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 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 Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

2006 ◽  
Vol 72 (1) ◽  
pp. 695-701 ◽  
Author(s):  
Vandana Sharma ◽  
Chris E. Noriega ◽  
John J. Rowe
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrate Reductase ◽  
Nitrate Uptake ◽  
Reductase Activity ◽  
Physiological Role ◽  
Genome Project ◽  
Anaerobic Growth ◽  
Wild Type ◽  
Uptake Rates ◽  
Nitrate And Nitrite

ABSTRACT Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ΔnarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ΔnarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ΔnarK2::Gm and ΔnarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

scholarly journals Compensatory periplasmic nitrate reductase activity supports anaerobic growth ofPseudomonas aeruginosaPAO1 in the absence of membrane nitrate reductase

2009 ◽  
Vol 55 (10) ◽  
pp. 1133-1144 ◽  
Author(s):  
Nadine E. Van Alst ◽  
Lani A. Sherrill ◽  
Barbara H. Iglewski ◽  
Constantine G. Haidaris
Keyword(s):  
Nitrate Reductase ◽  
Response Regulator ◽  
Reductase Activity ◽  
Transcriptional Start Site ◽  
Atp Synthesis ◽  
Anaerobic Growth ◽  
Start Site ◽  
Terminal Electron Acceptor ◽  
Periplasmic Nitrate Reductase ◽  
Growth Recovery

Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa . Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.

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.

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 Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1

2006 ◽  
Vol 189 (2) ◽  
pp. 656-662 ◽  
Author(s):  
Claribel Cruz-García ◽  
Alison E. Murray ◽  
Joel A. Klappenbach ◽  
Valley Stewart ◽  
James M. Tiedje
Keyword(s):  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Electron Acceptor ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Shewanella Oneidensis ◽  
Gene Encoding ◽  
Nitrate Ammonification ◽  
Sequential Reduction

ABSTRACT Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.

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 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.

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