Levels Of Nitrite Reductase Activity Research Articles

Negative Regulation of Expression of the Nitrate AssimilationnirAOperon in the Heterocyst-Forming CyanobacteriumAnabaenasp. Strain PCC 7120

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group

A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate. The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions. Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions. In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N 2O as electron acceptor. The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions. Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N 2O as electron acceptors. Others functioned best with N 2O during anaerobic thiosulfate oxidation. The latter strains appeared to have a lower level of nitrite reductase activity. Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor. The strains had DNA-DNA similarity levels higher than 30%. Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica. As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed ‘ Flavobacterium lutescens’ (ATCC 27951) is also a P. stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N 2O. The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P. stutzeri ‘superspecies’, can play a much more significant role in the biogeochemical cycles than was previously recognized.

Anaerobic oxidation of thiosulfate to tetrathionate by obligately heterotrophic bacteria, belonging to the Pseudomonas stutzeri group.

A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate. The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions. Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions. In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N(2)O as electron acceptor. The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions. Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N(2)O as electron acceptors. Others functioned best with N(2)O during anaerobic thiosulfate oxidation. The latter strains appeared to have a lower level of nitrite reductase activity. Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor. The strains had DNA-DNA similarity levels higher than 30%. Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica. As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed 'Flavobacterium lutescens' (ATCC 27951) is also a P. stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N(2)O. The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P. stutzeri 'superspecies', can play a much more significant role in the biogeochemical cycles than was previously recognized.

Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures

The nitrite ion content and activity of nitrate reductase and nitrite reductase were examined in scutellum-derived calluses of rice varieties using a modified R2 medium (medium A) and a medium derived from the modified R2 medium (medium B). In medium A, marked differences were observed in callus growth between the varieties. The calluses of the poor-growth varieties accumulated significantly more nitrite ions during the culture period than did the good-growth varieties. Callus growth rate was negatively correlated with the nitrite ion content, indicating that the calluses of the poor-growth varieties were injured by toxic nitrite ions, which lead to browning and inhibited growth. The calluses of the poor-growth varieties had significantly lower levels of nitrite reductase activity than good-growth varieties. On the other hand, no between-group differences were observed in the nitrate reductase activity. These results indicate that the higher nitrite ion levels observed in the poor-growth varieties resulted from a lower ability to reduce nitrite and that nitrite reductase activity is one of the physiological factors that correlates with differences between varieties in rice cell cultures. In medium B, the calluses of the poor-growth varieties grew as well as the good-growth varieties, but also had significantly lower levels of nitrite reductase. Nitrate reductase activity was repressed in the calluses of both varieties in medium B compared to culture in medium A. The results suggest that repressed nitrate reductase activity causes the calluses of poor-growth varieties to accumulate only trace amounts of nitrite ions despite lower nitrite reductase activity and as a result, callus growth improved in medium B.

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii.

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 bp in length. The nucleotide coding sequence could be aligned with the 3' end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3' untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Nitrite Reductase Cellular Levels in the Cyanobiont Nostoc ANTH

Summary The cellular nitrite reductase activity in response to different nitrogen sources has been studied in the cyanobiont Nostoc ANTH isolated from the liverwort Anthoceros. De novo protein synthesis of nitrite reductase occurred even in the absence of added nitrogen source, although enzyme activity was higher when nitrite or nitrate served as the sole nitrogen source. In tungstate-treated cells, nitrate did not show any positive effect on nitrite reductase activity, suggesting that the stimulatory effect was not due to nitrate itself but it requires its reduction to nitrite. Thus, nitrite may be the actual inducer in this case. Ammonium-grown cells showed reduced level of nitrite reductase activity. The nitrite reductase activity was freed from ammonium repression by L-methionine-DL-sulphoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Thus ammonium metabolism through GS is required for the ammonium repression of nitrite reductase to occur.

Synthesis of nitrite reductase is regulated differently in leaf and root of barley ( Hordeum vulgare L.)

Activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE) of extracts from nitrate-treated plants indicates the presence of a single isoform of nitrite reductase in the leaf and root of barley. Crossed and tandem-crossed immunoelectrophoresis, and immunoblotting, show that polyclonal antibodies raised against the purified leaf nitrite reductase protein recognise specifically the nitrite reductase protein in both leaf and root, suggesting that the leaf and root enzymes are immunologically similar, if not identical. Green, light-grown plants maintained in the absence of added nitrate possess low levels of nitrite reductase activity (measured with dithionite-reduced methyl viologen as electron donor) and nitrite reductase cross-reacting material in both leaf and root. Addition of nitrate in the light to green, light-grown plants leads to a concomitant increase in the level of nitrite reductase activity and cross-reacting material in both leaf and root. When green, light-grown plants are treated with nitrate and transferred immediately to the dark, there is a concomitant increase in nitrite reductase activity and cross-reacting-material in the root, but neither increase in the leaf. We conclude that the observed increases in nitrate reductase activity are due to the net synthesis of new nitrite reductase molecules and that their synthesis is regulated differently in leaf and root: both light and nitrate together are required in the leaf but nitrate alone is able to bring about synthesis of nitrite reductase molecules in the root. Inability to synthesise nitrite reductase molecules in leaves in the dark after treatment of plants with nitrate is not due to exclusion of nitrate from the leaves. Possible mechanisms which underlie these observations are discussed.

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.

Differences in nitrogen metabolism between cultivars of tomato and pepper during exposure to glasshouse atmosphere containing oxides of nitrogen

Metabolism of the products of atmospheric oxides of nitrogen (NO x) once they enter the plant cell involves normal nitrate assimilation pathways. Activities of two enzymes involved in this pathway have been investigated in two important horticultural plants (tomato and sweet pepper) exposed to atmospheric levels of nitric oxide (NO) or nitrogen dioxide (NO 2) commonly found during commercial cultivation under glass. In most cultivars of tomato, the response to NO x has been shown to be a significant induction of nitrite reductase (NiR) but in leaves of sweet pepper, exposure to either of the two gases reduced the level of in vitro NiR activities even though an accumulation of amine compounds in the tissue suggested that the rates of nitrate reduction had increased in vivo. This decrease in activity in pepper was not due to storage of nitrate or nitrite ions, contrasting sharply with the effect of NO x upon the levels of NiR activity from cultivars of tomato. No effect on nitrate reductase (NaR) activity of either of the two gases was detected in pepper although an increase in NaR activity in the presence of NO 2 was shown by one tomato cultivar. This increase, which was dependent on both the photon flux density and the NO 3 − supply, involved synthesis of NaR. On the other hand nitrate reduction in the roots of both species appears to be unaffected by exposure to NO 2.

Purification and molecular properties of nitrite reductase from Anabaena sp. 7119

Ferredoxin‐nitrite reductase (EC 1.7.7.1.) from the cyanobacteria Anabaena sp. 7119 has been purified 763‐fold with a specific activity of 21.5 units/nig protein (0.358 μkatals/mg). The enzyme has a molecular mass of 52,000 daltons with a Stokes radius of 3.09 nm and a sedimentation coefficient of 4.07 S. The cellular level of nitrite reductase activity gradually increases in response to the addition of increasing amounts of iron to the culture medium.When partially purified nitrite reductase preparations are subjected to sucrose‐density‐gradient centrifugation there is a dose correspondence between nitrite reductase activity and absorbance at 400 nm. This suggests the association of a heme chromophore with the enzyme. Furthermore, the presence of an iron‐sulfur center is suggested by a close association of acid‐labile sulfide with nitrite reductase activity. Carbon monoxide inhibits nitrite reductase activity. The nature and kinetics of this reaction are comparable to other siroheme‐containing nitrite reductases.

Molybdenum and iron as functional consitituents of the enzymes of the nitrate-reducing system of Azotobacter chroococcum.

The roles of molybdenum and iron in the enzymes of the assimilatory nitrate-reducing system from Azotobacter chroococcum have been investigated. 1. By adding 99 Mo-molybdate to a cell culture of A. chrocococcum with nitrate as the nitrogen source, it has been possible to incroporate the radioactive metal into a purified preparation of the enzyme nitrate reductase. 2. When 185 W-tungstate was supplied to a culture medium lacking added molybdate, a 185 W-labelled nitrate reductase preparation with negligible activity could be obtained. This in vivo incorporation of tungsten was competitively hindered by molybdenum. 3. The cellular level of nitrite reductase activity gradually increased in response to the addition of increasing amounts of iron to the culture medium. Under the same conditions, of the level of nitrate reductase activity was not affected.

Effect of iron supply on the activities of the nitrate-reducing system from Chlorella.

In Chlorella, as in most photosynthetic organisms, the reduction of nitrate to ammonia proceeds sequentially in two independent and well characterized steps, catalyzed by the enzymes of the nitrate-reducing system: 1. the reduction of nitrate to nitrite by the flavomolybdoprotein NADH-nitrate reductase, and 2. the reduction of nitrite to ammonia by the ironprotein ferredoxin-nitrite reductase. In this communication, it is shown that, in Chlorella, the cellular level of nitrite reductase activity specifically increases in response to the iron content of the culture medium. By contrast, the activity of nitrate reductase is apparently not affected by the concentration of iron in the nutrient solution under the same conditions.