The complete denitrification pathway of the symbiotic, nitrogen-fixing bacterium Bradyrhizobium japonicum

2005 ◽  
Vol 33 (1) ◽  
pp. 141-144 ◽  
Author(s):  
E.J. Bedmar ◽  
E.F. Robles ◽  
M.J. Delgado
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Bradyrhizobium Japonicum ◽  
Mutational Analysis ◽  
Control Level ◽  
Gene Clusters ◽  
Alternative Form ◽  
Nitrate Respiration ◽  
Regulatory Cascade ◽  
Denitrification Genes

Denitrification is an alternative form of respiration in which bacteria sequentially reduce nitrate or nitrite to nitrogen gas by the intermediates nitric oxide and nitrous oxide when oxygen concentrations are limiting. In Bradyrhizobium japonicum, the N2-fixing microsymbiont of soya beans, denitrification depends on the napEDABC, nirK, norCBQD, and nosRZDFYLX gene clusters encoding nitrate-, nitrite-, nitric oxide- and nitrous oxide-reductase respectively. Mutational analysis of the B. japonicum nap genes has demonstrated that the periplasmic nitrate reductase is the only enzyme responsible for nitrate respiration in this bacterium. Regulatory studies using transcriptional lacZ fusions to the nirK, norCBQD and nosRZDFYLX promoter region indicated that microaerobic induction of these promoters is dependent on the fixLJ and fixK2 genes whose products form the FixLJ–FixK2 regulatory cascade. Besides FixK2, another protein, nitrite and nitric oxide respiratory regulator, has been shown to be required for N-oxide regulation of the B. japonicum nirK and norCBQD genes. Thus nitrite and nitric oxide respiratory regulator adds to the FixLJ–FixK2 cascade an additional control level which integrates the N-oxide signal that is critical for maximal induction of the B. japonicum denitrification genes. However, the identity of the signalling molecule and the sensing mechanism remains unknown.

scholarly journals Bradyrhizobium japonicum NnrR, a Denitrification Regulator, Expands the FixLJ-FixK2 Regulatory Cascade

2003 ◽  
Vol 185 (13) ◽  
pp. 3978-3982 ◽  
Author(s):  
Socorro Mesa ◽  
Eulogio J. Bedmar ◽  
Astrid Chanfon ◽  
Hauke Hennecke ◽  
Hans-Martin Fischer
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Control Level ◽  
Nitric Oxide Reductase ◽  
Regulatory Cascade ◽  
Additional Control ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Maximal Induction ◽  
Nitrate And Nitrite ◽  
Denitrification Genes

ABSTRACT In Bradyrhizobium japonicum, a gene named nnrR was identified which encodes a protein with high similarity to FNR/CRP-type transcriptional regulators. Mutant strains carrying an nnrR null mutation were unable to grow anaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and nitrite reductase activities. Anaerobic activation of an nnrR′-′lacZ fusion required FixLJ and FixK2. In turn, N oxide-mediated induction of nir and nor genes encoding nitrite and nitric oxide reductase, respectively, depended on NnrR. Thus, NnrR expands the FixLJ-FixK2 regulatory cascade by an additional control level which integrates the N oxide signal required for maximal induction of the denitrification genes.

Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum

2011 ◽  
Vol 39 (1) ◽  
pp. 284-288 ◽  
Author(s):  
María J. Torres ◽  
Emilio Bueno ◽  
Socorro Mesa ◽  
Eulogio J. Bedmar ◽  
María J. Delgado
Keyword(s):  
Regulatory Network ◽  
Bradyrhizobium Japonicum ◽  
Control Level ◽  
Receptor Protein ◽  
Regulatory Cascade ◽  
Expression Of Genes ◽  
Bean Plants ◽  
Regulatory Cascades ◽  
Camp Receptor ◽  
Denitrification Genes

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.

scholarly journals Denitrifying metabolism of the methylotrophic marine bacteriumMethylophaga nitratireducenticrescensstrain JAM1

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4098 ◽  
Author(s):  
Florian Mauffrey ◽  
Alexandra Cucaita ◽  
Philippe Constant ◽  
Richard Villemur
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Isotopic Labeling ◽  
Gene Clusters ◽  
No Production ◽  
Gene Encoding ◽  
Anoxic Conditions ◽  
Denitrification Reactor ◽  
Denitrification Genes

BackgroundMethylophaga nitratireducenticrescensstrain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate (${\mathrm{NO}}_{3}^{-}$) to nitrite (${\mathrm{NO}}_{2}^{-}$). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities ofM. nitratireducenticrescensJAM1.MethodsSeries of oxic and anoxic cultures of strain JAM1 were performed with N2O, ${\mathrm{NO}}_{3}^{-}$ or sodium nitroprusside, and growth and N2O, ${\mathrm{NO}}_{3}^{-}$, ${\mathrm{NO}}_{2}^{-}$ and N2concentrations were measured. Ammonium (${\mathrm{NH}}_{4}^{+}$)-free cultures were also tested to assess the dynamics of N2O, ${\mathrm{NO}}_{3}^{-}$ and ${\mathrm{NO}}_{2}^{-}$. Isotopic labeling of N2O was performed in15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochromebc-type nitric oxide reductase (cnorB1andcnorB2) and nitrous oxide reductase (nosZ), and alsonnrSandnorRthat encode NO-sensitive regulators.ResultsStrain JAM1 can reduce NO to N2O and N2O to N2and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory ${\mathrm{NO}}_{2}^{-}$ reductase, ${\mathrm{NO}}_{3}^{-}$-amended cultures produce N2O, representing up to 6% of the N-input. ${\mathrm{NO}}_{2}^{-}$ was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1,cnorB2, nnrSandnorRduring the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures.DiscussionBy showing that all the three denitrification reductases are active, this demonstrates thatM. nitratireducenticrescensJAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated inEscherichia coli.M. nitratireducenticrescensJAM1 is the only reportedMethylophagaspecies that has the capacity to grow under anoxic conditions by using ${\mathrm{NO}}_{3}^{-}$ and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

scholarly journals Bradyrhizobium japonicumFixK2, a Crucial Distributor in the FixLJ-Dependent Regulatory Cascade for Control of Genes Inducible by Low Oxygen Levels

1998 ◽  
Vol 180 (19) ◽  
pp. 5251-5255 ◽  
Author(s):  
D. Nellen-Anthamatten ◽  
P. Rossi ◽  
O. Preisig ◽  
I. Kullik ◽  
M. Babst ◽  
...  
Keyword(s):  
Structural Gene ◽  
Bradyrhizobium Japonicum ◽  
Target Genes ◽  
Regulatory System ◽  
Heme Biosynthesis ◽  
Nitrate Respiration ◽  
Low Oxygen ◽  
Oxygen Levels ◽  
Regulatory Cascade ◽  
Two Component

ABSTRACT Bradyrhizobium japonicum possesses a secondfixK-like gene, fixK 2, in addition to the previously identified fixK 1 gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates fixK 2, whose product then activates fixK 1. While the target genes for control by FixK1 are unknown, there is evidence for activation of the fixNOQP, fixGHIS, andrpoN 1 genes and some heme biosynthesis and nitrate respiration genes by FixK2. FixK2 also regulates its own structural gene, directly or indirectly, in a negative way.

Experimental evidence for plasmid-bornenor-nirgenes inSinorhizobium melilotiJJ1c10

2004 ◽  
Vol 50 (9) ◽  
pp. 657-667 ◽  
Author(s):  
Yiu-Kwok Chan ◽  
Wayne A McCormick
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Sinorhizobium Meliloti ◽  
Nitrite Reduction ◽  
Nucleotide Sequence Analysis ◽  
Gene Products ◽  
Oxide Reduction ◽  
Accessory Gene ◽  
Genes Encoding ◽  
Denitrification Genes

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 N2fixer 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.Key words: denitrification genes, nitric oxide reductase, nitrite reductase, Rhizobiaceae, Sinorhizobium meliloti.

The Bradyrhizobium japonicum napEDABC genes are controlled by the FixLJ-FixK2-NnrR regulatory cascade

2006 ◽  
Vol 34 (1) ◽  
pp. 108-110 ◽  
Author(s):  
E.F. Robles ◽  
C. Sánchez ◽  
N. Bonnard ◽  
M.J. Delgado ◽  
E.J. Bedmar
Keyword(s):  
Nitrate Reductase ◽  
Bradyrhizobium Japonicum ◽  
Reductase Activity ◽  
Lacz Fusion ◽  
Nitrate Respiration ◽  
Regulatory Cascade ◽  
Genes Expression ◽  
Mutant Strains ◽  
Microaerobic Conditions ◽  
Nitrate And Nitrite

Nitrate respiration by the N2-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, PnapE-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The PnapE-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK2 9043 mutant strains. Microaerobic induction of the PnapE-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in β-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.

scholarly journals Transcriptional Regulation of the Flavohemoglobin Gene for Aerobic Nitric Oxide Detoxification by the Second Nitric Oxide-Responsive Regulator of Pseudomonas aeruginosa

2005 ◽  
Vol 187 (12) ◽  
pp. 3960-3968 ◽  
Author(s):  
Hiroyuki Arai ◽  
Michiko Hayashi ◽  
Azusa Kuroi ◽  
Masaharu Ishii ◽  
Yasuo Igarashi
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Mutant Strain ◽  
Regulatory Gene ◽  
Physiological Role ◽  
Nitrate Respiration ◽  
Binding Motif ◽  
Aerobic Conditions ◽  
Mutant Strains ◽  
Denitrification Genes

ABSTRACT The regulatory gene for a σ54-dependent-type transcriptional regulator, fhpR, is located upstream of the fhp gene for flavohemoglobin in Pseudomonas aeruginosa. Transcription of fhp was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the fhp promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for σ54 was found in the promoter region of fhp, but an FNR (fumarate nitrate reductase regulator) box was not. The fhp promoter was inactive in the fhpR or rpoN mutant strain, suggesting that the NO-sensing regulation of the fhp promoter was mediated by FhpR. The DNR-dependent denitrification promoters (nirS, norC, and nosR) were active in the fhpR or rpoN mutants. These results indicated that P. aeruginosa has at least two independent NO-responsive regulatory systems. The fhp or fhpR mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in P. aeruginosa is detoxification of NO under aerobic conditions.

Emissions of nitrous oxide and nitric oxide associated with the decomposition of arable crop residues on a sandy loam soil in Eastern England

Agronomie ◽  
2002 ◽  
Vol 22 (7-8) ◽  
pp. 731-738 ◽  
Author(s):  
Roland Harrison ◽  
Sharon Ellis ◽  
Roy Cross ◽  
James Harrison Hodgson
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Crop Residues ◽  
Sandy Loam ◽  
Sandy Loam Soil ◽  
Arable Crop ◽  
Loam Soil

scholarly journals Genotoxic and oxidative effect of duloxetine on mouse brain and liver tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Isela Álvarez-González ◽  
Scarlett Camacho-Cantera ◽  
Patricia Gómez-González ◽  
Michael J. Rendón Barrón ◽  
José A. Morales-González ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Dna Damage ◽  
Mouse Brain ◽  
Control Level ◽  
Dna Oxidation ◽  
Methyl Methanesulfonate ◽  
Control Group ◽  
Oxidative Potential ◽  
The Brain ◽  
Oxidative Effect

AbstractWe evaluated the duloxetine DNA damaging capacity utilizing the comet assay applied to mouse brain and liver cells, as well as its DNA, lipid, protein, and nitric oxide oxidative potential in the same cells. A kinetic time/dose strategy showed the effect of 2, 20, and 200 mg/kg of the drug administered intraperitoneally once in comparison with a control and a methyl methanesulfonate group. Each parameter was evaluated at 3, 9, 15, and 21 h postadministration in five mice per group, except for the DNA oxidation that was examined only at 9 h postadministration. Results showed a significant DNA damage mainly at 9 h postexposure in both organs. In the brain, with 20 and 200 mg/kg we found 50 and 80% increase over the control group (p ≤ 0.05), in the liver, the increase of 2, 20, and 200 mg/kg of duloxetine was 50, 80, and 135% in comparison with the control level (p ≤ 0.05). DNA, lipid, protein and nitric oxide oxidation increase was also observed in both organs. Our data established the DNA damaging capacity of duloxetine even with a dose from the therapeutic range (2 mg/kg), and suggest that this effect can be related with its oxidative potential.

scholarly journals Mutational analysis of genes implicated in LPS and capsular polysaccharide biosynthesis in the opportunistic pathogen Bacteroides fragilis

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Sheila Patrick ◽  
Simon Houston ◽  
Zubin Thacker ◽  
Garry W. Blakely
Keyword(s):  
Capsular Polysaccharide ◽  
Mutational Analysis ◽  
Bacteroides Fragilis ◽  
Gene Clusters ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Phase Variable ◽  
Multiple Gene ◽  
Polysaccharide Biosynthesis ◽  
Group 1

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

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