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. Glucoamlyase 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.

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Covalent linkages between chitin and P-glucan in the wall of Schizophyllum commune were indicated by the markedly changed solubility characteristics of the glucan when chitin was specifically removed either (i) by enzymic digestion with purified chitinase or (ii) by first deacetylating the chitin with alkali followed by depolymerization of the deacetylated chitin with nitrous acid. After depolymerization of the chitin, two types of P-glucans could be isolated: one was water-soluble and highly branched, the other was alkali-soluble with branches only one glucose unit long. Lysine (50%) and citrulline (20%) were the major amino acids in the R-glucan/chitin complex. By digesting 90% of the P-glucan in the R-glucan/chitin complex with (1+3)-/?-glucanase, a residue was obtained which, on hydrolysis with chitinase, yielded N-acetylglucosamine and a compound containing (N-acety1)-glucosamine, lysine and/or citrulline. A model is proposed for the R-glucan/chitin complex in which amino acids, especially lysine and citrulline, are involved in the linkages between glucan and chitin. I N T R O D U C T I O NThe alkali-insoluble portion of the hyphal wall of most fungi, except Phycomycetes, appears to contain chitin microfibrils embedded in a matrix of R-glucan, which mainly consists of (1+3)-and (1+6)-linked P-glucan (for a review, see Rosenberger, 1976). The results of a detailed study of wall architecture in the basidiomycete Schizophyllum commune were consistent with this view (van der Valk et al., 1977) but the results of a chemical analysis of the structure of R-glucan did not explain the extreme insolubility of the glucan . Therefore, covalent linkages between the highly insoluble chitin and R-glucan were proposed. The present study was aimed at obtaining more evidence for the existence of such linkages. The results indicate that a large proportion of the glucosamine residues in chitin are linked to P-glucan via amino acids, particularly lysine and citrulline. M E T H O D SPreparation of the R-glucanlchitin complex. This was isolated as the alkali-resistant residue from the walls of Schizophyllum commune strain 1-40 ( = 699), as described previously . R-glucan/ chitin labelled with 14C was isolated from mycelium cultivated in minimal medium supplemented with 0.1 mCi [U-14C]glucose per 100 ml medium.Isolation and purification of enzymes. Chitinase was produced by cultivating a strain of Streptomyces satsumaensis in a medium with colloidal chitin as a carbon source (containing, per litre, 5 g chitin, 0.5 g yeast extract, 2 g KH2P04, 0-5 g KCl, 1 g MgS04, 1 g peptone, 0.5 g NaCl and 1 g NaNO,; pH 6.5) for 5 d at 25 "C on a rotary shaker. The enzyme was isolated from the culture filtrate by adding 2 vol. acetone at -30 "C. The precipitate was suspended in water and dialysed against 0.005 M-acetate buffer pH 5 -8 for 24 h. After centrifugation to remove insoluble material, the supernatant was freeze-dried yielding the crude chitinase preparation. The chitinase was purified by adsorbing it twice on colloidal chitin as descri...

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.

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