Vincent Girard

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

Abstract The presence of chitin in hyphal cell walls and regenerating protoplast walls of Saprolegnia 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 radioactive N -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 μ M ). The reaction product was insoluble in 1 M KOH or 1 M 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 of Saprolegnia .

pH controls both transcription and post-translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis cinerea.

During pathogenesis, the ascomycete Botrytis cinerea secretes a range of cell-wall-degrading enzymes such as polygalacturonases, glucanases and proteases. We report the identification of a new member of the G1 family of proteases, BcACP1, which is secreted by B. cinerea during infection. The production of BcACP1 correlates with the acidification of the plant tissue, and transcriptional analysis of the Bcacp1 gene showed that it is only expressed under acidic growth conditions. Using a transcriptional reporter system, we showed that pH regulation of Bcacp1 is not mediated by the canonical PacC transcription factor binding site. Like other G1 proteases, BcACP1 is produced as a pro-enzyme. Trapping of the zymogen form allowed investigation of its maturation process. Evidence is presented for an autocatalytic proteolysis of the enzyme that is triggered by acidic pH. Environmental pH therefore controls Bcacp1 production at both the transcriptional and post-translational level.

β-1-4-and β-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5′-diphosphate (UDP) glucose for β-1-4-glucan synthase) and high (millimolar UDP glucose for β-1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm-3. Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm-3). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [3H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.

Comparative genomics, proteomics and transcriptomics give new insight into the exoproteome of the basidiomycete Hebeloma cylindrosporum and its involvement in ectomycorrhizal symbiosis

Extracellular proteins play crucial roles in the interaction between mycorrhizal fungi and their environment. Computational prediction and experimental detection allowed identification of 869 proteins constituting the exoproteome of Hebeloma cylindrosporum. Small secreted proteins (SSPs) and carbohydrate-active enzymes (CAZymes) were the two major classes of extracellular proteins. Twenty-eight per cent of the SSPs were secreted by free-living mycelia and five of the 10 most abundant extracellular proteins were SSPs. By contrast, 63-75% of enzymes involved in nutrient acquisition were secreted. A total of 150 extracellular protein-coding genes were differentially expressed between mycorrhizas and free-living mycelia. SSPs were the most affected. External environmental conditions also affected expression of 199 exoproteome genes in mycorrhizas. SSPs displayed different patterns of regulation in response to presence of a host plant or other environmental signals. Several of the genes most overexpressed in the presence of organic matter encoded oxidoreductases. Hebeloma cylindrosporum has not fully lost its ancestral saprotrophic capacities but rather adapted them not to harm its hosts and to use soil organic nitrogen. The complex and divergent patterns of regulation of SSPs in response to a symbiotic partner and/or organic matter suggest various roles in the biology of mycorrhizal fungi.

Separation and partial peptide characterization of β1-3 glucan synthase from Saprolegnia

Abstract The membrane-localized 1,3-β-glucan synthase (EC 2.4.1.34; UDPglucose: 1,3-β- d -glucan 3-β-glucosyltransferase) from Saprolegnia monoica was greatly enriched by a two-step purification procedure. Starting with a microsomal preparation, the enzyme was solubilized with a cholamidopropyl-dimethyl-ammonio-propanesulfonate (Chaps)/octylglucoside mixture and further purified by ultracentrifugation on a linear glycerol density gradient. The most purified enzyme preparation showed enrichment in 34, 48 and 50 kDa polypeptides. The true involvement of these three bands in the 1,3-β-glucan synthase structure was demonstrated by three experimental procedures. After enzyme purification, entrapment gave selective synthase sedimentation with its reaction product and subsequent SDS-PAGE electrophoresis showed the presence of these three polypeptides. Lectin binding on solubilized preparation provoked a shift in the density gradient of the synthase activity correlating with the distribution of these three bands. Western analysis also showed that the doublet 48, 50 kDa was glycosylated. Finally, the three bands were used to raise polyspecific antibodies which precipitated 1,3-β-glucan synthase activity.

Chitin Synthase Activity from Neocallimastix frontalis, an Anaerobic Rumen Fungus

Chitin synthase activity was detected in actively growing mycelium of Neocallimastix frontalis after mechanical disruption of the cells. Chitin formation in fungal extracts at 32 ° was linear with respect to time for at least 60 min, and with respect to protein concentration up to 750 μg ml-1. The optimum pH for enzyme activity was 8.5 using 10 mm-Tris/HC1 buffer. Mg2+ was necessary for maximum activity and 10 mm-MgC12 was routinely used during the assays. The apparent K m for the substrate UDP-GlcNAc was 2 mm. Polyoxin D was a competitive inhibitor of chitin synthesis with an apparent K i of 4 μm. Following treatment with trypsin (12.5 μg ml-1), the chitin synthase activity of the fungal extract increased by six-fold, indicating that most of the chitin synthase activity was zymogenic. The reaction product was insoluble in 1 m-KOH or 1 m-acetic acid, but it was solubilized by heating in 6 m-HC1 at 120 ° for 2.5 h and was hydrolysed by chitinase into diacetylchitobiose.

A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica

An apparently novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica has been characterized. The enzyme exhibits properties that differ markedly from those of the enzyme previously described [Fevre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca2+, Mg2+ and Mn2+, and appears to be located mainly in the apical part of the hypha. Taking into consideration the differences in pH optimum and effect of divalent ions, each enzyme activity could be assayed in the presence of the other. The insoluble polymeric product of the enzyme with alkaline pH optimum was characterized as a linear 1,3-β-glucan. Comparisons of the general properties of 1,3-β-glucan synthases suggest that enzymes from the oomycetes are more closely related to enzymes from higher plants than to those of true fungi, reflecting the fact that the oomycetes are highly divergent from chitinous fungi.

Synthesis in vitro of crystalline chitin by a solubilized enzyme from the cellulosic fungus Saprolegnia monoica

SUMMARY: Enriched preparations of chitin synthase were obtained from cell homogenates from Saprolegnia monoica. Chitin synthase was solubilized from a mixed membrane fraction by two successive digitonin treatments. Glycerol gradient centrifugation of the solubilized proteins separated the chitin synthase activity from the majority of proteins and from β-1,3 and β-1,4 glucan synthases. The properties of chitin synthase from this Oomycete fungus are similar to those reported for the enzymes of chitinous fungi. The solubilized enzyme catalysed the synthesis in vitro of spindle-like crystals of chitin. Biophysical analysis (by electron and X-ray diffractometry and infrared spectroscopy) demonstrated that the polymer synthesized in vitro was α-chitin. These results and those previously reported demonstrate unambiguously that chitin synthase and chitin are normal components of the cell wall of the cellulosic fungus S. monoica.

β-Glucan synthesis and glucan synthase activities during early stages of cell wall regeneration by protoplasts from Saprolegnia monoica

Protoplasts isolated from Saprolegnia monoica were used to study β-glucan synthases and polysaccharide synthesis during wall regeneration. (1→4)-β- and (1→3)-β-glucan synthase activities of plasma membranes and internal membranes increased during regeneration. Within minutes of cultivation, protoplasts transferred radioactivity from [14C]glucose to cellulose and other cell wall polymers. UDP[14C]glucose did not serve as substrate. Cellulose microfibrils were produced from the beginning of regeneration. The early phases of regeneration are realized by plasma membrane enzymes having remained intact during protoplast isolation and independently of the Golgi apparatus.

Cellulose and β-Glucan Synthesis in Saprolegnia

The biological and physiological properties of fungal cell walls reside in their chemical composition and also in the mode of spatial arrangement of the individual polymers in the wall. The general organization is that skeletal microfibrillar wall components are embedded in an amorphous matrix (Gooday and Trinci 1980). In Saprolegnia monoica, as in other Oomycetes, microcrystalline cellulose is overlayed by an outer layer of glucans containing β(1→3) and β(1→6)linkages (Sietsma 1969; Hunsley and Burnett 1970). During normal hyphal growth, synthesis and deposition of cell wall components must be highly co-ordinated in order to provide and maintain the cell wall architecture. However, the morphogenetic roles of the cell wall polysaccharides are of differential importance. Amorphous glucans have probably little morphogenetic significance. In contrast, microfibrillar polysaccharide synthesis seems to have a primary role. The importance of chitin in cell wall maintenance was explored using chitin deficient mutants of Aspergillus nidulans (Katz and Rosenberger 1971) and by inhibiting chitin synthesis. Polyoxin D, a potent inhibitor of chitin synthase, prevented primary septum formation in yeast (Bowers et al. 1974), inhibited stipe elongation of Coprinus fruit-bodies (Gooday 1972), and induced abnormal hyphal growth of Trichoderma (Benitez et al. 1976). Therefore, microfibrillar chitin synthesis plays a fundamental role in the ontogeny of the mycelial wall. Chitin crystallization and its cross-linking to β-glucan chains would lead to normal hyphal wall extension (Wessels 1984; see also Chapter 6).