Halococcus morrhuae: A sulfated heteropolysaccharide as the structural component of the bacterial cell wall

The qualitative and quantitative composition of purified cell walls of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heteroglycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.     

Relationship of the major constituents of the Neurospora crassa cell wall to wild-type and colonial morphology

Mahadevan, P. R. (The Rockefeller Institute, New York, N.Y.), and E. L. Tatum. Relationship of the major constituents of the Neurospora crassa cell wall to wild-type and colonial morphology. J. Bacteriol. 90:1073-1081. 1965.-The relationship of cell wall to morphology in Neurospora crassa was studied by correlating the levels of structural polymers of the cell wall with wild-type and colonial morphology. The cell wall of N. crassa contains at least four major complexes: a peptide-polysaccharide complex; two glucose polymers, one of which was found to be a laminarinlike beta-1,3-glucan; and, lastly, chitin. The levels of one or more of these structural polymers are consistently altered in single-gene mutants with colonial growth, and in sorbose-induced colonial growth. The proportions of these polymers, particularly of the peptide-polysaccharide complex and the beta-1,3-glucan, appear to be important to morphology.     

Revealing the structural and functional diversity of plant cell walls

The extensive knowledge of the chemistry of isolated cell wall polymers, and that relating to the identification and partial annotation of gene families involved in their synthesis and modification, is not yet matched by a sophisticated understanding of the occurrence of the polymers within cell walls of the diverse cell types within a growing organ. Currently, the main sets of tools that are used to determine cell-type-specific configurations of cell wall polymers and aspects of cell wall microstructures are antibodies, carbohydrate-binding modules (CBMs) and microspectroscopies. As these tools are applied we see that cell wall polymers are extensively developmentally regulated and that there is a range of structurally distinct primary and secondary cell walls within organs and across species. The challenge now is to document cell wall structures in relation to diverse cell biological events and to integrate this knowledge with the emerging understanding of polymer functions.     

Cell wall assembly in fucus zygotes: I. Characterization of the polysaccharide components

Fertilization triggers the assembly of a cell wall around the egg cell of three brown algae, Fucus vesiculosus, F. distichus, and F. inflatus. New polysaccharide polymers are continually being added to the cell wall during the first 24 hours of synchronous embryo development. This wall assembly involves the extracellular deposition of fibrillar material by cytoplasmic vesicles fusing with the plasma membrane. One hour after fertilization a fragmented wall can be isolated free of cytoplasm and contains equal amounts of cellulose and alginic acid with no fucose-containing polymers (fucans) present. Birefringence of the wall caused by oriented cellulose microfibrils is not detected in all zygotes until 4 hours, at which time intact cell walls can be isolated that retain the shape of the zygote. These walls have a relatively low ratio of fucose to xylose and little sulfate when compared to walls from older embryos. When extracts of walls from 4-hour zygotes are subjected to cellulose acetate electrophoresis at pH 7, a single fucan (F₁) can be detected. By 12 hours, purified cell walls are composed of fucans containing a relatively high ratio of fucose to xylose and high levels of sulfate, and contain a second fucan (F₂) which is electrophoretically distinct from F₁. F₂ appears to be deposited in only a localized region of the wall, that which elongates to form the rhizoid cell. Throughout wall assembly, the polyuronide block co-polymer alginic acid did not significantly vary its mannuronic (M) to guluronic (G) acid ratio (0.33-0.55) or its block distribution (MG, 54%; GG, 30%; MM, 16%). From 6 to 24 hours of embryo development, the proportion of the major polysaccharide components found in purified walls is stable. Alginic acid is the major polymer and comprises about 60% of the total wall, while cellulose and the fucans each make-up about 20% of the remainder. During the extracellular assembly of this wall, the intracellular levels of the storage glucan laminaran decreases. A membrane-bound β-1, 3-exoglucanase is found in young zygotes which degrades laminaran to glucose. It is postulated that hydrolysis of laminaran by this glucanase accounts, at least in part, for glucose availability for wall biosynthesis and the increase in respiration triggered by fertilization. The properties and function of alginic acid, the fucans, and cellulose are discussed in relation to changes in wall structure and function during development.     

Evidence against the presence of fibres or chemically distinct layers in the cell wall ofSaccharomyces

The gross chemical composition of material extracted from yeast cell walls with various solvents or enzymes was studied. Attempts were made to locate these materials in situ by comparing electron micrographs of negatively stained and sectioned cell walls with those of the residues of the extraction procedures. There are at least two chemically distinct species of carbohydrate polymers which can be extracted with strong alkali: one yielding mainly mannose and some amino acid on hydrolysis and the other yielding mannose, glucose and amino acid. The alkali-insoluble material also yielded glucose, mannose and amino acid on hydrolysis but the glucose/mannose ratio was much higher. It was shown that none of these polymers constituted a physically distinct layer in the yeast cell wall. However, there does seem to be a region at the outer surface with distinctive properties. This is not fibrillar in nature and after extraction with ethylene diamine forms a double-layered structure. Materials which react with KMnO₄ to produce an electron-dense material are located throughout the wall but tend to be concentrated in the outer and inner regions. Procedures which remove this material also remove up to 80% of the mannose, 40% of the glucose and 35 % of the protein of the original wall material. It was shown that fibres do not constitute a major fraction of the normal cell wall, except possibly in the region of the bud scars but may be produced fairly readily by certain specific treatments. The classical view of the yeast cell wall with the structural integrity being maintained by a fibrous network of 1–3, 1–6 linked glucose residues is challenged and evidence to support an alternative view is presented.The results in wthis paper were presented to the University of Manchester Institute of Science and Technology by JKB in a Thesis for the degree of M.Sc. (Bowden, 1966). The authors are indebted to Miss C. Backhouse and Miss B. Murphy for help in preparing the electron micrographs.     

Structural arrangement of polymers within the wall of Streptococcus faecalis.

The structure of the cell wall of Streptococcus faecalis was studied in thin sections and freeze fractures of whole cells and partially purified wall fractions. Also, the structures of wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan wall polymers were compared with wall preparations that possess a full complement of accessory polymers. The appearance of the wall varied with the degree of hydration of preparations and physical removal of the cell membrane from the wall before study. Seen in freeze fractures of whole cells, the fully hydrated wall seemed to be a thick, largely amorphic layer. Breaking cells with beads caused the cell membrane to separate from the wall and transformed the wall from a predominantly amorphic layer to a structure seemingly made up of two rows of "cobblestones" enclosing a central channel of lower density. Dehydration of walls seemingly caused the cobblestones to be transformed into two bands which continued to be separated by a channel. This channel was also observed in isolated wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan polymers. These observations are consistent with the interpretation that both peptidogylcan and non-peptidoglycan polymers are concentrated at the outer and inner surfaces of cell walls. These observations are discussed in relation to possible models of wall structure and assembly.     

Zelloberflächenstrukturen der Bacteria und Archaea

Cell wall types of Bacteria and Archaea The acaryote microorganisms are divided into the two domains Bacteria and Archaea. The third domain represent the Eukarya. There is no universal cell wall polymer found in all Bacteria and Archaea. Due to their morphology several cell wall types can be identified, but the chemical diversity of the individual polymers is considerably greater. Certain cell wall polymers are limited to one of the two domains of Bacteria or Archaea like the murein of the Bacteria or the pseudomurein of some methanogens. Peptidoglycans (murein, pseudomurein) do not occur in eukaryotes. On the other hand individual cell wall polymers possess similarities to polymers of other domains. The structural principle of the methanochondroitin is also implemented in the eukaryotic connective tissue. The cell wall polymers consist frequently of glycoconjugates in which the amino acid content (glycoproteins) or the glycan moiety (proteoglycan‐like polymers) predominate. Both components (carbohydrates, amino acids) can also occur in similar amounts (peptidoglycan). There exist also cell wall polymers, which consist only of glycans (slimes, methanochondroitin) or amino acids (proteins, poly‐γ‐D‐glutamyl polymers). Cell wall‐free species (Mycoplasma) also occur. The chemical composition of the cell surface polymers was one of the first phenotypic characteristics that supported the 16 sRNA concept of Carl Woese to assign acaryote organisms into the two domains Bacteria and Archaea. A common feature of all Archaea is the lack of muramic acid and an outer membrane. The later occurs in the gramnegative Bacteria. During the evolution of Bacteria and Archaea a great variety of chemically different cell wall polymers has been developed which allow the growth and interaction of Bacteria and Archaea in different habitats. In this paper, some important surface polymers of Bacteria and Archaea are presented according to their chemical composition.     

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

Background  

Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Adaptation and growth of tomato cells on the herbicide 2,6-dichlorobenzonitrile leads to production of unique cell walls virtually lacking a cellulose-xyloglucan network

Suspension-cultured cells of tomato (Lycopersicon esculentum VF 36) have been adapted to growth on high concentrations of 2,6-dichlorobenzonitrile, an herbicide which inhibits cellulose biosynthesis. The mechanism of adaptation appears to rest largely on the ability of these cells to divide and expand in the virtual absence of a cellulose-xyloglucan network. Walls of adapted cells growing on 2,6-dichlorobenzonitrile also differ from nonadapted cells by having reduced levels of hydroxyproline in protein, both in bound and salt-elutable form, and in having a much higher proportion of homogalacturonan and rhamnogalacturonan-like polymers. Most of these latter polymers are apparently cross-linked in the wall via phenolic-ester and/or phenolic ether linkages, and these polymers appear to represent the major load-bearing network in these unusual cell walls. The surprising finding that plant cells can survive in the virtual absence of a major load-bearing network in their primary cell walls indicates that plants possess remarkable flexibility for tolerating changes in wall composition.     

Self-assembly of plant cell walls

The object of this paper is to define criteria for distinguishing between self-assembly and template-based assembly in plant cell walls. The example of cellulose shows that cell wall polymers biosynthesized at a membrane may retain parallel chain packing arrangements that are thermodynamically unstable and cannot be reproduced in vitro, making the experimental testing of the self-assembly hypothesis difficult. Also, natural cellulose is ordered on a number of scales of pattern, each of which may be constructed by either self- or template-based assembly independently of the rest. These conceptual problems apply equally to the self-assembly of complete cell walls and other cell wall polymers. It is suggested that the self-assembly concept should be applied only to one stage or level in the synthesis of a cell wall, and that an additional concept of parallel assembly may be useful for understanding the synthesis of some polysaccharides.     

Cell wall composition of the aquatic fungusBlastocladiella emersonii

Cell walls fromBlastocladiella emersonii were isolated by repeated washing and centrifugation. Purity and uniformity of cell wall preparations were assessed by light and electron microscopy and chemical reproducibility. Electron microscopy showed the cell walls to consist of an inner microfibrillar network and an outer amorphous layer. Analyses by X-ray and infrared spectroscopy were consistent with chitin as the major wall component. Gross chemical analysis indicated that the cell walls were composed of 74.7% amino sugar (as anhydroN-acetylhexosamine), 10.7% neutral sugar (as anhydro hexose), 10.6% protein, and 4.2% lipid. Analysis of the neutral sugars showed that isolated cell walls contain 1.5% mannose, 3.0% galactose, and 3.0% glucose. Isolated cell walls were fractionated using a hot sodium dodecyl sulfate (SDS) extraction followed by either Pronase digestion or hot KOH extraction. The hot SDS extract was found to contain two polymer types, galactose- and/or glucose-containing polymers and glycoprotein. However, the residue from the hot SDS extraction still contained most of the neutral sugars and protein present in the isolated walls. Both Pronase digestion and the hot potassium hydroxide extraction removed all of the neutral sugars except glucose. The cell wall fractionation results indicate that the major wall component is microfibrillar chitin. The results further suggest that the SDS-solubilized glycoproteins and neutral sugar polymers may represent an outer amorphous layer.     

Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa

Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.     

A widespread family of bacterial cell wall assembly proteins

Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall.     

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis

MreB proteins play a major role during morphogenesis of rod‐shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane‐associated MreB polymers have been shown to be associated to elongation‐specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso‐diaminopimelate (m‐DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane‐associated cell wall synthesizing machineries.     

Enzymatic synthesis and purification of uridine diphospho-beta-l-arabinopyranose, a substrate for the biosynthesis of plant polysaccharides

Many plant cell wall components such as the polysaccharides xylans and pectins or the glycoproteins arabinogalactan proteins and extensins contain arabinosyl residues. The arabinosyl substituents are thought to be incorporated into these wall polymers by the action of arabinosyltransferases using UDP-l-arabinose as the precursor. UDP-l-arabinose is not commercially available and therefore a procedure for generating UDP-l-arabinose was developed for use in studies on the biosynthesis of the arabinose-containing polymers. In this procedure UDP-d-xylose is incubated with an enzyme preparation from wheat germ and the nucleotide sugars in the reaction mixture are extracted. High-performance anion-exchange chromatography of the extract resolves two major UV-absorbing components: one corresponding to UDP-xylose and a second that elutes earlier. TLC analysis of collected and hydrolyzed fractions demonstrated the presence of l-arabinose in the early eluting fraction. Further analysis by NMR identified the compound as UDP-beta-l-arabinopyranose. The procedure reported here provides an efficient method for preparing either radioactive UDP-l-[(14)C]arabinose or nonradioactive UDP-l-arabinose and can also be used as an assay for UDP-xylose-4-epimerase activity.     

The role of fungal polysaccharidases in the hydrolysis of cell wall materials from sunflower and palm-kernel meals

Main fractions from multi-component polysaccharidase preparations (Driselase, Gamanase and an experimental preparation of fungal origin), previously used for the enzymic treatment of cell wall materials from sunflower and palm-kernel meals, were sub-fractionated by different chromatographic techniques to evaluate the contribution of each of their constituent activities in cell wall degradation. Based on activity measurements, 5- to 10-fold purification was achieved for the major enzymes but residual side-activities were still detectable in most sub-fractions. Solubilization of non-starch polysaccharides from the cell wall materials by the resulting pectolytic, xylanolytic, cellulolytic and mannanolytic sub-fractions and by highly purified glucanases, arabinanases and xylanases was, when acting individually, very low (1% to 5%). With few exceptions, the solubilizing effect of the main fractions could only be slightly enhanced by supplementation with pectolytic, cellulolytic or mannanolytic sub-fractions or by highly purified enzymes. The extent of solubilization remained mostly lower than the sum of both individually obtained values. In the degradation of palm-kernel cell wall material, however, synergistic action of mannanases and glucanases was observed. The hydrolysis of pectic compounds in sunflower cell wall material was most effective when polygalacturonases, arabinanases and rhamnogalacturonan-degrading activities were applied together. The resistance of 4-O-methyl-glucuronoxylan, the major hemicellulosic polymer in the cell wall material from sunflower meal, to enzymic hydrolysis was not only caused by its location in the cell wall or interlinkage to other polymers but also by its primary structure. Neither purified endo-xylanase nor the crude parent preparation were able to achieve complete hydrolysis of this polysaccharide after extraction.