Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.     

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.     

On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.     

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.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.     

Isolation and characterization of the rodlet layer of Trichophyton mentagrophytes microconidial wall.

The rodlet layer of the microconidial wall of Trichophyton mentagrophytes was isolated and partially characterized. The purified microconidial walls were first extracted with urea (8M), mercaptoethanol (1%), and sodium dodecyl sulfate (1%) followed by enzymatic digestion with glusulase (snail intestinal enzymes) and purified (1 leads to 3)-beta-D-glucanase and chitinase. The purified rodlet layer was 15 to 30 nm thick and accounted for approximately 10% of the original wall weight. The pattern of rodlet patches, as revealed by electron microscopy of freeze-etched preparations of the isolated layer, was essentially the same as that observed on the intact microconidial wall. The rodlet layer was found to be resistant to most of the common organic solvents, cell wall lytic enzymes, mild acid treatments, and surface-active agents, but was solubilized in boiling 1 N NaOH with concomitant disorientation of the rodlet patterns. A melanin or melanin-like pigment appeared to be intimately associated with this rodlet layer and was solubilized during a hot-alkali treatment. Protein (80 to 85%) and glucomannan (7 to 10%) were the major components of the rodlet layer. The rodlet layer did not contain any appreciable amounts of lipid or phosphorus.     

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.     

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。     

Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia

Chitin samples in a alpha-form structure were isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia by treatment with 1 N HCl and 1 N NaOH. Chitosan was prepared by treating them in 40% NaOH containing NaBH(4). Chitin and chitosan were analyzed by X-ray, [13C]CP/MAS NMR, [13C]FT-NMR, and scanning electron microscopy (SEM) methods. Insect chitin degraded more readily than shrimp chitin when treated with 6 N HCl and the enzyme-chitinase. After treatment with 2 N HCl at 100 degrees C, the insect chitin crystallinity increased. N-deacetylation of insect chitin was easier than that of crustaceous chitin, and about 94% of the N-acetyl groups were removed in one treatment with 40% NaOH for 4 h at 110 degrees C. After treatment with 2 N HCl, 55% of the N-acetyl groups of silkworm chitin were removed under the same conditions. Beetle chitin showed a higher affinity for chitinase than shrimp chitin.     

THE CHEMICAL COMPOSITION OF THE HYPHAL WALLS OF THE FUNGUS ALLOMYCES

Aronson , Jerome M., and Leonard Machlis . (U. California, Berkeley.) The chemical composition of the hyphal walls of the fungus Allomyees. Amer. Jour. Bot. 46(4): 292–300. Illus. 1959.—The hyphal walls of Allomyces macrogynus were isloated by both alkaline digestion methods and by sonic oscillation. Both types of preparations showed the walls to consist of chitin, glucan, and ash. In addition, the mechanically isolated walls contained a protein fraction, the properties and significance of which were not determined. Hemicellulose-type polysaccharides, pectic substances, ether soluble lipids, and constituents giving rise to 3–0-α-earboxyethyl hexosamine were not found to be present in the walls. The walls of plants grown for 60–70 hr. under the prescribed conditions contain approximately 60% chitin, 15% glucan, 10% ash, and 10% protein intimately associated with the walls. The percentage of wall material in a mycelium, as well as the percentage of chitin in the walls, increases with the chronological age of the mycelium. These percentages were not, however, affected by variations in the composition of the nutrient medium. The chitin in the walls could be hydrolyzed in the presence of chitinase; lysozyme, however, had no detectable effect on the walls.     

The effects of temperature, incubation atmosphere, and medium composition on arthrospore formation in the fungus Trichophyton mentagrophytes.

Several growth conditions were found to allow abundant arthrospore formation in T. mentagrophytes. These included growth at 32--37 degrees C on Sabouraud's medium (1% neopeptone, 4% glucose) and growth at temperatures below 32 degrees C solely on neopeptone or other complex peptide sources without the addition of glucose, a supplementary carbon source. Sabouraud's medium did not allow arthropsore formation at 30 degrees C under normal atmospheric conditions. However, if oxygen tension were reduced by partial replacement of air with either N2 or CO2 arthrosporulation did occur on Sabouraud's medium at 30 degrees C. The rate of germ tube elongation was lower under those conditions which supported arthrospore formation, suggesting a correlation between decreased rate of hyphal extension and arthrospore formation. Stimulation of arthrospore formation by sublethal concentrations of several antifungal agents tends to support this hypothesis.     

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.     

The cell wall of Fusarium oxysporum.

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria

Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase‐containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate‐binding modules and the highest number of CBM33‐type lytic polysaccharide monooxygenases. A ctinobacteria do also have the highest fraction of genomes containing β‐1, 3‐glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase‐containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell‐wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.     

Spore differentiation in a clinical strain of Trichophyton mentagrophytes

Spore differentiation and, in particular, arthrosporogenesis in a clinical strain of T. mentagrophytes was investigated using a variety of methods and by altering environmental conditions. Results are discussed with reference to the in vivo situation. Arthrospores were obtained in the presence of increased CO2 tension but not increased N2 tension. High humidity was necessary for arthrospore formation but maturity (i.e. crops of single spores) was associated with conditions of reduced humidity. Desiccation reduced arthrospore viability. Glucose and peptone based media were suitable for arthrospore formation. Arthrospores were produced at 30 degrees C and 37 degrees C, but 30 degrees C is preferred since chlamydospores were prevalent at 37 degrees C. Conditions for production of arthrospore, microconidial and mycelial suspensions are presented.     

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.     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.