Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria

2003 ◽  
Vol 31 (1) ◽  
pp. 1-10 ◽  
Author(s):  
V. Stewart
Keyword(s):  
Nitrate Reductase ◽  
Functional Divergence ◽  
Anaerobic Respiration ◽  
Anaerobic Growth ◽  
Regulatory Systems ◽  
Two Component ◽  
Sensor Kinases ◽  
Membrane Bound ◽  
Nitrate And Nitrite ◽  
Evolution Function

Nitrate and nitrite are efficient respiratory oxidants for anaerobic growth. In Escherichia coli, the homologous nitrate reductase (Nar) two-component regulatory systems NarX–NarL and NarQ–NarP collaborate to control anaerobic respiratory gene expression in response to nitrate and nitrite. Several other species classified in the γ and β subdivisions of the proteobacteria contain only a single Nar two-component regulatory pair. This raises questions concerning the physiology of anaerobic respiration as well as the evolution, function and cross-regulation of two-component regulatory systems. Here, I focus on the sensor histidine kinases NarX and NarQ, and present a comparison of the deduced NarX and NarQ primary sequences from a broad sampling of proteobacteria. This comparison defines shared features, including a large central region of unknown function that appears to be unique to this family of sensor kinases. I then consider the phylogenetic distribution of narX and narQ genes in relation to anaerobic respiratory enzyme repertoire and physiological function. One noteworthy observation is that narXL genes are specifically associated with the structural genes for membrane-bound nitrate reductase, narGHJI, whereas organization and linkage of the narQ and narP genes is quite variable. I conclude with some speculative thoughts on the evolutionary and functional divergence of the NarX–NarL and NarQ–NarP regulatory systems. Overall, this analysis aims to provide a basis for future hypothesis and experimentation in this area.

Different responses to nitrate and nitrite by the model organism Escherichia coli and the human pathogen Neisseria gonorrhoeae

2006 ◽  
Vol 34 (1) ◽  
pp. 111-114 ◽  
Author(s):  
R.N. Whitehead ◽  
J.A. Cole
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Model Organism ◽  
Anaerobic Growth ◽  
Human Pathogen ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Regulatory Systems ◽  
Two Component ◽  
Nitrate And Nitrite

The ability of Escherichia coli to use both nitrate and nitrite as terminal electron acceptors during anaerobic growth is mediated by the dual-acting two-component regulatory systems NarX-NarL and NarQ-NarP. In contrast, Neisseria gonorrhoeae responds only to nitrite: it expresses only NarQ-NarP. We have shown that although N. gonorrhoeae NarQ can phosphorylate E. coli NarL and NarP, the N. gonorrhoeae NarP is unable to regulate gene expression in E. coli. Mutagenesis experiments have revealed residues in E. coli NarQ that are essential for nitrate and nitrite sensing. Chimaeric proteins revealed domains of NarQ that are important for ligand sensing.

scholarly journals Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum

2007 ◽  
Vol 189 (12) ◽  
pp. 4449-4455 ◽  
Author(s):  
Kelli L. Palmer ◽  
Stacie A. Brown ◽  
Marvin Whiteley
Keyword(s):  
Cystic Fibrosis ◽  
Nitrate Reductase ◽  
Mutational Analysis ◽  
Autosomal Recessive Disorder ◽  
Opportunistic Pathogen ◽  
Anaerobic Growth ◽  
Expression Of Genes ◽  
Membrane Bound ◽  
Lung Infections

ABSTRACT The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.

A composite biochemical system for bacterial nitrate and nitrite assimilation as exemplified by Paracoccus denitrificans

2011 ◽  
Vol 435 (3) ◽  
pp. 743-753 ◽  
Author(s):  
Andrew J. Gates ◽  
Victor M. Luque-Almagro ◽  
Alan D. Goddard ◽  
Stuart J. Ferguson ◽  
M. Dolores Roldán ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Source ◽  
Nitrite Reductase ◽  
Paracoccus Denitrificans ◽  
Gene Clusters ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Nitrate And Nitrite

The denitrifying bacterium Paracoccus denitrificans can grow aerobically or anaerobically using nitrate or nitrite as the sole nitrogen source. The biochemical pathway responsible is expressed from a gene cluster comprising a nitrate/nitrite transporter (NasA), nitrite transporter (NasH), nitrite reductase (NasB), ferredoxin (NasG) and nitrate reductase (NasC). NasB and NasG are essential for growth with nitrate or nitrite as the nitrogen source. NADH serves as the electron donor for nitrate and nitrite reduction, but only NasB has a NADH-oxidizing domain. Nitrate and nitrite reductase activities show the same Km for NADH and can be separated by anion-exchange chromatography, but only fractions containing NasB retain the ability to oxidize NADH. This implies that NasG mediates electron flux from the NADH-oxidizing site in NasB to the sites of nitrate and nitrite reduction in NasC and NasB respectively. Delivery of extracellular nitrate to NasBGC is mediated by NasA, but both NasA and NasH contribute to nitrite uptake. The roles of NasA and NasC can be substituted during anaerobic growth by the biochemically distinct membrane-bound respiratory nitrate reductase (Nar), demonstrating functional overlap. nasG is highly conserved in nitrate/nitrite assimilation gene clusters, which is consistent with a key role for the NasG ferredoxin, as part of a phylogenetically widespread composite nitrate and nitrite reductase system.

Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and NarP during anaerobic growth of Escherichia coli: new insights into microbial physiology

2006 ◽  
Vol 34 (1) ◽  
pp. 104-107 ◽  
Author(s):  
T.W. Overton ◽  
L. Griffiths ◽  
M.D. Patel ◽  
J.L. Hobman ◽  
C.W. Penn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Anaerobic Growth ◽  
Aerobic Growth ◽  
Two Component System ◽  
Strain Mg1655 ◽  
Microbial Physiology ◽  
Two Component ◽  
Nitrate And Nitrite ◽  
First Time

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

scholarly journals Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

2006 ◽  
Vol 72 (8) ◽  
pp. 5173-5180 ◽  
Author(s):  
Helen Ridley ◽  
Carys A. Watts ◽  
David J. Richardson ◽  
Clive S. Butler
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Selective Reduction ◽  
Enterobacter Cloacae ◽  
Anaerobic Growth ◽  
Elemental Selenium ◽  
Membrane Bound ◽  
Membrane Bound Enzymes ◽  
Distinct Membrane

ABSTRACT Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be ∼2 mM, with an observed V max of 500 nmol SeO4 2− min−1 mg−1 (k cat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

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 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.

scholarly journals Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

1998 ◽  
Vol 180 (16) ◽  
pp. 4192-4198 ◽  
Author(s):  
Andrew J. Darwin ◽  
Eva C. Ziegelhoffer ◽  
Patricia J. Kiley ◽  
Valley Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Binding Site ◽  
Anaerobic Growth ◽  
Periplasmic Nitrate Reductase ◽  
Operon Expression ◽  
Nitrate And Nitrite ◽  
K 12

ABSTRACT The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, thenapF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napFcontrol region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

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.

scholarly journals Specificity of induction of glycopeptide resistance genes in Enterococcus faecalis.

1996 ◽  
Vol 40 (10) ◽  
pp. 2291-2295 ◽  
Author(s):  
M Baptista ◽  
F Depardieu ◽  
P Courvalin ◽  
M Arthur
Keyword(s):  
Enterococcus Faecalis ◽  
Regulatory System ◽  
Penicillin G ◽  
Genetic Element ◽  
Glycopeptide Resistance ◽  
Regulatory Systems ◽  
Two Component ◽  
Membrane Bound ◽  
Constitutive Synthesis ◽  
Potential Signal

Regulation of VanA- and VanB-type glycopeptide resistance in enterococci is mediated by related two-component regulatory systems (VanR-VanS and VanRB-VanSB). The transglycosylase inhibitors vancomycin, teicoplanin, and moenomycin induced synthesis of the VanX D,D-dipeptidase in a VanA-type Enterococcus faecalis harboring transposon Tn1546. Inhibitors of reactions immediately preceding (ramoplanin) or following (penicillin G and bacitracin) transglycosylation were not inducers. These results identify accumulation of membrane-bound lipid intermediate II as a potential signal for induction of VanA-type resistance. In E.faecalis BM4281 harboring a wild vanB genetic element, D,D-dipeptidase synthesis was only inducible by vancomycin. Induction of the production of the VanB ligase by vancomycin was required for growth of a vancomycin-dependent derivative of BM4281, since introduction of a plasmid coding for constitutive synthesis of the VanA ligase eliminated the requirement of glycopeptide for growth. Both vancomycin and teicoplanin were able to induce D,D-dipeptidase synthesis in BM4281 derivatives that were vancomycin and teicoplanin resistant or vancomycin and teicoplanin dependent. Acquisition of teicoplanin resistance in the latter types of strains was due to alteration in induction specificity associated with an increase in the sensitivity of the regulatory system to vancomycin. Thus, the wild VanRB-VanSB system is unable or not sensitive enough to sense teicoplanin, although mutations can lead to recognition of this antibiotic.

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