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.     

Ammonium and glucose repression of the arginine catabolic enzymes in Aspergillus nidulans

Summary The synthesis of two enzymes of the arginine catabolic pathway, arginase and ornithine -transaminase (OTAse), in Aspergillus nidulans was found to be sensitive to both glucose and ammonium repression. The glucose and nitrogen starvation result in the identical derepression of OTAse synthesis and have no effects on arginase synthesis. Glucose and ammonium affect the kinetics of induction of both enzymes, however, the effect of ammonium is much stronger. Evidence was obtained for the direct involvement of ammonium in the repression phenomenon. The relations between glucose and ammonium repression are discussed.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

Nitrogen metabolite repression in Aspergillus nidulans

Summary In Aspergillus nidulans, mutations, designated areA^r, can result in the inability to utilise a wide variety of nitrogen sources including amino acids, purines, amides, nitrate, and nitrite, whilst not affecting growth on ammonium. Other allelic areA mutations, designated areA^d, lead to derepression of one or more activities which are ammonium repressible in wild type (areA⁺) strains, whilst not affecting their inducibility. Various areA mutations exhibit a wide variety of phenotypes: areA^r alleles can be temperature sensitive on some nitrogen sources while not on others, and different alleles can be temperature sensitive for utilisation of different nitrogen sources. areA^d alleles can be derepressed for one ammonium-repressible activity, be normally repressible for another, and lead to abnormally low levels for a third. Once again each areA^d allele has its own highly specific phenotype. The inability of areA^r strains to utilise most nitrogen sources is paralleled by low activities of certain ammonium-repressible enzymes. areA^r mutations appear to be epistatic to some but not all regulatory mutations leading to constitutive synthesis of inducible enzymes and also epistatic to gdhA mutations which lead both to loss of NADP-linked glutamate dehydrogenase and to derepression of ammonium-repressible activities. areA^r mutations do not interfere with repair of a large number of auxotrophies in double mutants. Furthermore, although areA^r mutations prevent utilisation of L-arginine, L-ornithine, and L--amino-n-butyrate as nitrogen sources, they do not prevent the metabolism of these compounds necessary for repairing auxotrophies for proline and isoleucine in the appropriate double mutants. Utilisation of acetamide and most amino acids as carbon or carbon and nitrogen sources is unaffected by areA^r mutations, and areA^r strains are able to utilise acetamide and L-proline (but not other amino acids) as nitrogen sources in the presence of non-catabolite-repressing carbon sources such as L-arabinose, glycerol, melibiose, and lactose. Suppressor mutations, designated creA^d, probably leading to loss of carbon catabolite repression, allow utilisation of acetamide and proline as nitrogen sources in areA^r double mutants in the presence of carbon catabolite-repressing carbon sources. creA^d mutations allow ethanol to serve as a source of acetate for pyruvate dehydrogenaseless (pdhA) strains in the presence of carbon catabolite-repressing carbon sources, whereas pdhA single mutants respond to ethanol as sole carbon source only in the presence of non-carbon catabolite-repressing carbon sources. Specific suppressor mutations, designated amd ^d and prn ^d, allow utilisation of acetamide or proline, respectively, in areA^r double mutants.The areA locus can be interpreted as specifying a protein which is capable of (and in most cases essential for) allowing the synthesis of a number of enzymes of nitrogen metabolism but which cannot function in the presence of ammonium (i.e., as specifying a positive regulatory element which mediates ammonium repression) although the possibility that the areA product also plays a negative regulatory role cannot at present be ruled out.     

Xylanase production in Aspergillus nidulans: induction and carbon catabolite repression

Abstract The induction of the synthesis of extracellular xylanases was investigated in the fungus Aspergillus nidulans using a number of compounds, including xylans of different origin, monosaccharides, xylooligosaccharides and xylose derivatives. Certain xylans (wheat arabinoxylan, oat spelt xylan, birchwood xylan and 4-O-methyl-D-glucurono-D-xylan) were found to be the most powerful inducers. Also, xylooligosaccharides such as xylobiose, xylotriose and xylotetraose served as inducers, their efficiency being directly related to their chain length. Xylose, on the contrary, was not a true inducer. Of the three endo-β-(1,4)-xylanases secreted by A. nidulans , that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.     

Improving extracellular production of food-use enzymes from Aspergillus nidulans

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.     

Physiological studies on xylose induction and glucose repression of xylanolytic enzymes in Aspergillus sydowii MG49

Abstract Synthesis of extracellular xylanase and intracellular β-xylosidase in Aspergillus sydowii is induced in the presence of both d- and l-xylose in addition to xylobiose and β-d-methyl xyloside. Glucose exhibits a transient catabolite repression which can be partially overcome by external addition of 100 μM dibutyryl 3',5'-cAMP but not by that of cAMP itself. In the presence of xylose or other inducers this cyclic nucleotide stimulates the rate of xylanolytic enzyme synthesis by 85% and 129% for xylanase and β-xylosidase, respectively.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Mutations simultaneously affecting ammonium and glucose repression of the arginine catabolic enzymes in Aspergillus nidulans

Summary Two kinds of mutants of Aspergillus nidulans with altered response of arginine catabolic enzymes to glucose and ammonium repression were obtained. Mutations in the suF locus result in the insensitivity of these enzymes to glucose and to one type of ammonium repression. Mutations in the AniA locus result in hypersensitivity to both types of repression. The enzymes studied can be induced by arginine in AniA mutants only when glucose or the nitrogen source is removed from the medium. The suF mutations are recessive while AniA are dominant. Double suF AniA mutants retain only the suF properties. The functions of both genes and their interrelations are discussed.     

Production of xylanolytic enzymes by strains of Aspergillus

Summary Production of hemicellulolytic enzymes required in the hydrolysis of different xylans was investigated using strains of seven species of Aspergillus. Of the strains producing highest levels of xylanolytic activities, a. foetidus VTT-D-71002 was apparently non-cellulolytic and could therefore be a possible source of cellulase-free hemicellulase for applications in the pulping industry. The non-metabolizable synthetic xylobiose analogue -methyl-D-xyloside was the best xylanase inducer of the materials tested. Batches of hemicellulase produced in laboratory scale fermentations on practical media were tested in the hydrolysis of both cellulosic and hemicellulosic substrates.     

A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae

By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.     

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.     

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.     

Carbon catabolite repression in Aspergillus nidulans involves deubiquitination

The best studied role of ubiquitination is to mark proteins for destruction by the proteasome but, in addition, it has recently been shown to promote macromolecular assembly and function, and alter protein function, thus playing a regulatory role distinct from protein degradation. Deubiquinating enzymes, the ubiquitin-processing proteases (ubps) and the ubiquitin carboxy-terminal hydrolases (uchs), remove ubiquitin from ubiquitinated substrates. We show here that the creB gene involved in carbon catabolite repression in Aspergillus nidulans encodes a functional member of the novel subfamily of the ubp family defined by the human homologue UBH1, thus implicating ubiquitination in the process of carbon catabolite repression. Members of the novel subfamily of ubps that include CreB are widespread amongst eukaryotes, with homologues present in mammals, nematodes, Drosophila and Arabidopsis, but mutations in the genes have only been identified in A. nidulans. From phenotypes of the A. nidulans mutants it is probable that this subfamily is involved in complex regulatory pathways. Mutations in the gene encoding the WD40 repeat protein CreC result in an identical phenotype, implicating both genes in this pathway.