Characterization of nitrate reductase-deficient barley mutants

Summary Ten nitrate reductase-deficient Hordeum vulgare mutants were characterized for NADH and FMNH₂ nitrate reductase (NR), cytochrome C reductase (CR) and nitrite reductase (NiR) activities. The mutants sort into four major groups. Group I represented by mutants Az 12, Az 23, Az 29 and Az 30 have low Nr and Cr activities. Group II represented by mutants Az 13, Az 31, Az 33 and Az 34 have low NR activities but intermediate CR activities. Group III represented by mutant Az 28 has low NR activity, but above normal CR activity. Group IV represented by Az 32 has low NADH-NR, low CR, but above normal FMNH₂-NR activity. All ten mutants have elevated NiR activities. None of the ten mutants were constitutive for nitrite reductase activity. Only Az 34 showed a definite high temperature sensitivity when the NADH nitrate reductase activity was compared in the 12 to 26° C range. The mutants Az 12, Az 13, Az 23, Az 28, Az 29, Az 30, Az 31, Az 32 and Az 33 are allelic and were assigned the locus designation nar1. Mutant Az 34 represents a different genetic locus designated nar2. The nar1 gene is codominant and the nar2 gene is recessive.Scientific Paper No. 5463. College of Agriculture Research Center, Washington State University, Pullman, Project Nos. 0233 and 0430. Supported in part by National Science Foundation Grants PCM 78-07649 and PCM 78-16025     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Diversity of culturable denitrifying bacteria

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.Abbreviations NiR Nitrite reductase - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

NO3 −/NO2 − assimilation in halophilic archaea: physiological analysis, nasA and nasD expressions

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT)

Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N(2)OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations, with an IC(50) for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N(2)OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N(2)OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N(2)OR was much more sensitive to TNT than NaR+NiR+NOR.     

Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes

Nitrite reduction is the key step in the denitrification reaction with two predominant types of nitrite reductase genes: nirS and nirK. The diversity of denitrifying bacteria in a municipal wastewater treatment plant is described by using both these genes. Of the cultured colonies, 22.5% contained the NirS gene and 12.5% the nirK gene. These nitrite reductase-containing colonies could be further divided into five different types by using both restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis. Phylogenetic analysis showed that these five types of denitrifying bacteria were phylogenetically diverse. Finally, one nirS gene was obtained and compared with the published sequences.     

Nitrosylation of c heme in cd1-nitrite reductase is enhanced during catalysis

The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d₁ heme per monomer (cd₁NiR), encoded by the nirS gene.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Isolation,sequence and expression in Escherichia coli of the nitrite reductase gene from the filamentous,thermophilic cyanobacterium Phormidium laminosum

The nitrite reductase (NiR) gene (nirA) has been isolated and sequenced from the filamentous, thermophilic non-N₂-fixing cyanobacterium Phormidium laminosum. Putative promoter-like and Shine-Dalgarno sequences appear at the 5 end of the 1533 bp long nir-coding region. The deduced amino acid sequence of NiR from P. laminosum corresponds to a 56 kDa polypeptide, a size identical to the molecular mass previously determined for the pure enzyme, and shows a high identity with amino acid sequences from ferredoxin-dependent NiR. This cyanobacterial NiR gene has been efficiently expressed in Escherichia coli DH5 from the E. coli lac promoter and probably from the P. laminosum NiR promoter.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - NiR nitrite reductase - NR nitrate reductase - NT nitrate transport - SiR sulfite reductase     

Effects of pentachlorophenol on the bacterial denitrification process

The use of pentachlorophenol (PCP) was banned or restricted in many countries worldwide because of its adverse influences on the ecological environment and humans. However, the potential disrupting effects of PCP on denitrifying microorganisms have warranted more analysis. In this study, the impacts of PCP on denitrification were investigated by using Paracoccus denitrificans as a model denitrifying bacterium. Compared with the control, the presences of 10 and 50 μM of PCP were found to significantly decrease the denitrification efficiencies from 98.5 to 87.2% and 68.7%, respectively. The mechanism studies showed that PCP induced the generation of reactive oxygen species, which decreased the vital enzymes activities related to glycolysis process, causing the disturbance of the metabolism of P. denitrificans utilizing carbon source (glucose) and the growth of the cell, and subsequently the generation of electron donor (NADH) for denitrification via NAD⁺ reduction was severely depressed. Further studies indicated that PCP also decreased the genes expression of several key enzymes responsible for denitrification, such as napA of nitrate reductase (NAR), nirS of nitrite reductase, norB of nitric oxide reductase, and nosZ of nitrous oxide reductase; however, there was only the enzyme activity of NAR was remarkably inhibited.     

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans,Hyphomicrobium denitrificans,and Hyphomicrobium zavarzinii

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.     

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

Enhanced biological denitrification of high concentration of nitrite with supplementary carbon source

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l^?1 nitrite at a rate of 0.27 mg NO₂–N g^?1?VSS h^?1. Notably, 1,500 mg l^?1 acetate and 700 mg l^?1 methanol or ethanol enhanced denitrification rates for 200 mg l^?1 nitrite at 2.07, 1.20, and 1.60 mg NO₂–N g^?1?VSS h^?1, respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37–41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol?>?acetate?>?ethanol on molar basis or acetate?>?ethanol?>?methanol on an added weight basis.