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This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of l-arabinose. Expression of the three known l-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of l-arabinose or l-arabitol. In addition, the intracellular activities of l-arabitol dehydrogenase and l-arabinose reductase, involved in l-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding d-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. l-Arabitol, most likely the true inducer of the arabinanolytic and l-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the l-arabinose catabolic pathway.

The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the ΔpkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus nidulans conidia and rapidly degraded upon induction of conidial germination. Furthermore, trehalose is accumulated in response to a heat shock or to an oxidative shock. The authors have characterized the A. nidulans tpsA gene encoding trehalose-6-phosphate synthase, which catalyses the first step in trehalose biosynthesis. Expression of tpsA in a Saccharomyces cerevisiae tps1 mutant revealed that the tpsA gene product is a functional equivalent of the yeast Tps1 trehalose-6-phosphate synthase. The A. nidulans tpsA-null mutant does not produce trehalose during conidiation or in response to various stress conditions. While germlings of the tpsA mutant show an increased sensitivity to moderate stress conditions (growth at 45 °C or in the presence of 2 mM H2O2), they display a response to severe stress (60 min at 50 °C or in the presence of 100 mM H2O2) similar to that of wild-type germlings. Furthermore, conidia of the tpsA mutant show a rapid loss of viability upon storage. These results are consistent with a role of trehalose in the acquisition of stress tolerance. Inactivation of the tpsA gene also results in increased steady-state levels of sugar phosphates but does not prevent growth on rapidly metabolizable carbon sources (glucose, fructose) as seen in Saccharomyces cerevisiae. This suggests that trehalose 6-phosphate is a physiological inhibitor of hexokinase but that this control is not essential for proper glycolytic flux in A. nidulans. Interestingly, tpsA transcription is not induced in response to heat shock or during conidiation, indicating that trehalose accumulation is probably due to a post-translational activation process of the trehalose 6-phosphate synthase.

The genepkaCencoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungusAspergillus niger.A probe for screeningA. nigerphage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) fromA. nigerhas a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that - except for the first 15 amino acids, which are homologous to theMagnaporthe griseaPKA-C - shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A.nigershows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any otherpkaChomologue inA. niger.The clonedpkaCwas used for transformation ofA. niger,leading to increased levels ofpkaCmRNA and PKA-C activity. Transformants overexpressingpkaCwere phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.