Vegetative Cell Wall Research Articles

The Yeast Spore Wall Enables Spores to Survive Passage through the Digestive Tract of Drosophila

In nature, yeasts are subject to predation by flies of the genus Drosophila. In response to nutritional starvation Saccharomyces cerevisiae differentiates into a dormant cell type, termed a spore, which is resistant to many types of environmental stress. The stress resistance of the spore is due primarily to a spore wall that is more elaborate than the vegetative cell wall. We report here that S. cerevisiae spores survive passage through the gut of Drosophila melanogaster. Constituents of the spore wall that distinguish it from the vegetative cell wall are necessary for this resistance. Ascospores of the distantly related yeast Schizosaccharomyces pombe also display resistance to digestion by D. melanogaster. These results suggest that the primary function of the yeast ascospore is as a cell type specialized for dispersion by insect vectors.

ChemInform Abstract: Synthesis of a Hexasaccharide Repeating Unit from Bacillus anthracis Vegetative Cell Walls.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of a Hexasaccharide Repeating Unit from Bacillus anthracis Vegetative Cell Walls

The first synthesis of hexasaccharide 1 representing a repeat unit of a polysaccharide specific to the vegetative cell wall of Bacillus anthracis is reported. The synthetic hexasaccharide is equipped with an n-pentenyl handle at the reducing terminus to allow for further functionalization. Key transformations during the synthesis are the conversion of a glucose into a mannosazide residue, a (2+2) coupling, followed by double alpha-galactosylation to furnish the hexasaccharide, and global deprotection under Birch conditions.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Chitosan, the Deacetylated Form of Chitin, Is Necessary for Cell Wall Integrity in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a "leaky melanin" phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.

The Structure of the Major Cell Wall Polysaccharide of Bacillus anthracis Is Species-specific

In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one- and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of -->6)-alpha-GlcNAc-(1-->4)-beta-ManNAc-(1-->4)-beta-GlcNAc-(1-->, in which the alpha-GlcNAc residue is substituted with alpha-Gal and beta-Gal at O-3 and O-4, respectively, and the beta-GlcNAc substituted with alpha-Gal at O-3. There is some variability in the presence of two of these three Gal substitutions. Comparison with the HF-PSs from B. cereus ATCC 10987 and B. cereus ATCC 14579 showed that the B. anthracis structure was clearly different from each of these HF-PSs and, furthermore, that the B. cereus ATCC 10987 HF-PS structure was different from that of B. cereus ATCC 14579. The presence of a B. anthracis-specific polysaccharide structure in its vegetative cell wall is discussed with regard to its relationship to those of other Bacillus species.

Extração aquosa enzimática de óleo de soja

The aqueous enzymatic treatment has showed efficient in the degradation of the vegetable cell walls, propitiating bigger yield in the oil extraction, improvements in the bran nutricional quality and increase in the product stability. This work evaluated the commercial enzyme performance (Alcalase - protease - and Celluclast - cellulase) in the aqueous process of soybean oil extration, as well as the influence of the enzymes concentration, the extraction time and the dilution of the sample (soybean) in the final yield. Four experiments of extration were carried through and the results had been gotten from an factorial planning 23. The statistics analysis showed that the most significant variable of all extration process was the sample dilution (1:20). The yield income of the process of aqueous extration was inferior to the process of solvent extraction. The increase in the enzymatic concentration and in the extraction time favored the yield. Of used enzymes, the Alcalase presented better efficiency in the extration, being that the use of two enzymes simultaneously propitiated significant increment on the process yield.

Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases.

LytF, LytE, and LytC are vegetative cell wall hydrolases in Bacillus subtilis. Immunofluorescence microscopy showed that an epitope-tagged LytF fusion protein (LytF-3xFLAG) in the wild-type background strain was localized at cell separation sites and one of the cell poles of rod-shaped cells during vegetative growth. However, in a mutant lacking both the cell surface protease WprA and the extracellular protease Epr, the fusion protein was observed at both cell poles in addition to cell separation sites. This suggests that LytF is potentially localized at cell separation sites and both cell poles during vegetative growth and that WprA and Epr are involved in LytF degradation. The localization pattern of LytE-3xFLAG was very similar to that of LytF-3xFLAG during vegetative growth. However, especially in the early vegetative growth phase, there was a remarkable difference between the shape of cells expressing LytE-3xFLAG and the shape of cells expressing LytF-3xFLAG. In the case of LytF-3xFLAG, it seemed that the signals in normal rod-shaped cells were stronger than those in long-chain cells. In contrast, the reverse was found in the case of LytE-3xFLAG. This difference may reflect the dependence on different sigma factors for gene expression. The results support and extend the previous finding that LytF and LytE are cell-separating enzymes. On the other hand, we observed that cells producing LytC-3xFLAG are uniformly coated with the fusion protein after the middle of the exponential growth phase, which supports the suggestion that LytC is a major autolysin that is not associated with cell separation.

A Mechanics Model for the Compression of Plant and Vegetative Tissues

The mechanics analysis of plant or vegetable tissue under a compressive stress has been developed based on large deformation elasticity theory. The tissue was treated as a lattice of regular perfect three-dimensional hexagonal cells. The cell walls were assumed to be impermeable under the time-scale of the loading. The cell walls of plants and vegetables are polymeric composite materials, consisting of a relatively amorphous matrix and a highly structured network of microfibrils embedded in the cell wall matrix. The micromechanical features of the individual cells have been related to the macroscopic properties of the whole tissue. The effects of microfibrillar stiffening factors k 1 and k 2, the cell wall matrix property α and the initial cell expansion ratio ν i on the compressive behaviour of a plant or vegetable tissue have been investigated. The predicted results have also been related to some experimental evidence.

A light and electron microscopy study on the formation, structure and germination of akinetes of Stigeoclonium tenue (Chaetophorales, Chlorophyceae)

Akinete formation in Stigeoclonium tenue (AG) KUTZ began under unfavourable conditions (exhaustion of nutrients and/or evaporation of the culture medium). Cells from both prostrate and erect portions produced akinetes. A gradual change in the shape of vegetative cells was observed. They lost their typical straight edges and acquired an oval contour with constrictions at the level of the transverse cell walls. At the ultrastructural level, the changes were: 1) accumulation of starch and lipids as reserve substances; 2) disorganization of the chloroplast; 3) reduction of the vacuole until it disappeared in mature akinetes; 4) thickening of the wall resulting from the synthesis of both an additional new own wall next to the parental wall and a mucilage external layer; 5) disorganization of the plasmodesmata in the transverse walls of vegetative cells. All of these drastic cytological changes are essential to the development of akinetes which provide S. tenue with an effective survival mechanism under unfavourable conditions.

Location of (1→3)- and (1→3),(1→4)-β-D-glucans in vegetative cell walls of barley (Hordeum vulgare) using immunogold labelling.

• (1→3),(1→4)-β-Glucans occur only in the cell walls of the family Poaceae (grasses and cereals) and related families, but little is known about their distribution among walls of different cell types or within walls. • The locations of (1→3)- and (1→3),(1→4)-β-glucans in the walls of the coleoptile, first leaf and root tip of barley (Hordeum vulgare) seedlings were determined using immunogold labelling. • All the walls were labelled with the (1→3),(1→4)-β-glucan antibody, except those of the outer root cap cells. Labelling of the primary walls was heavy in the coleoptile and leaf, but light or very light in the root tip. Two types of primary wall labelling occurred: in the coleoptiles (except the walls of the epidermis and two layers of parenchyma under this) and in the leaf, labelling was throughout the walls; in the root tips, labelling was only adjacent to the plasma membrane. Small amounts of labelling occurred with the (1→3)-β-glucan antibody, mostly over plasmodesmata. Both antibodies labelled cell plates. • (1→3),(1→4)-β-Glucans occur widely in the cell walls of vegetative organs of the barley seedlings, including the walls of meristematic tissues.

ULTRASTRUCTURE AND CYTOCHEMISTRY OF SPERMATANGIAL DEVELOPMENT AND FERTILIZATION IN AGLAOTHAMNION OOSUMIENSE (CERAMIACEAE, RHODOPHYTA)

Spermatial development and fertilization processes were investigated by electron microscopy in Aglaothamnion oosumiense Itono. The spermatium is composed of two parts, an ovoidal head and two appendages projecting from each distal end. The appendages originate from spermatangial vesicles (SVs) and follow a developmental sequence beginning as amorphous material and ending as a fully‐formed fibrous structure compressed within the SVs. SV formation is due to contributions initially from endoplasmic reticulum and later from dictyosome‐derived vesicles. Chemical differentiation of the spermatial wall occurs early in its development. Calcofluor white does not label spermatial walls, indicating an absence of cellulose polysaccharides, which are abundant in vegetative cell walls. Liberated spermatia had a prophase arrested nucleus with a pair of polar rings. The cytoplasm of the trichogyne was connected with that of spermatia at the fertilization canal. The cytoplasm of the trichogyne was empty when the nuclear fusion between spermatium and carpogonium occurred.

Cellular and molecular approaches to understanding primary adhesion in Enteromorpha: an overview

The attachment of motile spores of the green alga Enteromorpha to the substratum is an active process involving an irreversible commitment to adhesion and the secretion of an adhesive. This paper provides an overview of the spore adhesion processes and outlines the results of an experimental approach towards the molecular characterisation of the adhesive, based on the use of monoclonal antibody (mAb) technology. Hybridomas were produced to settled spores displaying secreted adhesive. Candidates producing mAbs to putative adhesive were selected using a range of criteria based on cellular localisation, time of secretion and functional inhibition of adhesion. MAb Ent 6 immunolabelled fibrillar material which was secreted during the early stages of adhesion and low (nM) concentrations of this mAb, or its F(ab)2 fragments, strongly inhibited the attachment of zoospores. A related antibody (Ent 1) also labelled the spore adhesive apparatus, but the antigen appeared to be secreted later during the adhesion process and was predominantly associated with the developing cell wall. Ent 1 also inhibited settlement in spore adhesion assays but the effect was most pronounced at later time points which suggests that this antigen does not have a role in the earliest stages of adhesion. Immunolocalisation showed that both antigens were absent from the cytoplasm or organelles of vegetative tissue but labelled the vegetative cell wall, suggesting a relationship between cell wall components and materials involved in primary adhesion. Both mAbs labelled the Golgi region of settled spores, suggesting continued synthesis of both antigens after adhesion. Both mAbs recognised a 110 kDa N‐linked polydisperse and heterogeneous glycoprotein in extracts of swimming spores under denaturing conditions. In native form the antigens behaved as high molecular weight aggregates (Mr>1.3 × 106). The antigens became progressively insoluble after zoospore attachment. Taken together, the data suggest that the two antibodies recognise closely related, polydisperse, self‐aggregating cell wall glycoproteins in which there is some structural variation to suit alternative roles in primary adhesion and cell wall formation. The two mAbs Ent 1 and Ent 6 partially discriminate between these structural and functional variants. A model for zoospore adhesion is discussed in which adhesion is viewed as an extension of cell wall synthesis, with cross‐links between glycoproteins and other cell wall matrix components providing a strong physical continuum between the cell and the adhesive at the substratum interface.

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation.

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.

Interaction between the mycoparasite Verticillium fungicola and the vegetative mycelial phase of Agaricus bisporus

Host-parasite interactions of Agaricus bisporus and Verticillium fungicola were studied in paired-cultures. Different growth reactions were found according to the culture medium used, but no apparent alterations could be detected by light and electron microscopy in A. bisporus vegetative mycelia. In vitro, different hydrolytic enzymes are produced by V. fungicola in cultures with A. bisporus vegetative mycelial cell walls as the only carbon source. In vitro, this enzyme preparation is able to partially digest isolated A. bisporus vegetative mycelial cell walls. These results suggest that although apparent attachment of the mycoparasite to the host cell surface seems to occur, there is no in vivo A. bisporus vegetative mycelial cell wall degradation.

Structural and physical properties of dietary fibres, and consequences of processing on human physiology

Dietary fibre encompasses very diverse macromolecules exhibiting a large variety of physico-chemical properties. They might be naturally present in the food (in cell walls of vegetables and fruits, for instance) or introduced in the food to improve their nutritional properties (e.g. bran in bread products) or their physical characteristics (e.g. gelling agents or improved resistance to storage). The properties that are nutritionally relevant are mainly the particle size and bulk volume, the surface area characteristics, the hydration and rheological properties, and the adsorption or entrapment of minerals and organic molecules. Amongst these properties, the viscosity and ion exchange capacity are the main contributors to metabolic effects (glucose and lipid metabolisms) whereas fermentation pattern, bulking effect and particle size are strongly involved in effects on colonic function. Technological treatments can modify the physico-chemical properties of the fibre. This can be further exploited to optimise both their techno-functional and physiological properties.

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

Aspects of material science in food processing: changes in plant cell walls of fruits and vegetables

Foods can be regarded as complex, dispersed systems which are normally metastable. Food processing causes state transitions (second-order transitions) when raw materials, food components or food systems are subjected to external stresses. The state transitions occurring during processing are detectable as changes in structure and properties of the investigated systems. The processing of fruits and vegetables is often connected with changes in cell walls. Cell wall materials in dispersed fruit and vegetable systems can be regarded as a model substrate of the dispersed phase. During processing, cell walls undergo modifications in terms of their physical state, macrostructure, microstructure, and composition, as well as structure-dependent changes in their functional and material properties. The interactions and connections (dependencies) between state transitions, and various changes in structure and properties, are very complex and multivariate and are not well understood as yet. For the evaluation of changing material properties during processing, examination of hydration, rheological (external mechanical stress) and thermal (external thermal stress) characteristics is important. The changes occurring during the processing of fruits and vegetables are determined by external factors (especially various mechanical and thermal stresses) and by internal factors. External stress in many cases causes solubilisation of the cell wall, loss of firmness and favours cell separation. Thermal processing increases pectin degradation by β-elimination. Internal factors such as pH and modified ionic strength, e.g. by applying soak solutions, can have an important influence on the changes in the cell wall during processing. So, calcium ions on the one hand can favour cell wall degradation by β-elimination and, on the other hand, after low temperature blanching and de-esterification of the pectin by activated pectin methyl esterase, can contribute to stabilisation of the texture by formation of a calcium-pectin complex. Knowledge about cell wall degradation mechanisms can be markedly improved by studies using model substrates such as pectin, or cell wall materials like the alcohol-insoluble residue and materials with cellular structure. This knowledge has been used to improve the technology used to process fruits and vegetables and to produce products with better properties. Moreover, testing and applying cell wall materials as ingredients for the production of textured foods and potential health-related foods is suggested.

The role of peptidoglycan structure and structural dynamics during endospore dormancy and germination.

Dormant, bacterial endospores are the most resistant living structures known. The spore cell wall (cortex) maintains dormancy, core dehydration, and heat resistance. The cortex peptidoglycan has a unique, spore specific structure that enables it to fulfill its role. The cross-linking index of spore cortex peptidoglycan is very low, occurring at only 2.9% of the muramic acid residues compared to 33% in vegetative cells. The level of cross-linking of the cortex may be important in maintaining spore dormancy and heat resistance. Approximately 50% of the muramic acid residues in spore cortex are substituted with muramic delta-lactam. This modification is spore specific and is the major characteristic feature of the cortex. The muramic delta-lactam has no apparent role in establishing core dehydration, maintaining dormancy or heat resistance. However, the muramic delta-lactam residues are necessary for spore cortex hydrolysis during germination. They constitute part of the substrate recognition profile of the germination specific lytic enzymes (GSLEs) which are responsible for cortex hydrolysis. Germination results in loss of dormant spore properties and hydrolysis of the cortex is essential for later germination events and outgrowth. Application of muropeptide analysis to determine peptidoglycan structural dynamics during germination has revealed an unexpected degree of complexity in peptidoglycan hydrolysis. At least three hydrolytic activities, an N-acetyl glucosaminidase, a lytic transglycosylase and a possible amidase, are involved. A non-hydrolytic activity, likely to be an epimerase of muramic acid also occurs early during germination. The lytic transglycosylase generates anhydro-muropeptides which are released during germination and may be recycled during outgrowth to form part of the new vegetative cell wall.

The Contribution of Symbiodinium Microadriaticum to the Symbiosome Membrane in the Anthozoan Host Aiptasia Pallida

Abstract Most endosymbionts remain separated from their host cell by a derivative of the phagocytic membrane. This membrane and its contents have been defined as a novel “organelle-like” structure called a symbiosome. However, in the case of many cnidarian-dinoflagellate symbioses the putative symbiosome is composed of multiple layers of apparent membranes (Fig. 1). In the past the origin of these membranes has been assigned to host and/or symbiont with very little experimental evidence to support either view; however, several interesting hypotheses have been proposed to explain the origin of the “membranes”. Trench and Blank suggest that occasionally the outer most layer of the vegetative symbiont's cell wall is sloughed off the surface of the cell. They concede that the outer most layer of “membranes” must be the host vacuolar membrane, but believe that it simply prevents the sloughed layer from moving away from the cell, (as would happen in culture), and that, over time, multiple sloughed layers accumulate beneath the host vacular membrane.