Nitrite Reductase Enzyme Research Articles

Preventing Nitrite Desorption via Switching Hydrogenation Position: A Dual-Site Approach for Selective Nitrate Reduction to Ammonia.

The electrochemical nitrate reduction reaction (NO3RR), which converts harmful nitrates into valuable ammonia (NH3) with zero carbon emission, is one of the most promising alternatives to the Haber-Bosch process. However, the NO3RR process is complex and involves multiple proton-coupled electron transfers that generate intermediates or byproducts, such as NO2 -, resulting in low ammonia yields and faradaic efficiency (FE). Herein, by constructing a FeCu bimetallic catalyst (FeCu-NC), the hydrogenation position of *NO3 is switched at the FeCu dual-atom site, preventing the desorption of *NO2 intermediate. Furthermore, electron transfer from Cu to Fe sites mimics the electron flow direction in natural nitrite reductase enzymes and accelerates the reduction of *NO2 to NH3, achieving efficient conversion of NO3 - to NH3. A 24-hour electrocatalytic experiment with FeCu-NC demonstrates negligible NO2 - formation throughout the NO3RR process, with an ammonia production rate of 6.13mgh-1mgcat -1 and an impressive FE of 95%, which are remarkably superior in comparison to most of the NO3RR electrocatalysts. This work opens new avenues for the fundamental understanding of catalytic mechanisms and the development of next-generation catalysts for sustainable ammonia production.

Acid-catalyzed Transformation of Nitrite to Nitric Oxide on Copper(II)-Cobalt(II) Centers in a Bimetallic Complex.

Nitrite (NO2 -) serves as a pool of nitric oxide (NO) in biological systems under hypoxic conditions, and it is transformed to NO by nitrite reductase (NiR) enzyme in the presence of acid (H+ ions). However, NO synthases (NOSs) generate NO via L-arginine oxidation in normoxic conditions. Previously, acid-induced NO2 - reduction chemistry was modeled on mono-metallic 3d-metals, generating metal-nitrosyls or NO(g) with H2O or H2O2 products. Herein, to understand the relative potency of a bimetallic system, we report the acid-induced reductive conversion of η2-bound NO2 - to NO on CuII-CoII centers of a hetero-bimetallic CuII-nitrito-CoII complex, [(LN8H)CuII-NO2 --CoII]3+ (CuII-NO2 --CoII, 2) bearing an octadentate N8-cryptand ligand (LN8H). The CuII-NO2 --CoII generates [CuII(LN8H)CoII]4+ (1) upon reaction with one equiv. acid (HClO4, H+ ions source) with NO(g) via a presumed transient nitrousacid (ONOH) intermediate species. Likewise, this NO2 - reduction was found to form H2O, which is believed to be from the decomposition of H2O2, an intermediate species. In addition, complex 2, in the presence of more than one equiv. H+ ions also showed the formation of NO(g) with H2O. Mechanistic investigations, using 15N-labeled-15NO2 -, 18O-labeled-18O14N16O- and 2H-labeled-DClO4 (D+ source), revealed that the N-atom and O-atom in the 14/15NO and 14N18O gases are derived from NO2 - ligand and H-atom in H2O derived from H+-source, respectively.

Interspecies electron transfer and microbial interactions in a novel Fe(II)-mediated anammox coupled mixotrophic denitrification system

This study effectively coupled anammox and mixotrophic denitrification at a high nitrogen load rate of 6.84 g N/L/d with 40 mg/L Fe(II). Fe(II) enhanced the activity of nitrate reductase, nitrite reductase, and hydrazine dehydrogenase enzymes, facilitating accelerated ATP synthesis. Through electrochemical experiments, interspecies electron transfer processes in coupled system were explored. Fe(II) promoted flavin mononucleotide secretion, enhancing electron-donating and electron-accepting capacity by 2.8 and 1.3 times, respectively. Fe(II) triggered the enrichment of autotrophic denitrifying bacteria (Azospira and Hydrogenophaga), transitioning from single organic nutrient to mixotrophic denitrification. Meanwhile, Fe(II) increased Candidatus_Kuenenia abundance from 35.2 % to 49.0 %, establishing the competitive advantage of anammox bacteria over completed denitrifying bacteria (Comamonas). The synergistic interactions between anammox and various denitrification pathways achieved a nitrogen removal rate of 5.88 g N/L/d, with anammox contribution rate of 88.3 %. This study provides insights into broadening the application of partial denitrification /anammox and electron transfer in multi-bacterial coupling systems.

Microbial community and extracellular polymeric substance dynamics in arid–zone temporary pan ecosystems

Microbial extracellular polymeric substances (EPS) are an important component in sediment ecology. However, most research is highly skewed towards the northern hemisphere and in more permanent systems. This paper investigates EPS (i.e., carbohydrates and proteins) dynamics in arid Austral zone temporary pans sediments. Colorimetric methods and sequence–based metagenomics techniques were employed in a series of small temporary pan ecosystems characterised by alternating wet and dry hydroperiods. Microbial community patterns of distribution were evaluated between seasons (hot–wet and cool–dry) and across depths (and inferred inundation period) based on estimated elevation. Carbohydrates generally occurred in relatively higher proportions than proteins; the carbohydrate:protein ratio was 2.8:1 and 1.6:1 for the dry and wet season respectively, suggesting that EPS found in these systems was largely diatom produced. The wet– hydroperiods (Carbohydrate mean 102 μg g−1; Protein mean 65 μg g−1) supported more EPS production as compared to the dry– hydroperiods (Carbohydrate mean 73 μg g−1; Protein mean 26 μg g−1). A total of 15,042 Unique Amplicon Sequence Variants (ASVs) were allocated to 51 bacterial phyla and 1127 genera. The most abundant genera had commonality in high temperature tolerance, with Firmicutes, Actinobacteria and Proteobacteria in high abundances. Microbial communities were more distinct between seasons compared to within seasons which further suggested that the observed metagenome functions could be seasonally driven. This study's findings implied that there were high levels of denitrification by mostly nitric oxide reductase and nitrite reductase enzymes. EPS production was high in the hot–wet season as compared to relatively lower rates of nitrification in the cool–dry season by ammonia monooxygenases. Both EPS quantities and metagenome functions were highly associated with availability of water, with high rates being mainly associated with wet– hydroperiods compared to dry– hydroperiods. These data suggest that extended dry periods threaten microbially mediated processes in temporary wetlands, with implications to loss of biodiversity by desiccation.

Microplastics shaped performance, microbial ecology and community assembly in simultaneous nitrification, denitrification and phosphorus removal process

The widespread use of microplastics (MPs) has led to an increase in their discharge to wastewater treatment plants. However, the knowledge of impact of MPs on macro-performance and micro-ecology in simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) systems is limited, hampering the understanding of potential risks posed by MPs. This study firstly comprehensively investigated the performance, species interactions, and community assembly under polystyrene (PS) and polyvinyl chloride (PVC) exposure in SNDPR systems. The results showed under PS (1, 10 mg/L) and PVC (1, 10 mg/L) exposure, total nitrogen removal was reduced by 3.38–10.15 %. PS and PVC restrained the specific rates of nitrite and nitrate reduction (SNIRR, SNRR), as well as the activities of nitrite and nitrate reductase enzymes (NIR, NR). The specific ammonia oxidation rate (SAOR) and activity of ammonia oxidase enzyme (AMO) were reduced only at 10 mg/L PVC. PS and PVC enhanced the size of co-occurrence networks, niche breadth, and number of key species while decreasing microbial cooperation by 5.85–13.48 %. Heterogeneous selection dominated microbial community assembly, and PS and PVC strengthened the contribution of stochastic processes. PICRUSt prediction further revealed some important pathways were blocked by PS and PVC. Together, the reduced TN removal under PS and PVC exposure can be attributed to the inhibition of SAOR, SNRR, and SNIRR, the restrained activities of NIR, NR, and AMO, the changes in species interactions and community assembly mechanisms, and the suppression of some essential metabolic pathways. This paper offers a new perspective on comprehending the effects of MPs on SNDPR systems.

Unravelling the interplay of different traits and parameters related to nitrogen use efficiency in wheat for climate‐resilient agriculture

AbstractEnhancing Nitrogen Use Efficiency (NUE) is extremely important towards mitigating climate change, especially in wheat where the NUE is less than 50%. Hence, optimizing grain yield under reduced application of nitrogenous fertilizer is a significant challenge. To address this challenge, a comprehensive study was conducted to investigate various agronomic traits and morphological, biochemical and molecular parameters related to NUE. This study explored their interrelationships and effects on grain yield, providing novel insights that were not previously reported. A set of 278 diverse wheat genotypes were assessed, encompassing eight NUE‐related field traits. All traits' values were reduced under stressed N (ranging from 7.5% to 77.5%) except Nitrogen Utilization Efficiency (NUtE) and NUE. Data analysis showed a significant positive correlation between grain yield and all other NUE‐related traits (r2 value ranged from .23 to 1.00), highlighting their relevance in comprehending the biological NUE of wheat plants. Principal component analysis (PCA) also revealed that N at head and N at harvest were more connected with gain yield, NUE and biomass under the optimum N condition, but less connected with gain yield and NUE under the stressed N condition. To complement the field data, representative genotypes were further subjected to a hydroponics experiment under absolute N control to study the different morphological parameters, photosynthetic pigments and the performance of essential N‐ and C‐metabolizing enzymes at the seedling stage. N stress had a detrimental impact on the majority of the parameters (−0.84% to −79.8%). Nitrite reductase (NiR), glutamate dehydrogenase (GDH) and isocitrate dehydrogenase (ICDH) enzymes as well as root length (RL), root fresh weight (RFW) and CS transcript, were positively affected by 5.9%–35.6%. The correlation analysis highlighted the substantial influence of four key N‐metabolizing enzymes, namely nitrate reductase (NR), glutamine synthetase (GS), glutamate oxo‐glutarate aminotransferase (GOGAT), and GDH on grain yield. Additionally, this study highlighted the direct and indirect associations between seedling parameters and field traits, where shoot and root length were found to be most significant for N acquisition, especially under N stress. In conclusion, these findings offer valuable insights into the intricate network of traits and parameters influencing wheat grain yield under varying N regimes.

Identification of a Strain Degrading Ammonia Nitrogen, Optimization of Ammonia Nitrogen Degradation Conditions, and Gene Expression of Key Degrading Enzyme Nitrite Reductase

In recent years, nitrogen pollutants have become one of the main causes of water pollution and eutrophication; thus, it is very important to increase the research on nitrogen removal in wastewater. In this study, a bacterium with outstanding ammonia nitrogen degradation capability was isolated from piggery wastewater and identified as Bacillus tequilensis (designated as A2). The ammonia nitrogen degradation rate of A2 reached the highest level (95%) when the incubation temperature was 42 °C, the initial pH was 7, the seed volume was 5%, the rotation speed was 160 r·min−1, the C/N was 10:1, and the carbon source was sodium citrate. A new nitrite reductase gene was successfully expressed in E. coli BL21 (DE3), and the result showed that the enzyme gene contained 2418 bp and 805 encoding amino acids, the recombinant enzyme was purified through an Ni2+ affinity chromatography column, it had a molecular weight of about 94 kDa, it displayed the maximum enzyme activity at 40 °C and pH 6.0, it exhibited good stability in the range of 25 °C to 35 °C, and it showed a pH of 6.0 to 7.0. A 1 mM concentration of Fe3+ promoted the enzyme activity, followed by a 1 mM concentration of Fe2+ and Mg2+. The kinetic parameters of Km, Kcat, and the Vmax of NiR-A2 were calculated to be 1.37 μmol·mL−1, 4.9 × 102 s−1, and 23.75 μmol·mg−1·min−1, respectively. This strain shows good prospects for wastewater treatment, especially in the treatment of high concentration ammonia nitrogen and nitrite degradation, because of its tolerance to and high degradation rate of high concentrations of ammonia nitrogen and high nitrite.

Research Progress of Nitrite Metabolism in Fermented Meat Products.

Nitrite is a common color and flavor enhancer in fermented meat products, but its secondary amines may transfer to the carcinogen N-nitrosamines. This review focuses on the sources, degradation, limitations, and alteration techniques of nitrite. The transition among NO3- and NO2-, NH4+, and N2 constitutes the balance of nitrogen. Exogenous addition is the most common source of nitrite in fermented meat products, but it can also be produced by contamination and endogenous microbial synthesis. While nitrite is degraded by acids, enzymes, and other metabolites produced by lactic acid bacteria (LAB), four nitrite reductase enzymes play a leading role. At a deeper level, nitrite metabolism is primarily regulated by the genes found in these bacteria. By incorporating antioxidants, chromogenic agents, bacteriostats, LAB, or non-thermal plasma sterilization, the amount of nitrite supplied can be decreased, or even eliminated. Finally, the aim of producing low-nitrite fermented meat products is expected to be achieved.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

Klebsiella oxytoca (EN-B2): A novel type of simultaneous nitrification and denitrification strain for excellent total nitrogen removal during multiple nitrogen pollution wastewater treatment

The poor total nitrogen (TN) removal rate achieved using microorganisms to treat wastewater polluted with multiple types of nitrogen was improved using a novel simultaneous nitrification and denitrification strain (Klebsiella oxytoca EN-B2). Strain EN-B2 rapidly eliminated ammonium, nitrate, and nitrite, giving maximum elimination rates of 4.58, 7.46, and 7.83 mg/(L h), respectively, equivalent to TN elimination rates of 4.35, 6.92, and 7.11 mg/(L h), respectively. The simultaneous nitrification and denitrification system gave ammonium and nitrite elimination rates of 7.14 and 9.17 mg/(L h), respectively, and a TN elimination rate ≥ 9.0 mg/(L h). Nitrogen balance calculations indicated that 51.22 %, 31.62 % and 46.82 % of TN in systems containing only ammonium, nitrite, and nitrate, respectively, were lost as nitrogenous gases. The ammonia monooxygenase, hydroxylamine oxidoreductase, nitrate reductase and nitrite reductase enzyme activities were determined. The results indicated that strain EN-B2 can be used to treat wastewater polluted with multiple types of nitrogen.

Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli

The assimilatory nitrite reductase enzyme NirB and small subunit NirD genes encoded in nir operon in Escherichia coli were cloned into the pET28a vector, and the recombinant enzyme was characterized for the first time. Docking of NirB with NirD, NADH, NO2-, NO3-, and CHO2- was performed using docking modeling programs. Methyl viologen and sodium dithionite were used as electron couples, and the amount of reduced nitrite was measured to calculate enzyme activity. NirB is the main enzyme and shows high activity with or without NirD. However, the inclusion of NirD into the enzyme solution at a ratio of 1NirD:2NirB resulted in 10% higher nitrite reductase activity. The enzyme tends to aggregate in the absence of β-mercaptoethanol, which causes the conversion of tetrameric NirB to monomeric form, and the NirB enzyme shows its highest activity in monomeric form. The optimum temperature for enzyme activity was 37°C and the optimum pH was found to be 7.0. Km and Vmax values of NirB were calculated as 9833μM and 416.67μmol NO2- reduced min-1 mg-1. Enzyme activity decreased by 55% and 50% in the presence of 100mM nitrate and formate, respectively. The presence of 25mM Cd2+ protected the enzyme at room temperature and the enzyme showed 10% higher activity in the presence of cadmium.

Homology modeling and virtual characterization of cytochrome c nitrite reductase (NrfA) in three model bacteria responsible for short-circuit pathway, DNRA in the terrestrial nitrogen cycle.

NrfA is the molecular marker for dissimilatory nitrate reduction to ammonium (DNRA) activity, catalysing cytochrome c nitrite reductase enzyme. However, the limited study has been made so far to understand the structural homology modeling of NrfA protein in DNRA bacteria. Therefore, three model DNRA bacteria (Escherechia coli, Wolinella succinogenes and Shewanella oneidensis) were chosen in this study for in-silico protein modeling of NrfA which roughly consists of similar length of amino acids and molecular weight and they belong to two contrasting taxonomicfamilies (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJoperon). Multiple bioinformatic tools were used to examine the primary, secondary, and tertiary structure of NrfA protein using three distinct homology modeling pipelines viz., Phyre2, Swiss model and Modeller. The results indicated that NrfA protein in E. coli, W. succinogenes and S. oneidensis was mostly periplasmic and hydrophilic. Four conserved Cys-X1-X2-Cys-His motifs, one Cys-X1-X2-Cys-Lys haem-binding motif and Ca ligand were also identified in NrfA protein irrespective of three model bacteria. Moreover, 11 identical conserved amino acids sequence was observed for the first time between serine and proline in NrfA protein. Secondary structure of NrfA revealed that α-helices were observed in 77.9%, 73.4%, and 77.4% in E. coli, W. succinogenes and S. oneidensis, respectively. Ramachandran plot showed that number of residue in favored region in E. coli, W. succinogenes and S. oneidensis was 97.03%, 97.01% and 97.25%, respectively. Our findings also revealed that among three pipelines, Modeller was considered the best in-silico tool for prediction of NrfA protein. Overall, significant findings of this study may aid in the identification of future unexplored DNRA bacteria containing cytochrome c nitrite reductase. The NrfA system, which is linked to respiratory nitrite ammonification, provides an analogous target for monitoring less studied N-retention processes, particularly in agricultural ecosystems. Furthermore, one of the challenging research tasks for the future is to determine how the NrfA protein responds to redox status in the microbial cells.

Changes and Driving Mechanism of Microbial Community Structure during Paocai Fermentation

Fermentation of paocai is a dynamic process of the microbial community structure, and the interaction between community structure and physicochemical factors endows paocai with unique taste and flavor. The study of bacterial and fungal community structure changes and the driving mechanism of physicochemical factors induced changes in community structure, showing that Pseudomonas belonging to Proteobacteria and Lactobacillus belonging to Firmicutes were the dominant bacteria in the process of paocai fermentation. The correlation analysis of physicochemical factors with bacterial community showed that titratable acid was significantly positively correlated with Lactobacillus and negatively correlated with Pseudomonas, while nitrite was the opposite. Redundancy analysis (RDA) showed that pH was positively correlated with the bacterial community in the early fermentation stage, amino acid nitrogen was positively correlated with the bacterial community in the middle fermentation stage, and titratable acid was positively correlated with the bacterial community in the late fermentation stage. Variance partitioning analysis (VPA) showed that environmental factors, pH and metabolites, were the main driving forces of bacterial community diversity, which jointly explained 32.02% of the bacterial community structure variation. To study the glucolysis and nitrogen metabolism in the process of paocai fermentation, we found that in the early stage of the fermentation, the nitrite reductase enzyme of Pseudomonas activity was high, with high nitrite content in the prophase, but by the end of fermentation, lactic acid bacteria rapidly increased, the content of L−lactic acid through the glycolysis pathway, making paocai fermentation environment become acidic, then Pseudomonas decreased. Ascomycota and Basidiomycota were the main phylum fungi in the fermentation process. RDA analysis showed that the fungal community was positively correlated with pH, nitrite, and soluble protein at the early fermentation stage, amino acid nitrogen was positively correlated with the fungal community at the middle fermentation stage, titratable acid and reducing sugar were positively correlated with the fungal community at the late fermentation stage. VPA analysis showed that metabolites were the main driving force of fungal community diversity and accounted for 45.58% of fungal community diversity. These results had a certain guiding significance for the production and preservation of naturally fermented paocai.

Integrated evaluation for advanced removal of nitrate and phosphorus in novel PHBV/ZVI-based biofilters: Insight into functional genes and key enzymes

Effective control of nitrogen and phosphorus simultaneously is of great significance to satisfy the strict requirement of the ecological health of receiving waters. In this study, PHBV/ZVI composites made from solid carbon (poly-3-hydroxybutyrate-cohyroxyvelate, PHBV) and zero-valent iron (ZVI) were proposed to be functional fillers in biofilters for advanced wastewater treatment. Results showed that high-rate treatment performance was obtained with nitrate and phosphorus removal efficiencies of 79–97% and 97–98% in the biofilters packed with PHBV/ZVI composites. Lower N2O and CH4 emission (56.3–129.2 μg m−2 h−1) were also achieved simultaneously, further indicating the superiority of PHBV/ZVI composites applied in the wastewater treatment. High-throughput quantitative-PCR (HT-qPCR) results uncovered that the existence of ZVI could enrich carbon-degradation genes (manA, gam and mxa) and facilitate denitrifier utilize organic matters more efficiently, as evidenced by up-regulations of genes involved in nitrate reduction (nirS and nosZ). Meanwhile, higher Fe concentration and less functional genes inducing lower activities of phosphate metabolism and in PHBV/ZVI systems indicated ZVI corrosion and coprecipitation were the main pathway of phosphorus removal. Network and redundancy analysis highlighted the role of ZVI in the removal of pollutants with keystone genes changed (pox and napA) and genes distribution remodeled compared to single PHBV fillers. Further, the activities of dehydrogenase (DHA) and nitrite reductase (Nir) enzymes also increased by the modulation of microbes, which explicitly interpreted the synergistic promotion of PHBV and ZVI on the denitrification process. These findings provided an alternative for the advanced treatment of wastewater and improve the understanding of C, N and P cycling in the co-occurrence of PHBV and ZVI.

Nitrogen Advanced Treatment of Urban Sewage by Denitrification Deep-Bed Filter: Removal Performance and Metabolic Pathway.

This study aimed to explore the performance of denitrification deep-bed filter (DN-DBF) to treat municipal sewage for meeting a more stringent discharge standard of total nitrogen (TN) (10.0 mg L–1). A lab-scale DN-DBF was conducted to optimize operation parameters and reveal the microbiological mechanism for TN removal. The results showed that more than 12.7% TN removal was obtained by adding methanol compared with sodium acetate. The effluent TN concentration reached 6.0–7.0 mg L–1 with the optimal influent carbon and nitrogen ratio (C/N) and hydraulic retention time (HRT) (3:1 and 0.25 h). For the nitrogen removal mechanism, Blastocatellaceae_Subgroup_4 and norank_o_JG30-KF-CM45 were dominant denitrification floras with an abundance of 6–10%. Though large TN was removed at the top layer of DN-DBF, microbial richness and diversity at the middle layer were higher than both ends. However, the relative abundance of nitrite reductase enzymes (EC1.7.2.1) gradually increases as the depth increases; conversely, the relative abundance of nitrous oxide reductase gradually decreased.

Role of oxylipin on Luffa seedlings exposed to NaCl and UV-B stresses: An insight into mechanism

Nowadays, among several abiotic stresses, salt, especially NaCl and UV-B are of major concern. They lead to deleterious effects on plant growth and ultimately affect crop productivity. The present study was planned to find out some ameliorative solution against these stresses. Here, the modulatory action of two oxylipins, namely, methyl jasmonate (MeJA) and 12-Oxo-phytodienoic acid (OPDA) on growth, photosynthetic performance, nitrate/ammonia assimilating enzymes, and nutritive values of Luffa Mill. seedlings grown under NaCl (20 and 40 mM) and/or enhanced UV-B stresses (ambient: 8.2 kJ m−2 d−1 + additional: 2.2 kJ m−2 s−1) were analyzed. Both the stresses when given alone, negatively affected the fresh mass, root/shoot ratio, leaf area, photosynthetic pigments content, photosynthetic oxygen yield and, chlorophyll a fluorescence kinetic parameter. This decline was further aggravated upon combined exposure to the stressors. However, supplementation of MeJA/OPDA effectively counteracted the negative impact on important growth-regulating processes. The activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) enzymes, as well as the contents of inorganic nitrogen, protein, and carbohydrate, were increased with the supplementation of MeJA/OPDA. The increase in the Na+ and Cl‾ contents due to NaCl or/and UV-B was depreciated by MeJA or OPDA. Ameliorating behaviour of MeJA or OPDA is correlated with improved photosynthetic activity and nitrogen metabolism. These findings, point out that supplementation of MeJA/OPDA, particularly OPDA more favourably regulated the growth-promoting activities, which can be linked with the mitigation of NaCl and UV-B stress.

Effect of LED Spectrum on the Quality and Nitrogen Metabolism of Lettuce Under Recycled Hydroponics.

Light quality optimization is an efficient method for improving the growth and quality of lettuce in plant factories. In this study, lettuce seedlings were illuminated under different light-emitting diode (LED) lights, namely, red-blue (RB), red-blue-green (RBG), red-blue-purple (RBP), and red-blue-far-red (RBF) LED lights, to investigate the effect of light quality on growth, quality, and nitrogen metabolism. The combination of 75% red and 25% blue light was set as the basic light source, and 20% of green, purple and far-red light were added to basic light source, respectively. All the treatments were set to 200 μmol m–2 s–1. Results showed that the fresh weight and dry weight of aboveground lettuce under RBG, RBP, and RBF treatments were significantly lower than those under the RB treatment because of the decrease in the effective photon flux density for chlorophyll absorption. The vitamin C content of the lettuce leaves was increased by about 23% with the addition of purple light. For nitrate reduction, the addition of green light significantly increased the nitrite content of the lettuce leaves. It also promoted the reduction from nitrite to ammonium through the activation of the nitrite reductase (NiR) expression and enzyme activity. The nitrate and ammonium content decreased with the addition of purple light because of the inhibited NR and NiR expression and enzyme activity. For nitrogen assimilation, individual (e.g., Asp, Glu, and Leu) and total amino acids were induced to increase by adding green, purple, and far-red light. The addition of light was hypothesized to have inhibited protein biosynthesis, thereby causing the accumulation of amino acids. Correlation analysis showed that the relative expression levels between HY5 and NR/NiR presented a significantly negative correlation. Transcription factor HY5 might mediate the regulation of light quality on nitrogen metabolism by inhibiting NR and NiR expressions. It might also exert a negative effect on nitrate reduction. Further studies via genome editing techniques on the identification of HY5 functions for nitrate assimilation will be valuable. Nevertheless, the results of this work enrich the understanding of the effect of light quality on nitrate metabolism at the level of gene expression and enzyme activity.

Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.

A Mechanistic Investigation of Cytochrome c Nitrite Reductase Catalyzed Reduction of Nitrite to Ammonia: The Search for Catalytic Intermediates

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were done with the wild type enzyme (wtccNiR) as well as with R103Q, Y206F, and H257Q variants. A UV/Vis stopped-flow investigation of the reaction between nitrite-loaded wtccNiR and TMPD revealed for the first time that the reaction proceeds via a transient 1-electron reduced intermediate. Generation of this species is pH-independent, whereas its decay to a previously characterized 2-electron reduced intermediate is fastest at pH 6.8 and significantly slower at higher pH. The pH dependence is ascribed to the rate-limiting cleavage of the nitrite N – O bond, which requires a prior di-protonation predicted to be slow in earlier computational studies. Under steady-state conditions, S. oneidensis ccNiR catalyzed the slow 1-electron reduction of nitrite to nitric oxide, which was monitored by tracking the concomitant appearance of the colored TMPD+ radical product. The rate of TMPD+ formation was found to be directly proportional to the concentration of TMPD+ at low TMPD concentrations, providing important insights about the mechanism of NO˙ release. The steady-state studies also showed that nitrite is a substrate inhibitor of NO˙ release when TMPD is the electron source, probably because it blocks exit by NO˙ through the nitrite entry channel. The H257Q variant of ccNiR was found to have 1/400th of the wild type enzyme's nitrite reductase activity in the standard assay in which methyl viologen monocation radical is the electron source, but nearly normal hydroxylamine reductase activity. This demonstrated that H257 is essential for nitrite reduction but not for reduction of hydroxylamine, a putative intermediate in the catalytic process. UV/Vis spectropotentiometry showed that the nitrite-loaded active site of H257Q still reduced at fairly high applied potential, but the reduction was by one electron, whereas the wild type is reduced in a concerted 2-electron step. The experiments with the variant, coupled with the pH-dependent stopped flow experiments with wtccNiR, confirm H257's importance in facilitating nitrite reduction, but suggest that its role is to modulate the pKa values of one or more of the waters that form a complex hydrogen bonding network in the active site. This view is more conservative than earlier theoretical predictions of direct histidine involvement in nitrite diprotonation leading to N – O bond cleavage.

Trace metal and nitrogen concentrations differentially affect bloom forming cyanobacteria of the genus Dolichospermum

The role of nitrogen and phosphorus in promoting cyanobacterial blooms has been researched for many decades. In contrast, much less is known about the effect of trace metals on regulating cyanobacterial bloom formation. This is particularity relevant for nitrogen-fixing species, as some trace metals act as co-factors in nitrogenase and nitrite reductase enzymes. In the present study, we assessed the response of two nitrogen-fixing species, Dolichospermum lemmermannii and Dolichospermum planctonicum, to copper (Cu), cobalt (Co), molybdenum (Mo), iron (Fe) and nitrogen (N) using a culture-based approach. Response surface modelling showed growth rates in D. planctonicum were positively influenced by increased Co and Fe (p < 0.05), whereas Fe (p < 0.001) and Mo (p < 0.05) increased growth rates in D. lemmermannii. Heterocyst frequency in D. planctonicum was associated with nitrogen (negative response, p < 0.001) and all trace metals tested (positive; p < 0.001 or < 0.05) except Mo. Nitrogen had a strong negative effect (p < 0.001), and Fe a positive (p < 0.01) impact on heterocyst frequency in D. lemmermannii. These results indicate that in addition to regulating nitrogen and phosphorus inputs, management efforts should consider minimising trace metals from entering waterbodies.