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 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.

scholarly journals Linked Expressions ofnapandnosGenes in a Bradyrhizobium japonicum Mutant with Increased N2O Reductase Activity

2013 ◽  
Vol 79 (13) ◽  
pp. 4178-4180 ◽  
Author(s):  
Cristina Sánchez ◽  
Manabu Itakura ◽  
Hisayuki Mitsui ◽  
Kiwamu Minamisawa
Keyword(s):  
Nitrate Reductase ◽  
Bradyrhizobium Japonicum ◽  
Enrichment Culture ◽  
Reductase Activity ◽  
Content Type ◽  
Periplasmic Nitrate Reductase ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
N2o Reductase

ABSTRACTTo understand the mechanisms underlying the increased N2O reductase activity in theBradyrhizobium japonicum5M09 mutant from enrichment culture under N2O respiration, we analyzed the expression of genes encoding denitrification reductases and regulators. Our results suggest a common regulation ofnap(encoding periplasmic nitrate reductase) andnos(encoding N2O reductase).

scholarly journals Characterization of the membrane-bound nitrate reductase activity of aerobically grown chlorate-sensitive mutants of escherichia coli K12

FEBS Letters ◽  
1978 ◽  
Vol 95 (2) ◽  
pp. 290-294 ◽  
Author(s):  
Gérard Giordano ◽  
Alec Graham ◽  
David H. Boxer ◽  
Bruce A. Haddock ◽  
Edgard Azoulay
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Escherichia Coli K12 ◽  
Membrane Bound

scholarly journals Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

2002 ◽  
Vol 184 (5) ◽  
pp. 1314-1323 ◽  
Author(s):  
Valley Stewart ◽  
Yiran Lu ◽  
Andrew J. Darwin
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Null Alleles ◽  
E Coli ◽  
Periplasmic Nitrate Reductase ◽  
Nitrate Concentrations ◽  
K 12

ABSTRACT Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

scholarly journals Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase

1979 ◽  
Vol 184 (1) ◽  
pp. 45-50 ◽  
Author(s):  
E Cadenas ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Selection Method ◽  
Separation Procedure ◽  
Physical Separation ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93–101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).

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.

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