Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa.

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate.     

Oxidation of Neurospora crassa glutamine synthetase.

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.     

Neurospora crassa mutant impaired in glutamine regulation.

The final products of the catabolism of arginine that can be utilized as nitrogen sources by Neurospora crassa are ammonium, glutamic acid, and glutamine. Of these compounds, only glutamine represses arginase and glutamine synthetase. We report here the isolation and characterization of a mutant of N. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glnI). This mutant has a greater capacity than the wild type (glns) to accumulate most of the arginine and some of the glutamine in osmotically sensitive compartments while growing exponentially. Nonetheless, the major part of the glutamine remains soluble and metabolically available for repression. We propose that the lower repression of glutamine synthetase by glutamine in this mutant could be a necessary condition for sustaining the higher flow of nitrogen for the accumulation of amino acids observed in ammonium excess and that, if glutamine is the nitrogen signal that regulates the arginine accumulation of the vesicle, the glnr mutant has also escaped this control. Finally, in the glnr mutant, some glutamine resynthesis is necessary for arginine biosynthesis and accumulation.     

Genetics and physiology of Neurospora crassa glutamine auxotrophs.

This work reports on the isolation and characterization of two glutamine auxotrophs in Neurospora crassa. The mutations responsible for the glutamine-requiring phenotype were very closely linked, and one of them proved to be recessive to wild type. The mutations impaired the conversion of glutamic acid to glutamine and resulted in changes of both the activity and oligomeric structure of the enzyme glutamine synthetase.     

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.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

Regulation of thymidine metabolism in Neurospora crassa.

The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'-hydroxylase. Qualitatively similar effects on these enzymes were bought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Regulation of hypoxanthine transport in Neurospora crassa.

Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.     

Heterogeneity of glutamine synthetase polypeptides in Neurospora crassa

Purified preparations of Neurospora crassa glutamine synthetase contain two nonidentical polypeptides that can be separated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 7 M urea. These polypeptides are synthesized both in vivo and in a heterologous cell-free protein-synthesizing system. The data presented indicate that both polypeptides contain an active site for glutamine synthetase activity and suggest that there is not a precursor-product relationship between them.     

Omega-amidase pathway in the degradation of glutamine in Neurospora crassa.

Evidence for the participation of the glutamine transaminase-omega-amidase pathway in the utilization of glutamine in Neurospora crassa was obtained. Its participation is indicated by the in vitro activities of glutamine transaminase and omega-amidase, the in vivo accumulation of alpha-ketoglutaramate when an inhibitor of transamidases is present, and the inhibition by aminooxyacetic acid and 6-diazo-5-oxo-L-norleucine of the ammonium excreted in the presence of glutamine by a mutant strain that lacks glutamate dehydrogenase and glutamate synthase.     

Regulation of Neurospora crassa nucleosidases by some environmental factors.

Enzymic cleavage of N-glycosidic bonds of AMP, GMP, and inosine by the cell-free extracts of Neurospora crassa has been studied. The enzymic activities with these substrates appear to be discrete from one another. The levels of these enzymes in the cell vary with age, and are dependent upon the inoculum size, aeration rate, and phosphate level in the medium. Glucose (or ribose) controls the phosphate-mediated repression of all the three nucleosidases of this fungus.     

Regulation of polyamine synthesis in relation to putrescine and spermidine pools in Neurospora crassa.

Polyamine pools were measured under various conditions of high and low concentrations of cytosolic ornithine with the wild-type and mutant strains of Neurospora crassa. In minimal medium, the wild-type strain has 1 to 2 nmol of putrescine and approximately 14 nmol of spermidine per mg (dry weight); no spermine is found in N. crassa. Exogenous ornithine was found to cause a rapid, but quickly damped, increase in the rate of polyamine synthesis. This effect was greater in a mutant (ota) unable to catabolize ornithine. No turnover of polyamines was detected during exponential growth. Exogenous spermidine was not taken up efficiently by N. crassa; thus, the compound could not be used directly in studies of regulation. However, by nutritional manipulation of a mutant strain, aga, lacking arginase, cultures were starved for ornithine and thus ultimately for putrescine and spermidine. During ornithine starvation, the remaining putrescine pool was not converted to spermidine. The pattern of polyamine synthesis after restoration of ornithine to the polyamine-deprived aga strain indicated that, in vivo, spermidine regulates polyamine synthesis at the ornithine decarboxylase reaction. The results suggest that the regulatory process is a form of negative control which becomes highly effective when spermidine exceeds its normal level. The possible relationship between the regulation of polyamine synthesis and the ratio of free to bound spermidine is discussed.