Nitrite Reductase

The two key enzymes of denitrification, the nitrate and nitrite reductases, were studied in a marine organism, Pseudomonas nautica, strain 617. Both enzymes were grown under anaerobic conditions with either nitrate or nitrous oxide as electron acceptor. The effect of sodium on these enzymes was studied. Only sodium activated the membranous and purified nitrate reductase, and none of the salts affected nitrite reductase.

In the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245, nitric oxide produced by denitrification could be a signal involved in stimulation of root branching, and the dissimilatory nitrite reductase gene nirK is upregulated on wheat roots. Here, it was found that Sp245 did not contain one copy of nirK but two (named nirK1 and nirK2), localized on two different plasmids, including one plasmid prone to rearrangements. Their deduced protein sequences displayed 99.2% identity but their promoter regions and upstream genetic environment differed. Phylogenetic studies revealed that nirK1 and nirK2 clustered next to most β-proteobacterial sequences rather than in the vicinity of other Azospirillum spp. and most α-proteobacterial sequences, regardless of whether DNA or deduced protein sequences were used. This points to past horizontal gene transfers. Analysis of the number of nonsynonymous and synonymous substitutions per site indicated that nirK has been subjected to neutral selection in bacteria. The use of transcriptional fusions with egfp, encoding an enhanced green fluorescent protein variant, revealed that both nirK1 and nirK2 promoter regions were upregulated in vitro under microaerobiosis or the presence of nitrite as well as on wheat roots. The analysis of nirK1 and nirK2 mutants revealed that the two genes were functional. Overall, results suggest that nirK has been acquired horizontally by A. brasilense Sp245 from a distant relative and underwent subsequent duplication; however, both paralogs remained functional and retained their upregulation by the plant partner.

In order to assess the potential of Spirulina (Arthospira) platensis as a source of abundant, thermostable nitrate assimilatory enzymes, the specific activities and thermal tolerance of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) were compared with those of rice in crude extracts in vitro. The results show that Spirulina enzymes have relatively higher thermotolerance. When the extracts were pre-exposed to 80 °C for 1 hr, Spirulina enzymes retained higher activities by 3.4, 1.7 and 3.7 fold, respectively than corresponding enzymes in rice. This property was not due to salts and other small proteins/molecules, as their removal by gel filtration (G-25) did not affect their thermotolerance.

Spirulina platensis, a cyanobacterium whose N-metabolic pathway is similar to that of higher plants like rice (Oryza sativa), produces tenfold more protein, indicating a higher capacity for nitrate utilization/removal. Our in vitro analyses in crude extracts revealed that this can be attributed, at least in part, to the higher specific activities (3-6 fold) and half lives (1.2-4.4 fold) of the N-assimilating enzymes, nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in Spirulina.

The influence of nitrate and its metabolites on the nitrate reductase (NR) gene expression and its relationship with phytochrome (Pfr) regulation of NR in etiolated maize leaves is examined. Nitrate inSuction and Pfr stimulation are brought about by independent signalling phenomena. Phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), mimicked the effect of red light but could not replace the nitrate requirement for the induction of NR transcript accumulation. This suggests that while PKC-type enzymes may be involved in mediating the Pfr signal, nitrate may follow an independent signalling mechanism. Experiments with 5-hydroxytryptamine (5HT) and lithium ions (Li+), which are known to modulate phosphoinositide (PI) turnover, indicated that in addition to generating Pfr-induced second messengers for PKC activation, PI cycle may also generate other signals which mediate nitrate induction of NR gene expression in the dark. The products of nitrate reduction ie, nitrite and ammonium ion had inhibitory and stimulatory effects respectively, on NR transcript accumulation. They work mainly at the level of nitrate induction.

In the context of climate change and biodiversity loss, rehabilitation of degraded urban soils is a means of limiting artificialization of terrestrial ecosystems and preventing further degradation of soils. Ecological rehabilitation approaches are available to reinitiate soil functions and enhance plant development. However, little is known about the long-term stability of rehabilitated soils in terms of soil functions when further natural or anthropogenic perturbations occur. Based on rehabilitated urban soils, the present study sought to evaluate the resistance and resilience of soil functions linked to carbon cycling and phosphate dynamics in addition to nitrogen cycling and related microbial communities after a heat and drought stress. A laboratory experiment was conducted in microcosms under controlled temperature conditions, with four contrasted soils collected from a rehabilitated urban brownfield; an initial, non-rehabilitated soil (IS), a technosol with a high organic matte...

Rhizobium etli CFN42 is not capable of growing anaerobically with nitrate but it grows with nitrite as a terminal electron acceptor. This bacterium contains the nirK gene encoding the copper-containing Nir (nitrite reductase), which is located on the cryptic plasmid pCFN42f. Mutational analysis has demonstrated that a nirK deficient mutant was not capable of growing under nitrite-respiring conditions. Moreover, microaerobic growth of this mutant was inhibited by the presence of nitrite. Nir activity and nitrite uptake were highly diminished in a nirK mutant, compared with the wild-type levels after incubation under anaerobic conditions. Our results suggest that the copper-containing Nir may have both a respiratory and a nitrite-detoxifying role in R. etli.

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O2) tension and nitrate (NO3(-)), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK2-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK2-NnrR circuit, we analyzed β-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK2 and nnrR mutant backgrounds. While microoxic conditions (2% O2 at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK2, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK2 and NnrR transcription factors. Purified FixK2 protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from ...

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux perme...

Nitrogen pollution has been serious problem in environmental water particularly in freshwater ecosystems. A heterotrophic nitrifier and aerobic denitrifier was isolated and characterised from domestic wastewater. Based on phenotypic and phylogenetic characteristics, the isolate was identified as Klebsiella pneumonia. The strain had the capability to utilise ammonia, nitrate and nitrite as a sole nitrogen source. The maximum growth rate (m max ) of isolate was 0.55 h À1 when ammonia used as nitrogen source. The isolate could express hydroxylamine oxidase (hao), periplasmic nitrate reductase (nap) and nitrite reductase (nir) which are essential for heterotrophic nitrification and aerobic denitrification. Both hao and napA genes were amplified in isolated strain by PCR. Furthermore, the isolate showed capacities of extracellular polymeric substances (EPS) secretion and auto-aggregation. Results demonstrated that the isolation of heterotrophic nitrifier and aerobic denitrifier favoured the bioremediation of nitrogenous compounds from domestic wastewater.

The development of the latent phenotype of Mycobacterium tuberculosis (Mtb) in the human lungs is the major hurdle to eradicate Tuberculosis. We recently reported that exposure to nitrite (10 mM) for six days under in vitro aerobic conditions completely transforms the bacilli into a viable but non-cultivable phenotype. Herein, we show that nitrite (beyond 5 mM) treated Mtb produces nitric oxide (NO) within the cell in a dose-dependent manner. Our search for the conserved sequence of NO synthesizing enzyme in the bacterial system identified MRA2164 and MRA0854 genes, of which the former was found to be significantly up regulated after nitrite exposure. In addition, the purified recombinant MRA2164 protein shows significant nitrite dependent NO synthesizing activity. The knockdown of the MRA2164 gene at mRNA level expression resulted in a significantly reduced NO level compared to the wild type bacilli with a simultaneous return of its replicative capability. Therefore, this study fir...

Ammonium regulation of cellular nitrate and nitrite reductases (NR;NiR) was studied in Phormidium uncinatum. Diminished to low activities were found with ammonia and development of the enzymes in its absence required de novo protein synthesis but not nitrate. While an inhibition of ammonium metabolism via glutamine synthetase (GS) activity reversed the ammonium effect on NR. NiR was non-responsive. Development of NiR in such cultures in absence of ammonia however needed new protein synthesis. These data suggest that aran~nia by itself and through assi~mlation exerts repression control on NiR and NR respectively, which are otherwise derepressed

Nitrite ions have emerged as one of the major problems in environmental and pollution issuesnowadays. Food products are the major source of these ions to preserve color. However, thereis a maximum permissible limit (MPL) for the use of nitrite in food. A lot of works in theliterature have been carried out on nitrite quantification and most of them are using simple andquick analysis methods. Recently, the advent of biosensor has allowed the fabrication ofsensors for the detection of nitrite. This paper specifically reviews the interest in the usage ofthe nitrite reductase enzyme as a biorecognition element for the application of nitritebiosensors.

Degenerate primers to detect nrfA were designed by aligning six nrfA sequences including Escherichia coli K-12, Sulfurospirillum deleyianum and Wolinella succinogenes. These primers amplified a 490 bp fragment of nrfA. The ability of these primers to detect nrfA was tested with chromosomal DNA isolated from a variety of bacteria: they could distinguish between bacteria in which the gene is known to be present or absent. The positive reference organisms spanned the various classes of Proteobacteria, suggesting that these primers are probably generic. The primer pair F1 and R1 was also used successfully to analyse nrfA diversity from community DNA isolated from a sulphate reducing bioreactor, and from two established Anammox reactors (for anaerobic ammonia oxidation, in which nitrite is reduced by ammonia to dinitrogen gas). The nrfA clones isolated from these three sources grouped with the Bacteroidetes phylum. The nrfA primers also amplified 570 bp fragments from the Anammox community DNA. These fragments encoded a protein with four haem-binding motifs typical of a c-type cytochrome, but were unrelated to the NrfA nitrite reductase. A BLAST search failed to reveal similarity to any known proteins. However, similarity was found to one sequence, which was annotated as rapC (response regulator aspartate phosphatase), in the genome of the planctomycete Rhodopirellula baltica. These sequences possibly belong to a new class of c-type cytochrome that might be specific to members of the order Planctomycetales. The data are consistent with the proposal that cytochrome c nitrite reductases, present in the periplasm of Gram-negative bacteria, are widely distributed in many different environments where they provide a short circuit in the biological nitrogen cycle by reducing nitrite directly to ammonia.

This work reports the evaluation of a set of multi-walled carbon nanotubes (MWCNT) presenting different surface chemistries, as interfaces for the direct electrochemistry of the multihemic nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC27774 (Dd). The carbon nanotubes dispersions were prepared in aqueous media and deposited on pyrolytic graphite (PG) macroelectrodes, following a layer-by-layer methodology. The resulting MWCNT bed was coated with ccNiR and studied by cyclic voltammetry. Interestingly, although small non-catalytic cathodic waves were detected in all carbon nanotubes bioconjugates, the complexity of these electrochemical signals was partially deconvoluted in some materials, the less acidic ones emphasizing the contribution of the catalytic centre. Consistently, these MWCNT were the most favourable for enzyme catalysis, highlighting the importance of the surface oxide functionalities to enzyme reactivity.

Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b5. On SDS gels, the fraction showed two heme-containing bands of -10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b5 starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b54ike cytochromes.

A cobalt-tripeptide complex (CoGGH) is developed as an electrocatalyst for the selective sixelectron, eight-proton reduction of nitrite to ammonium in aqueous buffer near neutral pH. The onset potential for nitrite reduction occurs at -0.65 V vs. Ag/AgCl (1 M KCl). Controlled potential electrolysis at -0.90 V generates ammonium with a faradaic efficiency of 90 ± 2% and a turnover number of 3,550 ± 420 over 5.5 hours. CoGGH also catalyzes the reduction of the proposed intermediates nitric oxide and hydroxylamine to ammonium. These results reveal that a simple metallopeptide is an active functional mimic of the complex enzymes cytochrome c nitrite reductase and siroheme-containing nitrite reductase.

A cobalt-tripeptide complex (CoGGH) is developed as an electrocatalyst for the selective sixelectron, eight-proton reduction of nitrite to ammonium in aqueous buffer near neutral pH. The onset potential for nitrite reduction occurs at -0.65 V vs. Ag/AgCl (1 M KCl). Controlled potential electrolysis at -0.90 V generates ammonium with a faradaic efficiency of 90 ± 2% and a turnover number of 3,550 ± 420 over 5.5 hours. CoGGH also catalyzes the reduction of the proposed intermediates nitric oxide and hydroxylamine to ammonium. These results reveal that a simple metallopeptide is an active functional mimic of the complex enzymes cytochrome c nitrite reductase and siroheme-containing nitrite reductase.

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ⌬narK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants were found to be severely restricted in nitratedependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ⌬narK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15 NO 2 -to 15 NO, the oxidation of hydroxylamine ( 15 NH 2 OH) to 15 NO, and the production of 14-15 N 2 O from 15 NH 2 OH and 14 NO 2 -. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO3– in a hydroponic system, growth and N‐assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH4+, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO3– supplied plants. NO3– was not detected in plant material that had been solely supplied with NH4+, there was no NO3– contamination of the hydroponic system, and the NH4+‐induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO3– or NH4+, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO3– assimilation was i...

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N 2 O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N 2 O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.

Purpose: Ensifer meliloti, the endosymbiont of alfalfa, contains all the denitrification genes but the capacity of alfalfa root nodules to produce N2O is not known. In this work, N2O emissions as well as the influence of bacteroidal denitrification on nodulation competitiveness and N2O release from alfalfa nodules has been investigated. Methods: Medicago sativa cv. Victoria plants were inoculated with E. meliloti 1021 and napA-, nap+ and nosZ- mutants. Plants were grown in the presence of different nitrate and copper treatments and subjected to flooding during one week before harvesting. MV+-NR and MV+-NIR enzymatic activities were measured in isolated bacteroids by analysing the capacity of the cells to produce or consume nitrite, respectively. Bacteroidal nitrous reductase (N2OR) activity was determined by measuring N2O consumption capacity. N2O was analysed by using a gas cromatograph. Results: Alfalfa root nodules are able to produce N2O in response to nitrate and flooding. Over...

The CuA centers of cytochrome c oxidases are unique examples of a new type of binuclear copper cluster. X -ray crystallography of enzymes from beef heart, Paracoccus, and the engineered cyoA fragment of the quinol oxidase of E. Coli have provided a structural description of the site. The coppers are bridged by two cysteine ligands, and have an extremely short Cu-Cu distance of

Ynt1 is the only high-affinity nitrate uptake system in Hansenula polymorpha. Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels. Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild-type. Nitrite uptake in a wild-type strain was partially inhibited by nitrate to levels shown by a YNT1disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate. It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite-specific transporter(s). The nitrite-specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes. Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s). At pH 5.5, the contribution of Ynt1 to high-affinity nitrate and nitrite uptake was around 95% and 60%, respectively. The apparent K m of Ynt1 for nitrate and nitrite is in the µM range, as is the specific nitrite uptake system for nitrite. The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite-specific system.

The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0-10 cm), horizon B (45-55 cm), and horizon C (120-130 cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P < 0.03). Denitrifier gene copy concentrations per gram of soil (GCC) varied in response to carbon type amendment (P < 0.01). Denitrifier GCCs were high (ca. 10(7)) and the bac:nirK, bac:nirS, bac:nir (T) , and bac:nosZ ratios were low (ca. 10(-1)/10) in ...

Polyamines and guanidines enhanced the growth of radish seedlings grown in dark or light, irrespective of the supply of nitrogen. All the compounds inhibited ntirate reducatase and glutamine synthetase in the cotyledons of light-grown but not in darkgrown seeds. Nitrite reductase and glutamate dehydrogenase were not affected. Protease activity was enhanced by all the compounds in dark-as well as in light-grown seeds. Alanine aminotransferase activity was increased only in the light-grown seeds. The inhibition of nitrate reductase was not due to decreased nitrate uptake but was due to a decreased metabolic pool of nitrate and a decline in enzyme synthesis. The inhibition of glutamine synthetase and activation of alanine aminotransferase by the compounds was found only in the chloroplast fraction. The activation of protease was due to the release or activation of preexisting enzyme while that of alanine aminotransferase was dependent on the de novo protein synthesis which was abolished by cycloheximide.

supplemented with chromium (0.75 mM). After 120-d of growth under chromium (Cr) stress, Carbon (C) and nitrogen (N) metabolism in leaves were studied. In the C metabolism study sucrose, glucose, fructose, starch contents, and the enzymatic activities of acid invertase, neutral invertase, sucrose synthetase, sucrose phosphatesynthetase, fructokinase (FK), hexokinase (HK), phosphofructokinase, pyruvate kinase were investigated. Nitrogen metabolism study was comprised of determination of nitrate, nitrite and ammonium content and the nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase activities. Basically, in the C metabolism study the Cr enhanced the enzymatic activities and sugar contents, and decreased starch. Chromium stress caused a decrease in nitrate and nitrite, and an increase in ammonia; it also inhibited all measured enzymatic activities except glutamate dehydrogenase which was increased. The responses of Kinnow mandarin plants grafted on 2x or 4x for any rootstock followed a similar pattern, although the effects of Cr toxicity were more drastic in 2x grafted plants. Glycolysis was the only metabolic process which had a different behavior between 2x and 4x grafted plants under Cr stress: FK and HK activities under Cr stress were significantly increased in 4x grafted plants than in 2x grafted plants. These data suggest that in Kinnow mandarin plants grafted 4x rootstocks the C metabolism is modified thereby strengthening the plants to adapt Cr stress, but N metabolism was inhibited by Cr toxicity.

Bacterial denitrification results in the loss of fertilizer nitrogen and greenhouse gas emissions as nitrous oxides, but ecological factors in soil influencing denitrifier communities are not well understood, impeding the potential for mitigation by land management. Communities vary in the relative abundance of the alternative dissimilatory nitrite reductase genes nirK and nirS, and the nitrous oxide reductase gene nosZ; however, the significance for nitrous oxide emissions is unclear. We assessed the influence of different long-term fertilization and cultivation treatments in a 160year-old field experiment, comparing the potential for denitrification by soil samples with the size and diversity of their denitrifier communities. Denitrification potential was much higher in soil from an area left to develop from arable into woodland than from a farmyard manure-fertilized arable treatment, which in turn was significantly higher than inorganic nitrogen-fertilized and unfertilized arable plots. This correlated with abundance of nirK but not nirS, the least abundant of the genes tested in all soils, showing an inverse relationship with nirK. Most genetic variation was seen in nirK, where sequences resolved into separate groups according to soil treatment. We conclude that bacteria containing nirK are most probably responsible for the increased denitrification potential associated with nitrogen and organic carbon in this soil.

Microbial communities in marine oxygen deficient zones (ODZs) are responsible for up to half of marine N loss through conversion of nutrients to N 2 O and N 2 . This N loss is accomplished by a consortium of diverse microbes, many of which remain uncultured. Here, we characterize genes for all steps in the anoxic N cycle in metagenomes from the water column and >30 µm particles from the Eastern Tropical North Pacific (ETNP) ODZ. We use an approach that allows for both phylogenetic identification and semi-quantitative assessment of gene abundances from individual organisms, and place these results in context of chemical measurements and rate data from the same location. Denitrification genes were enriched in >30 µm particles, even in the oxycline, while anammox bacteria were not abundant on particles. Many steps in denitrification were encoded by multiple phylotypes with different distributions. Notably three N 2 O reductases (nosZ), each with no cultured relative, inhabited distinct niches; one was free-living, one dominant on particles and one had a C terminal extension found in autotrophic S-oxidizing bacteria. At some depths >30% of the community possessed nitrite reductase nirK. A nirK OTU linked to SAR11 explained much of this abundance. The only bacterial gene found for NO reduction to N 2 O in the ODZ was a form of qnorB related to the previously postulated "nitric oxide dismutase," hypothesized to produce N 2 directly while oxidizing methane. However, similar qnorB-like genes are also found in the published genomes of many bacteria that do not oxidize methane, and here the qnorB-like genes did not correlate with the presence of methane oxidation genes. Correlations with N 2 O concentrations indicate that these qnorB-like genes likely facilitate NO reduction to N 2 O in the ODZ. In the oxycline, qnorB-like genes were not detected in the water column, and estimated N 2 O production rates from ammonia oxidation were insufficient to support the observed oxycline N 2 O maximum. However, both qnorB-like and nosZ genes were present within particles in the oxycline, suggesting a particulate source of N 2 O and N 2 . Together, our analyses provide a holistic view of the diverse players in the low oxygen nitrogen cycle.

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (α2β2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1Δ/Δ) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/Δ36-39). In EC Hba1Δ/Δ or Hba1WT/Δ36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of th...

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l -1 nitrite at a rate of 0.27 mg NO 2 -N g -1 VSS h -1 . Notably, 1,500 mg l -1 acetate and 700 mg l -1 methanol or ethanol enhanced denitrification rates for 200 mg l -1 nitrite at 2.07, 1.20, and 1.60 mg NO 2 -N g -1 VSS h -1 , respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37-41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol>acetate>ethanol on molar basis or acetate>ethanol>methanol on an added weight basis.

Experimental evidence has shown that nitrite anion plays a key role in one of the proposed mechanisms for hypoxic vasodilation, in which the erythrocyte acts as a NO generator and deoxygenated hemoglobin in pre-capillary arterioles reduces nitrite to NO, which contributes to vascular smooth muscle relaxation. However, because of the complex reactions among nitrite, hemoglobin, and the NO that is formed, the amount of NO delivered by this mechanism under various conditions has not been quantified experimentally. Furthermore, paracrine NO is scavenged by cell-free hemoglobin, as shown by studies of diseases characterized by extensive hemolysis (e.g., sickle cell disease) and the administration of hemoglobin-based oxygen carriers. Taking into consideration the free access of cell-free hemoglobin to the vascular wall and its ability to act as a nitrite reductase, we have now examined the hypothesis that in hypoxia this cell-free hemoglobin could serve as an additional endocrine source of NO. In this study, we constructed a multicellular model to characterize the amount of NO delivered by the reaction of nitrite with both intraerythrocytic and cell-free hemoglobin, while intentionally neglecting all other possible sources of NO in the vasculature. We also examined the roles of hemoglobin molecules in each compartment as nitrite reductases and NO scavengers using the model. Our calculations show that: (1) $0.04 pM NO from erythrocytes could reach the smooth muscle if free diffusion were the sole export mechanism; however, this value could rise to $43 pM with a membrane-associated mechanism that facilitated NO release from erythrocytes; the results also strongly depend on the erythrocyte membrane permeability to NO; (2) despite the closer proximity of cell-free hemoglobin to the smooth muscle, cell-free hemoglobin reaction with nitrite generates approximately 0.02 pM of free NO that can reach the vascular wall, because of a strong self-capture effect. However, it is worth noting that this value is in the same range as erythrocytic hemoglobin-generated NO that is able to diffuse freely out of the cell, despite the tremendous difference in hemoglobin concentration in both cases (lM hemoglobin in plasma vs. mM in erythrocyte); (3) intraerythrocytic hemoglobin encapsulated by a NO-resistant membrane is the major source of NO from nitrite reduction, and cell-free hemoglobin is a significant scavenger of both paracrine and endocrine NO.

The genes that encode the bc‐type nitric‐oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1‐type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu‐wenhoek 66, 111–127]. norC and norB encode the cytochrome‐c ‐containing subunit II and cytochrome‐b ‐containing subunit I of nitric‐oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP‐binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonus aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane α‐helices and has similarity to CoxIII, the third subunit of the aa3...

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane‐bound protein with six transmembrane helices, a large periplasmic domain and cysteine‐rich cytoplasmic domains that resemble the binding sites of [4Fe‐4S] clusters in many ferredoxin‐like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI‐deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N‐oxides. The NirX‐deficient mutant transiently accumulated nitrite in the growth medium, but it had a...

Production of nitric oxide (NO) and nitrous oxide (N 2 O) by ammonia (NH 3 )-oxidizing bacteria in natural and man-made habitats is thought to contribute to the undesirable emission of NO and N 2 O into the earth's atmosphere. The NH 3 -oxidizing bacterium Nitrosomonas europaea expresses nitrite reductase (NirK), an enzyme that has so far been studied predominantly in heterotrophic denitrifying bacteria where it is involved in the production of these nitrogenous gases. The finding of nirK homologues in other NH 3 -oxidizing bacteria suggests that NirK is widespread among this group; however, its role in these nitrifying bacteria remains unresolved. We identified a gene, nsrR , which encodes a novel nitrite (NO 2 -)-sensitive transcription repressor that plays a pivotal role in the regulation of NirK expression in N. europaea . NsrR is a member of the Rrf2 family of putative transcription regulators. NirK was expressed aerobically in response to increasing concentrations of NO 2 -and decreasing pH. Disruption of nsrR resulted in the constitutive expression of NirK. NsrR repressed transcription from the nirK gene cluster promoter (P nir ), the activity of which correlated with NirK expression. Reconstruction of the NsrR-P nir system in Escherichia coli revealed that repression by NsrR was reversed by NO 2 -in a pH-dependent manner. The findings are consistent with the hypothesis that N. europaea expresses NirK as a defence against the toxic NO 2 that is produced during nitrification.

This study evaluated the effects of light intensity and the spectrum produced by light-emitting diodes (LEDs) on the growth and nitrate assimilation of tatsoi (Brassica rapa var. rosularis), grown in a controlled environment. A system of high-powered solid-state lighting modules with blue 445, red 640 and 660 and far-red 731 nm LEDs were used for illumination. Two experiments were performed seeking to explore the light parameter effects on tatsoi growth, nitrate, nitrite, total protein, and free amino acid contents, as well as soluble sugar contents and chlorophyll indexes: (1) investigation of the effects of a photosynthetically active photon flux density (PPFD) of 100, 200, 300, 400 and 500 μmol m-2 s-1 and (2) evaluation of the effects of different combinations of red (R660 and R660 + R640), red supplemented with blue (R660 + B445 and R660 + R640+B445) and far red (R660 + R640+B445+FR731) LEDs. A deficient light intensity resulted in nitrate and nitrite accumulation in tatsoi leaves; under the exposure of 100 μmol m-2 s-1 PPFD, tatsoi contained 3.4 times higher NO 3 and 34 times higher NO 2 contents, compared to that under 200 μmol m-2 s-1. The photosynthetic photon flux density of ∼300 μmol m 2 s −1 allowed the obtaining of an optimal tatsoi production with high biomass and minimal nitrate and nitrite contents in the leaves. Long term exposure of sole red light was stressful for tatsoi growth; however the addition of blue light to the red 660-and 640-nm LEDs resulted in the highest total protein and essential amino acid contents, leaf area and biomass, and significantly reduced nitrate but increased nitrite content.

Nitrite (NO2−) recycling to nitric oxide (NO) is catalysed by a number of enzymes and induces a protective vasodilation effect under hypoxia/ischaemia. In the present work, we tested the in vitro ability of the three NOS (nitric oxide synthase) isoforms to release NO from nitrite under anoxia using electrochemical detection, chemiluminescence and absorption spectroscopy. The release of free NO from anoxic nitrite solutions at 15 μM was specific to the endothelial NOS isoform (eNOS) and did not occur with the neuronal (nNOS) or inducible (iNOS) isoforms. Unlike xanthine oxidase, the eNOS reductase domain did not recycle nitrite to NO, and wild-type eNOS did not reduce nitrate. Our data suggest that structural and, by inference, dynamic differences between nNOS and eNOS in the distal haem side account for eNOS being the only isoform capable of converting nitrite into NO at pH 7.6. In human dermal microvascular endothelial cells under careful control of oxygen tension, the rates of NO ...

The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (encoding NO 2 − →NO reductases) and couples growth to NO 3 − reduction to NH 4 + (respiratory ammonification) and to N 2 O reduction to N 2 . A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO 2 − to form N 2 O (i.e., chemodenitrification). Following the addition of 100 μmol of NO 3 − or NO 2 − to Fe(III)-grown axenic cultures of A. dehalogenans , 54 (±7) μmol and 113 (±2) μmol N 2 O-N, respectively, were produced and subsequently consumed. The conversion of NO 3 − to N 2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans . The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NC...

Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have been studied and found to be dominated by pronounced interactions between the c and the d 1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime example of intraprotein control of the electrontransfer rates by allosteric interactions.

When Suljia-ospirillum deleyianum [l], was cultivated under anoxic conditions with nitrate as terminal electron acceptor (reduced to ammonia via nitrite as only liberated intermediate), both the soluble and the membrane fraction contained nitrite reductase (NiR) activity [2]: N02-+ 6 e' + 8 H+-> NHq+ + 2 H20 NiR was isolated from the soluble and the membrane fraction of S. deleyianum and from the membrane fraction of W. succinogenes. The NiR activity of the membrane was purified as a monomer (57 kDa by SDS-PAGE and gelfiltration) and heterooligomer (250 kDa by gelfiltration), whereas the soluble NiR was obtained only as a monomer (57 kDa by SDS-PAGE and gelfiltration). The membrane-bound NiR complex of both bacteria consisted of 4 x 57 kDa plus 1 x 22 kDa (gelfiltration, cross-linking, SDS-PAGE). The different forms of NiR presented identical epitopes (Western blot). However, the NiR from W. succinogenes only exhibited a weak cross reactivity with the antiserum raised against the soluble NiR from S. deleyianum. The UVMS spectra are typical for c-type cytochromes including a high-spin heme band at 610 nm (oxidized). EPR spectroscopy revealed additional interactions in the membrane-bound NiR complex that were not observed for the monomer. A redox titration followed by EPR gave ferric heme signals with redox potentials lower than-0.35 V in the membrane-bound NiR complex. However, these signals were not observed for the monomer. Rapid scan stopped-flow reduction kinetics of the NiR monomer in the ms time scale exhibited a biphasic time course. This biphasic time course was also observed for the NiR complex. The second order rate constants for the fast phase and the slow phase are different for the monomer and the complex. These results, in combination with new data on the molecular composition, will be discussed in terms of a mechanistic model. [l] W.

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c 8 with low-and high-midpoint potentials, and cytochrome c 4 as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wildtype cells. This cytochrome c has a redox midpoint potential of about + 280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c 8 of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd 1-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c 8 in this bacterium is NirM. The two other cytochromes c 8 , of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/ nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.