaments of insect flight muscles.

Transverse sections (approximately 140 nm thick) of solid myosin filaments of the flight muscles of the fleshfly, Phormia terrae-novae, the honey bee, Apis mellifica, and the waterbug, Lethocerus uhleri, were photographed in a JEM model 200A electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. In each of these filaments it was found that the symmetry of the core and the wall was not the same. The power spectra of the images showed sixfold symmetry for the wall and threefold symmetry for the core of the filaments. The images of the filaments in each muscle were superimposed according to the sixfold center of the wall. These averaged images for all three muscles showed six pairs of subunits in the wall similar to those found in the wall of tubular filaments. From serial sections of the fleshfly filaments, we conclude that the subunits in the wall of the filaments represent subfilaments essentially parallel to the long axis of the filament. In each muscle there are additional subunits in the core, closely related to the subunits in the wall. Evaluation of serial sections through fleshfly filaments suggests that the relationship of the three subunits observed in the core to those in the wall varies along the length of the filaments. In waterbug filaments there are three dense and three less dense subunits for a total of six all closely related to the wall. Bee filaments have three subunits related to the wall and three subunits located eccentrically in the core of the filaments. The presence of core subunits can be related to the paramyosin content of the filaments.