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.     

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.     

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.     

Distinctive properties of glutamine synthetase from the cyanobacterium Anacystis nidulans.

The intracellular levels of glutamine synthetase (GS) in Anacystis nidulans grown under different conditions were determined using a whole-cell assay. Nitrate-grown cells have 64% more GS than cells grown in ammonium sulfate. Nitrogen starvation does not affect GS levels appreciably. Incubation of nitrate-grown cells with ammonium sulfate does not change the ratio of gamma-glutamyl transferase activities stimulated by Mg2+ and Mn2+ ions. An in vitro test of adenylylation indicates that algae do not have an endogenous adenylyl transferase (ATase) and that algal GS is not adenylylatable by the Klebsiella aerogenes ATase. Some characteristics of the GS-membrane complex were determined by centrifugation of the complex under varying conditions of pH and ionic strength. In this way, it was shown that acid pH (4.5) stabilizes the complex and high ionic strength tends to solubilize the enzyme. A simple partial purification of GS (89-fold) was developed based on the sedimentation properties of GS.     

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.     

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.     

Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression.

Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis.     

Isolation and characterization of an analogue-resistant aminoacyl-tRNA synthetase mutant in Aspergillus nidulans.

Six mutants resistant to p-fluorophenylalanine (FPA) were selected on a medium containing aspartate as the sole source of nitrogen using a phenylalanine-requiring (phenA)auxotroph of A. nidulans as the wild type. The mutants, on the basis of genetic characterization, were found to be alleilic and located on the left arm of the linkage group III, approximately 13 map unit left to meth H locus, henceforth assigned to the symbol fpaV. At a fixed concentration of phenylalanine (23 micrograms/ml), the LD50 value of FPA for all the six mutants was found to be about three times more than that for the wild type strain. Affinity chromatographic purification of the enzyme phenylalanyl-tRNA (Phe-tRNA) synthetase from the mutant as well as the wild type strains, revealed that the wild type enzyme had about 1.4-fold higher affinity for phenylalanine as compared to that for FPA, both in the affinity column and in the catalytic reaction. However, the mutant enzyme showed almost a similar affinity for both in columns but a greatly reduced affinity for FPA in the catalytic reaction.     

Catabolite repression in Enterococcus faecalis

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.     

Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans.

The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.