Regulation of Nitrate Reductase in Neurospora crassa: Stability In Vivo

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.     

Intracellular Localization of Nitrate Reductase in Neurospora crassa

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.     

Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.     

Quantum Requirement for Photoreactivation of Inactivated Nitrate Reductase of Neurospora crassa Strain al-2, bd

Chemically inactivated nitrate reductase of Neurospora crassa strain al-2, bd can be photoreactivated by blue light. The quantum requirement for this reaction in the presence of exogenous FMN was determined with different light intensities. The results are discussed with the alternate assumptions that free FMN or photoactivated FAD bound to the nitrate reductase molecule is the reactivating species.     

In Vitro Activities of the Multifunctional RNA Silencing Polymerase QDE-1 of Neurospora crassa

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases.     

Bacteriochlorophyll, Fatty-Acid, and Protein Synthesis in Relation to Thylakoid Formation in Mutant Strains of Rhodospirillum rubrum

Mutant strains of Rhodospirillum rubrum are isolated which are blocked in different stages of pigment synthesis. In these strains the morphogenesis of thylakoids and the pigment production are investigated. Concerning bacteriochlorophyll synthesis two groups of mutants are separable. The members of the first group synthesize bacteriochlorophyll. Some of these mutants excrete bacteriopheophytin. The strains of the second group are not able to synthesize bacteriochlorophyll. Members of both groups excrete bacteriochlorophyll precursors into the cultural medium. These pigments were identified by their spectral properties as Mg-2,4-divinyl-pheoporphyrin a(5)-monomethylester, pheophorbide a, and 2-devinyl-2-hydroxyethyl-pheophorbide a. Thylakoids are only formed by those strains which are able to synthesize bacteriochlorophyll. However, small amounts of bacteriochlorophyll can be produced without a concomitant thylakoid synthesis. The fatty-acid pattern in some mutants is modified quantitatively. However, the results do not indicate any correlation between disturbance of thylakoid morphogenesis and a deviation of fatty-acid composition. Fatty acids seem to have no special functions in thylakoid morphogenesis. The membranes of the mutants were isolated, split into protein subunits, and these were separated by disc electrophoresis. A characteristic protein pattern, first of all a high content of fraction E, is correlated with the ability to form thylakoids. In addition, all mutants which synthesize bacteriochlorophyll contain a fast-migrating membrane protein (zone G). The results suggest that the whole bacteriochlorophyll-protein complex is necessary for thylakoid formation.     

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.     

Changes in Leaf Nitrate Reductase Activity In Vivo and In Vitro During Light-Dark Transitions

Nitrate reductase activity in the leaves of a number of plants after transfer from light to dark was assayed both by in vivo and in vitro methods. The initial activity persisted during the dark phase for a considerable length of time and declined gradually. After exposure to light again, the NR activity increased rapidly. The possibility of nitrate assimilation in complete darkness is discussed.     

Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa

We used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of Neurospora crassa. Restriction fragment length polymorphism mapping indicated that the cosmid DNA insert was derived from linkage group IL, between 5S rDNA locus 12 and mt, the region of the N. crassa genome that contains nit-2. We conclude that the cosmid carries the nit-2 gene.     

Purification of PII and PII-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in Rhodospirillum rubrum

The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Identification and Characterization of a Nitrate Transporter Gene in Neurospora crassa

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.     

The lipopolysaccharides of Rhodospirillum rubrum,Rhodospirillum molischianum,and Rhodopila globiformis

The cell wall lipopolysaccharides from three phototrophic species of the alpha₁-group of Proteobacteria, Rhodospirillum rubrum, Rhodospirillum molischianum, and Rhodopila globiformis were isolated and chemically characterized. Sodium deoxycholate polyacrylamide gel electrophoresis patterns revealed that the lipopolysaccharides of all three species possess O-chains. They are composed of repeating units only in R. molischianum and R. globiformis. The presence of l-glycero-d-mannoheptose and 2-keto-3-deoxyoctonate indicated core structures in all three lipopolysaccharides. Glucosamine was found as backbone amino sugar in lipid A of R. molischianum and R. rubrum, while R. globiformis has 2,3-diaminoglucose as backbone amino sugar. The latter species also differed from the two former ones in its content of hydroxy fatty acids (3-OH-14:0, 3-OH-16:0 in R. rubrum and R. molischianum and 3-OH-14:0, 3-OH-18:0 and 3-OH-19:0 (possibly iso- or anteisobranched) in R. globiformis).Abbreviations DOC-PAGE sodium deoxycholate polyacrylamide gel electrophoresis - GC/MS combined gas-liquid chromatography/mass spectrometry - KDO 2-keto-3-deoxyoctonate