The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation. Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium. The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family. The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane alpha-helices linked by an extensive polar region. Sequence comparisons suggest that the acuH gene of A. nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man. The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH. In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily. The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids.

Vitamin B6 (pyridoxal 5′-phosphate) is an essential cofactor of many metabolic enzymes. Plants biosynthesize the vitamin de novo employing two enzymes, pyridoxine synthase1 (PDX1) and PDX2. In Arabidopsis (Arabidopsis thaliana), there are two catalytically active paralogs of PDX1 (PDX1.1 and PDX1.3) producing the vitamin at comparable rates. Since single mutants are viable but the pdx1.1 pdx1.3 double mutant is lethal, the corresponding enzymes seem redundant. However, the single mutants exhibit substantial phenotypic differences, particularly at the level of root development, with pdx1.3 being more impaired than pdx1.1. Here, we investigate the differential regulation of PDX1.1 and PDX1.3 by identifying factors involved in their disparate phenotypes. Swapped-promoter experiments clarify the presence of distinct regulatory elements in the upstream regions of both genes. Exogenous sucrose (Suc) triggers impaired ethylene production in both mutants but is more severe in pdx1.3 than in pdx1.1. Interestingly, Suc specifically represses PDX1.1 expression, accounting for the stronger vitamin B6 deficit in pdx1.3 compared with pdx1.1. Surprisingly, Suc enhances auxin levels in pdx1.1, whereas the levels are diminished in pdx1.3. In the case of pdx1.3, the previously reported reduced meristem activity combined with the impaired ethylene and auxin levels manifest the specific root developmental defects. Moreover, it is the deficit in ethylene production and/or signaling that triggers this outcome. On the other hand, we hypothesize that it is the increased auxin content of pdx1.1 that is responsible for the root developmental defects observed therein. We conclude that PDX1.1 and PDX1.3 play partially nonredundant roles and are differentially regulated as manifested in disparate root growth impairment morphologies.

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 x 10(7) viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole-) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole- mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim- (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 x g supernatant, but produced wild-type levels of penicillin.

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.