A Novel C1-Using Denitrifier Alcaligenes sp. STC1 and Its Genes for Copper-containing Nitrite Reductase and Azurin

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O₂) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O₂-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.     

Electrochemical Behaviour of Copper-Containing Nitrite Reductase from Alcaligenes Faecalis Strain-6

Indirect and direct electrochemical reactions of copper containing nitrite reductase (NIR) from Alcaligenes faecalis strain-6 are described. The reactivity of mediators, including blue protein from the same organism (the native redox partner of NIR, AfBP), in electrocatalytic reactions (EC') involving a mediator, NIR and nitrite was investigated. Several types of EC were observed and AfBP was found to be an effective mediator in spite of its high redox potential. Direct electrochemistry was observed at an Indium Tin Oxide electrode (ITO) and an edge plane oriented pyrolytic graphite electrode (PGE). Observation of the redox activity of NIR at an ITO in an optically transparent thin layer electrode cell (OTTLE) showed that it underwent reversible changes in absorbance that corresponded to the applied potential. The electrochemically adsorbed NIR at PGE showed fast electrochemical kinetics in cyclic voltammetry. It is suggested that the weak affinity of NIR to the PGE electrode may prevent complete denaturation of NIR in the adsorbed state.     

Impact of Long-Term Fertilization on the Composition of Denitrifier Communities Based on Nitrite Reductase Analyses in a Paddy Soil

The effect of long-term fertilization on soil-denitrifying communities was determined by measuring the abundance and diversity of the nitrite reductase genes nirK and nirS. Soil samples were collected from plots of a long-term fertilization experiment started in 1990, located in Taoyuan (110°72″ E, 28°52″ N), China. The treatments were no fertilizer (NF), urea (UR), balanced mineral fertilizers (BM), and BM combined with rice straw (BMR). The abundance, diversity, and composition of the soil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of nirK and nirS genes. There was a pronounced difference in the community composition and diversity of nirK-containing denitrifiers responding to the long-term fertilization regimes; however, less variation was observed in communities of nirS-containing denitrifiers, indicating that denitrifiers possessing nirK were more sensitive to the fertilization practices than those with nirS. In contrast, fertilization regimes had similar effects on the copy numbers of nirK and nirS genes. The BMR treatment had the highest copy numbers of nirK and nirS, followed by the two mineral fertilization regimes (UR and BM), and the lowest was in the NF treatment. Of the measured soil parameters, the differences in the community composition of nirK and the abundance of nir denitrifiers were highly correlated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on the community structure of nirK populations only, and total organic carbon was the dominant factor in relation to the variations of nir community sizes.     

Presence of Two Different Active nirS Nitrite Reductase Genes in a Denitrifying Thauera sp. from a High-Nitrate-Removal-Rate Reactor

The nirS nitrite reductase genes were studied in two strains (strains 27 and 28) isolated from two denitrifying reactors and characterized as Thauera according to their 16S rRNA gene sequences. Strain 28 contains a single nirS sequence, which is related to the nirS of Thauera mechernichensis, and strain 27 contains two nirS sequences; one is similar to the nirS sequence from Thauera mechernichensis (gene 2), but the second one (gene 8) is from a separate clade with nirS from Pseudomonas stutzeri, Azoarcus species, Alcaligenes faecalis, and other Thauera species. Both genes were expressed, but gene 8 was constitutively expressed while gene 2 was positively regulated by nitrate.     

Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

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

Proton translocation during denitrification by a nitrifying-denitrifying Alcaligenes sp.

A heterotrophic nitrifying Alcaligenes sp. from soil was grown as a denitrifier on nitrate and subjected to oxidant pulse experiments to ascertain the apparent effeciencies of proton translocations during O₂ and nitrogen-oxide respirations. With endogenous substrate as the reducing agent the H⁺/2e^– ratios, extrapolated to zero amount of oxidant per pulse, were 9.4, 3.7, 4.3 and 3.5 for O₂, nitrate, nitrite and N₂O, respectively. The value for O₂ and those for the N-oxides are, respectively, somewhat larger and smaller than corresponding values for Paracoccus denitrificans. None of the three permeant ions employed with the Alcaligenes sp. (valinomycin-K⁺, thiocyanate and triphenylmethylphosphonium) was ideal for all purposes. Thiocyanate provided highest ratios for O₂ but abolished the oxidant pulse response for nitrate and N₂O. Valinomycin was slow to penetrate to the cytoplasmic membrane and relatively high concentrations were required for optimal performance. Triphenylmethylphosphonium enhanced passive proton permeability and diminished proton translocation at concentrations required to realize the maximal oxidant pulse response.     

Regulation of Nitrite Reductase Activity under CO2 Limitation in the Cyanobacterium Synechococcus sp. PCC7942

During photoautotrophic growth under CO2-limited conditions, cells of Synechococcus sp. PCC7942 excreted into the medium about 30% of the nitrite produced by reduction of nitrate. No nitrite was excreted under CO2-sufficient conditions. After transfer of high-CO2-grown cells to CO2-limited conditions, nitrite reductase activity started to decline within 0.5 h and decreased to 50% of the initial level in 3 h, whereas nitrate reductase activity was virtually unchanged. Nitrite started to accumulate in the medium about 3 h after the transfer of the cells to CO2-limited conditions and reached a concentration of >0.4 mM at 17 h. These findings suggested that the nitrite excretion was due to an imbalance of the activities of nitrite reductase and nitrate reductase. Since ammonium, the product of nitrite reduction, was not detected in the medium, it was concluded that the step of nitrite reduction limits the rate of nitrate assimilation under CO2-limited conditions. The extent of decrease in nitrite reductase activity under CO2-limited conditions was much larger than that caused by rifampicin (an inhibitor of RNA synthesis) treatment under high-CO2 conditions. Addition of CO2, in the form of sodium bicarbonate, to the CO2-limited culture increased the nitrite reductase activity, but rifampicin inhibited this increase. These findings suggested the presence of a mechanism that irreversibly inactivates nitrite reductase under CO2-limited conditions.     

Nitrite and nitrate synthesis from pyruvic-oxime by anAlcaligenes sp.

AnAlcaligenes sp. isolated from soil was characterized as to its ability to oxidize and grow on pyruvic-oxime. Abundant nitrification of pyruvic-oxime was demonstrated with maximal nitrite and nitrate production of 1867 mg NO₂ ^–-N per liter and 42 mg NO₃ ^–-N per liter. TheAlcaligenes sp. oxidized hydroxylamine and this metabolism was stimulated when either acetate or pyruvate was present. This organism was also capable of limited pyruvic-oxime oxidation when cultured in an acidic medium. The metabolism of pyruvic-oxime and nitrification by theAlcaligenes sp. in the environment are discussed.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

Dissimilatory Nitrate and Nitrite Reduction under Aerobic Conditions by an Aerobically and Anaerobically Grown Alcaligenes sp. and by Activated Sludge

The oxygen uptake of an Alcaligenes sp., isolated from activated sludge, was inhibited by small amounts of nitric oxide. The occurrence of this inhibition was dependent on the growth conditions and the pretreatment of the cells. Anaerobically grown cells, which had subsequently been aerated in a nitrogen-free medium, accumulated nitric oxide, after the addition of nitrate or nitrite. When the oxygen uptake was inhibited by nitric oxide, dissimilatory reduction of nitrate and nitrite proceeded under aerobic conditions at the same rate as in the absence of oxygen. Activated sludge removed nitric oxide actively under aerobic conditions and as a consequence the oxygen uptake of the sludge was not inhibited in the presence of nitrite. The rate of nitrate reduction under aerobic conditions was about 20% of that in the absence of oxygen.     

产碱菌麦芽四糖淀粉酶的底物特异性

产碱菌Ｇ４Ａ仅能水解由α－１,４－葡萄糖苷键连接的麦芽寡糖、淀粉或糖原。淀粉和糖原的水解产物为Ｇ４;麦芽寡糖Ｇ５,Ｇ６和Ｇ７的水解产物分别为Ｇ４＋Ｇ、Ｇ４＋Ｇ２和Ｇ４＋Ｇ３,水解速度为Ｇ５〈Ｇ６〈Ｇ７。直链淀粉的水解限度为１００％,其他淀粉为６０￣７４％,糖原仅３４％,表明该酶为从麦芽寡糖、淀粉或糖原非还原末端顺序切割第４个α－１,４－葡萄糖苷键的外切型淀粉酶。Ｇ４Ａ对Ｇ５、Ｇ６、Ｇ７及直链淀粉、     

Characterization of a Nitrite Reductase Involved in Nitrifier Denitrification

Nitrifier denitrification is the conversion of nitrite to nitrous oxide by ammonia-oxidizing organisms. This process, which is distinct from denitrification, is active under aerobic conditions in the model nitrifier Nitrosomonas europaea. The central enzyme of the nitrifier dentrification pathway is a copper nitrite reductase (CuNIR). To understand how a CuNIR, typically inactivated by oxygen, functions in this pathway, the enzyme isolated directly from N. europaea (NeNIR) was biochemically and structurally characterized. NeNIR reduces nitrite at a similar rate to other CuNIRs but appears to be oxygen tolerant. Crystal structures of oxidized and reduced NeNIR reveal a substrate channel to the active site that is much more restricted than channels in typical CuNIRs. In addition, there is a second fully hydrated channel leading to the active site that likely acts a water exit pathway. The structure is minimally affected by changes in pH. Taken together, these findings provide insight into the molecular basis for NeNIR oxygen tolerance.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V_max) of 0.066 and 0.003 μmol of N × min⁻¹ × mg of protein⁻¹, respectively, at 22°C. K_m values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.     

Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from Arabidopsis thaliana

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M(-1) s(-1), respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.     

Further Characterization of Ferredoxin Nitrite Reductase and the Relationship between the Enzyme and Methyl Viologen-dependent Nitrite Reductase

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.

Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to ¹⁴N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.

On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.

From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.     

The Purification and Properties of a Copper Nitrite Reductase from Haloferax denitrificans

A dissimilatory nitrite reductase from Haloferax denitrificans was purified to apparent electrophoretic homogeneity. The overall purification was 125-fold with about a 1% recovery of activity. The enzyme, which had a molecular mass of 127 kDa, was composed of a 64-kDa subunit as determined by SDS-PAGE. Although maximum activity occurred in the presence of 4 M NaCl, no activity was lost when the enzyme was incubated in the absence of NaCl. The absorption spectrum had maxima at 462, 594, and 682 nm, which disappeared upon reduction with dithionite. Diethyldithiocarbamate (DDC) was inhibitory, and the addition of copper sulfate to DDC-inhibited enzyme partially restored activity. These results suggest this enzyme is a copper-containing nitrite reductase and is the first such nitrite reductase to be described in an Archeon.