Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Effect of paraquat exposure on nitric oxide-responsive genes in rat mesencephalic cells

When neural cells are exposed to paraquat, nitric oxide generation increases primarily due to an increase in the expression of the inducible isoform of nitric oxide synthase. The nitric oxide generated has controversial actions in paraquat exposure, as both protective and harmful effects have been described previously. While the actions mediated by nitric oxide in neural cells have been well described, there is evidence that nitric oxide may also be an important modulator of the expression of several genes during paraquat exposure. To better understand the actions of nitric oxide and its potential role in paraquat-induced gene expression, we examined changes in GCH1, ARG1, ARG2, NOS1, NOS2, NOS3, NOSTRIN, NOSIP, NOS1AP, RASD1, DYNLL1, GUCY1A3, DDAH1, DDAH2 and CYGB genes whose expression is controlled by or involved in signaling by the second messenger nitric oxide, in rat mesencephalic cells after 3, 6, 12 and 24 h of paraquat exposure. A qPCR strategy targeting these genes was developed using a SYBR green I-based method. The mRNA levels of all the genes studied were differentially regulated during exposure. These results demonstrate that nitric oxide-related genes are regulated following paraquat exposure of mesencephalic cells and provide the basis for further studies exploring the physiological and functional significance of nitric oxide-sensitive genes in paraquat-mediated neurotoxicity.     

Nitric oxide-induced salt stress tolerance in plants: ROS metabolism,signaling, and molecular interactions

Nitric oxide (NO), a non-charged, small, gaseous free-radical, is a signaling molecule in all plant cells. Several studies have proposed multifarious physiological roles for NO, from seed germination to plant maturation and senescence. Nitric oxide is thought to act as an antioxidant, quenching ROS during oxidative stress and reducing lipid peroxidation. NO also mediates photosynthesis and stomatal conductance and regulates programmed cell death, thus providing tolerance to abiotic stress. In mitochondria, NO participates in the electron transport pathway. Nitric oxide synthase and nitrate reductase are the key enzymes involved in NO-biosynthesis in aerobic plants, but non-enzymatic pathways have been reported as well. Nitric oxide can interact with a broad range of molecules, leading to the modification of protein activity, GSH biosynthesis, S-nitrosylation, peroxynitrite formation, proline accumulation, etc., to sustain stress tolerance. In addition to these interactions, NO interacts with fatty acids to form nitro-fatty acids as signals for antioxidant defense. Polyamines and NO interact positively to increase polyamine content and activity. A large number of genes are reprogrammed by NO; among these genes, proline metabolism genes are upregulated. Exogenous NO application is also shown to be involved in salinity tolerance and/or resistance via growth promotion, reversing oxidative damage and maintaining ion homeostasis. This review highlights NO-mediated salinity-stress tolerance in plants, including NO biosynthesis, regulation, and signaling. Nitric oxide-mediated ROS metabolism, antioxidant defense, and gene expression and the interactions of NO with other bioactive molecules are also discussed. We conclude the review with a discussion of unsolved issues and suggestions for future research.     

Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum

Bradyrhizobium japonicum is a Gram-negative soil bacterium symbiotically associated with soya bean plants, which is also able to denitrify under free-living and symbiotic conditions. In B. japonicum, the napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide respectively are required for denitrification. Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide. In B. japonicum, a sophisticated regulatory network consisting of two linked regulatory cascades co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). The involvement of the FixLJ/FixK2 regulatory cascade in the microaerobic induction of the denitrification genes is well established. In addition, the FNR (fumarase and nitrate reduction regulator)/CRP(cAMP receptor protein)-type regulator NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level. A role for NifA is suggested in this process by recent experiments which have shown that it is required for full expression of denitrification genes in B. japonicum. The present review summarizes the current understanding of the regulatory network of denitrification in B. japonicum.     

Gene expression study of the flavodi-iron proteins from the cyanobacterium Synechocystis sp. PCC6803

The flavodi-iron proteins, also named FDPs, are an extensive family of enzymes able to reduce dioxygen to water and/or nitric oxide to nitrous oxide. These proteins are formed by a metallo-β-lactamase-like module with a di-iron catalytic site fused to a flavodoxin-like module bearing an FMN. However, in cyanobacteria, which are oxygenic photosynthetic organisms widespread in Nature, FDPs have an extra NAD(P)H:flavin reductase-like domain as a C-terminal extension. Interestingly, cyanobacteria contain more than one gene encoding FDP-like proteins, with the genome of Synechocystis sp. PCC6803 containing four genes coding for putative FDPs. However, the function of those proteins remains unclear. In the present study, we have analysed the expression profile of these genes under oxidative and nitrosative stress conditions. The results indicate that one of the flavodi-iron genes, the so-called flv1, is induced in cells exposed to nitrosative stress. By conducting a broad analysis on the primary sequences of FDPs, we have identified that the FDPs of cyanobacteria and oxygenic photosynthetic eukaryotes may be divided into multiple types (1-12), according to the amino acid residues of the di-iron catalytic site.     

Partial and complete denitrification in Thermus thermophilus: lessons from genome drafts

We have obtained draft genomic sequences of PD (partial denitrificant) and CD (complete denitrificant) strains of Thermus thermophilus. Their genomes are similar in size to that of the aerobic strains sequenced to date and probably contain a similar megaplasmid. In the CD strain, the genes encoding a putative cytochrome cd1 Nir (nitrite reductase) and ancillary proteins were clustered with a cytochrome c-dependent Nor (nitric oxide reductase), and with genes that are probably implicated in their regulation. The Nar (nitrate reductase) and associated genes were also clustered and located 7?kb downstream of the genes coding for the Nir. The whole nar-nir-nor denitrification supercluster was identified as part of a variable region of a megaplasmid. No homologues of NosZ were found despite nitrogen balance supports the idea that such activity actually exists.     

硫化氢信号能够通过生物钟调控拟南芥对冷胁迫的响应

硫化氢(hydrogen sulfide, H_2S)是继一氧化氮(nitric oxide, NO)与一氧化碳(carbon oxide, CO)之后的第3种气体信号分子,在动植物中均发挥着重要的生理功能。生物钟是生物体的内在计时器,对动植物适应环境和生长发育至关重要。鉴于H_2S与生物钟调控的生理过程有较大的相关性,本文以拟南芥(Arabidopsis thaliana)为实验材料,对二者之间的关系进行了探索。结果发现,外源NaHS(H_2S供体)处理能够上调生物钟相关基因CCA1(circadian clock associated 1)和PRR9(pseudo-response regulator 9)的表达,而且在H_2S生成关键酶编码基因缺失的双突变体lcd/des1中,CCA1与PRR9的峰值表达时间明显滞后。CBFs(c-repeat binding factors)是受CCA1调控的冷胁迫响应基因,其表达也受H_2S的调控。lcd/des1中CBF1和CBF3的峰值表达时间延迟,同时在lcd/des1中CBF1、CBF2和CBF3都下调表达。lcd/des1幼苗对冷胁迫表现出更高的敏感性。本文也对拟南芥内源H_2S生成关键酶L-半胱氨酸脱硫基酶(L-cysteine desulfhydrase, LCD)与脱硫基酶1(desulfhydrase 1, DES1)编码基因的转录水平节律性进行了初步的探索。LCD的表达在1 d内未见明显的变化,而DES1的表达有明显的节律性,在早上8:00达到峰值。综上所述,H_2S能够通过调节CCA1与PRR9基因的表达调控生物钟,进而影响下游靶标CBFs基因的表达以增加拟南芥对冷胁迫的耐受性。     

铝诱导植物程序性细胞死亡信号转导的研究进展

铝是制约酸性土壤上作物生产的主要因素。铝诱导氧化胁迫产生大量活性氧/一氧化氮,引起胞质钙超载,通过线粒体信号转导途径激发相关凋亡基因,从而引起细胞主动死亡,以减轻铝对植物的进一步毒害。本文综述了铝诱导程序性细胞死亡的信号分子、相关基因以及信号转导途径,对未来的研究方向提出了展望,为深入研究植物铝毒害机理和耐铝机制提供参考。     

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca²⁺/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation. shear stress; nitric oxide; endothelial cells; protein kinases     

Experimental evidence for plasmid-borne nor-nir genes in Sinorhizobium meliloti JJ1c10

In denitrification, nir and nor genes are respectively required for the sequential dissimilatory reduction of nitrite and nitric oxide to form nitrous oxide. Their location on the pSymA megaplasmid of Sinorhizobium meliloti was confirmed by Southern hybridization of its clones with specific structural gene probes for nirK and norCB. A 20-kb region of pSymA containing the nor-nir genes was delineated by nucleotide sequence analysis. These genes were linked to the nap genes encoding periplasmic proteins involved in nitrate reduction. The nor-nir-nap segment is situated within 30 kb downstream from the nos genes encoding nitrous oxide reduction, with a fix cluster intervening between nir and nos. Most of these predicted nor-nir and accessory gene products are highly homologous with those of related proteobacterial denitrifiers. Functional tests of Tn5 mutants confirmed the requirement of the nirV product and 1 unidentified protein for nitrite reduction as well as the norB-D products and another unidentified protein for nitric oxide reduction. Overall comparative analysis of the derived amino acid sequences of the S. meliloti gene products suggested a close relationship between this symbiotic N2 fixer and the free-living non-N2-fixing denitrifier Pseudomonas G-179, despite differences in their genetic organization. This relationship may be due to lateral gene transfer of denitrification genes from a common donor followed by rearrangement and recombination of these genes.     

Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides

Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.