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As the association of denitrification with global warming and nitrogen removal from ecosystems has gained attention in recent decades, numerous studies have examined denitrification rates and the distribution of denitrifiers across different environments. In this minireview, reported studies focused on coastal saline environments, including estuaries, mangroves, and hypersaline ecosystems, have been analysed to identify the relationship between denitrification and saline gradients. The analyses of the literature and databases stated the direct effect of salinity on the distribution patterns of denitrifiers. However, few works do not support this hypothesis thus making this topic controversial. The specific mechanisms by which salinity influences denitrifier distribution are not fully understood. Nevertheless, several physical and chemical environmental parameters, in addition to salinity, have been shown to play a role in structuring the denitrifying microbial communities. The prevalence of nirS or nirK denitrifiers in ecosystems is a subject of debate in this work. In general terms, in mesohaline environments, the predominant nitrite reductase is NirS type and, NirK is found predominantly in hypersaline environments. Moreover, the approaches used by different researchers are quite different, resulting in a huge amount of unrelated information, making it difficult to establish comparative analysis. The main techniques used to analyse the distribution of denitrifying populations along salt gradients have been also discussed.

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.

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.

We analyzed changes in activities of enzymes of phases I and II of xenobiotic biotransformation and parameters of NO metabolism in liver microsomes of rats with toxic CCl4-induced hepatitis after a 14-day course of sesquiterpene lactones from Artemisia leucodes (10 mg/kg). It was found that toxic hepatitis was associated with significant inhibition of NADPH-cytochrome c-reductase, benzo(a)pyrene hydroxylase, and NADPH-diaphorase, reduced cytochrome P-450 content, and enhanced induction of nitrate/nitrite reductase with accumulation of NO metabolites in the liver. Administration of sesquiterpene lactones stimulated activity of the studied components of the cytochrome P-450 system and promoted recovery of the NOergic system components; the effects were most pronounced in 7 and 14 days after treatment.

Duckweed plants play important roles in aquatic ecosystems worldwide. They rapidly accumulate biomass and have potential uses in bioremediation of water polluted by fertilizer runoff or other chemicals. Here we studied the assimilation of two major sources of inorganic nitrogen, nitrate (NO3− ) and ammonium (NH4+), in six duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna turionifera, Lemna minor, and Wolffia globosa. All six duckweed species preferred NH4+ over NO3− and started using NO3− only when NH4+ was depleted. Using the available genome sequence, we analyzed the molecular structure and expression of eight key nitrogen assimilation genes in S. polyrhiza. The expression of genes encoding nitrate reductase and nitrite reductase increased about 10-fold when NO3− was supplied and decreased when NH4+ was supplied. NO3− and NH4+ induced the glutamine synthetase (GS) genes GS1;2 and the GS2 by 2- to 5-fold, respectively, but repressed GS1;1 and GS1;3. NH4+ and NO3− upregulated the genes encoding ferredoxin- and NADH-dependent glutamate synthases (Fd-GOGAT and NADH-GOGAT). A survey of nitrogen assimilation gene promoters suggested complex regulation, with major roles for NRE-like and GAATC/GATTC cis-elements, TATA-based enhancers, GA/CTn repeats, and G-quadruplex structures. These results will inform efforts to improve bioremediation and nitrogen use efficiency.

Fluorides, being hazardous contaminants of soil and drinking water, can get in excessive amounts into human and animal bodies. This is especially true for regions where the fluoride content in soils is very high, for example, Poltava, Dnipropetrovsk, and Kirovohrad regions in Ukraine. Excessive fluoride intake can change the rate of nitric oxide production. The impact of fluorides on changes in nitric oxide production and metabolism in the heart and the role of redox-sensitive transcription factors in these changes are poorly understood. The aim of this study was to determine the effect of activation of κB transcription factors and activator protein 1 on the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, nitrite and nitrate reductase, arginase, concentration of nitrites, peroxynitrite and nitrosothiols in the heart of rats during chronic fluoride intoxication. Materials and methods. The study was performed on 24 adult male Wistar rats weighing 220-260 grams. Animals were randomly divided into 4 groups of 6 animals in each (control, chronic fluoride intoxication group, κB blockade group and activator protein 1 blockade group). The experiment lasted 30 days. We determined the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, the concentration of peroxynitrite alkali and alkaline earth metals, the concentration of nitrites and nitrosothiols, the activity of nitrite reductase, nitrate reductase and arginase. Results. Chronic fluoride intoxication increases the activity of inducible NO-synthase by 1.74 times, does not affect the activity of constitutive isoforms and reduces the activity of arginase by 35.68% compared with the control group of animals. The concentration of nitrites in the heart of rats increases 1.73 times, peroxynitrite 1.43 times, and the concentration of nitrosothiols doubled. The use of κB transcription factor blockers and activator protein 1 reduces nitric oxide production from NO synthases and reduces the concentrations of all nitric oxide metabolites in the heart of rats under conditions of chronic fluoride intoxication. Conclusions. Activation of κB transcription factors and activator protein 1 during chronic excessive intake of fluoride leads to hyperproduction of nitric oxide in the heart of rats due to increased activity of inducible NO-synthase and nitrite reductases. Excess production of nitric oxide under chronic fluoride intoxication leads to the accumulation of nitrites, peroxynitrite and nitrosothiols in the heart of rats.

As the overuse of nitrogen fertilizer has caused series of environmental problems, improving the nitrogen use efficiency of rice crop has been drawing much attention. Since developing nitrogen-efficient rice genotypes is a feasible solution, illustrating the microbiological characteristics of the nitrogen-efficient rice genotypes is of great importance. We performed a field experiment, and planted 16 genotypes of rice. The nitrogen utilization efficiencies and yields of rice were examined. Real-time PCR and Illumina sequencing were employed to investigate the abundance of the nitrogen-cycle related genes and the bacterial community structure of the rhizosphere. The results revealed that rice genotypes enriched specific bacterial communities to inhabit in the rhizosphere at both the taxonomic and functional levels. The enriched bacteria were mainly involved in the functions of nitrogen transformation, ferric iron reduction, organic compound biodegradation, and sulfur transformation. We observed a strong correlation between the yield and the abundance of rhizospheric functional bacteria, especially the groups of Proteobacteria and Chloroflexi. The analysis of nitrite reductase and denitrifying genes showed that the denitrification activity in the rhizosphere inversely correlate with the efficiency of nitrogen utilization. We reason that a smaller denitrifying bacteria population leads to less the nitrogen loss in the rhizosphere, thus an increase of available nitrogen and a higher rice yield. This research revealed how functional bacteria assembled in the rhizosphere of rice genotypes with varied nitrogen use efficiencies, which may provide valuable information with regard to the improvement of nitrogen use efficiency and reduction of fertilizer application.

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.

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 number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10–HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

Nitrification is a key step in the global nitrogen cycle. Compared with autotrophic nitrification, heterotrophic nitrification remains poorly understood. In this study, Halomonas venusta MA-ZP17-13, isolated from seawater in shrimp aquaculture (Penaeus vannamei), could simultaneously undertake nitrification and denitrification. With the initial ammonium concentration at 100 mg/L, the maximum ammonium-nitrogen removal rate reached 98.7% under the optimal conditions including C/N concentration ratio at 5.95, pH at 8.93, and NaCl at 2.33%. The corresponding average removal rate was 1.37 mg/(L·h) (according to nitrogen) in 3 d at 11.2°C. By whole genome sequencing and analysis, nitrification- and denitrification-related genes were identified, including ammonia monooxygenase, nitrate reductase, nitrite reductase, nitric oxide dioxygenase and nitric oxide synthase; while no gene encoding hydroxylamine oxidase was identified, it implied the existence of a novel nitrification pathway from hydroxylamine to nitrate. These results indicate heterotrophic bacterium H. venusta MA-ZP17-13 can undertake simultaneous nitrification and denitrification at low temperature and has potential for NH 4 + -N/NH3-N removal in marine aquaculture systems.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

Melatonin (MT) and salicylic acid (SA) are known to improve plant tolerance to environmental stresses; however, no reports have evaluated the effect of their combined treatment on plants under both normal and stressful conditions. For the first time, the present study aimed to investigate MT and SA’s potential role in regulating polyamine and nitrogen metabolism in wheat plants subjected to salty soils, and counterbalancing oxidative damage induced by salt stress. Moreover, this research aimed to elucidate a possible link between melatonin, salicylic acid, nitrogen and polyamine levels. Wheat (Triticum aestivum L. cv. Sids 14) plants were grown under non-saline or saline conditions (6.0 and 12.0 dS m–1) and were foliar sprayed with 70 µM MT and/or 75 mg l−1 SA. The injury impacts of salt stress on wheat growth and production were significantly alleviated by exogenous MT and/or SA treatments. This was evidenced by increasing polyamines content through accelerating the metabolic flow from the precursor amino acids arginine and methionine to polyamines, decreasing the polyamines degradation, and enhancing the polyamines biosynthesis. Furthermore, MT and/or SA are involved in promoting nitrogen metabolism through up-regulating the activities of N uptake and metabolism related enzymes (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase) as well as enhancing the content of nitrogen, nitrate and protein in salt-stressed plants. The mitigation was also detected in reduced lipid peroxidation, hydrogen peroxide content, carbonyl content, and protease activity in stressed treated plants. Notably, the best response was registered for the combined treatment of MT and SA. Therefore, the co-application of MT and SA is a promising way for ameliorating salt toxicity in sustainable agricultural systems.

Soil salinization is one main environmental factor restricting plant growth and agricultural productivity. However, phytoremediation is one of the important means to improve saline-alkali soil by planting halophytes or salt-tolerant plants. In order to study whether there are differences among soil microorganisms in different phytoremediation, the effects of four plants, including alfalfa (MX), oil sunflower (YK), maize (YM) and ryegrass (HMC) on soil physicochemical properties, enzyme activity and microbial community diversity and composition were investigated in this study and the relationships between microbial community structure and soil physicochemical properties, enzyme activity were analyzed. The results showed that all plants treatments significantly decreased pH, TS (total saltinity) and BD (bulk density), while increased OM (organic matter), TN (total nitrogen), AN (available nitrogen), TP (total phosphorus), AP (available phosphorus), TK (total potassium) and TPOR (total porosity), and the number of nitrite bacteria reduced by planting at the same time. Except for YM, other treatments significantly increased the number of nitrifying and denitrifying bacteria compared with CK, while only YK increased that of fungi. Additionally, all plants increased the activity of nitrite reductase and decreased that of urease. More interestingly, plants treatments shifted microbial community compositions, and only YM significantly decreased the bacterial diversity and increased the fungal diversity. Redundancy analysis suggested that TK, pH, BD, TS, AN, OM and nitrite reductase, lignin peroxidase were the key environmental factors that shaped the bacterial community structure, while that of fungi was mainly driven by OM, nitrite reductase, urease and lignin peroxidase. The results indicated that MX and YM are the best choice for remediation of saline-alkali soil. These data can provide certain theoretical basis for the further restoration of saline-alkali land.HIGHLIGHTSThe effects of different phytoremediation on microbial diversity and community structure were different.Phytoremediation can significantly decreased pH, TS and BD, while increased OM, TN, AN, TP, AP, TK and TPOR in saline-alkali soil.All plants increased the activity of nitrite reductase and decreased the activity of urease. The effects of different phytoremediation on microbial diversity and community structure were different. Phytoremediation can significantly decreased pH, TS and BD, while increased OM, TN, AN, TP, AP, TK and TPOR in saline-alkali soil. All plants increased the activity of nitrite reductase and decreased the activity of urease.

In the present, the consequences of nitrate pollution of the environment are very pronounced. In humans and animals, microorganisms can reduce nitrates to nitrites, which cause cancer. Purified and homogeneous xanthine oxidase (XO) of cow's milk can restore these compounds, which makes the article extremely relevant. The purpose of the article is to determine the effect of antioxidants on the activity of xanthine oxidase in fresh ovine milk. Various natural and artificial antioxidants were examined for the detection of xanthine oxidase (XO) activity in ovine milk. Among the natural antioxidants, L-cysteine was more effective in the stabilization of XO in heated milk. XO of sheep milk activated by heat treatment in the presence of cysteine and molybdenum became able to convert nitrate and nitrite to nitric oxide (NO). Therefore, L-cysteine was used for double purposes: as the protector of enzyme active center against the oxidation during heat treatment of milk and as a reagent for S-nitrosothiol formation. Hypoxanthine, as a natural substrate of XO, is an effective electron donor for nitrate reductase (NR) and nitrite reductase (NiR) activities. Heat treatment of the milk in the presence of exogenous lecithin increased the activity of NR and NiR of XO and CysNO formation. Thus, during the heat treatment: a) excess of exogenous phospholipids disintegrates the structure of milk fat globule membrane (MFGM) and b) enzyme molecules denatured partially and their active center became available for exogenous cysteine, molybdenum, hypoxanthine, and nitrate or nitrite.

In the present study, improved moving bed biofilm reactor (MBBR) was applied to enhance the nutrient removal ability of the municipal wastewater. A total of 18 indigenous bacterial isolates were screened from the sewage sludge sample and nitrate reductase, nitrite reductase and hydroxylamine oxidase was analyzed. The strains Pseudomonas aeruginosa NU1 and Acinetobacter calcoaceticus K12 produced 0.87 ± 0.05 U/mg and 0.52 ± 0.12 U/mg hydroxylamine oxidase, 1.023 ± 0.062 U/mg and 1.29 ± 0.07 U/mg nitrite reductase, and 0.789 ± 0.031 U/mg and 1.07 ± 0.13 U/mg nitrate reductase. Nitrogen and phosphate removal improved by the addition of nutrient sources and achieved > 80% removal rate. pH and temperature of the medium also affected nutrient removal and improved removal was achieved at optimum level (p

Background Jujube is a typical fruit tree species from China. ‘Muzao’, a cracking-susceptible cultivar, and ‘Linhuang No. 1’, a cracking-resistant cultivar, were selected in a previous study as contrasting research materials. Whole-genome resequencing and transcriptomic analysis of ‘Linhuang No. 1’ and ‘Muzao’ allowed the identification of differentially expressed genes with different gene structures between the two cultivars and could be helpful in explaining the differences and similarities between the two cultivars. Results Resequencing identified 664,129 polymorphic variable sites between ‘Linhuang No. 1’ and ‘Muzao’. To determine the genetic relationship among ‘Linhuang No. 1’, ‘Muzao’ and the jujube genome reference cultivar ‘Dongzao’, the characteristic polymorphic variable sites were analysed by principal component analysis. The genetic relationship between ‘Linhuang No. 1’ and ‘Muzao’ was closer than that of either variety and ‘Dongzao’. Nineteen differentially expressed genes were identified by combining transcriptomic analysis with resequencing analysis. LOC107427052 (encoding a nitrite reductase) was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for further study. The identified insertion was not in the domain region of the LOC107427052 gene coding sequence (CDS) region and was verified by the finding that the insertion did not affect translation of the protein. The LOC107427052 gene expression levels, nitrite reductase activities and nitrite contents of ‘Muzao’ were significantly higher than the corresponding values of ‘Linhuang No. 1’ at the young fruit stage. There was no significant difference in the quantity of the product of nitrite reductase, namely, ammonia, between the two cultivars. Conclusions The present study was the first to explore the differences between different jujube cultivars (‘Linhuang No. 1’ and ‘Muzao’) by combining genome resequencing and transcriptomics. LOC107427052 (encoding a nitrite reductase) was characterized by KEGG enrichment analysis. The insertion in the CDS region of the LOC107427052 gene provides a new direction for the study of nitrogen metabolism in jujube. Our study has laid a foundation for the comparative analysis of nitrite metabolism between the jujube cultivars ‘Linhuang No. 1’ and ‘Muzao’.

Nitrate reductases (NRs) are molybdoenzymes that reduce nitrate (NO3−) to nitrite (NO2−) in both mammals and plants. In mammals, the salival microbes take part in the generation of the NO2− from NO3−, which further produces nitric oxide (NO) either in acid-induced NO2− reduction or in the presence of nitrite reductases (NiRs). Here, we report a new approach of VCl3 (V3+ ion source) induced step-wise reduction of NO3− in a CoII-nitrato complex, [(12-TMC)CoII(NO3−)]+ (2,{CoII–NO3−}), to a CoIII–nitrosyl complex, [(12-TMC)CoIII(NO)]2+ (4,{CoNO}8), bearing an N-tetramethylated cyclam (TMC) ligand. The VCl3 inspired reduction of NO3− to NO is believed to occur in two consecutive oxygen atom transfer (OAT) reactions, i.e., OAT-1 = NO3− → NO2− (r1) and OAT-2 = NO2− → NO (r2). In these OAT reactions, VCl3 functions as an O-atom abstracting species, and the reaction of 2 with VCl3 produces a CoIII-nitrosyl ({CoNO}8) with VV-Oxo ({VV Created by potrace 1.16, written by Peter Selinger 2001-2019 O}3+) species, via a proposed CoII-nitrito (3, {CoII–NO2−}) intermediate species. Further, in a separate experiment, we explored the reaction of isolated complex 3 with VCl3, which showed the generation of 4 with VV-Oxo, validating our proposed reaction sequences of OAT reactions. We ensured and characterized 3 using VCl3 as a limiting reagent, as the second-order rate constant of OAT-2 (k2/) is found to be ∼1420 times faster than that of the OAT-1 (k2) reaction. Binding constant (Kb) calculations also support our proposition of NO3− to NO transformation in two successive OAT reactions, as Kb(CoII–NO2−) is higher than Kb(CoII–NO3−), hence the reaction moves in the forward direction (OAT-1). However, Kb(CoII–NO2−) is comparable to Kb{CoNO}8, and therefore sequenced the second OAT reaction (OAT-2). Mechanistic investigations of these reactions using 15N-labeled-15NO3− and 15NO2− revealed that the N-atom in the {CoNO}8 is derived from NO3− ligand. This work highlights the first-ever report of VCl3 induced step-wise NO3− reduction (NRs activity) followed by the OAT induced NO2− reduction and then the generation of Co-nitrosyl species {CoNO}8., Single metal-induced reduction of NO3− → {NO2−} → NO via oxygen atom transfer reaction.

The present study assessed the effect of α-ketoglutarate supplementation on Solanum melongena L. seedlings grown under As stress by evaluating growth performance, photosynthetic pigments, carbonic anhydrase activity, photochemistry of PSII, nitrogen metabolism and oxidative stress. Growth, pigment contents, photosynthetic oxygen yield and carbonic anhydrase activity declined with AsV stress in dose dependent manner. As compared to control, As1 (5 µM As) and As2 (25 µM As) caused decline in fresh weight by 13 and 29% and dry weight by 14 and 39%, respectively. Furthermore, photochemistry of PSII parameters as quantum yield of primary photochemistry, yield of electron transport per trapped excitation, the quantum yield of electron transport and performance index of PSII declined with AsV stress while parameters related to energy fluxes per reaction center were enhanced. Not withstanding to this, respiration rate and ammonium content enhanced significantly due to more accumulation of As content in test seedlings. The As accumulation at As1 and As2 treated seedlings was found to be 39.75 ± 0.69 and 158.62 ± 2.75 µg As g−1 DW in root and 30.93 ± 0.54 and 141.37 ± 2.45 µg As g−1 DW in shoot, respectively. In addition, nitrate and ammonium assimilating enzymes: nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activity were adversely affected by AsV stress while glutamate dehydrogenase activity showed reverse trend. Such damaging effect of AsV could be due to enhanced oxidative stress as evidenced by increased SOR, H2O2 and MDA equivalents contents and electrolyte leakage in turn. However, negative effects of AsV on these parameters were significantly mitigated by α-Ketoglutarate, therefore, considerable improvement in growth performance of S. melongena seedlings was noticed. The findings suggest that α-Ketoglutarate, a key intermediate of the Krebs cycle and nitrogen metabolism reduces the AsV toxicity in S. melongena L. seedlings by downregulating As accumulation and oxidative stress biomarkers, activation of antioxidant defense system which collectively protect PSII photochemistry leading to improved growth in S. melongena seedlings.

In the current study, the role of chlorophyll precursor, 5-aminolevulinic acid (ALA) and its effect on growth attributes, photosynthetic pigments, indices of chlorophyll a fluorescence, nitrogen metabolism, and contents of protein and carbohydrate in seedlings of pigeon pea varities BAHAR and KB-17 under the enhanced ultraviolet (UV-B; 2.2 kJ m−2d−1) radiations was investigated. Ultraviolet radiations (UV-B) adversely affected the fresh mass of shoot and root, shoot and root length, photosynthetic pigments content (Chl a + b and ratios of Chl a/b and Chl/Car), photosynthetic oxygen yield and chlorophyll a fluorescence parameters: i.e., Fv/Fm (Phi_P0), ψ0 (Psi_0), φE0 (Phi_E0) and performance index of PS II (PIABS) along with Fv/F0 while increased the energy flux parameters, i.e., ABS/RC, TR0/RC, ET0/RC, and DI0/RC along with F0/Fv and also consequently raised the dark respiratory rate in test seedlings; however, exogenous application with both the doses of ALA (low dose: ALA1; 25 µM and high dose: ALA2; 100 µM) through seed priming notably counteracted UV-B induced negative effects on growth attributes and other important growth-regulating processes. Additionally, inorganic nitrogen contents, i.e., nitrate (NO3−) and nitrite (NO2−) and the activities of nitrate assimilating enzymes, viz., nitrate reductase and nitrite reductase and ammonia assimilating enzymes, viz., glutamine synthetase and glutamate synthase (GOGAT) along with carbohydrate and protein contents were rigorously affected and declined under UV-B toxicity; while in present study, ammonium (NH4+) content and glutamate dehydrogenase (GDH) activity in both BAHAR and KB-17 varieties exhibited reverse trend. Furthermore, ALA priming induced significant alliveation against UV-B stress on these parameters except NH4+ content and GDH activity, which exhibited an apparent reduction under similar conditions. Our results suggest that ALA priming, particularly high dose (ALA2) more efficiently protects Cajanus cajan seedlings by attenuating UV-B toxicity in both the varieties (BAHAR and KB-17) by maintaining carbon and nitrogen status of seedlings through regulating photosynthesis and nitrogen metabolism.

The treatment of ion-type rare earth mine (REM) wastewater by a modified constructed rapid infiltration system (CRIS) was explored. Meanwhile, the activities of hydroxylamine oxidase, nitrite reductase and urease in the column were measured, and the microorganisms in the system were analyzed. When the hydraulic load was 0.1 m3 m−2·d-1, the column height had a significant influence on the NH4+-N and TN removal rates. Specifically, the average NH4+-N and TN removal rates were 29.06 % and 14.83 %, respectively, for the CRIS with a 100 cm single-layer inorganic filler layer. While the average NH4+-N and TN removal rates increased to 69.70 % and 38.90 %, respectively, for the CRIS with a 140 cm double-layer inorganic filler layer. After increasing the hydraulic load from 0.1 m3 m−2·d-1 to 0.2 m3 m−2·d-1, the average NH4+-N and TN removal rates increased by 29.61 % and 15.84 %, respectively, in the long CRIS. The activities of hydroxylamine oxidase, nitrite reductase and urease in the column also improved. Nitrite reductase increased to 0.605 nitrite/min mg protein and hydroxylamine oxidase increased to 0.470 U/ mL g in the lower layer of the long CRIS. High-throughput sequencing analysis showed that the dominant bacterial phyla was mainly Proteobacteria and Planctomycetes. Pseudomonas, Bacillus and other aerobic denitrifying bacteria were detected at the genus level, as were the anaerobic denitrifying bacteria Comamonas and Thauera. These results illustrated that aerobic denitrification, anaerobic denitrification and anaerobic ammoxidation simultaneously existed during the CRIS treatment of REM wastewater to achieve TN removal. Furthermore, when using two full-scale CRIS in a series to treat actual REM wastewater, the NH4+-N removal rate reached 99.14 %.

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

To elucidate the direct effects of biochar on denitrification metabolism at the cellular level, the global response of model denitrifying soil bacterium (Paracoccus denitrificans) to biochar addition was investigated by physiological, proteomic, and metabolomics analyses. The enhancement effect on denitrification was positively correlated with its pyrolysis temperatures (300-500 °C) and dosages (0.1-1%), regardless of precursors [corn straw (CS) or wheat straw). Moreover, the stimulating effect of CS biochar made at 500 °C (CS-500) was mainly attributed to the bulk particles rather than the released soluble compounds. Without direct contact with cells, bulk CS-500 particles might directly modulate the carbon metabolism by the adsorption of extracellular metabolites. Since carbon flux to storage was shifted to oxidative catabolism and growth assimilation, more share of the produced reducing power was used for nitrogen reduction. Meanwhile, except for nitrate reductase, both protein expressions and enzyme activities of nitrite reductase, nitric oxide reductase, and nitrous oxide reductase were up-regulated. Accordingly, the accumulation of N2O was reduced by 98% due to the optimized electron distribution among denitrifying enzymes. Eventually, the growth rate of Pc. denitrificans enhanced because of the improved energy utilization efficiency. These results updated the regulation mechanism of biochar on denitrification metabolism and N2O mitigation.

Bacterial genetic material can be horizontally transferred between microorganisms via outer membrane vesicles (OMVs) released by bacteria. Up to now, the application of vesicle-mediated horizontal transfer of “degrading genes” in environmental remediation has not been reported. In this study, the nirS gene from an aerobic denitrification bacterium, Pseudomonas stutzeri, was enclosed in a pET28a plasmid, transformed into Escherichia coli (E. coli) DH5α and expressed in E. coli BL21. The E. coli DH5α released OMVs containing the recombination plasmid pET28a–nirS-EGFP. When compared with the free pET28a–nirS-EGFP plasmid’s inability to transform, nirS in OMVs could be transferred into E. coli BL21 with the transformation frequency of 2.76 × 106 CFU/g when the dosage of OMVs was 200 µg under natural conditions, and nirS could express successfully in recipient bacteria. Furthermore, the recipient bacteria that received OMVs containing pET28a–nirS-EGFP could produce 18.16 U/mL activity of nitrite reductase.

Sugarcane, the commercial sugar-yielding crop, frequently suffers serious damage by trunk borers. The neonicotinoid insecticide imidacloprid is often used for disturbing insect nervous system to defense pests, while the molecular regulation of plant response controlling the imidacloprid is still unclear. Here, using the RNA-seq method, we detected the transcription level of sugarcane roots under the imidacloprid treatment for 24 h and 48 h, respectively. Results showed that a total of 1544 and 966 differentially expressed genes (DEGs) were detected under 24 h (1045 up-regulated and 499 down-regulated) and 48 h imidacloprid treatment (539 up-regulated and 427 down-regulated), respectively. Then the accuracy of RNA-seq data was verified by qRT-PCR assay, and a significant positive correlation between RNA-seq data and qRT-PCR results was detected. Crucial genes encoding the peroxidase (Sspon.03G0025980-4P, Sspon.08G0008080-2B) and the ZIM TFs (Sspon.01G0017230-1A, Sspon.01G0042730-3D) were prominently up-regulated in the 24 h treatment, while genes encoding nitrite reductase (Sspon.04G0002730-1A, Sspon.04G0002730-3D) were significantly down-regulated in the 24 h stress. In all, our results not only provide a whole expression analysis of imidacloprid on sugarcane but also are helpful in exploring crucial genes related to plant response on imidacloprid.

A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth ofB. subtilis, the native promoter PsrfAof the surfactin operon in strainB. subtilisJABs24 was replaced by promoters PnarGand PnasDto induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native PsrfAexpression. As hypothesized, activity of PnarGand PnasDincreased with lower oxygen levels and surfactin was not produced by PsrfA::PnarGas well as PsrfA::PnasDmutant strains under conditions with highest oxygen availability. PnarGshowed expressions similar to PsrfAat lowest oxygen availability, while maximum value of PnasDwas more than 5.5-fold higher. Although the promoter exchange PsrfA::PnarGresulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying PsrfA::PnasDreached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overallYP/Xof 1.007 g/g. This value also surpassed theYP/Xof the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying PsrfA::PnasDpromoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter PnasDrepresents a promising tool for anaerobic-inducible bioprocesses inBacillus.

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

Whereas prospects of bioremediation for a vanadium(V) [V(V)]-contaminated environment are widely recognized, reported functional species are extremely limited, with the vast majority of Gram-negative bacteria in Proteobacteria. Herein, the effectiveness of V(V) reduction is proved for the first time by Lactococcus raffinolactis, a Gram-positive bacterium in Firmicutes. The V(V) removal efficiency was 86.5 ± 2.17% during 10-d operation, with an average removal rate of 4.32 ± 0.28 mg/L·d in a citrate-fed system correspondingly. V(V) was bio-reduced to insoluble vanadium(IV) and distributed both inside and outside the cells. Nitrite reductase encoded by gene nirS mainly catalyzed intracellular V(V) reduction, revealing a previously unrecognized pathway. Oxidative stress induced by reactive oxygen species from dissimilatory V(V) reduction was alleviated through strengthened superoxide dismutase and catalase activities. Extracellular polymeric substances with chemically reactive hydroxyl (-OH) and carboxyl (-COO-) groups also contributed to V(V) binding and reduction as well as ROS scavenging. This study can improve the understanding of Gram-positive bacteria for V(V) bio-detoxification and offer microbial resources for bioremediation of a V(V)-polluted environment.

The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter activities in living cells. In this report, we established a firefly Luc reporter assay system in the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter-Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity was also significantly increased by the overexpression of the MYB1 gene, which encodes a transcription factor responsible for NIR promoter activation. Thus, our results demonstrate the utility of the Luc reporter system in C. merolae.

Perfluorooctane sulfonic acid (PFOS) has been widely detected in wastewater treatment plants (WWTPs). Here, we investigated the nitrogen removal efficiency of activated sludge and related stress mechanisms in a sequencing batch reactor (SBR) after 8 h exposure to PFOS at 10, 100, and 1000 μg L−1. At 10 μg L−1, no significant effect (p > 0.05) was observed on total nitrogen (TN) removal. However, the TN removal efficiency significantly increased from 79.24% (control) to 81.99% and 86.21% at high concentrations (100 and 1000 μg L−1), respectively. In addition, the activities of nitrogen removal enzymes such as ammonia monooxygenase, nitrite oxidoreductase, nitrate reductase, and nitrite reductase were promoted by PFOS, which had variation trends similar to the TN removal rates. High throughput sequencing revealed that acute-term exposure to PFOS had little influence on the microbial richness (Chao) and diversity (Shannon) of activated sludge, but the relative abundance of most nitrogen-removing bacteria increased (Paracoccus, Thauera, and Comamonas increased from 0.43%, 1.78%, and 1.52% in the control to 0.60%, 2.13%, and 1.74% in SBR3, respectively), which could explain the increased nitrogen removal efficiency. This might be due to the protection of relevant bacteria by extracellular polymeric substances (EPSs), because the EPS evidently increased at 100 and 1000 μg L−1. However, an extremely significant (p < 0.01) increase of reactive oxygen species production and lactate dehydrogenase release at 100 and 1000 μg L−1 PFOS showed that PFOS caused oxidative damage and destroyed the integrity of microbial cell membranes. This indicated that PFOS has an acute bio-augmentation effect on the nitrogen removal process in WWTPs, but causes cytotoxicity after acute exposure.

Microplastic pollution has become an increasingly pervasive issue worldwide, but little is known about its effects on the soil environment. A soil microcosm experiment was conducted using low-density polyethylene microplastics to estimate the effect of microplastic pollution on soil nutrient cycling and the soil microbial community structure. The results showed that microplastic addition significantly promoted soil carbon dioxide emissions but not soil nitrous oxide emissions. Soil pH, dissolved organic carbon, ammonia nitrogen, the contents of total phospholipid fatty acid (PLFA), and the ratios of gram-positive bacteria to gram-negative bacteria and saturated to monounsaturated PLFAs significantly increased. In addition, nitrate nitrogen and the ratios of fungi to bacteria, total iso-branched fatty acids to total anteiso-branched fatty acids, and cyclopropyl to precursor significantly decreased with increasing microplastic addition. The addition of microplastics decreased the abundance of ammonia oxidizing bacteria and nitrite reductase (nirS) but had little effect on the functional genes of ammonia oxidizing archaea, nitrite reductase (nirK), and nitrous oxide reductase. A principal coordinate analysis of the bacterial 16S ribosomal RNA gene and fungal internal transcribed spacer in the microplastic addition treatments revealed that the bacterial and fungal communities formed an obvious cluster. The average abundance of some microbial species with tolerance and degradability to microplastics, such as Nocardioidaceae, Amycolatopsis, Aeromicrobium, Cytophagaceae, Betaproteobacteria, Rhodoplanes, and Mortierella, in the microplastic addition treatments was significantly higher than that of the control treatment. The results suggested that microplastics have obvious influences on microbial communities and may affect global carbon and nitrogen cycles. Environ Toxicol Chem 2021;40:352-365. © 2020 SETAC.

AIMS The aim of this study was to obtain cold-adapted denitrifying fungi that could be used for bioaugmentation in woodchip bioreactors to remove nitrate from agricultural subsurface drainage water. METHODS AND RESULTS We isolated a total of 91 nitrate-reducing fungal strains belonging to Ascomycota and Mucoromycota from agricultural soil and a woodchip bioreactor under relatively cold conditions (5 and 15°C). When these strains were incubated with 15 N-labelled nitrate, 29 N2 was frequently produced, suggesting the occurrence of co-denitrification (microbially mediated nitrosation). Two strains also produced 30 N2 , indicating their ability to reduce N2 O. Of the 91 nitrate-reducing fungal strains, fungal nitrite reductase gene (nirK) and cytochrome P450 nitric oxide reductase gene (p450nor) were detected by PCR in 34 (37%) and 11 (12%) strains, respectively. Eight strains possessed both nirK and p450nor, further verifying their denitrification capability. In addition, most strains degraded cellulose under denitrification condition. CONCLUSIONS Diverse nitrate-reducing fungi were isolated from soil and a woodchip bioreactor. These fungi reduced nitrate to gaseous N forms at relatively low temperatures. These cold-adapted, cellulose-degrading and nitrate-reducing fungi could support themselves and other denitrifiers in woodchip bioreactors. SIGNIFICANCE AND IMPACT OF THE STUDY The cold-adapted, cellulose-degrading and nitrate-reducing fungi isolated in this study could be useful to enhance nitrate removal in woodchip bioreactors under low-temperature conditions.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Metalloenzymes are efficient biological catalysts that inspire the creation of coordination compounds that mimic their active site. In this work, the synthesized complexes were inspired by two enzymes: Nitrite Reductase containing Copper (Cu-NiRs) and Carbonic Anhydrase. CuNiRs catalyze the nitrite reduction to nitric oxide, whereas the enzyme Carbonic Anhydrase, acts on the hydration of CO2. Both of these enzymes contain the active site with the same binding environment “NNN”. Therefore, three new tridentate ligands were synthesized, which differ among them by the phenyl, n-butyl and n-propanol substituent groups. These ligands gave rise to the three Cu2+ complexes and three Zn2+ complexes mimetic of the Cu-NiRs and carbonic anhydrase enzymes, respectively. All complexes were characterized by microanalysis, FTIR, conductivity and UV-Vis spectroscopy. The copper complexes were also analyzed by EPR, whereas the zinc complexes were studied by NMR spectroscopy. Both classes of complex, Cu2+ and Zn2+, have the tridentate ligands coordinated to the metals forming pentacoordinate structures with two water molecules completing the coordination sphere. The catalytic activities were dependent on the different substituent groups of the ligands. In the catalytic tests with the copper complexes, it was observed that the NO2‾ coordinates in the bidentate form. After the addition of benzoic acid, EPR and UV-Vis spectroscopies indicate that the nitrite changes the coordination mode from κ2-ONO to κ1-NO2. Although all three copper complexes were able to reduce nitrite to NO both chemically and electrocatalytically, the most efficient catalyst was the one containing the n-propanol group in the second coordination sphere. Our analyses reveal that n-propanol is capable of transferring protons in a proton-coupled electron transfer mechanism, aiding the reaction. For the carbonic anhydrase catalysis, all complexes were also able to hydrate CO2, and the complex with the n-propanol substituents showed a faster bicarbonate formation because its OH group helps in the coordinated water deprotonation step. Therefore, the complexes proved to be good enzyme mimetic catalysts. As metaloenzimas são eficientes catalisadores biológicos e inspiram a criação de compostos de coordenação que mimetizem seu sítio ativo. Neste trabalho, os complexos sintetizados foram inspirados em duas enzimas: a Nitrito Redutase contendo Cobre (Cu-NiRs), que catalisa a redução do nitrito para óxido nítrico, e a Anidrase Carbônica, que contém zinco no seu sítio ativo, e atua nos processos de transporte da hidratação do CO2. Essas enzimas contém o sítio ativo com o mesmo ambiente de coordenação “NNN”. Para obter um mimético com característica similar foram sintetizados três novos ligantes tridentados. Os ligantes são baseados em triazol, prolina e piridina, diferindo-se pelos grupos substituintes fenil, n-butil e n-propanol. Esses ligantes deram origem a três complexos de Cu2+ e três complexos de Zn2+ miméticos das enzimas Cu-NiRs e Anidrase Carbônica, respectivamente. Todos os complexos foram caracterizados por análise elementar, FTIR, condutividade e espectroscopia UV-Vis. Os complexos de cobre também foram analisados por EPR, enquanto os complexos de zinco foram estudados por espectroscopia de RMN. As análises de caracterização indicam que os ligantes tridentados se coordenam aos metais Cu2+ e Zn2+, formando estruturas pentacoordenadas sendo que duas moléculas de água completam a esfera de coordenação. Para ambas as classes de miméticos observou-se que as atividades catalíticas foram dependentes dos diferentes grupos substituintes dos ligantes. Nos testes catalíticos com os complexos de cobre, foi observado que o NO2‾ coordena-se na forma bidentada. As análises de EPR e UV-Vis indicam que após a adição de ácido benzóico, o modo de coordenação do nitrito muda de κ2-ONO para κ1-NO2. Os três complexos foram capazes de reduzir o nitrito a NO. A redução foi realizada usando ascorbato de sódio ou por eletrocatálise. Em ambas as metodologias, o catalisador mais eficiente foi o que continha o grupo n-propanol na segunda esfera de coordenação, capaz de transferir prótons em um mecanismo de transferência de elétrons acoplado a prótons. Os ensaios catalíticos com os complexos de zinco mostram que os três complexos foram eficientes para a fixação do CO2 e geração do íon bicarbonato. O complexo com o substituinte n-propanol apresentou mais rápida formação de bicarbonato, devido seu grupo OH ajudar na etapa de desprotonação da água coordenada. Portanto, os complexos mostraram-se bons catalisadores miméticos das enzimas. 88882.332767/2019-01

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions. Fil: Cristaldi, Julio César. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ferroni, Felix Martín. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Duré, Andrea Belén. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ramirez, Cintia Soledad. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Dalosto, Sergio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; Argentina Fil: Rizzi, Alberto Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: González, Pablo Javier. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivas, Maria Gabriela. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Brondino, Carlos Dante. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Catalytic reduction of nitrite by an iron complex in water near neutral pH to form hydroxylamine and ammonium is reported. The catalyst is an iron center coordinated by the pentadentate macrocycle ...

One popular and relevant proposed function for cyanobacterial hemoglobin (Synechocystis Hb) is anaerobic nitrite reductase in vivo. During such reduction reactions, the hexacoordinated heme iron atom of SynHb is oxidized from the ferrous (Fe+2) to ferric (Fe+3) state and prevent damage by limiting the concentration of toxic metabolites such as nitrite. In order to perform these functions in vivo, there must be a mechanism that converts inactive Fe+3-SynHb back to the active Fe+2-SynHb to accomplish the nitrite reductase function. Here, we report a cognate reductase protein for Synechocystis hemoglobin which can reduce the Fe+3-SynHb to Fe+2-SynHb, thus lending a support to the proposed nitrite reductase function. This reductase is also able to reduce pentacoordinate Hbs such as myoglobin but with lower affinity compared to hexacoordinate SynHb. Insilico model of reductase protein-cyanobacterial hemoglobin complex revealed that the heme active site of Hb faces the catalytic center of the reductase protein and several amino acids in the interface interacts non-covalently thus favoring their interaction. Overall, our in vitro study provides the basic foundation for the understanding of the specific molecular mechanism of action and interaction of the SynHb reductase protein, which need to be investigated in further detail.

Dopamine plays numerous physiological roles in plants. We explored its role in the regulation of growth, nutrient absorption, and response to nitrogen (N) deficiency in Malus hupehensis Rehd. Under low N condition, plant growth slowed, and the net photosynthetic rates, chlorophyll contents, and maximal quantum yield of PSII (Fv/Fm) decreased significantly. However, the application of 100 μmol L−1 exogenous dopamine significantly reduced the inhibition of low N stress on plant growth. In addition to modifying root system architecture under low N supply, exogenous dopamine also changed the uptake, transport, and distribution of N, P, and K. Furthermore, exogenous dopamine enhances the tolerance to low nitrogen stress by increasing the activity of enzymes (nitrate reductase, nitrite reductase, glutamic acid synthase and glutamine synthetase) involved in N metabolism. We also found that exogenous dopamine promoted the expression of ethylene signaling genes (ERF1, ERF2, EIL1, ERS2, ETR1, and EIN4) under low N stress. Therefore, we hypothesized that ethylene might be involved in dopamine response to low N stress in M. hupehensis. Our results suggest that exogenous dopamine can mitigate low N stress by regulating the absorption of mineral nutrients, possibly through the regulation of the ethylene signaling pathway.

Biodegradable carrier are vital for the solid-phase denitrification (SPD) systems for treating nitrate-rich water. Two solid-phase denitrification reactors were developed with both 200 g L−1 of single (polycaprolactone, PCL) (R1) and hybrid solid carbon sources (PCL/polylactic acid (PLA) /polyhydroxyalkanoates (PHA)) (R2) to examine the denitrification performance, denitrifying community and functional genes to various oxytetracycline (OTC) exposure in this study, respectively. Complete denitrification performance was achieved in the both SPD systems at low stress of OTC (1 mg L−1), but then dramatically reduced to less than 20% of nitrate reduction efficiency after one-month high OTC stress (10 mg L−1), and rapidly recovered to stable nitrate removal rates of 76.77 ± 5.48% (R1) and 40.68 ± 4.40% (R2) after the next day of no OTC stress. However, the reactor R1 with single PCL carriers acquired more efficient nitrate removal rate than that of reactor R2 at the high OTC stress and recovery phase with OTC stress, mainly due to the more organics availability from the single PCL carriers. The richness and diversity of nirK and nirS deintrifiers significantly declined at high OTC stress, and much more of those occurred in biofilm R1 with more organics availability. Besides, biofilm R1 achieved much more abundant periplasmic nitrate reductase, nitrite reductase genes and tetracycline resistance genes after high OTC stress, which explained the potential resistance to OTC and rapid recovery efficiency after no stress of OTC. Thus, the organics availability played an important role in assuring SPD system to be efficient under high OTC stress.

Increased use of nitrogen fertilizers has deleterious impact on the environment. Increase in yield potential at low nitrogen supply is regarded as a cereal breeding goal for future agricultural sustainability. Although natural variations of nitrogen transporters have been investigated, key genes associated with assimilation remain largely unexplored for nitrogen use efficiency (NUE) enhancement. Here, we identified a NIN‐like protein NLP4 associated with NUE through a GWAS in rice. We found that OsNLP4 transactivated OsNiR encoding nitrite reductase that was critical in nitrogen assimilation in rice. We further constructed quadrupling NREs (Nitrate‐responsive cis‐elements) in the promoter of OsNiR (p4xNRE:OsNiR) and enhanced nitrogen assimilation significantly. We demonstrated that OsNLP4‐OsNiR increased tiller number and yield through enhancing nitrogen assimilation and NUE. Our discovery highlights the genetic modulation of OsNLP4‐OsNiR signalling cascade as a strategy for high NUE and yield breeding in rice.

Nitrogen (N) deficiency adversely affects tree growth. Additionally, γ-aminobutyric acid (GABA) is closely associated with growth and stress responses because of its effects on carbon (C) and N metabolism. However, little is known about its roles related to plant adaptations to N-deficient conditions. In this study, we analyzed the effects of GABA (0, 2 and 10 mM) applications on the growth traits and physiological responses of poplar (Populus alba × P. glandulosa ‘84K’) seedlings under high N (HN) and low N (LN) conditions. We found that the added GABA interacted with N to affect more than half of the studied parameters, with greater effects in LN plants than in HN plants. Under LN conditions, the GABA application tended to increase poplar growth, accompanied by increased xylem fiber cell length and xylem width. In stems, exogenous GABA increased the abundance of non-structural carbohydrates (starch and sugars) and tricarboxylic acid cycle intermediates (succinate, malate and citrate), but had the opposite effect on the structural C contents (hemicellulose and lignin). Meanwhile, exogenous GABA increased the total soluble protein contents in leaves and stems, accompanied by significant increases in nitrate reductase, nitrite reductase and glutamine synthetase activities in leaves, but significant decreases in those (except for the increased glutamate synthetase activity) in stems. A multiple factorial analysis indicated that the nitrate assimilation pathway substantially influences poplar survival and growth in the presence of GABA under LN conditions. Interestingly, GABA applications also considerably attenuated the LN-induced increase in the activities of leaf antioxidant enzymes, including peroxidase and catalase, implying that GABA may regulate the relative allocation of C and N for growth activities by decreasing the energy cost associated with stress defense. Our results suggest that GABA enhances poplar growth and adaptation by regulating the C and N metabolic flux under N-deficient conditions.

Nitric oxide (NO) signaling has been studied in the eye, including in the pathophysiology of some eye diseases. While NO production by nitric oxide synthase (NOS) enzymes in the eye has been characterized, the more recently described pathways of NO generation by nitrate (NO3−) and nitrite (NO2−) ions reduction has received much less attention. To elucidate the potential roles of these pathways, we analyzed nitrate and nitrite levels in components of the eye and lacrimal glands, primarily in porcine samples. Nitrate and nitrite levels were higher in cornea than in other eye parts, while lens contained the least amounts. Lacrimal glands exhibited much higher levels of both ions compared to other organs, such as liver and skeletal muscle, and even to salivary glands which are known to concentrate these ions. Western blotting showed expression of sialin, a known nitrate transporter, in the lacrimal glands and other eye components, and also xanthine oxidoreductase, a nitrate and nitrite reductase, in cornea and sclera. Cornea and sclera homogenates possessed a measurable amount of nitrate reduction activity. These results suggest that nitrate ions are concentrated in the lacrimal glands by sialin and can be secreted into eye components via tears and then reduced to nitrite and NO, thereby being an important source of NO in the eye.

Dissimilarity nitrate reduction to ammonium (DNRA) is of significance in agriculture ecosystems as the process is beneficial to N retention in soils. However, how fertilization regimes influence DNRA rates and functional microbes in agriculture was rarely estimated. In the present study, a 2-year pot experiment was conducted in two contrasting paddy soils to evaluate the effects of straw and nitrogen addition on DNRA process and the related functional microbes, using stable isotope tracer and molecular ecology techniques. The results showed that the abundance and transcription activity of nitrite reductase encoding gene (nrfA) involved in DNRA process and DNRA rates were significantly higher in alkaline soils than in acidic soils. Straw incorporation significantly enhanced nrfA gene abundance and transcription activity, with a greater effect in alkaline soil than in acidic soil. The rates of DNRA, abundance and transcription activity of nrfA gene positively correlated to soil C/N and C/NO3− induced by straw application. Sequencing analysis based on nrfA gene transcript showed that Deltaproteobacteria was the most dominant group in both soil types (30.9%–67.4%), while Gammaproteobacteria, Chloroflexi, Actinobacteria were selectively enriched by straw incorporation. These results demonstrated that DNRA activity can be improved by straw return practice in paddy soils while the effect will vary among soil types due to differentiated functional microbial communities and edaphic properties.

Injudicious use of chemical fertilizers particularly nitrogen to crops has increased dramatically in the last half-century and, therefore, identifying crop varieties with improved nitrogen efficiency is urgent for sustainable development. In this study, a pot experiment was conducted to screen the intrinsic nitrogen efficiency of eleven cabbage genotypes on the basis of various morpho-physiological and biochemical parameters related to nitrogen metabolism. High genotypic variation was found in nitrogen metabolizing enzymes. A significant variation in nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase activity was found between contrasting (low and high) nitrogen efficient genotypes in all the nitrogen treatments. The experimental data were subjected to Principal component analysis and Agglomerate hierarchical clustering. Based on the data, Early Golden Acre and Pusa Drumhead were screened as low and high nitrogen efficiency genotypes respectively. Our study identifies low and high nitrogen efficient genotypes in cabbage which can be used in breeding programs and could help in addressing the problem of nitrate pollution in the environment and also could lessen the economic burden of fertilizers on farmers.

Silicon (Si) can increase salt tolerance of plants, and previous studies have focused on NaCl stress; whereas in protected facilities, nitrate (but not NaCl) accumulation is one of the major causes of secondary soil salinization. However, information on Si's effect on plant growth under nitrate stress is very limited, and the underlying mechanism is unknown. Here, we investigated Si's effect on plant growth, nitrogen assimilation and chlorophyll synthesis in cucumber. Cucumber seedlings (‘Jinyou 1’) were subjected to 200 mM nitrate stress without or with addition of 2 mM Si. The results showed that root application, but not foliar application of Si, could improve cucumber growth under nitrate stress. Root addition of Si increased photosynthetic rate and decreased oxidative damage of stressed plants. Under nitrate stress, Si addition decreased the accumulation of nitrate, nitrite and ammonium, and promoted the activities of nitrate reductase, nitrite reductase, glutamine synthase, glutamine-2-oxoglutarate aminotransferase and glutamate dehydrogenase in leaves. The concentrations of glutamic acid, 5-aminolevulinic acid, porphobilinogen and uroporphyrinogen Ⅲ were increased under nitrate stress, while these were decreased by added Si. Added Si increased the levels of chlorophyll and its precursors (protoporphyrin Ⅸ, Mg-protoporphyrin Ⅸ and protochlorophyllide), and expressions of genes encoding enzymes in chlorophyll synthesis (CHLH, POR and CAO) under nitrate stress. These results suggest that Si could improve cucumber growth under nitrate stress by enhancing nitrogen assimilation and chlorophyll synthesis, and imply an application of Si fertilizer in solving secondary soil salinization in protected facilities.

Planting density and genotype choice are important agronomic techniques to achieve high crop yields. This study explored how planting density and genotypes affect cotton leaf nitrogen metabolism and lint yield. Treatments included three planting densities (low, 3.00 × 104, moderate, 6.00 × 104, and high 9.00 × 104 plants ha−1) and two genotypes (Zhongmiang16 and J‐4B). A randomized complete block design with split plot arrangement was used. Planting density of 6.00 × 104 ha−1 positively influenced leaf physio‐biochemical attributes and boll production. The high planting density reduced lint yield by suppressing leaf soluble sugar, protein, amino acid, chlorophyll a, b, nitrate nitrogen and nitrite nitrogen content, nitrite reductase and nitrate reductase activity. Moderate density crop produced 15.9% and 10.7% more lint yield than low and high planting densities, respectively. This high yield was the result of improved leaf level functioning e.g. increased pigmentation and nitrogen metabolism during late reproductive growth, which increased carbon assimilation. Favorable leaf microclimate could also have sustained activities of leaf nitrate and nitrite reductase and increased amino acid and protein contents with better distribution of nitrogen metabolic assimilates. Late reproductive structure development closely allied with leaf physiological attributes under a short growing season. Cotton genotypes, irrespective of their architectural variation, produced higher lint yield when grown at 6 × 104 ha−1 in a short cropping season.

Anaerobic ammonium oxidation (anammox) and denitrification are two different microbial reactions that form nitrogen gas. The initial step in the anammox reaction—reduction of nitrite to nitric oxide—is thought to be catalyzed by homologs of dissimilatory nitrite reductase, which is known to be involved in denitrification. Here, we reveal the crystal structure of the copper-containing nitrite reductase (CuNIR) of strain KSU-1, an anammox bacterium. CuNIR had a unique homohexameric structure with three disulfide bridges between homotrimers, although the trimer was similar to that of known CuNIRs. Kinetic and mutagenesis analyses suggested that the hexameric structure is important for the electron transfer reaction.

Trichloroethylene (TCE) is the most ubiquitous halogenated organic pollutant in the environment, it is one of the 129 priority control pollutants. In order to clarify the influence of TCE on microorganisms and nitrogen transformation in Mollisol is the core purpose of this study. Results showed that 10 mg kg−1 TCE is the concentration limit of ammonification in Mollisol. When the concentration of TCE reached 10 mg kg−1 and the effect lasted for over 7 days, the process of ammonia oxidation to nitric acid in Mollisol will be affected. TCE affected the process of nitrate (NO3−) transformation into nitrite (NO2−) by affecting the activity of nitrate reductase, thereby affected the denitrification process in soil. When the concentration of TCE is more than 10 mg kg−1 it reduced the ability of soil microorganisms to obtain nitrogen, thereby affecting soil nitrogen transformation. RDA (Redundancy analysis) showed that the activity of nitrate reductase and the number of nitrifying bacteria and denitrifying bacteria in soil was negatively correlated with the incubation of TCE. In addition, soil nitrate reductase, nitrite reductase, peroxidase activity, ammonifying bacteria, nitrifying bacteria and denitrifying bacteria were negatively correlated with TCE concentration. Beyond that PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) of functional gene structure depend on KEGG (Kyoto Encyclopedia of Genes and Genomes) showed that 20 mg kg−1 TCE significantly inhibited the metabolism of energy and other substances in Mollisol. Based on the above, it is found that TCE significantly affected nitrification and denitrification in Mollisol, thus the nitrogen transformation in Mollisol was affected by TCE contamination.