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 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.

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.

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.

In vivo Nitrate Reductase Activity in Leaves of Pearl Millet, Pennisetum americanum (L.) Leeke

1987 ◽  
Vol 14 (2) ◽  
pp. 125 ◽  
Author(s):  
SV Chanda ◽  
AK Joshi ◽  
PN Krishnan ◽  
YD Singh
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Potassium Phosphate ◽  
Limiting Factor ◽  
Growth Stages ◽  
In Vivo Assay ◽  
Rate Limiting ◽  
Nr Activity

In the in vivo assay of nitrate reductase (NR) in P. americanum leaves, addition of 1% (v/v) Triton X-100, potassium phosphate buffer (80 mM, pH 7.4) and 1.13 mM NADH to the assay medium resulted in maximum activity. With increasing concentration of NADH, saturation-type kinetics were observed. Based on this data metabolic pool concentration for NADH and apparent Km for nitrate reductase were determined. In field studies with cultivars BJ-104, J-104 and 5141-A of P. americanum, the relative limitation of NO3-, NADH and nitrate reductase in NO3- assimilation was determined. NR activity was measured by four modifications of the in vivo assay technique (with NO3-, with NADH, without NO3- and NADH and with both NO3- and NADH additions to the reaction mixture) and with one in vitro technique. For all the cultivars, NADH was the major rate-limiting factor for in vivo assay during early growth stages, while at later stages, NO3- was limiting. At no stage was NR rate-limiting. It is concluded that NR activity alone may not serve as biochemical marker for improved efficiency of utilisation of nitrogen in P. americanum.

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