L-histidine utilization in Aspergillus nidulans.

Histidase activity rather than uptake of L-histidine is the limiting factor for the utilization of histidine as the sole nitrogen source for Aspergillus nidulans. Histidine cannot act as the sole carbon source, and evidence is presented indicating that this is attributable to an inability to convert histidine to L-glutamate in vivo. It has been shown that this fungus lacks an active urocanase enzyme and that histidine is quantitatively converted to urocanate, which accumulates in the extracellular medium. The use of histidine as a nitrogen source is regulated by nitrogen metabolite repression control of histidase synthesis. In addition, evidence for a requirement for a carbon source for histidase synthesis and for a minor form of control by nitrate is presented. The activity of the histidase enzyme is inhibited by micromolar concentrations of the product urocanate and by physiological levels of L-glutamate and L-glutamine.     

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.     

Induction of the acetamidase of Aspergillus nidulans by acetate metabolism.

Growth tests and enzyme determinations strongly suggest that the acetamidase of Aspergillus nidulans is induced by a product of acetate metabolism rather than the substrate, acetamide. The cis-dominant mutation, amdI9, which is closely linked to amdS, the structural gene for the acetamidase, results in greatly increased sensitivity to induction by acetate metabolism. Propionate, L-threonine, and ethanol also result in acetamidase induction. Mutations in the facA, facB, and facC genes, which lead to low levels of acetyl-coenzyme A synthase, are epistatic to the amdI9 mutation for strong growth on acetamide medium and abolish acetamide and propionamide induction of the acetamidase and isocitrate lyase enzymes. Acetate, L-threonine, and ethanol, however, can induce these enzymes in strains containing facA and facC lesions but not in strains containing a facB lesion. The evidence suggests that acetamidase and isocitrate lyase may be induced by a similar mechanism.     

Survey of representative species of Aspergillus for regions of DNA homology to Aspergillus nidulans developmental genes

Summary This study surveyed five representative species of Aspergillus for regions of homology with previously cloned A. nidulans developmental genes. Areas of hybridization were found for all A. nidulans genes in the DNA of all of the Aspergillus species examined. All five species had a high level of homology with the tubC gene, but varied in degree of homology with the brlA gene and the SpoC1 gene cluster. These results suggest that DNA sequences analogous to A. nidulans developmental genes are found in other members of the genus and support the hypothesis that genetic investigations of A. nidulans could serve as model systems for genetic studies of other aspergilli.     

Chromosome-specific recombinant DNA libraries from the fungus Aspergillus nidulans.

Development of physical genomic maps is facilitated by identification of overlapping recombinant DNA clones containing long chromosomal DNA inserts. To simplify the analysis required to determine which clones in a genomic library overlap one another, we partitioned Aspergillus nidulans cosmid libraries into chromosome-specific subcollections. The eight A. nidulans chromosomes were resolved by pulsed field gel electrophoresis and hybridized to filter replicas of cosmid libraries. The subcollections obtained appeared to be representative of the chromosomes based on the correspondence between subcollection size and chromosome length. A sufficient number of clones was obtained in each chromosome-specific subcollection to predict the overlap and assembly of individual clones into a limited number of contiguous regions. This approach should be applicable to many organisms whose genomes can be resolved by pulsed field gel electrophoresis.     

Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans.

Genetic analysis of 119 acetate non-utilizing (acu) mutants in Aspergillus nidulans revealed ten new loci affecting acetate metabolism in addition to the three previously recognized on the basis of resistance to fluoroacetate and acetate non-utilization. The enzyme lesions associated with mutations at seven of the acu loci are described. These are: facA (= acuA), acetyl-CoA synthase; acuD, isocitrate lyase; acuE, malate synthase; acuF, phosphoenolpyruvate carboxykinase; acuG, fructose 1,6-diphosphatase; acuK and acuM, malic enzyme. The acu loci have been mapped and are widely distributed over the genome of A. nidulans. Close linkage has only been found between acuA and acuD (less than 1% recombination). There is no evidence for any pleiotropic mutation in that region affecting the expression of both these genes. Poor induction of the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in mutants lacking acetyl-CoA synthase, and also in the other two classes of fluoroacetate-resistant mutants, indicates that the inducer, acetate, may be metabolized to a true metabolic inducer, perhaps acetyl-CoA, to effect formation of the enzymes. There is no evidence of any other class of pleiotropic recessive acu mutations affecting the expression of the acuD and acuE genes, which are therefore thought to be subject to negative rather than positive control.     

Two unlinked genes for the pyruvate dehydrogenase complex in Aspergillus nidulans.

The activity of the overall pyruvate dehydrogenase complex was found to be similar in extracts of Aspergillus nidulans after growth on either sucrose or acetate. Eight mutants lacking the activity of this complex were found among some 200 glycolytic mutants selected for their inability to grow on sucrose. The absence of pyruvate dehydrogenase complex activity was also confirmed for a mutant, g6 (pdhA1), isolated previously. Studies with the mutants supported the existence of two unlinked genes, pdhA and pdhB, controlling the function of the complex. In vivo and in vitro complementation between mutations at the two loci were shown by the ability of forced heterokaryons to grow on sucrose and by the restoration of overall pyruvate dehydrogenase complex activity in mixed cell-free extracts. The mutations were recessive to their wild-type alleles, and the pdhA and pdhB loci were assigned to linkage groups I and V, respectively.     

Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis.

Strains of Aspergillus nidulans carrying the orlA1 or tse6 allele are deficient in cell wall chitin and undergo lysis at restrictive temperatures. The strains are remediable by osmotic stabilizers or by the presence of N-acetylglucosamine (GlcNAc) in the medium. The remediation by GlcNAc suggests that the lesion(s) in chitin synthesis resides in the amino sugar biosynthetic pathway prior to the synthesis of N-acetylglucosamine-6-phosphate. orlA1 strains grown at permissive temperature exhibit an abnormally low specific activity for L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), the first enzyme unique to amino sugar synthesis. In addition, the enzyme produced is temperature sensitive in vitro. tsE6 strains grown at permissive temperature show virtually no amidotransferase activity. This finding is consistent with an extremely labile enzyme which is destroyed by cell breakage and extract preparation. The enzyme must be active in vivo at permissive temperatures since GlcNAc is not required for growth. Thus, two structural genes (orlA and tsE) are necessary for the amidotransferase activity. bimG11 strains are temperature sensitive for a type 1 protein phosphatase involved in cell cycle regulation and arrest in mitosis. Like orlA1 and tsE6 strains, conidia from bimG11 strains swell excessively when germinated and lyse; the germlings produced are deficient in chitin content. The amidotransferase from wild-type and mutant strains is sensitive to feedback inhibition by uridine diphosphate-N-acetylglucosamine. The sensitivity of the amidotransferase from bimG11 strains is dependent on growth temperature, while that from wild-type strains is independent of temperature. The enzyme can be desensitized in vitro under conditions consistent with a protein phosphatase reaction. It is proposed that amino sugar (and chitin biosynthesis) is partially regulated by phosphorylation-dephosphorylation of the amidotransferase or a protein regulator of the enzyme.