The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development.

The chsB gene from Aspergillus nidulans encodes a class III chitin synthase, an enzyme class found in filamentous fungi but not in yeast-like organisms. Using a novel method, we isolated haploid segregants carrying a disrupted chsB allele from heterozygous diploid disruptants. The haploid disruptants grow as minute colonies that do not conidiate. Hyphae from the disruptants have enlarged tips, a high degree of branching, and disorganized lateral walls. The mycelium is not deficient in chitin content and shows no evidence of lysis. The disruptant phenotype is not remedied by osmotic stabilizers. The results indicate that chitin synthesized by the chsB-encoded enzyme does not substantially contribute to the rigidity of the cell wall but is necessary for normal hyphal growth and organization. The properties of the A. nidulans disruptant are similar to those for Neurospora crassa strains with a disrupted chs-1 gene, which also encodes a class III chitin synthase. The morphology of an A. nidulans heterokaryon containing both the wild-type and the disrupted chsB alleles indicates that chsB acts in local areas of the mycelium. The heterokaryon produces conidia of both parental genotypes in nearly equal numbers, indicating that the wild-type chsB gene is not necessary for conidium formation. In addition, we identified and sequenced a second, previously undescribed, homolog of chsB from the closely related opportunistic pathogen, A. fumigatus. The finding of two class III chitin synthase genes in A. fumigatus and a single gene of this class in A. nidulans illustrates limitations of using A. nidulans as a genetic model for A. fumigatus.

The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis.

Two chitin synthase genes, chsD and chsE, were identified from the filamentous ascomycete Aspergillus nidulans. In a region that is conserved among chitin synthases, the deduced amino acid sequences of chsD and chsE have greater sequence identity to the polypeptides encoded by the Saccharomyces cerevisiae CHS3 gene (also named CSD2, CAL1, DIT101, and KTI1) and the Candida albicans CHSE gene than to other chitin synthases. chsE is more closely related to the CHS3 genes, and this group constitutes the class IV chitin synthases. chsD differs sufficiently from the other classes of fungal chitin synthase genes to constitute a new class, class V. Each of the wild-type A. nidulans genes was replaced by a copy that had a substantial fraction of its coding region replaced by the A. nidulans argB gene. Hyphae from both chsD and chsE disruptants contain about 60-70% of the chitin content of wild-type hyphae. The morphology and development of chsE disruptants are indistinguishable from those of wild type. Nearly all of the conidia of chsD disruption strains swell excessively and lyse when germinated in low osmotic strength medium. Conidia that do not lyse produce hyphae that initially have normal morphology but subsequently lyse at subapical locations and show ballooned walls along their length. The lysis of germinating conidia and hyphae of chsD disruptants is prevented by the presence of osmotic stabilizers in the medium. Conidiophore vesicles from chsD disruption strains frequently swell excessively and lyse, resulting in colonies that show reduced conidiation. These properties indicate that chitin synthesized by the chsD-encoded isozyme contributes to the rigidity of the walls of germinating conidia, of the subapical region of hyphae, and of conidiophore vesicles, but is not necessary for normal morphology of these cells. The phenotypes of chsD and chsE disruptants indicate that the chitin synthesized by each isozyme serves a distinct function. The propensity of a chsD disruptant for osmotically induced lysis was compared to that of strains carrying two other mutations (tsE6 and orlA::trpC) which also result in reduced chitin content vegetative cell lysis. The concentration of osmotic stabilizer necessary to remedy the lysis of strains carrying the three mutations is inversely related to the chitin content of each strain. This finding directly demonstrates the importance of chitin to the integrity of the cell wall and indicates that agents that inhibit the chsD-encoded chitin synthase could be useful anti-Aspergillus drugs.