Nitrogen catabolic gene expression in Saccharomyces cerevisiae has been reported to be regulated by three GATA family proteins, the positive regulators Gln3p and Gat1p/Nil1p and the negative regulator Dal80p/ Uga43p. We show here that a fourth member of the yeast GATA family, the Dal80p homolog Deh1p, also negatively regulates expression of some, but not all, nitrogen catabolic genes, i.e., GAP1, DAL80, and UGA4 expression increases in a deh1⌬ mutant. Consistent with Deh1p regulation of these genes is the observation that Deh1p forms specific DNA-protein complexes with GATAA-containing UGA4 and GAP1 promoter fragments in electrophoretic mobility shift assays. Deh1p function is demonstrable, however, only when a repressive nitrogen source such as glutamine is present; deh1⌬ mutants exhibit no detectable phenotype with a poor nitrogen source such as proline. Our experiments also demonstrate that GATA factor gene expression is highly regulated by the GATA factors themselves in an interdependent manner. DAL80 expression is Gln3p and Gat1p dependent and Dal80p regulated. Moreover, Gln3p and Dal80p bind to DAL80 promoter fragments. In turn, GAT1 expression is Gln3p dependent and Dal80p regulated but is not autogenously regulated like DAL80. DEH1 expression is largely Gln3p independent, modestly Gat1p dependent, and most highly regulated by Dal80p. Paradoxically, the high-level DEH1 expression observed in a dal80::hisG disruption mutant is highly sensitive to nitrogen catabolite repression.
In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDPmannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located~9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.
As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.
As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.
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