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.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The mycelium from a surface culture of the fungusAspergillus niger was mechanically destroyed in a mixer. By this action three distinct particle types arose: isolated hyphae, hyphae agglomerates and granules of cytoplasm. The hyphae agglomerates were removed by fractional centrifugation, the hyphae were separated from the granules by repeated filtration over a sieve with openings 0.063 mm. The isolated hyphae were then deprived of the cytoplasm by the action of pancreatin. Thus obtained cell walls preserved the cell shape and were free of cell contents. The chemical composition of the walls exhibits a dispersion after the mycelium origin. The walls give a positive reaction on chitin, and contain 55 to 85% of the total carbohydrate contents (cellulose 2.5 to 5.5%), 1.5 to 2.1% of total N₂ (0.8 to 0.9% of the amino-N₂) and 8% of the lipides. After an acid hydrolysis, the presence of 45 to 59% of glucose, 9.5 to 14.5% of glucosamine, 1.5 to 6% of galactose, and small amounts of arabinose and mannose were determined. By qualitative chromatography, several aminoacids were ascertained.     

Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress

In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.     

Colonial growth and ramihyphin A — Induced changes in cell walls ofNeurospora sitophila

Colonial growth ofNeurospora sitophila phenotypically induced by ramihyphin A is accompanied by marked changes in the contents of DNA, RNA and proteins in the mycelium, and in the relative proportion of hexoses in cell wall hydrolysates. The glucosamine/glucose ratio is also characteristic for colonial growth. X-ray analysis of cell walls showed that ramihyphin A suppresses the crystalline arrangement of chitin in cell walls. A combination of microbiological, biochemical and physico-chemical methods yielded a general picture of the changes accompanying the colonial growth ofNeurospora sitophila.     

Carbohydrates from the hyphal walls of some Oomycetes

Chemically extracted walls of seven species of Oomycetes were found to contain glucosamine in small amounts, possibly originating from chitin. The species belonging to the order Peronosporales seem to contain less glucosamine than the Saprolegniales.     

Improved Colorimetric Determination of Cell Wall Chitin in Wood Decay Fungi

The Svennerholm modification of the Elson-Morgan method for glucosamine analysis was evaluated for its applicability to the rapid determination of chitin in wood decay fungi. The evaluation included extent of chromogen interference, sensitivity, color stability, and hydrolysis conditions for maximum release of glucosamine from fungal cell walls. With our further modification, the Svennerholm method was shown to be suitable for rapid quantitative determination of fungal chitin without chromatographic separation of hydrolysate chromogens.     

Screening microorganisms for chitin hydrolysis and production of ethanol from amino sugars

Seventy-two strains of bacteria representing 39 genera and one yeast (Candida albicans) were screened for ability to hydrolyze chitin. Chitin hydrolysis was determined by a clear zone surrounding colonies growing on the surface of chitin agar. Species with the largest clear zone to colony size (CZ/CS) ratio were further compared for chitinolysis by assaying the level of reducing sugar produced in broth culture. Three yeasts and one bacterial strain known to produce ethanol from glucose were compared for their abilities to produce ethanol from amino sugars. Of the 72 strains screened, 23 produced CZ/CS ratios ranging from 0·38 to 2·5. The highest ratios were observed for strains in the genera: Bacillus and Serratia, followed by Micrococcus, Aeromonas, Vibrio, Clostridium and Plesiomonas. The other species examined produced ratios of less than 1 or were unable to hydrolyze chitin.Hansenula anomala, Pachysolen tannophilus, Saccharomyces cerevisiae, and Zymomonas mobilis were compared for their abilities to grow on and produce ethanol from glucose, glucosamine, and N-acetylglucosamine (NAG). Saccharomyces cerevisiae and H. anomala produced ethanol only from glucose. Pachysolen tannophilus and Z. mobilis produced ethanol from glucose, glucosamine and NAG. The highest concentration of ethanol produced from amino sugar was 598 μg ml⁻¹ from 10 mg ml⁻¹ glucosamine by Z. mobilis. This level was achieved only when yeast extract was included in the medium. Saccharomyces cerevisiae did not grow on glucosamine and Z. mobilis did not grow well on NAG.     

Composition chimique de la paroi d'une levure du genre Torulopsis apres culture sur methanol et ethanol

The nature of the carbon source, methanol or ethanol, influences the cell wall composition of the same strain of yeast. Chemical treatment of the cell walls with ethylenediamine indicates that the parietal architecture varies. The analysis of amino acids, sugars and hexosamines reveals quantitative differences with a modification of the elaboration of the polymers. The methanol source favours the synthesis of chitin and mannan polymers, while the ethanol source favours the elaboration of glucan and glucomannan polymers. The chitin percentage rises in a ratio from 1 to 5 and simultaneously the protein percentage rises from 1 to 1.7; consequently the culture on methanol favours the formation of nitrogen compounds.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Lysis of cell walls ofMucor ramannianus möller by aStreptomyces sp.

A study has been made of some chemical and ultrastructural changes that occur in the hyphal, arthrospore and sporangiospore walls ofMucor ramannianus during lysis by a soil streptomycete.Arthrospore and hyphal walls, which were shown to contain chitin, chitosan, other polysaccharides and phosphate (principally as polyphosphate), were lysed by culture fluid of the streptomycete after this organism had been grown on the same material. Alcohol-insoluble material found in the supernatants of the incubation mixtures gave on hydrolysis glucosamine, galactose, mannose and fucose. No laminarinase activity was detected in these culture fluids. Culture fluids of the streptomycete after growth on chitin and chitosan were also found to lyse the walls of arthrospores and hyphae.Despite the chemical similarities the walls were very different in thin section.A major component in the sporangiospore walls was glucan and an active laminarinase was shown to be present in the culture fluids of the streptomycete after growth on them. Further, ultrathin sections showed that an inner fibrillar layer of the sporangiospore wall was lysed leaving an outer electron-dense layer.     

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K_d of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.     

A microapparatus for liquid hydrogen fluoride solvolysis: Sugar and amino sugar composition of Erysiphe graminis and Triticum aestivum cell walls

The assembly and use of a simple and safe apparatus for HF solvolysis of microgram amounts of cell walls, polysaccharides, or glycoproteins are described. Using this apparatus the cell wall composition of Erysiphe graminis was compared with that of its wheat host. The HF solvolysis combined with TFA posthydrolysis considerably increased sugar yields compared with TFA hydrolysis alone, due mainly to increased yields of glucose from wheat, and glucosamine from Erysiphe, corresponding to cellulose and chitin, respectively. A potentially useful method for determining amounts of fungal hyphae in plant tissue is also provided.     

Comparative Study of the Cell Walls of the Yeastlike and Mycelial Phases of Histoplasma capsulatum

Cell walls were prepared from the yeastlike and mycelial phases (YP and MP) of Histoplasma capsulatum and from Saccharomyces cerevisiae by mechanical disruption and washing. Lipids were extracted with methanol-ether, chloroform, and acidified methanol:ether; a final extraction was made with ethylenediamine. The lipid contents of H. capsulatum YP and MP walls were about the same. Qualitative and quantitative analyses were made of the products obtained from treatment of the cell walls, or fractions from them, with weak acid or with enzymatic preparations containing glucanase and chitinase activities. YP walls contained much larger quantities of chitin and smaller quantities of mannose and amino acids than the MP walls. H. capsulatum MP was shown to resemble S. cerevisiae by low chitin content and by the presence of a mannose polymer, soluble in ethylenediamine and water. H. capsulatum MP chitin appeared to be intimately associated with glucose in the wall, since enzymatic hydrolysis of the residue after mild acid hydrolysis of cell walls or fractions from them resulted in the release of glucose and acetylglucosamine; only acetylglucosamine was released from YP walls with such treatment. By electron microscopic observations, the unextracted MP cell walls were much thinner than the YP, and neither wall appeared laminated.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Estimation of mycelial growth of basidiomycetes by means of chitin determination

After hydrolysis of chitin in 6 M HCl, the glucosamine produced was assayed colorimetrically. The pH of the hydrolysate was adjusted to a value close to three by addition of Na acetate; this procedure avoids the elimination of excess acid by evaporation under reduced pressure or freeze-drying. Under these conditions, the amount of glucosamine determined by the assay represented an average of 90% of the amount which would result from a total hydrolysis of the chitin. The method was used to assay the chitin in the mycelia of basidomycetes obtained in vitro. The measured amount of glucosamine was proportional to the mycelial biomass and allowed the estimation of fungal growth.     

Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin

Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched β-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice.     

Fluorescence and infrared spectrometric study of cell walls from Candida, kluyveromyces, Rhodotorula and schizosaccharomyces yeasts in relation with their chemical composition

Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls. A small amount of a beta(1-->4) linear homopolymer of GlcNAc in the cell wall forms chitin. To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied. Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions. The results were compared to chemical assays. IR spectra allow identifying the structural types of polysaccharides in yeast walls. Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods. The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.     

On the cell wall composition ofSaccharomycopsis guttulata

Summary Cells ofSaccharomycopsis guttulata were ruptured by sonic oscillation and the resulting cell walls were purified by washing and centrifugation. The walls contained 43.7% carbohydrate (expressed as glucose), 39.6% protein and a trace of chitin. Paper chromatography of hydrolyzed cell walls showed that glucose and an unknown reducing compound make up the bulk of the carbohydrate fraction. Mannose and glucosamine were present in small amounts. The cell wall composition ofS. guttulata appears to differ considerably from that ofS. cerevisiae.     

Changes in glucosamine and galactosamine levels during conidial germination in Neurospora crassa.

The levels of glucosamine and galactosamine were determined in conidia, germinating conidia, and vegetative mycelia of Neurospora crassa. In the vegetative mycelia about 90% of the amino sugars were shown to be components of the cell wall. The remaining 10% of the amino sugars were tentatively identified as the nucleotide sugars uridine diphospho-2-acetamido-2-deoxy-D-glucose and uridine diphospho-2-acetamido-2-deoxy-D-galactose. Conidia and vegetative mycelia contained about the same levels of glucosamine. During the first 9 h after the initiation of germination, the total glucosamine content had increased 3.1-fold, whereas the residual dry weight of the culture had increased 7.7-fold. This led to a drop in the glucosamine concentration from 100 mumol/g of residual dry weight to 42 mumol/g. During this time, all of the conidia had germinated and the surface area of the new germ tubes had increased to 10 times that of the conidia. Either germ tubes were initially produced without glucosamine-containing polymers, or these polymers (probably chitin) were deposited only at low densities in the germ tube cell walls. The chitin precursor uridine diphospho-2-acetamido-2-deoxy-D-glucose was present at all times during conidial germination. Conida contained very low levels of galactosamine. During germination, galactosamine could not be detected until the culture had reached a cell density of about 0.6 mg of residual dry weight per ml of growth medium. This was observed regardless of the time required to reach this cell density or the fold increase in dry weight. The accumulation of galactosamine-containing polymers does not appear to be necessary for germ tube formation. The levels of soluble galactosamine (uridine diphospho-2-actamido-2-deoxy-D-galatose) were very low in conidia and increased during germination at the same time that galactosamine appeared in the cellular polymers. In addition, under certain culture conditions, the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously.