A cosmid carrying the orlA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26360 Da) that is homologous to a 211-amino-acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli otsB-encoded polypeptide. TPS2 and otsB each specify a trehalose-6-phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose-6-phosphate and have reduced trehalose-6-phosphatate phosphatase levels, indicating that the gene encodes a trehalose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32 degrees C. When germinated at 42 degrees C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N-acetylglucosamine (GlcNAc). The activity of glutamine:fructose-6-phosphate amido-transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orlA disruption strains. The findings are consistent with the hypothesis that trehalose-6-phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.

A fragment from the open reading frame of the cloned chsA gene from Aspergillus nidulans was deleted and replaced with the argB gene. The resulting construct was used to replace the wild-type chsA gene in an argB deletion strain. The growth and morphology of the vegetative hyphae from the resulting chsA disruptant strain were indistinguishable from those of a wild-type strain but the chitin content of the hyphae from the disruptant was reduced to approximately 90% of that of wild-type. The disruptant showed reduced ability to produce the asexual spores (conidia) that are formed by differentiated aerial hyphae called conidiophores. The ability to form undifferentiated aerial hyphae was not impaired in the disruptant. The conidiophores and conidia produced by the disruptant were indistinguishable from those of wild-type. Conidium formation by the disruptant grown on a variety of media was reduced to about 30% of the wild-type. A chsE null strain did not show a defect in conidiation but a strain in which both chsA and chsE were inactivated produced about 3% of the conidia of wild-type. That finding supports the hypothesis that chsA and chsE encode a partially redundant function necessary for conidiophore development.