Nitrite Assimilation Research Articles

Altered photosynthetic electron channelling into cyclic electron flow and nitrite assimilation in a mutant of ferredoxin:NADP(H) reductase

The mechanism by which plants regulate channelling of photosynthetically derived electrons into different areas of chloroplast metabolism remains obscure. Possible fates of such electrons include use in carbon assimilation, nitrogen assimilation and redox signalling pathways, or return to the plastoquinone pool through cyclic electron flow. In higher plants, these electrons are made accessible to stromal enzymes, or for cyclic electron flow, as reduced ferredoxin (Fd), or NADPH. We investigated how knockout of an Arabidopsis (Arabidopsis thaliana) ferredoxin:NADPH reductase (FNR) isoprotein and the loss of strong thylakoid binding by the remaining FNR in this mutant affected the channelling of photosynthetic electrons into NADPH- and Fd-dependent metabolism. Chlorophyll fluorescence data show that these mutants have complex variation in cyclic electron flow, dependent on light conditions. Measurements of electron transport in isolated thylakoid and chloroplast systems demonstrated perturbed channelling to NADPH-dependent carbon and Fd-dependent nitrogen assimilating metabolism, with greater competition in the mutant. Moreover, mutants accumulate greater biomass than the wild type under low nitrate growth conditions, indicating that such altered chloroplast electron channelling has profound physiological effects. Taken together, our results demonstrate the integral role played by FNR isoform and location in the partitioning of photosynthetic reducing power.

Altered photosynthetic electron channeling into cyclic electron flow and nitrite assimilation and stress signaling in a mutant of ferredoxin:NADP(H) reductase.

Altered photosynthetic electron channeling into cyclic electron flow and nitrite assimilation and stress signaling in a mutant of ferredoxin:NADP(H) reductase.

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO2-) or nitrate (NO3-) as the sole nitrogen source and aerated with air or with CO2-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 mu M NaNO3 as nitrogen source. Accordingly, NO2- concentration reached 10.4 mu M after 95 h at the low CO2 condition (aerated with air); while the maximum NO2- concentration was only around 2.0 mu M at the high CO2 condition (aerated with 5% CO2 in air), furthermore, after 30 h it decreased to no more than 1.0 mu M. NO2- was almost assimilated in 80 h when C. muelleri was cultured at the high CO2 condition with 100 mu M NaNO2 as sole nitrogen source. At the high CO2 condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO2 condition. It was indicated that enriched CO2 concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO2 might be an effective way to control nitrite content in aquaculture systems.

Sulfite Reduction in Mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.

Lack of Control of Nitrite Assimilation by Ammonium in an Oceanic Picocyanobacterium, Synechococcus sp. Strain WH 8103

In cyanobacteria, the transcriptional activator NtcA is involved in global nitrogen control and, in the absence of ammonium, regulates the expression of genes involved in the assimilation of alternative nitrogen sources. The oceanic picocyanobacterium Synechococcus sp. strain WH 8103 harbors a copy of ntcA, but in the present study, we show that unlike other marine cyanobacteria that have been investigated, this strain is capable of coassimilating nitrite when grown in the presence of ammonium. Transcript levels for the genes encoding the nitrate/nitrite-bispecific permease NrtP and nitrate reductase (NarB) were substantially down-regulated by ammonium, whereas the abundances of nitrite reductase (NirA) transcripts were similar in nitrite- and ammonium-grown cells. The growth of Synechococcus sp. strain WH 8103 in medium containing both ammonium and nitrite resulted in only minor changes in the expression profile in comparison to that of nitrite-grown cells with the exception that the gene encoding the high-affinity ammonium transporter Amt1 was down-regulated to the levels seen in ammonium-grown cells. Whereas the expression of nrtP, narB, and amt1 appears to be NtcA dependent in this marine cyanobacterium, the transcription and expression of nirA appear not to be. The ability to coassimilate nitrite and reduced-nitrogen sources like ammonium may be an adaptive trait that enables oceanic strains like Synechococcus sp. strain WH 8103 to exploit the low nitrite concentrations found in oceanic surface waters that are not available to their principal and more numerous competitor, Prochlorococcus.

The glucose and nitrogen starvation response of Bacillus licheniformis

The glucose and nitrogen starvation stimulons of Bacillus licheniformis were determined by transcriptome and proteome analyses. Under both starvation conditions, the main response of B. licheniformis was a switch to the usage of alternative nutrient sources. This was indicated by an induction of genes involved in the metabolism of C-2 substrates during glucose limitation. In addition, B. licheniformis seems to be using other organic substances like amino acids and lipids as carbon sources when subjected to glucose starvation. This observation is supported by the induction of a high number of genes coding for proteins involved in amino acid and lipid degradation. During nitrogen starvation, genes for several proteases and peptidases involved in nitrate and nitrite assimilation were induced, which enables this bacterium to recruit nitrogen from alternative sources. Both starvation conditions led to a down-regulation of transcription of most vegetative genes, which was subsequently reflected by a reduced synthesis of the corresponding proteins. A selected set of genes was induced by both starvation conditions. Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response.

The gene of is involved in nitrite assimilation

To establish a basis for genetic and molecular studies of nitrite assimilation in the methylotrophic yeast Hansenula polymorpha, we isolated and characterised six nitrite-negative mutants still capable of growing on nitrate. Gene isolation work yielded the NII2 gene, encoding a membrane protein homologous to the Saccharomyces cerevisiae Pho86p. Sequence analysis revealed an ORF of 860 bp encoding a 286-amino-acid protein with a predicted molecular mass of 32.8 kDa. This protein is shorter than its S. cerevisiae homologue, and is predicted to lack an ER-retention signal. Cell suspension work revealed that the null mutant is unable to take up nitrite from the medium.

Bacterial Degradation of Cyanide and Its Metal Complexes under Alkaline Conditions

A bacterial strain able to use cyanide as the sole nitrogen source under alkaline conditions has been isolated. The bacterium was classified as Pseudomonas pseudoalcaligenes by comparison of its 16S RNA gene sequence to those of existing strains and deposited in the Coleccion Espanola de Cultivos Tipo (Spanish Type Culture Collection) as strain CECT5344. Cyanide consumption is an assimilative process, since (i) bacterial growth was concomitant and proportional to cyanide degradation and (ii) the bacterium stoichiometrically converted cyanide into ammonium in the presence of l-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. The bacterium was able to grow in alkaline media, up to an initial pH of 11.5, and tolerated free cyanide in concentrations of up to 30 mM, which makes it a good candidate for the biological treatment of cyanide-contaminated residues. Both acetate and d,l-malate were suitable carbon sources for cyanotrophic growth, but no growth was detected in media with cyanide as the sole carbon source. In addition to cyanide, P. pseudoalcaligenes CECT5344 used other nitrogen sources, namely ammonium, nitrate, cyanate, cyanoacetamide, nitroferricyanide (nitroprusside), and a variety of cyanide-metal complexes. Cyanide and ammonium were assimilated simultaneously, whereas cyanide strongly inhibited nitrate and nitrite assimilation. Cyanase activity was induced during growth with cyanide or cyanate, but not with ammonium or nitrate as the nitrogen source. This result suggests that cyanate could be an intermediate in the cyanide degradation pathway, but alternative routes cannot be excluded.

The signal transducer P(II) and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation.

The amino acid sequence of the signal transducer P(II) (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria. P(II) of P. marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942. Moreover, P. marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite. This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (C(i); K(m,app) 240 and 4 micro M, respectively), a result consistent with the unphosphorylated form of P(II) acting as a sensor for the control of C(i) acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803. The present data are discussed in relation to the genetic information provided by the P. marinus MED4 genome sequence.

Cooperation of photosystems II and I in leaves as analyzed by simultaneous measurements of chlorophyll fluorescence and transmittance at 800 nm.

Parallel measurements of CO2 assimilation, Chl fluorescence and 800 nm transmittance were carried out on intact leaves of wild type and cytochrome b6/f deficient transgenic tobacco grown at two different light intensities and temperatures, with the aim to diagnose processes limiting quantum yield of photosynthesis and investigate their adaptations to growth conditions. Relative optical cross-sections of PSII and PSI antennae were calculated from measured gas exchange rates and fluorescence-related losses at PSII and P700 oxidation-related losses at PSI. In nonstress conditions (high light grown wild type and low light grown antisense type) optimal relative optical cross-section of PSII (aII) was 0.48-0.51 and that of PSI (aI) was 0.38-0.40, leaving a non-photosynthetic absorption cross-section (a0) of 0.09-0.14 for nitrite assimilation and absorption in PSII beta and other photosynthetically inactive pigments. Stress conditions (low light grown wild type and high light grown antisense type, elevated growth temperatures) tend to increase a0 and decrease PSII antenna cross-section more than that of PSI antenna, but this rule is reversed during senescence.

Studies on nitric oxide (NO) formation by the green alga Scenedesmus obliquus and the diazotrophic cyanobacterium Anabaena Doliolum

This study provides preliminary evidence that NO production could be a general attribute of algae. Anabaena doliolum was found to be a better NO producer than Scenedesmus and Synechoccocus. Experiments conducted with inhibitors of photosynthesis (DCMU), ATP synthesis (DCCD), and the uncoupler (2,4-DNP) and its analog arsenate clearly revealed that inhibition of nitrite assimilation through the blockage of nitrite reductase (NiR) is primarily responsible for NO emission. A linear relationship between nitrite concentration in the culture medium and NO in the exhaust gas supports the view that accumulation of nitrite is responsible for NO formation. A failure of Scenedesmus, grown in the medium substituted with W for Mo, to produce either NO/NO − 2 in light 1688 or a ‘light-off’ peak, and a resumption of these activities upon the addition of Mo proved beyond doubt that a functional nitrate reductase (NR) is necessary for the production of nitrite and NO by algae grown on nitrate as the nitrogen source. Moreover, the appearance of a NO peak immediately after nitrite supplementation under dark conditions in W-substituted cultures with or without glucose ruled out an enzymatic role of NR in NO emission.

Co-ordination of the nitrate and nitrite assimilation, the glutathione and free radical metabolisms, and the pentose phosphate pathway in Penicillium chrysogenum

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3— and NO2—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

Co-ordination of the nitrate and nitrite assimilation, the glutathione and free radical metabolisms, and the pentose phosphate pathway inPenicillium chrysogenum

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO3— and NO2—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H2O2, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.

3-nitroadipate, a metabolic intermediate for mineralization of 2, 4-dinitrophenol by a new strain of a Rhodococcus species.

The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a Rhodococcus species closely related to Rhodococcus opacus. Rhodococcus sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by 1H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.

NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al.

Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the delta nasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.

Nitrogen isotope fractionation during the uptake and assimilation of nitrate, nitrite, ammonium, and urea by a marine diatom

Knowledge of the fractionation of nitrogen isotopes by phytoplankton is a key requirement for the calibration of the new δ15N paleotracer. An essential part of information required in this calibration concerns the magnitude of isotopic fractionation during the incorporation of N substrates by phytoplankton. To this end, the δ15N of batch cultures of Thalassiosira pseudonana grown on nitrate, nitrite, ammonium, and urea was determined. This paper reports the first δ15N study of phytoplankton growth on urea (e.g. organic N substrate). The δ15N of the particulate nitrogen (PN) collected during the logarithmic growth phase, thus for N‐sufficient cells, was lower than the δ15N of the source due to kinetic isotope fractionation. With increasing drawdown of the N substrate, the δ15N of the accumulating PN increased in accordance with the Rayleigh distillation model. Enrichment factors (ε) derived from a least‐squares analysis of the accumulated δ15NPN data were 5.2 ± 0.2‰, 0.9 ± 0.6‰, 20 ± 1‰, and 0.8 ± 0.6‰ for NO3−, NO2−, NH4+, and urea incorporation, respectively. Overall, ε values for nitrate incorporation were consistent with field estimates of ~6‰ and could be used to estimate past relative nitrate utilization as estimated from the 615N of bulk sedimentary organic matter.

Antitermination of transcription of catabolic operons.

Antitermination of transcription mediated by proteins interacting with mRNA sequences is described for nine operons/regulons. Eight of the systems are catabolic, while the ninth, the Klebsiella pneumoniae nas regulon, is involved in the assimilation of nitrate and nitrite. Six of the catabolic operons/regulons are found in Bacillus subtilis, one is found in Escherichia coli, and one in Pseudomonas aeruginosa. The antitermination system of five of the operons/regulons (E. coli blg, and sacPA, sacB, bgl, and lic from B. subtilis) are assigned to the bgl-sac family on the basis of extensive similarities with regard to antiterminator proteins and the sequences of the antiterminators. Other members of the bgl-sac family are the arb operon of Erwinia chrysanthemi and a presumed bgl operon of Lactococcus lactis. The antitermination systems of the other four operons/regulons (B. subtilis glp, B. subtilis hut, P. aeruginosa ami, and K. pneumoniae nas) seem to be unrelated both to the bgl-sac family and to each other. The antiterminator protein of the B. subtilis glp regulon has been found not only to cause antitermination but also to stabilize the resultant mRNA and to mediate glucose repression. If other antiterminator proteins, and antitermination factors, also prove to have additional functions, it will broaden the impact of antitermination as a means of controlling gene expression.

Nitrite assimilation by the yeast Candida nitratophila

summaryNitrite uptake in the nitrate‐assimilating yeast, Candida nitratophila, was inhibited completely by addition of nitrate or ammonium to cultures at pH 8.0). Nitrite assimilation in these cultures was also completely dependent on the availability of a suitable carbon source (glucose), In cultures at pH 5.0, neither nitrate nor ammonium inhibited nitrite uptake completely. In these cultures, some nitrite was assimilated in the absence of a carbon source. Carbon‐deficient cultures at pH 5.0 were able to reduce nitrite to ammonium but excreted this ammonium without further assimilation, Evidence for the uptake of nitrite by passive diffusion and by active transport via the nitrate uptake system is discussed.

Molecular Biology of Nitrogen Nutrition in Plants. : 2. Molecular Biology of Nitrate and Nitrite Assimilation in Higher Plants

Molecular Biology of Nitrogen Nutrition in Plants. : 2. Molecular Biology of Nitrate and Nitrite Assimilation in Higher Plants

Targeted Disruption of the NIT8 Gene in Chlamydomonas reinhardtii

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.