Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans

Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnADelta mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans.

The areA gene, which mediates nitrogen metabolite repression in the fungus Aspergillus nidulans, lies sufficiently close to a telomere that no indispensable gene can be distal to it. We were able therefore to exploit the existence of a near terminal pericentric inversion to devise a method for cloning areA plus the region beyond it towards the telomere. In crosses heterozygous for this inversion a class of duplication-deficient progeny lacking areA and the region centromere-distal to it is obtained. We, therefore, sought clones from an A. nidulans gene library in lambda Charon 4 able to hybridize to total genomic DNA from a wild-type strain but not to that from a duplication-deficiency strain. A clone, containing an 11.6-kb insert, which hybridised weakly to duplication-deficiency DNA, overlapped chromosome breakpoints of three different aberration-associated areA alleles and was able to transform an areA mutant to areA+. Southern blotting and genetic analysis established that the transforming sequence had integrated in the region centromere distal to areA. The cloning method yielded other clones from the region centromere-distal to areA which were used to show that the translocation associated with a mutant areA allele is reciprocal rather than non-reciprocal, a fact which could not be established by classical genetics. Finally, analysis of the cloned portion of the dispensable region centromere-distal to areA indicates that this region contains at least 0.5% of the A. nidulans genome.     

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA , is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creA^d 30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA .C1, that is responsible for direct CreA repression in vivo . Using the creA^d 30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.     

Regulation of pyrimidine salvage in Aspergillus nidulans: a role for the major regulatory gene are A mediating nitrogen metabolite repression

Summary The synthesis of thymine 7-hydroxylase, an -ketoglutarate dependent dioxygenase, is subject both to nitrogen metabolite repression and to oxygen repression, while synthesis of the other pyrimidine salvage pathway dioxygenase, pyrimidine deoxyribonucleoside 2-hydroxylase, is subject to neither. areA300, an allele of the positive acting regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans, considerably elevates levels of thymine 7-hydroxylase, probably alleviating at least partly both nitrogen metabolite repression and oxygen repression. areA300 has little or no effect on levels of pyrimidine deoxyribonucleoside 2-hydroxylase but does elevate net uptake capacities for thymine, thymidine and deoxyuridine two-fold. areA300 was selected as allowing thymine to supplement a pyrimidine auxotrophy and was found to allow supplementation by thymidine, other pyrimidine nucleosides and pyrimidine salvage intermediates as well. This is the first reported evidence for areA control over an activity(-ies) not directly concerned with nitrogen source utilization.     

Catabolite repression in Aspergillus nidulans; the role of glutamine synthetase.

A mutant (nit8) with a lowered activity of glutamine synthetase (GS) was isolated in Aspergillus nidulans. The levels of GS and of an arginine catabolic enzyme, ornithine transaminase (OTA) were assayed under a variety of growth conditions leading to repression, depression and induction of OTA in the wild type, nit8 and several regulatory mutants. The results obtained appear to exclude the possibility of involvement of GS in the regulation of arginine catabolism in A. nidulans.     

Coordinate control of secondary metabolite production and asexual sporulation in Aspergillus nidulans

Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.     

A near terminal pericentric inversion leads to nitrogen metabolite derepression in Aspergillus nidulans

Summary The mutation xprD-1, previously shown to be an allele of the areA gene and to lead to nitrogen metabolite derepression in Aspergillus nidulans, is shown to be associated with a near terminal pericentric inversion in linkage group III. The left arm break-point is between the adI and sC genes, and the right arm break-point is between the ornC and areA genes but just centromere proximal to areA. In crosses of xprD-1 strains to inversion-free strains one class of duplication-deficiency progeny is recovered. These progeny have two copies of the distal portion of the left arm beginning just before sC but lack a copy of areA and the region distal to it on the right arm. The viability of these duplication-deficiency progeny indicates that no indispensable gene can lie distal to areA, suggesting proximity of areA to the telomere. The inversion might increase expression of areA which, given the positive acting nature of this regulatory gene, would result in nitrogen metabolite derepression. If increased expression be the result of fusion to (or creation of) a more efficient promoter and/or ribosome binding sequence, areA must be transcribed towards the right arm telomere.     

Metabolite repression and inducer exclusion in the proline utilization gene cluster of Aspergillus nidulans

The clustered prnB, prnC, and prnD genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in cis only on the permease gene (prnB) while derepression of prnD and prnC is largely the result of reversal of inducer exclusion.     

Characterization of the Aspergillus nidulans nmrA Gene Involved in Nitrogen Metabolite Repression

The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively acting areA gene.     

Nitrogen metabolite repression of fluoropyrimidine resistance and pyrimidine uptake in Neurospora crassa

Summary Wild type Neurospora crassa was shown to be more resistant to 5-fluoro-uracil, 5-fluoro-uridine and 5-fluoro-2-deoxyuridine in the presence of ammonia than in its absence. This differential resistance may in part be accounted for by the observation that both uracil and uridine uptake in germinating conidia is under nitrogen metabolite regulation. The uptake of uracil and uridine is increased on poor nitrogen sources in wild type, is unaffected by nitrogen source in a nit-2 mutant strain while a gln-1 mutant strain showed intermediate behaviour.Wild type also showed increased resistance to all three fluoropyrimidines with increased temperature and both uracil and uridine uptake in the wild type increased with temperature.Supported by S.R.C. grant GR/A/64655F.P. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Tubulins in Aspergillus nidulans

The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.