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 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.

scholarly journals Nitrogen and Oxygen Regulation of Bacillus subtilis nasDEF Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE

1998 ◽  
Vol 180 (20) ◽  
pp. 5344-5350 ◽  
Author(s):  
Michiko M. Nakano ◽  
Tamara Hoffmann ◽  
Yi Zhu ◽  
Dieter Jahn
Keyword(s):  
Bacillus Subtilis ◽  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Nitrogen Limitation ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Nitrate Respiration ◽  
Nitrite Reductase Activity ◽  
Nitrate And Nitrite

ABSTRACT The nitrate and nitrite reductases of Bacillus subtilishave two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded bynarGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC(encoding assimilatory nitrate reductase) and nasF(required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC andnasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation,nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEFrequired the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.

The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3395-3403 ◽  
Author(s):  
María J. Delgado ◽  
Nathalie Bonnard ◽  
Alvaro Tresierra-Ayala ◽  
Eulogio J. Bedmar ◽  
Peter Müller
Keyword(s):  
Nitrate Reductase ◽  
Bradyrhizobium Japonicum ◽  
Nitrate Reductase Activity ◽  
Transmembrane Protein ◽  
Reductase Activity ◽  
Protein Band ◽  
Anaerobic Growth ◽  
Nitrate Respiration ◽  
Western Blots ◽  
Periplasmic Nitrate Reductase

The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66·5 bp downstream of the centre of a putative FNR-like binding site.

Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum

2005 ◽  
Vol 33 (1) ◽  
pp. 127-129
Author(s):  
N. Bonnard ◽  
A. Tresierra-Ayala ◽  
E.J. Bedmar ◽  
M.J. Delgado
Keyword(s):  
Nitrate Reductase ◽  
Transport System ◽  
Bradyrhizobium Japonicum ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Lacz Fusion ◽  
Periplasmic Nitrate Reductase ◽  
High Affinity ◽  
System A ◽  
Membrane Bound

The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE–lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

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 Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration of Thermus thermophilus HB8

2000 ◽  
Vol 182 (8) ◽  
pp. 2179-2183 ◽  
Author(s):  
Sandra Ramírez ◽  
Renata Moreno ◽  
Olga Zafra ◽  
Pablo Castán ◽  
Cristina Vallés ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Average Distance ◽  
Thermus Thermophilus ◽  
Major Facilitator Superfamily ◽  
Nitrate Respiration ◽  
Mobilizable Plasmid ◽  
Excretion Rates ◽  
Major Facilitator ◽  
Nitrate And Nitrite

ABSTRACT Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.

scholarly journals Role of narK2X and narGHJI inHypoxic Upregulation of Nitrate Reduction byMycobacteriumtuberculosis

2003 ◽  
Vol 185 (24) ◽  
pp. 7247-7256 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Lawrence G. Wayne
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Whole Cells ◽  
Hypoxic Conditions ◽  
Cell Extracts ◽  
Insertional Inactivation ◽  
Nitrate And Nitrite

ABSTRACT Mycobacterium tuberculosis is one of the strongest reducers of nitrate in the genus Mycobacterium. Under microaerobic conditions, whole cells exhibit upregulation of activity, producing approximately eightfold more nitrite than those of aerobic cultures of the same age. Assays of cell extracts from aerobic cultures and hypoxic cultures yielded comparable nitrate reductase activities. Mycobacterium bovis produced only low levels of nitrite, and this activity was not induced by hypoxia. M. tuberculosis has two sets of genes, narGHJI and narX of the narK2X operon, that exhibit some degree of homology to prokaryotic dissimilatory nitrate reductases. Each of these were knocked out by insertional inactivation. The narG mutant showed no nitrate reductase activity in whole culture or in cell-free assays, while the narX mutant showed wild-type levels in both assays. A knockout of the putative nitrite transporter narK2 gene produced a strain that had aerobic levels of nitrate reductase activity but failed to show hypoxic upregulation. Insertion of the M. tuberculosis narGHJI into a nitrate reductase Escherichia coli mutant allowed anaerobic growth in the presence of nitrate. Under aerobic and hypoxic conditions, transcription of narGHJI was constitutive, while the narK2X operon was induced under hypoxia, as measured with a lacZ reporter system and by quantitative real-time reverse PCR. This indicates that nitrate reductase activity in M. tuberculosis is due to the narGHJI locus with no detectable contribution from narX and that the hypoxic upregulation of activity is associated with the induction of the nitrate and nitrite transport gene narK2.

scholarly journals The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated

1996 ◽  
Vol 317 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Nélida BRITO ◽  
Julio AVILA ◽  
M. Dolores PEREZ ◽  
Celedonio GONZALEZ ◽  
José M. SIVERIO
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Hansenula Polymorpha ◽  
Enzymic Activity ◽  
Wild Type ◽  
Reading Frame ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Nitrate And Nitrite

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

scholarly journals Molecular Components of Nitrate and Nitrite Efflux in Yeast

2013 ◽  
Vol 13 (2) ◽  
pp. 267-278 ◽  
Author(s):  
Elisa Cabrera ◽  
Rafaela González-Montelongo ◽  
Teresa Giraldez ◽  
Diego Alvarez de la Rosa ◽  
José M. Siverio
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Uptake ◽  
Reductase Activity ◽  
Hansenula Polymorpha ◽  
Nitrate Assimilation ◽  
Nitrate Reductase Gene ◽  
Content Type ◽  
Nitrite Toxicity ◽  
Essential Components ◽  
Nitrate And Nitrite

ABSTRACTSome eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeastHansenula polymorphawas used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking eitherSSU2orNAR1along with the nitrate reductase geneYNR1showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-knownSaccharomyces cerevisiaesulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.

scholarly journals Disruption of narG, the Gene Encoding the Catalytic Subunit of Respiratory Nitrate Reductase, Also Affects Nitrite Respiration in Pseudomonas fluorescens YT101

1999 ◽  
Vol 181 (16) ◽  
pp. 5099-5102 ◽  
Author(s):  
Jean-François Ghiglione ◽  
Laurent Philippot ◽  
Philippe Normand ◽  
Robert Lensi ◽  
Patrick Potier
Keyword(s):  
Nitrate Reductase ◽  
Pseudomonas Fluorescens ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Α Subunit ◽  
Regulatory Link ◽  
Nitrite Respiration ◽  
Nitrate And Nitrite ◽  
Respiratory Nitrate Reductase

ABSTRACT The Pseudomonas fluorescens YT101 genenarG, which encodes the catalytic α subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar− mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N2O respiration was not affected, anaerobic growth with NO2 as the sole electron acceptor was delayed for all of the Nar− mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

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