Structure of the cell wall proteogalactomannan from Neurospora crassa. I. Purification of the proteoheteroglycan and characterization of alkali-labile oligosaccharides

Proteoheteroglycan (PHG) was prepared from Neurospora crassa cells by extraction with hot water followed by cetyltrimethylammoniumbromide fractionation. The polymer was purified by DEAE-cellulose chromatography followed by gel filtrations. The PHG was fractionated into five subfractions containing carbohydrate (65-88%), protein (19-36%), and a trace amount of phosphate (0.3-1.9%). The sugar compositions of the fractions were similar to each other (D-mannose, 47-60%, D-galactose, 35-50%, D-glucose, 2-5%) while the fractions showed significant heterogeneity in molecular size. Mild alkali treatment of the PHG in the presence of sodium borohydride yielded three kinds of reduced oligosaccharides. Structural studies using a methylation-GC-MS method, and proton and carbon NMR indicated that the tetrasaccharide fragment is beta-D-Galf(1-5)-beta-D-Galf(1-2)-alpha-D-Manp(1-2)man nitol, the trisaccharide is beta-D-Galf(1-2)-alpha-D-Manp(1-2)mannitol, and the disaccharide is alpha-D-Manp(1-2)mannitol.     

Role of cell wall bound calcium in Neurospora crassa

Cell wall bound calcium constitutes a significant fraction (25%) of total mycelia calcium in Neurospora crassa. Wall bound calcium increases as a function of growth and calcium concentration, while cell wall bound calcium decreases in Ca-free medium. Removal of wall bound calcium causes its rapid replacement from intracellular pool, inhibited by verapamil, nifedipine, concanamycin A, and wortmanin in a vacuolar mutant (Vma-5), but is unaffected by trifluoropyrazine, and calmidizoluim in a calcineurin mutant (Cnb-1) of N. crassa. Ca²⁺ removal from surface with EGTA resulted in leakage of periplasmic enzymes invertase and alkaline phosphatase. Scanning and transmission electron microscopy revealed gross abnormalities represented by giant vacuoles. Toxic metal ions bound to wall fraction by displacing calcium. Our data underline the physiological importance of wall bound calcium in N. crassa.     

Structural studies of the polysaccharide part of the cell wall lipopolysaccharide from Bacteroides fragilis NCTC 9343

The structure of the polysaccharide part of the lipopolysaccharide from Bacteroides fragilis NCTC 9343 has been determined using sugar and methylation analysis as the principal tools. Phenol--water extraction followed by a phenol--chloroform--light petroleum extraction yielded a lipopolysaccharide suitable for structural analysis. Analysis of sugars using alditol acetates showed that the polysaccharide contained L-rhamnose, D-galactose and D-glucose in the approximate molar ratios of 1:5:1. After weak acid hydrolysis, two polysaccharide fractions were isolated by gel permeation chromatography: PSI and PSII with the sugar molar ratios 1:5:1 and 1:2:1 respectively. Chromium trioxide oxidation revealed that all galactosyl residues have the beta configuration, and that the rhamnosyl and glucosyl residues have the alpha configuration. From methylation analysis of lipopolysaccharide and the PS I and PS II fractions the following structures could be deduced.     

Genetic control of a structural polymer of the Neurospora crassa cell wall.

The heteropolysaccharide present in fraction 1 of the Neurospora crassa cell wall has been characterized in wild-type and morphological mutant strains of this fungus. Single and double mutations have been studied to determine possible genetic interactions controlling the chemical composition of such heteropolysaccharides . Single mutations studied were peak-2, scumbo ( FGSC 49), ragged ( FGSC 296), and crisp -1 ( FGSC 488). Double mutations studied were peak-2, scumbo ( FGSC 419), and ragged crisp -1. In all these strains, the main constituents of the heteropolysaccharide were glucose, mannose and galactose. Glycosidic linkages binding these neutral sugars have been identified by gas-liquid chromatography. A chemical structure of fraction I heteropolysaccharide is proposed. The results obtained with double mutants suggest the existence of genetic interactions, such as complementation or additive effects of lesions of different genes, to control the chemical composition and structure of the cell wall and the morphology of N. crassa mycelium.     

In vivo chitin-cadmium complexation in cell wall of Neurospora crassa

Fungal cell wall, mainly composed of chitin, an N-acetylglucosamine polymer, is known to participate in heavy metal detoxification. In the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of Neurospora crassa. Based on the results of physical techniques, such as solid-state 13C-NMR, X-ray diffraction, IR and molecular modeling, a structure was proposed for the chitin-cadmium complex. The ring and C-3 hydroxyl oxygens of N-acetylglucosamine were implicated in the complexation of cadmium by the chitin of the fungal cell wall. The studies further revealed that the conformation of chitin did not alter after cadmium complexation.     

Structure of D-prephenyllactate. A carboxycyclohexadienyl metabolite from Neurospora crassa

A novel natural product structurally related to prephenate and arogenate was isolated from a mutant of Neurospora crassa. This D-beta-(1-carboxy-4-hydroxy-2,5-cyclohexadiene-1-yl)-lactic acid is herein given the trivial name of D-prephenyllactate. The new metabolite is even more acid labile than is prephenate and is quantitatively converted to phenyllactate at mildly acidic pH. The structure characterization of prephenyllactate was performed using spectroscopic techniques (ultraviolet, 1H NMR, 13C NMR, two-dimensional heteronuclear experiments and mass spectrometry). Circular dichroism proved conclusively the R configuration of the asymmetric carbon at C-8 of prephenyllactate. Enzymatic utilization of prephenyllactate by cyclohexadienyl dehydratase and by cyclohexadienyl dehydrogenase from Klebsiella pneumoniae was demonstrated.     

Large scale analysis of sequences from Neurospora crassa.

After 50 years of analysing Neurospora crassa genes one by one large scale sequence analysis has increased the number of accessible genes tremendously in the last few years. Being the only filamentous fungus for which a comprehensive genomic sequence database is publicly accessible N. crassa serves as the model for this important group of microorganisms. The MIPS N. crassa database currently holds more than 16 Mb of non-redundant data of the chromosomes II and V analysed by the German Neurospora Genome Project. This represents more than one-third of the genome. Open reading frames (ORFs) have been extracted from the sequence and the deduced proteins have been annotated extensively. They are classified according to matches in sequence databases and attributed to functional categories according to their relatives. While 41% of analysed proteins are related to known proteins, 30% are hypothetical proteins with no match to a database entry. The entire genome is expected to comprise some 13000 protein coding genes, more than twice as many as found in yeasts, and reflects the high potential of filamentous fungi to cope with various environmental conditions.     

Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa.

An inositol deficiency in the inositol-requiring (inl) mutant of Neurospora crassa led to changes in the composition of the inositol-containing lipids and the cell wall. On deficient levels of inositol, phosphatidyl inositol decreased by 23-fold, di(inositolphosphoryl) ceramide decreased by 4-fold, and monoinositolphosphoryl ceramide increased slightly. The inositol deficiency also led to an aberrant hyphal morphology and changes in both the amount of cell wall and the amino sugar content of the cell wall. The glucosamine content of the cell wall decreased by 50%, the galactosamine increased by 50%, but no significant changes were found in the content of the cell wall amino sugar precursors, or in the amino acid, glucose, or total hexose content of the cell wall. Inositol-containing compounds were found associated with purified cell wall material. These compounds were bound tightly to the cell wall but could be removed by treatment with alkali, a treatment which disrupts the cell wall integrity. Possible mechanisms of how changes in lipid composition can affect cell wall biosynthesis are discussed.     

Characterization of the carbohydrate component of fraction I in the Neurospora crassa cell wall.

The carbohydrate portion of fraction I of the Neurospora crassa cell wall has been analyzed for sugar composition by gas-liquid chromatography and colorimetric methods. The analysis was performed comparatively in a wild-type strain (RL 3-8A) and three morphological mutants: scumbo (FGSC 49), peak-2a (a mutant known to be allelic to biscuit), and ragged (FGSC 296). Fraction I of all strains studied contains glucose, mannose, and galactose as the main sugars. Uronic acids and amino sugars are also present in small amounts. The glycosidic linkages binding the neutral sugars were analyzed by Lindberg's combined gas chromatography-mass spectrometry techniques for identification of the partially methylated alditol acitate sugar derivatives. The main polymeric portion of fraction I seems to be a linear glucan with the glucose residues linked by 1 leads to 3 and 1 leads to 4 bonds. A mannan portion with a branched configuration is also present, with galactose as the sugar residue which serves as branches in the molecule(s). The branched mannan portion appears to increase in amount in correlation with more drastic morphological changes of the mycelia. In this respect, the mutant ragged has the lowest mycelial growth rate and the largest amount of mannan. The importance of the polysaccharide structure of fraction I on the colonial morphology of the mycelia is discussed.     

Structure of a cell wall polysaccharide isolated from Hypocrea gelatinosa.

The structure of a polysaccharide isolated from the cell wall of Hypocrea gelatinosa has been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysacharide has an irregular structure, idealized as follows: [carbohydrate structure in text].     

Structural studies of a cell wall polysaccharide of Trichosporon asahii containing antigen II.

The structure of a cell-wall polysaccharide containing antigen II from Trichosporon asahii was investigated. A purified glucuronoxylomannan (GXM) antigen was found to contain O-acetyl groups that contribute to the serological reactivity. The structure of GXM was analyzed by partial acid hydrolysis, methylation analysis, controlled Smith degradation, NMR studies, and fluorophore-assisted carbohydrate electrophoresis. GXM has an alpha-(1-->3)-D-mannan backbone with a beta-D-glucopyranosyluronic acid residue bound to O-2 of a mannopyranosyl residue and the same number of beta-D-xylopyranosyl residues as mannose. Side chains of beta-D-xylopyranosyl-D-xylopyranose, forming a nonreducing terminus, and beta-D-xylopyranosyl residues were attached to O-2, O-4, and O-6 of the mannose residues.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.     

The mitochondrial ribosomes of Neurospora crassa. II. Comparison of the proteins from Neurospora crassa mitochondrial ribosomes with ribosomal proteins from Neurospora cytoplasm, from rat liver mitochondria and from bacteria.

1. It has been shown by Datema et al. (Datema, R., Agsteribbe, E. and Kroon, A.M. (1974) Biochim. Biophys. Acta 335, 386--395) that Neurospora mitochondria isolated in a Mg2+-containing medium (or after homogenization of the mycelium in this medium and subsequent washing of the mitochondria in EDTA-containing medium) possess 80-S ribosomes; mitochondria homogenized and isolated in EDTA medium yield 73-S ribosomes. The ribosomal proteins of the subunits of 80-S and 73-S ribosomes were compared by two-dimensional electrophoresis. The protein patterns of the large, as well as of the small subunits are very similar but not completely identical; the most conspicuous difference is that the large subunit of 80 S contains about eight more proteins than the large subunit of 73 S. 2. The contamination by Neurospora cytoplasmic 77-S ribosomes in the 80-S preparations, if present, is only minor. 3. Neurospora cytoplasmic ribosomes contain 31 proteins in the large, and 21 proteins in the small subunit. 4. Neurospora 80- mitochondrial ribosomes contain 39 proteins in the large, and 30 proteins in the small subunit 30 proteins. 5. Rat liver mitochondrial ribosomes contain 40 proteins in the large and at least 30 proteins in the small subunit. About 50% of these proteins has an isoelectric point below pH 8.6. 6. The pattern of Paracoccus denitrificans is very similar to that of other bacterial ribosomes, the large subunit contains 29, the small subunit 18 proteins.     

Structural gene for ornithine decarboxylase in Neurospora crassa.

To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. Four mutants, PE4, PE7, PE69, and PE85, were isolated. They were able to grow slowly at 25 degrees C on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees C. The mutants did not complement with one another or with ODC-less spe-1 mutants isolated in earlier studies. In all of the mutants isolated to date, the mutations map at the spe-1 locus on linkage group V. Strains carrying mutations PE4, PE7, and PE85 displayed a small amount of residual ODC activity in extracts. None of them had a temperature-sensitive enzyme. The enzyme of the PE85 mutant had a 25-fold higher Km for ornithine (5mM) than did the enzyme of wild-type or the PE4 mutant (ca. 0.2 mM). The enzyme of this mutant was more stable to heat than was the wild-type enzyme. These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25 degrees C and weakly at 35 degrees C with ornithine supplementation. This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. We conclude that spe-1 is the structural gene for the ODC.     

The Structure of Neurospora crassa Glycogen

The mycelia of a wild type strain of Neurospora crassa (6068, IFO) contain a polysaccharide which is stained reddish brown by iodine. The polysaccharide purified by repeated precipitation with ethanol is made up of d-glucose and has a molecular weight of about (more than) 2 × 10⁷, 101 S on ultracentrifugation analysis, an average chain length of 10, β-amylolysis limit of 33.6%, and α-amylolysis limit of 58.3%. The highly branched structure, therefore, resembles to that of a typical glycogen. The properties of the glycogen from N. crassa are discussed in comparison with the commercial glycogens from shellfish and rabbit liver.