A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

A RELATIONSHIP BETWEEN CELL WALL STRUCTURE AND COLONIAL GROWTH IN NEUROSPORA CRASSA

de Terra, Noël, and E. L. Tatum. (Rockefeller Inst., New York, N. Y.) A relationship between cell wall structure and colonial growth in Neurospora crassa . Amer.Jour. Bot. 50(7): 669–677. Illus. 1963.—Cell walls were isolated from morphological mutants of Neurospora crassa and from their corresponding wild-type strains. Acid hydrolysates prepared from these cell walls were then studied, qualitatively and quantitatively, with respect to their reducing sugar content. Paper chromatography revealed the presence of glucose and glucosamine in the cell walls of all strains studied. Quantitative analysis has shown that a group of 4 colonial mutants which strongly resemble each other in morphology all have significantly less glucose and more glucosamine per unit weight of cell wall than do their corresponding wild-type strains. These data strongly suggest that a particular type of morphological aberration in Neurospora is associated with similar changes in cell wall composition.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.     

A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation

AIM: The polysaccharide composition of the Saccharomyces cerevisiae cell wall was measured under various growth conditions and was compared with the cell wall structure. METHODS AND RESULTS: Chemical and enzymatic methods were used to determine levels of beta-1,3-glucan and 1,6-glucan, mannan and chitin of the yeast cell wall, whereas the structure/resistance of the wall was qualitatively assessed by the sensibility to the lytic action by zymolyase. It was found that the dry mass and polysaccharides content of the cell wall could vary by more than 50% with the nature of the carbon source, nitrogen limitation, pH, temperature and aeration, and with the mode of cell cultivation (shake flasks vs controlled fermentors). While no obvious correlation could be found between beta-glucan or mannan levels and the susceptibility of whole yeast cells to zymolyase, increase of beta-1,6-glucan levels, albeit modest with respect to the growth conditions investigated, and to a lesser extent that of chitin, was associated with decreased sensitivity of yeast cells to the lytic action by zymolyase. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that the cell wall structure is merely determined by cross-linking between cell wall polymers, pointed out the role of beta-1,6-glucan in this process. Hence, this study reinforces the idea that enzymes involved in these cross-linking reactions are potential targets for antifungal drugs.     

Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.     

KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties

The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.     

Correlation Between Reduced Nicotinamide Adenine Dinucleotide Phosphate Levels and Morphological Changes in Neurospora crassa

A colonial mutant of Neurospora crassa, previously shown to be altered in the structure of glucose-6-P dehydrogenase [a reduced nicotinamide adenine dinucleotide phosphate (NADPH) producing reaction], contained only 40% as much NADPH in extracts as did the wild type. A partial revertant strain, when grown at 23 C, had the same total NADPH content as the wild type, but, at 34 C, had lower levels of NADPH as well as a colonial morphology. A revertant with complete wild-type morphology had wild-type levels of NADPH. Two different colonial mutants, which have also been reported to be altered in NADPH-generating reactions, were found to have a lower content of NADPH, whereas other colonial mutants had wild-type levels. The wild-type strain, when grown under conditions in which it contained a lower total content of NADPH, had a morphology similar to that of a colonial mutant. The evidence indicates that lowered NADPH content leads to a dramatic alteration in the morphology of Neurospora, but not necessarily vice versa. The possible pleiotropic effects of the NADPH deficiency are discussed.     

Analysis of the beta-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes beta-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of beta-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular beta-1,3-glucanases upon induction with laminarin, a soluble beta-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible beta-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-beta-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Yeast glucan in the cyst wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.     

Photodynamic induction of a bacterial cell surface polypeptide.

A mutation in Aspergillus nidulans led to a loss of both melanin and alpha-(1,3)-glucan, a major wall polysaccharide. In addition, the mutation prevented the formation of cleistothecia. Mutant walls contained increased amounts of beta-(1,3)-glucan and galactose polymers, and electron micrographs indicated that they had lost the outermost wall layer. Such walls were more readily digested by lytic enzymes, and this increased susceptibility to hydrolysis was due to the absence of alpha-(1,3)-glucan and not of melanin. The pleiotropic effects of the mutation are discussed, with particular reference to the hypothesis that alpha-(1,3)-glucan acts as the endogenous carbon source for biosynthetic processes in the stationary phase of growth. In this view, glucan synthesis would be the primary target of the mutation, and the absence of glucan would result in the lack of melanin and cleistothecia, formed after nutrients are exhausted. Two other mutations that lowered themycelial alpha-(1,3)-glucan content also inhibited melanin and cleistothecia production.     

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.     

Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan.

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.     

THE RELATIONSHIP OF M-INOSITOL TO MORPHOLOGY IN NEUROSPORA CRASSA

Shatkin , A. J., and E. L. Tatum . (The Rockefeller Institute, New York 21, New York.) The relationship of m-inositol to morphology in Neurospora crassa. Amer. Jour. Bot. 48(9): 760–771. Illus. 1961.—The role of m-inositol and its relationship to morphology in Neurospora crassa have been examined. The growth pattern of the inositolless mutant has been found to be a function of concentration of carbon and nitrogen sources and m-inositol, utilizability of carbon source, ratio of concentrations of sugar to m-inositol, and size of conidial inoculum. Inhibition of mycelial m-inositol uptake has been demonstrated for a number of carbon sources, and the ability of a particular compound to inhibit m-inositol uptake found to be directly related to both its effectiveness as an energy source and its effect on the morphology of the inositol-requirer. Results of cell-fractionation experiments and autoradiography have shown that phospholipid inositol is widely distributed in the hyphae and not bound in a single subcellular organelle. Electron micrographs of cell fractions indicate that inositol is a structural constituent of N. crassa lipoprotein membranes, including plasmalemma, nuclear envelope, mitchondrial membranes, and endoplasmic reticulum. Normal inositol-requiring and wild-type hyphae are similar in ultrastructure. However, sub-optimally cultured, colonial, inositolless hyphae contain large lipid droplets, and the cellular membranes are in various stages of degeneration. Chemical evidence indicates that colonial hyphae do not synthesize more fat than normal hyphae, suggesting that the lipid droplets are products of membrane catabolism. It is proposed that the colonial morphology of the inositol-requiring mutant of N. crassa is due to a metabolic imbalance between membrane synthesis and the synthesis of other cellular constituents.     

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans.

Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific beta-1,6-glucan antiserum but not with a beta-1,3-glucan antiserum. They lost their beta-1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that beta-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure beta-1,3-glucanase, several mannoproteins that were recognized by both the beta-1,6-glucan antiserum and the beta-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that beta-1,3-glucan and beta-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of beta-glucans in the retention of cell wall proteins is discussed.     

Structural and functional definition of the human chitinase chitin-binding domain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.     

beta-1,6-Glucan synthesis in Saccharomyces cerevisiae

beta-1,6-Glucan is an essential fungal-specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to beta-1,3-glucan and chitin. Genetic approaches have identified genes that upon disruption result in beta-1,6-glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of beta-1,3-glucan and chitin synthesis, it is likely that the bulk of beta-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.     

Mutagen Formation in the Nitrite—Piperic Acid Reaction

A morphological mutant of Neurospora crassa, which showed great changes in cell wall β-glucan structures, was obtained. The mutant lacked spore-forming ability. Chemical analysis indicated that the mutant cell walls had more carbohydrates and less proteins than the wild type. In the structural polymers of cell walls, heteroglycan and chitin were not apparently changed in their sugar composition and structures. On the other hand, the alkali-soluble β-glucan of this mutant showed significant changes in the chemical structure, particularly, the number and length of branches. The mutant glucan had about 2.5 times as many branches as that from wild type and the number of 1,3-linked glucose residues was greatly reduced.     

Phagocytosis by human neutrophils is stimulated by a unique fungal cell wall component

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.     

(1,3)-β-D-Glucan Synthase Activity in Mycelial and Cell Wall-less Phenotypes of the fz,sg, os-1 ("Slime") Mutant Strain of Neurospora crassa

Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa. Experimental Mycology 19, 35-47. The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-βD-glucan synthase activity. fz, sg, os-1 segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, K_m app, V_max, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.