New Mutational Variants of Neurospora Nadp-Specific Glutamate Dehydrogenase

The am locus of Neurospora codes for NADP-dependent glutamate dehydrogenase (GDH). Four new am mutants that produced mutationally altered GDH have been characterized. Mutant am₁₁₉ is a CRM-negative, complementing mutant that maps between am₂ and am₁. The other three mutants are CRM formers that produce varieties of GDH that can be activated by glutamate or succinate. The GDH of am₁₃₀ and am₁₃₁ is similar in terms of activation properties to that of am₃. The GDH of am₁₂₂ requires very high concentrations of dicarboxylate for activity. The mutation in am₁₃₀ maps between am₁₄ and am₂ and resulted in a replacement at residue 75 of the GDH (pro → ser). The mutation in am₁₂₂ maps near am₁₁ and apparently resulted in the replacement of the tryptophan residue at position 389 with an unknown amino acid. The mutation in am₁₃₁ maps between am₂ and am₁.     

Nitrogen regulation of arginase in Neurospora crassa.

The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.     

Nitrogen regulation of glutamine synthetase in Neurospora crassa.

A higher activity of glutamine synthetase (EC 6.3.1.2) was found in Neurospora crassa when NH4+ was limiting as nitrogen source than when glutamate was limiting. When glutamate, glutamine or NH4+ were in excess, a lower activity was found. Immunological titration and sucrose gradient sedimentation of the enzyme established that under all these conditions enzyme activity corresponded to enzyme concentration and that the octamer was the predominant oligomeric form. When N. crassa was shifted from nitrogen-limiting substrates to excess product as nitrogen source, the concentration of glutamine synthetase was adjusted with kinetics that closely followed dilution by growth. When grown on limiting amounts of glutamate, a lower oligomer was present in addition to the octameric form of the enzyme. When the culture was shifted to excess NH4+, glutamine accululated at a high rate; nevertheless, there was only a slow decrease in enzyme activity and no modification of the oligomeric pattern.     

Nitrogen regulation of acid phosphatase in Neurospora crassa.

Neurospora crassa possesses a repressible acid phosphatase with phosphodiesterase activity which appears to permit it to utilize ribonucleic acid as a phosphorus and as a nitrogen source. This acid phosphatase, which is specified by the pho-3 locus, is derepressed approximately eightfold during nitrogen limitation and to an even greater extent during phosphorus limitation, but is unaffected by sulfur limitation. Derepression of the enzyme did not occur when adenosine 5'-monophosphate was the sole phosphorus or nitrogen source. Synthesis of the acid phosphatase is not under the control of the nit-2 locus, which regulates the expression of a large number of other nitrogen catabolic enzymes. The structural gene of the acid phosphatase appears to be a member of both the phosphorus and nitrogen regulatory circuits.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Glutamate dehydrogenase from wild type Neurospora crassa; inactivation by photo-oxidation

The NADP dependent glutamate dehydrogenase from wild type Neurospora crassa is inactivated by exposure to light in the presence of the dye, Methylene Blue. Photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. Data obtained suggests that the modified group is the same as that reactive to the histidine reagent, diethylpyrocarbonate.     

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.     

Pyrimidine Synthesis in Neurospora crassa: Gene-Enzyme Relationships

A new series of pyrimidine-requiring mutants of Neurospora has been isolated and all enzymes involved in pyrimidine biosynthesis are represented by at least one mutant. Among these mutants is included a single isolate for a new locus, pyr-6. This mutant is deficient in dihydroorotase (DHOase) and represents the only enzymatic step in orotate synthesis for which no mutant previously had been found. This mutant, which mapped genetically on the right arm of linkage group V, is unlinked to any of the other pyrimidine mutants. The DHOase-deficient mutant is also characterized by an unexpected growth behavior. The pyr-1 locus has been specifically associated with a lack of dihydroorotate dehydrogenase (DHOdehase). Mutants isolated in this series for other pyrimidine loci have been related to previously isolated mutants by allelism, recombination, and accumulation studies.     

Malonate Metabolism, Stimulation of Conidiation and Succinate Dehydrogenase Activity in Neurospora crassa

Malonate was studied for its effect on succinate dehydrogenase activity and conidiation. It Was found to stimulate the succinate dehydrogenase activity and also conidiation of Neurospora crassa. The efficiency of sucrose metabolization for cellular synthesis was improved in malonate supplemented cultures. High Concentration (0.5 M) had a distinct toxic effect on conidiation and economic efficiency. Teratological structures were observed at 0.5 M malonate concentration. A stimulation of the glyoxylate cycle is considered to be one of the factors responsible for the conidiogenic effect.     

Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.     

Nitrogen source regulates glutamine synthetase mRNA levels in Neurospora crassa.

Neurospora crassa glutamine synthetase mRNA was measured by its capacity to direct the synthesis of the specific protein in a cell-free system derived from rabbit reticulocytes. N. crassa cultures grown on glutamate as the sole nitrogen source had higher mRNA activities than did those grown on glutamine. The differences were about 10-fold when polysomal RNA was used for translation and about 5-fold when either total cellular RNA or polyadenylic acid-enriched cellular RNA was used. These data indicate that in exponentially growing N. crassa, the nitrogen source regulates glutamine synthetase by adjusting specific mRNA levels.     

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Conidiation in Neurospora crassa

Summary Conidiation in Neurospora crassa has been studied in vivo by time-lapse microphotography and shown to be most generally (in aerial, dry conditions) a budding-fission process. Such a two-phase process is characterized by an initial basifugal budding of proconidial elements which are then secondarily separated as maturing conidia by interconidial septa. Dry macroconidia of Neurospora are thus blasto-arthrospores, i.e. blastospores basifugally budded on conidiophores and secondarily disarticulated from the proconidial chain as arthrosporal elements. Inception and median splitting of the interconidial septum have been electron microphotographed.In the vegetative hyphae, ethanol dehydrogenase has been cytochemically detected by oxidative assay and demonstrates a dense, uniform distribution of activity except at the hyphal tips. In the conidiating hyphae, the ethanol dehydro-genase becomes less dense in distribution, especially in the budding apices. Cytochrome oxidase activity, localized in the mitochondria, is confined in the subapical zone of vegetative hyphae while at the initiation of conidiation it becomes dispersed throughout the proconidial buds.     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.