Cellular distribution of COT1 kinase in Neurospora crassa.

The Neurospora crassa cot-1 gene encodes a Ser/Thr protein kinase, which is involved in hyphal elongation. Many vacuoles, abnormally shaped mitochondria, and nuclei, along with differences in the structure of the cell wall and hyphal septa, were observed in hyphae of the cot-1 mutant shortly after a shift to the restrictive temperature. Immunolocalization experiments indicated that COT1 was associated with the cytoplasmic membrane; COT1 was also detected in the cytoplasm. The membrane-associated COT1 was absent from the cot-1 mutant when shifted to the restrictive temperature, as was a lower molecular weight isoform of COT1. We propose that COT1 may be involved in several cellular processes, and the spatial and temporal regulation of COT1 activity involves trafficking of the kinase within the fungal cell and its possible interaction with additional proteins.

Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune.

Disruption of the SC3 hydrophobin gene of Schizophyllum commune (DeltaSC3 strain) affected the composition of the cell wall. Compared to a wild-type strain the amount of mucilage (i.e., water-soluble (1-3)beta-glucan with single glucose residues attached by (1-6)beta-linkages) increased considerably, while the amount of alkali-resistant glucan (linked to chitin) decreased. Reintroduction of the SC3 gene or other hydrophobins genes expressed behind the SC3 promotor restored wild-type cell wall composition. However, addition of purified SC3 protein to the medium or growing the DeltaSC3 strain in spent medium of the wild-type strain had no effect. In young cultures of wild-type strains of S.commune, not yet expressing SC3, the amount of mucilage was also relatively high. These data show that hydrophobins not only function at hydrophilic/hydrophobic interfaces, as shown previously, but also affect wall composition.

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.

The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa.

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

The linkage of (1-3)-beta-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae.

Pulse-chase experiments with [14C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1-3)-beta-glucan serves as a precursor for alkali-insoluble (1-3)-beta-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan-chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.

Chemical and immunological analysis of the Aspergillus fumigatus cell wall.

Hyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1-->3)-alpha-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6.2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-beta-D-galactofuranosidase. The alkali-insoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1-->3)-beta-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin.

Localization of growth and secretion of proteins in Aspergillus niger.

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.

The occurrence of glucosaminoglycan in the wall of Schizosaccharomyces pombe.

The major part of the wall of Schizosaccharomyces pombe consists of (1----3)-alpha-glucan and (1----3)-beta-glucan with some (1----6)-beta-linkages. Although in hydrolysed samples only a minute amount of glucosamine could be detected, this amino sugar may play an essential role as an integral part of a glucosaminoglycan/glucan complex. Treatment of the wall with either nitrous acid or chitinase changed the solubility properties of the beta-glucan, which suggests that the glucosaminoglycan/glucan complex is essentially similar to that found in walls of other fungi. An enzyme with properties similar to that of chitin synthase of other fungi, and probably responsible for the synthesis of the glucosaminoglycan, was detected in a mixed-membrane fraction.

Introduction of hygromycin B resistance into Schizophyllum commune: preferential methylation of donor DNA.

Cotransformation of a trp1 strain of Schizophyllum commune with the homologous TRP gene and the Escherichia coli HPT gene was used to study the feasibility of transformation of S. commune to hygromycin B resistance. Southern blot analysis showed that 75% of the TRP transformants contained multiple integrated copies of the HPT gene. However only 7% of the transformants were resistant to 25 micrograms/ml hygromycin B and direct selection for hygromycin B resistance was hampered by the high incidence of spontaneously arising resistant colonies. Rescue of the HPT gene was possible with E. coli JA221 (mcr-) but not with JM83, suggesting methylation of the integrated donor DNA. Isoschizomer analyses confirmed heavy methylation in the HPT gene and flanking vector sequences but not in the homologous donor TRP gene and its flanking vector sequences. Also cotransforming S. commune Sc4 gene and flanking vector sequences were not methylated. A fusion between the S. commune TRP1 and the E. coli HPT genes resulted in only slight or no methylation of both vector and HPT sequences and in a higher hygromycin B resistance level. This suggests that transformation with DNA exclusively containing foreign sequences results in integration into regions where methylation occurs, possibly entailing poor transcription. Methylation of the HPT gene was also indicated by the stimulation of growth by 5-azacytidine of transformants on hygromycin B containing medium.

Solubility of (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan in fungal walls: importance of presumed linkage between glucan and chitin.

In Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans and Coprinus cinereus most of the alkali-insoluble (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan of the wall can be extracted with dimethyl sulphoxide. The same fraction, and in Saccharomyces cerevisiae a small additional fraction, can be extracted by a destructive procedure involving 40% NaOH at 100 degrees C. The small fraction of the glucan which resists this treatment becomes soluble after a subsequent treatment with HNO2 indicating that it is covalently linked to chitin in the wall. In contrast, in Schizophyllum commune and Agaricus bisporus, nearly all the (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan appears to be held insoluble by linkage to chitin.

Chemical analysis of the hyphal wall of Schizophyllum commune.

1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.

Ultrastructural aspects of wall regeneration by Pythium protoplasts.

Electron microscope studies were made of wall regeneration by Pythium protoplasts. Wall regeneration began with the formation of a loose network of fibrils on the surface of the protoplast followed by increase in density of the fibrillar mesh and deposition of granular mitrix material. The majority of the protoplasts did not develop beyond the loose fibrillar network stage, however a small percentage were able to complete wall formation and to form hyphal tubes. A clear zone of demarcation was visible between the fibrillar surface of the protoplast and the smooth surface at the base of the developing hyphal tube.