Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

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.

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes.

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 crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans.

BACKGROUND: Isocitrate lyase catalyses the first committed step of the carbon-conserving glyoxylate bypass, the Mg(2+)-dependent reversible cleavage of isocitrate into succinate and glyoxylate. This metabolic pathway is an inviting target for the control of a number of diseases, because the enzymes involved in this cycle have been identified in many pathogens including Mycobacterium leprae and Leishmania. RESULTS: As part of a programme of rational drug design the structure of the tetrameric Aspergillus nidulans isocitrate lyase and its complex with glyoxylate and a divalent cation have been solved to 2.8 A resolution using X-ray diffraction. Each subunit comprises two domains, one of which adopts a folding pattern highly reminiscent of the triose phosphate isomerase (TIM) barrel. A 'knot' between subunits observed in the three-dimensional structure, involving residues towards the C terminus, implies that tetramer assembly involves considerable flexibility in this part of the protein. CONCLUSIONS: Difference Fourier analysis together with the pattern of sequence conservation has led to the identification of both the glyoxylate and metal binding sites and implicates the C-terminal end of the TIM barrel as the active site, which is consistent with studies of other enzymes with this fold. Two disordered regions of the polypeptide chain lie close to the active site, one of which includes a critical cysteine residue suggesting that conformational rearrangements are essential for catalysis. Structural similarities between isocitrate lyase and both PEP mutase and enzymes belonging to the enolase superfamily suggest possible relationships in aspects of the mechanism.

The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier.

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.

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy.

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide.

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method.

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.

Purification and properties of beta-galactosidase from Aspergillus nidulans.

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Analysis of the regulation of the Aspergillus nidulans acuD gene, encoding isocitrate lyase, by construction of a hybrid promoter.

In order to confirm functionally that a 208 bp fragment of the 5'-flanking sequence of the acuD gene of Aspergillus nidulans is the region responsible for acetate inducibility and catabolite repression, a hybrid promoter was constructed by insertion of this fragment into the promoter of the (highly expressed) oliC gene of A. nidulans. Analysis of expression of the lacZ reporter gene fused to the oliC/acuD promoter showed induction by acetate at much higher levels than wild-type acuD expression. Acetate inducibility of the hybrid promoter was dependent on the facB gene, demonstrating that a facB-dependent upstream activating sequence (UAS) for acetate must be located in the 208 bp acuD fragment. In parallel, partial relief of the transcriptional repression of acetate inducibility by sucrose and glucose was observed in a creA- background, showing that the 208 bp acuD fragment also responds to the creA gene. In addition, the results show that combination of a regulatory element from a low-expression promoter (acuD) with a high-expression constitutive promoter (oliC) leads to amplification of the level of regulated expression.

Regulation of the expression of the isocitrate lyase gene (acuD) of Aspergillus nidulans.

Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acuD) of Aspergillus nidulans is described. Transcription start sites were detected at positions -163, -170 and approximately -281 upstream of the ATG. Transcription analysis showed that the acuD gene is transcribed during growth on acetate but not on hexoses or glycerol. Expression of the acuD gene was studied under inducing and repressing conditions in cre+, creA, creB and creC mutant strains, showing that the creA(d)-1 mutation led to slight derepression of isocitrate lyase. Regulation of expression of the acuD gene was also studied using an in-frame fusion with the lacZ gene of Escherichia coli. Several deletions were made in order to identify the regions responsible for acetate induction and repression. A deletion of the -412 to -200 bp upstream region resulted in loss of all promoter activity and a smaller deletion within this region abolished most of the acetate inducibility.

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans.

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.