Xanthine dehydrogenase expression inNeurospora crassa does not require a functionalnit-2 regulatory gene

Xanthine dehydrogenase (XDH) is the initial enzyme in the purine catabolic pathway ofN. crassa. Secondary nitrogen sources such as purines are metabolized when preferred sources of reduced nitrogen (ammonium or glutamine) are unavailable. XDH synthesis is regulated by glutamine repression and uric acid induction. Thenit-2 locus is believed to encode atrans-acting positive regulator essential for the expression of genes encoding enzymes involved in secondary pathways of nitrogen acquisition, such as XDH and nitrate reductase. However, immunoblot analyses and enzyme assays reveal that XDH protein is synthesized and XDH activity is expressed innit-2 mutants. Nevertheless, XDH responds to nitrogen metabolite repression. The generality thatnit-2 is an obligate control element in nitrogen metabolite repression is questioned. Additionally, mutants defective in XDH activity, namely,xdh-1 and the molybdenum cofactor mutantsnit-1, -7, -8 and -9, are observed to grow on xanthine but not hypoxanthine.This research was supported in part by National Science Foundation Grant DMB 8516203.     

DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: Analysis with mutated binding sites

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UAS_NTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5 or 3 sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.     

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.     

Nitrogen and carbon regulation of lignin peroxidase and enzymes of nitrogen metabolism inPhanerochaete chrysosporium

The regulation of nitrogen metabolism pathways was examined inPhanerochaete chrysosporium in relation to the repression of lignin peroxidase by nitrogen or carbon in this fungus. Under conditions of nitrogen derepression,P. chrysosporium synthesizes the amidohydrolases, formamidase (EC 3.5.1.9) and acetamidase (EC 3.5.1.4) and the enzymes of purine catabolism uricase (EC 1.7.3.3), allantoinase (EC 3.5.2.5), and allantoicase (EC 3.5.3.4). Formamidase is repressed to low levels in the presence of ammonium and there is no apparent control of this enzyme by carbon catabolite repression. Although formamide is a nitrogen source, it is not a carbon source forP. chrysosporium. Glutamate totally represses formamidase. Uricase, allantoinase, and allantoicase are also regulated by nitrogen repression but not carbon catabolite repression. Urease is synthesized at similar levels irrespective of the nitrogen or carbon conditions. The sensitivity of uricase, allantoinase, and allantoicase to nitrogen repression is less than that of formamidase. In contrast to formamidase, glutamate is not a more powerful repressor of uricase, allantoinase, and allantoicase compared with ammonium. No pathway-specific induction is required for the synthesis of formamidase, uricase, allantoinase, and allantoicase. Altogether these features indicate that nitrogen metabolism inP. chrysosporium is similar to that inAspergillus nidulans in its regulation, despite the absence of pathway-specific induction of the enzymes examined. These results are consistent with the existence of a regulatory gene mediating nitrogen catabolite repression similar to theA. nidulans areA gene inP. chrysosporium. Although glycerol acts as a nonrepressive carbon source for lignin peroxidase production (except when used at high concentrations), glutamate totally represses lignin peroxidase even in cultures with glycerol. This indicates that carbon regulation and nitrogen regulation of lignin peroxidase may not be separated inP. chrysosporium.     

Biochemical studies on the Nit mutants of Neurospora crassa

Summary One allele at each of the five nit loci in Neurospora crassa together with the wild type strain have been compared on various nitrogen sources with regard to (i) their growth characteristics (ii) the level of nitrate reductase and its associated activities (reduced benzyl viologen nitrate reductase and cytochrome c reductase) (iii) the level of nitrite reductase and (iv) their ability to take up nitrite from the surrounding medium. Results are consistent with the hypothesis that nit-3 is the structural gene for nitrate reductase, nit-1 specifies in part a molybdenum containing moiety which is responsible for the nit-3 gene product dimerising to form nitrate reductase, nit-4 and nit-5 are regulator genes whose products are involved in the induction of both nitrate reductase and nitrite reductase and nit-2 codes for a generalised ammonium activated repressor protein. Studies on the induction of nitrate reductase (and its associated activities) and nitrite reductase in wild type, nit-1 and nit-3 in the presence of either nitrate or nitrite suggest that each enzyme may be regulated independently of the other and that nitrite could be true co-inducer of the assimilatory pathway. Nitrite uptake experiments with nit-2, nit-4 and nit-5 strains show that whereas nit-4 and nit-5 are freely permeable to this molecule, it is unable to enter the nit-2 mycelium.     

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.     

Nitrate assimilation in Neurospora crassa: Enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures

Summary The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are ₂type homodimers. Enzymebound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. One, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. Three modes of regulation are imposed on the expression of nitrate assimilation, namely: nitrogen metabolite repression, nitrate induction and autogenous regulation by nitrate reductase. In this study, nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved proteins from crude extracts of the wild type and specific nitrate-nonutilizing (nit) mutants were examined for material cross-reactive with antibodies against nitrate reductase and nitrite reductase. The polyclonal antibody preparations used were rendered monospecific by reverse affinity chromatography. Growth conditions which alter the regulatory response of the organism were selected such that new insight could be made into the complex nature of the regulation imposed on this pathway. The results indicate that although nitrate reductase and nitrite reductase are coordinately expressed under specific nutritional conditions, the enzymes are differentially responsive to the regulatory signals.     

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Regulation by inducer exclusion

Summary The ornithine transaminase (EC.2.6.1.13) of Saccharomyces cerevisiae is induced by arginine, ornithine, and their analogs. Genetic regulatory elements which are involved in this induction process have been defined due to the isolation of specific mutants. Two classes of OTAse operator mutants have previously been described; three unlinked genes are presumed to code for a specific repressor, CARGR of both of the arginine catabolic enzymes, arginase, and ornithine transaminase. The level of transaminase of cells grown on ammonia plus arginine is much lower than it is when arginine is the sole nitrogen source. Ammonia thus seems to limit the amount of enzyme synthesized when arginine is present in the growth medium. Nevertheless, all attempts to disclose a nitrogen catabolite repression process in OTAse synthesis have failed; neither the action of mutations that release this regulation on arginase and other catabolic enzymes, nor the use of derepressing growth conditions, affect OTAse synthesis. A decrease of the cells' arginine pool when amonia or aminoacids (serine, glutamate) are added to arginine as a nitrogen nutrient results in a progressive reduction of transaminase synthesis. This suggests that arginine is the only physiological effector in those conditions: ammonia or some aminoacids would reduce the enzyme synthesis because of an inducer exclusion. The first stage of OTAse induction would then be operated by the CARGR repressor, and an additional regulatory element might take part in the full scale process. Preliminary data favoring the involvment of such an element are presented.     

A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi

In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X₂-CX₁₇-C-X₂-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X₂-C-X₁₈-C-X₂-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.     

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter--glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.     

Analysis of conventional and in vitro generated mutants of nmr, the negatively acting nitrogen regulatory gene of Neurospora crassa

Summary Thenmr gene is the major negative regulatory gene in the nitrogen control circuit ofNeurospora crassa, which, together with positive regulatory genes, governs the expression of multiple unlinked structural genes of the circuit. Possible functional domains of the NMR protein were investigated by mutational analyses using three different approaches. First, the polymerase chain reaction was used to clone thenmr locus from two conventional mutants, V2M304 and MS5, and the mutant amino acid codons were identified. A single point mutation was shown to be responsible for the mutant phenotype in each of these strains. The V2M304 allele contains a nonsense codon, and in the MS5 allele an aspartate has been substituted for glycine at residue 386. Our second approach studied possible functionally important regions in thenmr gene by the use of site-directed mutagenesis. The region containing the naturally occurring substitution in MS5 appears to be essential for function whereas a region in the N-terminal part of the protein does not seem important for NMR function. Finally, over 50% of the protein coding region was randomly mutagenized and amino acid residues that are essential for function and others that are functionally unimportant were identified.     

Genetic and metabolic regulation of purine base transport in Neurospora crassa.

Summary Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. The early purine catabolic enzymes in this organism are regulated by induction and by ammonium repression. Studies were undertaken to investigate purine base transport and its regulation in Neurospora. The results of competition experiments with uric acid and xanthine transport strongly suggest that uric acid and xanthine share a common transport system. It was also shown that the common transport system for uric acid and xanthine is distinct from a second transport system shared by hypoxanthine, adenine and guanine, and apparently also distinct from the transport system(s) for adenosine, cytosine and uracil. Regulation of the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system was studied. The results reveal that the uric acid-xanthine transport system is regulated by ammonium repression, but does not require uric acid induction. Neither ammonium repression nor uric acid induction controls the hypoxanthine-adenine-guanine transport system. A gene, designated amr, which is believed to be a positive regulatory gene for nitrogen metabolism of Neurospora crassa, was found to dramatically affect both the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system. A model for the action of the amr locus as a positive regulatory gene and for the interaction between the amr gene product and its recognition sites will be discussed.     

The uaY positive control gene of Aspergillus nidulans: fine structure, isolation of constitutive mutants and reversion patterns

Summary The product of the uaY gene of Aspergillus nidulans is necessary for the expression of at least eight genes coding for enzymes and permeases of the purine utilisation pathway. A detailed fine structure map has been constructed of this gene involving 13 presumed point mutations and eight deletions. Gene conversion of these deletions was demonstrated. A technique was devised to select for constitutive mutations and two were obtained which map within the uaY gene. We have shown that the most centromere proximal allele reverts to a number of different phenotypes. The properties of this allele suggest that it may map in the open reading frame of the uaY gene, in a domain that could be altered in a way that would differentially affect the expression of genes under uaY control.     

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli

High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium. We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR. However, filamentation occured even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway. Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.     

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

Summary The nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argF _po), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argF _po. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argF _po —strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru^–/Oru^– phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.Abbreviations Arg^– arginine auxotrophy - Aru arginine utilization - Oru ornithine utilization     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.