RESUMO
Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.
Assuntos
Cobre , Nitritos , Nitritos/química , Cobre/química , Ligantes , Biomimética , Nitrito Redutases/química , Espectroscopia de Ressonância de Spin Eletrônica , Catálise , Oxirredução , Cristalografia por Raios XRESUMO
A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a â¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.
Assuntos
Cobre , Sinorhizobium meliloti , Cobre/química , Nitritos/química , Sinorhizobium meliloti/química , Sinorhizobium meliloti/metabolismo , Histidina/química , Domínio Catalítico , Oxirredução , Ligantes , Glicina , Espectroscopia de Ressonância de Spin Eletrônica , Nitrito Redutases/químicaRESUMO
We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key ß-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.
Assuntos
Azurina/química , Proteínas de Bactérias/química , Nitrito Redutases/química , Sinorhizobium meliloti/enzimologia , Cristalografia por Raios X , Domínios ProteicosRESUMO
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 â 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 â 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 â Cu2+ LMCT transitions.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/metabolismo , Grupo dos Citocromos c/metabolismo , Nitrito Redutases/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Clonagem Molecular , Cobre/metabolismo , Grupo dos Citocromos c/genética , Nitrito Redutases/genética , Oxirredução , Regulação para CimaRESUMO
The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s-1) lower than that of wt SmNir (kcat=240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.
Assuntos
Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Nitrito Redutases/metabolismo , Sinorhizobium meliloti/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cobre/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Nitrito Redutases/química , Nitrito Redutases/genética , Nitritos/metabolismo , Oxirredução , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes/metabolismo , Sinorhizobium meliloti/genética , Espectrofotometria UltravioletaRESUMO
Nitrite and nitrate restore deficient endogenous nitric oxide (NO) production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR) mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion) to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite (15N-nitrite) by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Nitrate exerted no effect on aortic accumulation of 15N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in the Michaelis constant. Together, our results show that nitrate inhibits XOR-mediated NO production from nitrite, and this mechanism may explain how nitrate attenuates the vascular and blood pressure responses to nitrite.
Assuntos
Nitratos/metabolismo , Nitrito Redutases/metabolismo , Nitrito de Sódio/metabolismo , Xantina Desidrogenase/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Masculino , Modelos Biológicos , Nitratos/administração & dosagem , Óxido Nítrico/metabolismo , Compostos Nitrosos/farmacologia , Ratos , Nitrito de Sódio/administração & dosagemRESUMO
Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ54, was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.
Assuntos
Proteínas de Bactérias/genética , Bradyrhizobium/metabolismo , Nitrato Redutase/metabolismo , Nitrito Redutases/metabolismo , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Desnitrificação/fisiologia , Nitrato Redutase/genética , Nitrito Redutases/genética , Glycine max/microbiologiaRESUMO
Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas de Ligação ao Ferro/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/análise , Cloroplastos/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Deleção de Genes , Proteínas de Ligação ao Ferro/análise , Proteínas de Ligação ao Ferro/genética , Microscopia Confocal , Mitocôndrias/química , Proteínas Mitocondriais/fisiologia , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Plantas Geneticamente Modificadas , Protoplastos/metabolismo , Protoplastos/ultraestrutura , RNA Mensageiro/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.
Assuntos
Azurina/metabolismo , Elétrons , Nitrito Redutases/metabolismo , Sinorhizobium meliloti/química , Azurina/química , Azurina/genética , Oxirredução , Sinorhizobium meliloti/metabolismo , TermodinâmicaRESUMO
O nitrogênio possui um papel-chave em sistemas aquáticos, especialmente nos ambientes de transição entre águas marinhase doces como os estuários. Assim sendo, muitos organismos, como as ostras, podem ser afetados pelo aumentoda concentração em determinadas épocas, limitando assim seu cultivo. O objetivo deste estudo foi avaliar aspectos daqualidade da água das áreas utilizadas para o cultivo da ostra do mangue Crassostrea spp. em Cananeia (SP, Brasil) e desua adequabilidade para o cultivo dos moluscos com relação às concentrações de nitrogênio amoniacal, nitrito e nitrato.O nitrogênio amoniacal variou de 0,8 a 3,2 μmol/L, o nitrito, de 0,1 a 3,2 μmol/L, e o nitrato, de 0,1 a 1,9 μmol/L. Ao analisaresses dados em todas as estações, verificou-se que o nitrogênio amoniacal, o nitrito e o nitrato foram estatisticamentediferentes entre as marés de quadratura e alta de sizígia. Como não houve diferença entre as estações que fazem a engordadas ostras e a estação Agrossolar (controle), concluiu-se que a atividade no estuário de Cananeia não causa incrementosignificativo nas concentrações de amônia, nitrito e nitrato. As concentrações registradas em Cananeia são similares àsobservadas para outras regiões tropicais e subtropicais estuarinas do mundo.
Nitrogen plays a key role in aquatic systems, especially in environments which are influenced both by marine and freshwater,such as estuaries. The aim of this study was to evaluate aspects of water quality of Crassostrea spp. aquaculture sites inCananeia (SP, Brazil) and adequacy thereof for the cultivation of mollusks in relation to concentrations of total ammoniacalnitrogen, nitrite and nitrate. Variations in concentrations of total ammoniacal nitrogen were 0.8 to 3.2 μmol/L, nitrite 0.1to 3.2 μmol/L, and nitrate from 0.1 to 1.9 μmol/L. Analyzing these data in all seasons together, we found that average totalammoniacal nitrogen and nitrite levels were significantly different for the tides and neap tide. These concentrations recordedat Cananeia are similar to those observed for other estuarine tropical and subtropical regions of the world.
Assuntos
Animais , Amônia , Crassostrea , Nitratos , Nitrito RedutasesRESUMO
Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.
Assuntos
Beggiatoa/enzimologia , Beggiatoa/metabolismo , Citocromos/metabolismo , Pigmentos Biológicos/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromos/isolamento & purificação , Sedimentos Geológicos/microbiologia , México , Nitrito Redutases/isolamento & purificação , Nitrito Redutases/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs.
Assuntos
Proteínas de Bactérias/química , Cobre/química , Óxido Nítrico/química , Nitrito Redutases/química , Subunidades Proteicas/química , Sinorhizobium meliloti/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cianetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli , Expressão Gênica , Cinética , Dados de Sequência Molecular , Peso Molecular , Óxido Nítrico/biossíntese , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Nitritos/química , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinorhizobium meliloti/químicaRESUMO
Assimilatory nitrate reduction (ANR) is a pathway wherein NO(3)(-) is reduced to NH(4)(+), an N species that can be incorporated into the biomass. There is little information about the ANR genes in Archaea and most of the known information has been obtained from cultivable species. In this study, the diversity of the haloarchaeal assimilatory nitrate-reducing community was studied in an extreme saline alkaline soil of the former lake Texcoco (Mexico). Genes coding for the assimilatory nitrate reductase (narB) and the assimilatory nitrite reductase (nirA) were used as functional markers. Primers to amplify and detect partial narB and nirA were designed. The analysis of these amplicons by cloning and sequencing showed that the deduced protein fragments shared >45% identity with other NarB and NirA proteins from Euryarchaeota and <38% identity with other nitrate reductases from Bacteria and Crenarchaeota. Furthermore, these clone sequences were clustered within the class Halobacteria with strong support values in both constructed dendrograms, confirming that desired PCR products were obtained. The metabolic capacity to assimilate nitrate by these haloarchaea seems to be important given that at pH 10 and higher, NH(4)(+) is mostly converted to toxic and volatile NH(3), and NO(3)(-) becomes the preferable N source.
Assuntos
Biodiversidade , Euryarchaeota/classificação , Euryarchaeota/metabolismo , Nitratos/metabolismo , Microbiologia do Solo , Proteínas Arqueais/genética , Análise por Conglomerados , Primers do DNA/genética , DNA Arqueal/química , DNA Arqueal/genética , Euryarchaeota/genética , Euryarchaeota/isolamento & purificação , México , Dados de Sequência Molecular , Nitrato Redutase/genética , Nitrito Redutases/genética , Oxirredução , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
There is recent evidence suggesting that nitrite anion (NO 2 (-)) represents the major intravascular NO storage molecule whose transduction to NO is facilitated by a reduction mechanism catalyzed by deoxygenated hemoglobin (deoxy-Hb). In this work, we provide a detailed microscopic study of deoxy-Hb nitrite reductase (NIR) activity by combining classical molecular dynamics and hybrid quantum mechanical-molecular mechanical simulations. Our results point out that two alternative mechanisms could be operative and suggest that the most energetic barriers should stem from either reprotonation of the distal histidine or NO dissociation from the ferric heme. In the first proposed mechanism, which is similar to that proposed for bacterial NIRs, nitrite anion or nitrous acid coordinates to the heme through the N atom. This pathway involves HisE7 in a one or two proton transfer process, depending on whether the active species is nitrite anion or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and methemoglobin. In the second mechanism, the nitrite anion coordinates to the heme through the O atom. This pathway requires only one proton transfer from HisE7 and leads directly to the formation of a hydroxo Fe(III) complex and NO.
Assuntos
Ânions/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Óxido Nítrico/metabolismo , Nitritos/química , Nitritos/metabolismo , Ânions/química , Sítios de Ligação , Catálise , Histidina/química , Histidina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Óxido Nítrico/química , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Conformação ProteicaRESUMO
In the plant growth-promoting rhizobacterium Azospirillum brasilense Sp245, nitric oxide produced by denitrification could be a signal involved in stimulation of root branching, and the dissimilatory nitrite reductase gene nirK is upregulated on wheat roots. Here, it was found that Sp245 did not contain one copy of nirK but two (named nirK1 and nirK2), localized on two different plasmids, including one plasmid prone to rearrangements. Their deduced protein sequences displayed 99.2% identity but their promoter regions and upstream genetic environment differed. Phylogenetic studies revealed that nirK1 and nirK2 clustered next to most beta-proteobacterial sequences rather than in the vicinity of other Azospirillum spp. and most alpha-proteobacterial sequences, regardless of whether DNA or deduced protein sequences were used. This points to past horizontal gene transfers. Analysis of the number of nonsynonymous and synonymous substitutions per site indicated that nirK has been subjected to neutral selection in bacteria. The use of transcriptional fusions with egfp, encoding an enhanced green fluorescent protein variant, revealed that both nirK1 and nirK2 promoter regions were upregulated in vitro under microaerobiosis or the presence of nitrite as well as on wheat roots. The analysis of nirK1 and nirK2 mutants revealed that the two genes were functional. Overall, results suggest that nirK has been acquired horizontally by A. brasilense Sp245 from a distant relative and underwent subsequent duplication; however, both paralogs remained functional and retained their upregulation by the plant partner.
Assuntos
Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Nitrito Redutases/genética , Plasmídeos/genética , Triticum/microbiologia , Sequência de Aminoácidos , Azospirillum brasilense/enzimologia , Azospirillum brasilense/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Southern Blotting , Modelos Genéticos , Dados de Sequência Molecular , Nitrito Redutases/classificação , Nitrito Redutases/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Replicon/genética , Homologia de Sequência de AminoácidosRESUMO
Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Sedimentos Geológicos/análise , Sedimentos Geológicos/microbiologia , Nitrito Redutases/genética , Biologia Marinha/métodos , Nitratos/análise , Oceanos e Mares , FilogeniaRESUMO
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.
Assuntos
Reatores Biológicos , Regulação Bacteriana da Expressão Gênica , Nitratos/metabolismo , Nitrito Redutases/genética , Thauera/enzimologia , Thauera/crescimento & desenvolvimento , DNA Bacteriano/análise , Dados de Sequência Molecular , Nitrito Redutases/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Thauera/genéticaRESUMO
The major sites of water column denitrification in the ocean are oxygen minimum zones (OMZ), such as one in the eastern South Pacific (ESP). To understand the structure of denitrifying communities in the OMZ off Chile, denitrifier communities at two sites in the Chilean OMZ (Antofagasta and Iquique) and at different water depths were explored by terminal restriction fragment length polymorphism analysis and cloning of polymerase chain reaction (PCR)-amplified nirS genes. NirS is a functional marker gene for denitrification encoding cytochrome cd1-containing nitrite reductase, which catalyses the reduction of nitrite to nitric oxide, the key step in denitrification. Major differences were found between communities from the two geographic locations. Shifts in community structure occurred along a biogeochemical gradient at Antofagasta. Canonical correspondence analysis indicated that O2, NO3-, NO2- and depth were important environmental factors governing these communities along the biogeochemical gradient in the water column. Phylogenetic analysis grouped the majority of clones from the ESP in distinct clusters of genes from presumably novel and yet uncultivated denitrifers. These nirS clusters were distantly related to those found in the water column of the Arabian Sea but the phylogenetic distance was even higher compared with environmental sequences from marine sediments or any other habitat. This finding suggests similar environmental conditions trigger the development of denitrifiers with related nirS genotypes despite large geographic distances.
Assuntos
Bactérias/genética , Genes Bacterianos , Nitrito Redutases/genética , Oxigênio/análise , Água do Mar/microbiologia , Bactérias/enzimologia , Bactérias/crescimento & desenvolvimento , DNA Bacteriano/análise , Oceano Pacífico , Filogenia , Polimorfismo de Fragmento de Restrição , Água do Mar/químicaRESUMO
To understand the composition and structure of denitrifying communities in the oxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) and Pseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp. strain G-179 (98 to 99%), Bradyrhizobium japonicum (91%), Blastobacter denitrificans (83%), and Alcaligenes xylosoxidans (96%). The rest of the clones, however, were less than 80% similar to nirS and nirK sequences available in sequence databases. The results of a principal-component analysis (PCA) based on the percentage of OTUs and biogeochemical data indicated that the nitrate concentration and oxygen have an effect on the denitrifying communities. The communities at the stations in oxygen-deficient zones were more similar than the communities at the stations in the oxygenated zone. The denitrifying communities were more similar at the stations that were closer together and had similar nitrate levels. Also, the results of PCA based on biogeochemical properties suggest that geographic location and biogeochemical conditions, especially the nitrate and oxygen levels, appear to be the key factors that control the structure of denitrifying communities.