Architecture and chemistry of microconidial walls of Trichophyton mentagrophytes.

The ultrastructure and chemical composition of the walls of Trichophyton mentagrophytes microconidia were investigated with particular emphasis on the localization of the major structural components within the walls. The walls consisted of carbohydrate (56.1% neutral polysaccharide, and 16.0% chitin), protein (22.6%), lipid (6.5%), ash (1.7%), and trace amounts of melanin (0.2%) and phosphorus (0.2%). in thin sections, three distince layers were recognized. The electron-transparent pellicle (15 to 20 nm thick) covering the outermost surface of the wall consisted of a glycoprotein-lipid complex and was mostly extracted by sodium phosphate buffer (0.1 M, pH 6.5) containing 8 M urea, 1% (vol/vol) mercaptoethanol, and 1% (wt/vol) sodium dodecyl sulfate. The middle electron-dense layer (30 to 50 nm thick) represented the proteinaceous rodlet layer embedded in polysaccharides and could be completely solubilized by hot alkali extraction (1 N NaOH, 100 DEGREES C, 1 h). The thick inner layer (200 to 300 nm thick) was relatively resistant to the above treatments and was found to consist of amorphous glucans and microfibrillar chitin. Approximately half of the inner wall glucans was susceptible to (1 leads to 3)-beta-glucanase.     

Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea

The ascomycete Botrytis cinerea is a destructive and ubiquitous plant pathogen and represents a model organism for the study of necrotrophic fungal pathogens. Higher fungi possess a complex and dynamic multilayer cell wall involved in crucial aspects of fungal development, growth and pathogenicity. Plant resistance to microbial pathogens is determined often by the capacity of the plant to recognize molecular patterns associated with the surface of an interacting microbe. Here we report the chemical characterization of cell walls from B. cinerea during axenic growth. Neutral sugars and proteins constituted most of the mass of the B. cinerea cell walls, although chitin and uronic acids were detected. Glucose was the most abundant neutral sugar, but arabinose, galactose, xylose and mannose also were present. Changes in cell wall composition during culture were observed. As the culture developed, protein levels declined, while chitin and neutral sugars increased. Growth of B. cinerea was associated with a remarkable decline in the fraction of its cell wall material that was soluble in hot alkali. These results suggest that the cell wall of B. cinerea undergoes significant modifications during growth, possibly becoming more extensively covalently cross-linked, as a result of aging of mycelia or in response to decreasing nutrient supply or as a consequence of increasing culture density.     

Quantitative microanalysis of cell walls of Saprolegnia diclina humphrey and Tremella mesenterica fries

Cell walls of the fungi Saprolegnia diclina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (±3% for amino acids and amino sugars, and ±5–10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition.Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this imino acid from the cell wall of a Basidiomycete.     

Growth of Neocallimastix sp. Strain R1 on Italian Ryegrass Hay: Removal of Neutral Sugars from Plant Cell Walls

The anaerobic fungus Neocallimastix sp. strain R1 was grown for up to 5 days on a medium containing autoclaved Italian ryegrass hay as the carbon source. Culture supernatants and digested cell walls were harvested at 12-h intervals. Supernatants were analyzed for the fermentation products formate and acetate, and residual cell walls were analyzed for dry-matter and neutral-sugar losses. Fungal growth was accompanied by the digestion of plant cell walls and the accumulation of fermentation products in culture media. Dry-matter losses were accounted for by removal of four major neutral sugars (arabinose, galactose, glucose, and xylose) from the plant cell walls. First-order reaction kinetics could be used to describe the loss of each sugar. All cell wall sugars, including arabinose and galactose, which are not fermented by Neocallimastix sp. strain R1 were removed simultaneously. Although the rates of removal of individual sugars were similar, there were significant differences in their extents of removal: the extent of removal of arabinose exceeded that of the other three sugars, and xylose was the least digestible. This study provides the first account of simultaneous (nonpreferential) removal of neutral sugars from plant cell walls by an anaerobic fungus. Although in vitro techniques were used, these results indicate a potentially significant role for the anaerobic fungi as fiber digesters in the rumen.     

"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.     

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.     

Formation of a new cell wall by protoplasts of Candida albicans: effect of papulacandin B, tunicamycin and Nikkomycin

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.     

Chemical composition of the cell wall of the tree fungusCyttaria harioti Fischer

Isolated cell walls of stromata ofCyttaria harioti Fischer, collected in the field, accounted for ca. 67% of the fungus dry weight and were composed of neutral sugars (81%), 2-amino-2-deoxyglucose (0.2%), aminoacids (3.4%), and lipids (6.8%). Alkaline treatment gave a major soluble β-(1 → 3)-glucan and a minor insoluble one, structurally related, which in total accounted for 89% of the cell wall preparation. The absence of chitin is a remarkable feature since it is present in almost all the Ascomycetes previously studied.     

Cell wall composition of the yeast and mycelial forms of Yarrowia lipolytica

The cell walls of the yeast and mycelial forms of Yarrowia lipolytica were isolated and purified. Electron microscopy studies showed no differences between both types of cell walls. Chemical analysis revealed that the yeast cell wall contained 70% neutral carbohydrate, 7% amino sugars, 15% protein, 5% lipids and 0.8% phosphorus. Mycelial cell walls contained 70% carbohydrate, 14% aminosugars, 6% protein, 5% lipids and 0.6% phosphorus. Three polysaccharides: -glucan, mannan and chitin were detected. Proteins were solubilized from both cell wall fractions and separated by polyacrylamide gel electrophoresis. About 50 protein bands were detected, four of them corresponding to glycoproteins. The cell walls of the yeast and mycelial forms of Y. lipolytica were qualitatively similar and only quantitative differences were found.Abbreviations GlcNAc N-acetylglucosamine - FITC-WGA fluorescein isothiocyanate-wheat germ agglutinin - PAS periodic acid Schiff     

Structural mannoproteins released by β-elimination from Candida albicans cell walls

Abstract Mild alkaline solutions (β-elimination), after removing the non-covalently bonded wall materials by hot SDS, released 13% and 26% of remaining wall proteins from mycelial and yeast cells of Candida albicans , respectively. When the β-elimination was carried out after digestion of the walls with chitinase, four-fold more proteinaceous materials were released from mycelium and a similar amount in yeast walls. The solubilized materials were shown to be highly polydisperse, and endo-glycosidase H reduced their polydispersity and molecular masses, revealing different electrophoretic patterns in yeast and mycelial cell walls. The solubilized mycelial proteins carried N-glycosidic sugar chains and the epitopes recognized by two monoclonal antibodies were preserved, although showing a different behaviour in yeast walls. These results are consistent with the idea that significant amounts of intrinsic O-glycosylated mannoproteins are interconnected in the walls of C. albicans .     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisiae

Isolated cell walls of the yeast Saccharomyces cerevisiae were treated by either chemical (alkali and acid) or enzymatic (protease, mannanase or beta-glucuronidase) processes to yield partially purified products. These products were partially characterized by infrared analysis. They were subsequently reacted with heavy metal cation solutions and the quantity of metal accumulated by the cell wall material determined. The Cu(2+) ion (0.24, 0.36, 1.12, and 0.60 mumol/mg) was accumulated to a greater extent than either Co(2+) (0.13, 0.32, 0.43, and 0.32 mumol/mg) or Cd(2+) (0.17, 0.34, 0.39, and 0.32 mumol/mg) by yeast cell walls, glucan, mannan, and chitin, respectively The isolated components each accumulated greater quantities of the cations than the intact cell wall. Removal of the protein component of the yeast cell walls by Pronase caused a 29.5% decrease in metal accumulation by yeast cell walls per mass, indicating the protein is a heavy metal accumulating component. The data indicate that the outer mannan-protein layer of the yeast cell wall is more important than the inner glucan-chitin layer in heavy metal action accumulation. (c) 1994 John Wiley & Sons, Inc.     

Chemical and ultrastructural studies of isolated cell walls of Epidermophyton floccosum: Presence of chitin inferred from X-ray diffraction analysis and electron microscopy

Cell walls of Epidermophyton floccosum were isolated in high purity after mechanical breakage in the Ribi fractionator, followed by sonication and sodium dodecyl sulfate treatment. Major carbohydrate components of cell wall hydrolsates were glucose (35.2%) and glucosamine (30.9%), with lesser amounts of mannose and galactose.After treating isolated cell walls with acid and alkali, the glucosamine polymer was isolated in the form of insoluble residues, and was shown to be compared of chitin fibers by X-ray diffraction analysis and electron microscopy. The surface architecture of isolated cell walls, observed by scanning and shadowing electron microscopy, revealed some remarkable differences in the length and thickness of the fibrils, and also in the orientation of the network, between the internal and external surfaces of the cell wall. A possible involvement of chitin in cell wall integrity is discussed.     

Purification and characterization of a soluble β-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182–191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.     

Wall ultrastructure in regenerating protoplasts ofAspergillus nidulans

Summary The ultrastructure of the wall formed by regenerating protoplasts ofAspergillus nidulans was investigated by electron microscopy using shadowing and freeze-etching techniques. The first evidence of wall formation by the protoplasts was seen in the development of a microfibrillar network composed of chitin. As the protoplasts develop further amorphous material was deposited on the outer surface of the skeletal net.     

Cell Wall Metabolism in Ripening Fruit (IX. Synthesis of Pectic and Hemicellulosic Cell Wall Polymers in the Outer Pericarp of Mature Green Tomatoes (cv XMT-22)

Discs of outer pericarp were excised from mature green tomato (Lycopersicon esculentum Mill.) fruit and kept in sterile tissue culture plates for 4 d, including 2 d of incubation with D-[U-13C]glucose. Cell walls were prepared and the water-soluble, pectic, and hemicellulosic polymers were extracted. Cell wall synthetic capacity was determined by gas chromatography-mass spectrometry analysis of incorporation of the heavy isotope label. The "outer" 2-mm pericarp region, which included the cuticle, had a lower cell wall synthetic capacity than the "inner" 2-mm region immediately below it (closer to the locules), based on the percentage of labeling of the neutral sugars. There were no significant differences in relative abundance of glycosidic linkages in the two tissue regions. Label was incorporated into neutral sugars and linkages typical for each polysaccharide class were identified in the cell wall preparations. Galacturonic acid and glucuronic acid were labeled to an extent similar to that of the neutral sugars in each tissue region.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.