Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light

Nitrite reductase has been purified almost 3000-fold, in 35% yield, to a specific activity of 77 units (mg protein)-1 from wheat leaves using a multi-step procedure with affinity chromatography on ferredoxin-Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60 500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross-react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark-grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)-rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.     

Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3′ end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3′ untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures

The nitrite ion content and activity of nitrate reductase and nitrite reductase were examined in scutellum-derived calluses of rice varieties using a modified R2 medium (medium A) and a medium derived from the modified R2 medium (medium B). In medium A, marked differences were observed in callus growth between the varieties. The calluses of the poor-growth varieties accumulated significantly more nitrite ions during the culture period than did the good-growth varieties. Callus growth rate was negatively correlated with the nitrite ion content, indicating that the calluses of the poor-growth varieties were injured by toxic nitrite ions, which lead to browning and inhibited growth. The calluses of the poor-growth varieties had significantly lower levels of nitrite reductase activity than good-growth varieties. On the other hand, no between-group differences were observed in the nitrate reductase activity. These results indicate that the higher nitrite ion levels observed in the poor-growth varieties resulted from a lower ability to reduce nitrite and that nitrite reductase activity is one of the physiological factors that correlates with differences between varieties in rice cell cultures. In medium B, the calluses of the poor-growth varieties grew as well as the good-growth varieties, but also had significantly lower levels of nitrite reductase. Nitrate reductase activity was repressed in the calluses of both varieties in medium B compared to culture in medium A. The results suggest that repressed nitrate reductase activity causes the calluses of poor-growth varieties to accumulate only trace amounts of nitrite ions despite lower nitrite reductase activity and as a result, callus growth improved in medium B. Received: 28 July 1998 / Revision received: 14 October 1998 / Accepted: 27 October 1998     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.     

Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

沼泽红假单胞菌Rhodopseudomonas palustris 2-8的亚硝酸盐还原酶基因克隆和序列分析

沼泽红假单胞菌2-8具有亚硝酸盐还原能力, 根据不同类型亚硝酸盐还原酶保守序列设计引物, 通过PCR扩增的方法对2-8菌株的亚硝酸盐还原酶类型进行鉴定, 发现该菌株的亚硝酸盐还原酶为Cu型亚硝酸盐还原酶。从2-8菌株基因组中克隆出编码该Cu型亚硝酸盐还原酶的基因(nirK), 该基因由1 154个碱基对组成, 在GenBank数据库的登录号为GU332847, 与沼泽红假单胞菌(Rhodopseudomonas palustris TIE和CGA009) 的nirK序列相似性为90%。互联网数据库及生物信     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.     

Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize ( Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-₂ and SO^2-₃ In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S^2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.     

A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans

The denitrifying bacterium Paracoccus denitrificans can grow aerobically or anaerobically using nitrate or nitrite as the sole nitrogen source. The biochemical pathway responsible is expressed from a gene cluster comprising a nitrate/nitrite transporter (NasA), nitrite transporter (NasH), nitrite reductase (NasB), ferredoxin (NasG) and nitrate reductase (NasC). NasB and NasG are essential for growth with nitrate or nitrite as the nitrogen source. NADH serves as the electron donor for nitrate and nitrite reduction, but only NasB has a NADH-oxidizing domain. Nitrate and nitrite reductase activities show the same Km for NADH and can be separated by anion-exchange chromatography, but only fractions containing NasB retain the ability to oxidize NADH. This implies that NasG mediates electron flux from the NADH-oxidizing site in NasB to the sites of nitrate and nitrite reduction in NasC and NasB respectively. Delivery of extracellular nitrate to NasBGC is mediated by NasA, but both NasA and NasH contribute to nitrite uptake. The roles of NasA and NasC can be substituted during anaerobic growth by the biochemically distinct membrane-bound respiratory nitrate reductase (Nar), demonstrating functional overlap. nasG is highly conserved in nitrate/nitrite assimilation gene clusters, which is consistent with a key role for the NasG ferredoxin, as part of a phylogenetically widespread composite nitrate and nitrite reductase system.     

Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions.

Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively).