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 Effect of tungsten on nitrate and nitrite reductases in Azospirillum brasilense Sp7

1991 ◽  
Vol 37 (10) ◽  
pp. 744-750 ◽  
Author(s):  
Christian Chauret ◽  
Roger Knowles
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Azospirillum Brasilense ◽  
Nitrate Reductase Activity ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Whole Cells ◽  
Azospirillum Brasilense Sp7 ◽  
Nitrite Reductase Activity ◽  
Nitrate And Nitrite

Tungstate, at concentrations that completely suppressed nitrate reductase activity in Paracoccus denitrificans, caused only partial inhibition of nitrate reductase in Azospirillum brasilense Sp7. Nitrate reductase activity in cell-free extracts was much more sensitive than whole cells to tungstate, suggesting that there may be a barrier to its transport. Nitrite reductase activity was partially inhibited by tungstate in both whole cells and cell-free extracts. Azospirillum brasilense apparently scavenged enough contaminating molybdenum from molybdenum-limited medium to allow maximum nitrate reductase activity, which was not stimulated by added molybdate. Cells grown in molybdenum-depleted medium could not reduce nitrate. Nitrate concentrations less than 0.25 mM inhibited activity, but not synthesis, of nitrite reductase and caused significant accumulation of nitrite during reduction of nitrate. Key words: Azospirillum brasilense, Paracoccus denitrificans, nitrate reductase, nitrite reductase, tungsten, molybdenum, denitrification.

scholarly journals Heterologous NNR-Mediated Nitric Oxide Signaling inEscherichia coli

2000 ◽  
Vol 182 (22) ◽  
pp. 6434-6439 ◽  
Author(s):  
Matthew I. Hutchings ◽  
Neil Shearer ◽  
Sarah Wastell ◽  
Rob J. M. van Spanning ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Culture Media ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Nitric Oxide Signaling ◽  
Cell Extracts ◽  
Nitrate And Nitrite

ABSTRACT The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melRpromoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO+ donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a “positive control” phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

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 Salivary Nitrate, Nitrite and Nitrate Reductase Activity in Relation to Risk of Oral Cancer in Egypt

1998 ◽  
Vol 14 (2) ◽  
pp. 91-97 ◽  
Author(s):  
Alaa F. Badawi ◽  
Gehan Hosny ◽  
Mohamed El-Hadary ◽  
Mostafa H. Mostafa
Keyword(s):  
Oral Cancer ◽  
Nitrate Reductase ◽  
Odds Ratio ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Healthy Individuals ◽  
Oral Cancer Patients ◽  
Nitrate And Nitrite ◽  
Increased Activity

It has been suggested that nitrate and nitrite may play a role in the etiology of human oral cancer. We investigated whether salivary nitrate and nitrite and the activity of nitrate reductase (NRase) may affect the risk of oral cancer in Egypt, an area with high levels of environmental nitrosating agents. Levels of salivary nitrite (8.3 ± 1.0 μg/ml) and nitrate (44 ± 3.7 μg/ml) and activity of NRase (74 ± 10 nmol/ml/min) were significantly (P< 0.05) higher in oral cancer patients (n= 42) compared to control Egyptian healthy individuals (n= 40, nitrite = 5.3 ± 0.3 μg/ml, nitrate = 27 ± 1.2 μg/ml, and NRase activity = 46 ± 4 nmol/ml/min). The adjusted odds ratio (OR) and the 95% confidence intervals (C.I.) for risk of oral cancer, categorized by the levels of salivary nitrate and nitrite and NRase activity, showed a higher cancer risk associated with nitrite > 7.5 μg/ml (OR: 3.0, C.I.: 1.0–9.3), nitrite > 40 μg/ml (OR: 4.3, C.I.: 1.4–13.3) and NRase activity > 50 nmol/ml/min (OR: 2.9, C.I.: 1.1–7.4). Our findings suggest that increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. Together with the increased activity of salivary NRase, these observations may explain, at least in part, the role of nitrate and nitrite in the development of oral cancer in individuals from an area with a high burden ofN-nitroso precursors.

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.

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 Nitrate reductase activity in chosen mosses: a case of study in skalny colliery waste tip / Aktywność reduktazy azotanowej u wybranych mchów na przykładzie hałdy powęglowej skalny

2013 ◽  
Vol 18 (1-2) ◽  
pp. 61-68
Author(s):  
Marek Krywult ◽  
Anna Salachna ◽  
Damian Chmura ◽  
Jan Żarnowiec
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Stream Water ◽  
Reductase Activity ◽  
Dry Mass ◽  
Control Area ◽  
High Dispersion ◽  
Contaminated Waste ◽  
Nitrate And Nitrite ◽  
City Park

Abstract Two species of mosses in relation to nitrogen metabolism were examined. This subject is little known in this group of plant. Investigations of nitrate reductase activity in green tissues of Brachythecium rutabulum (Hedw.) Schimp. and Atrichum undulatum (Hedw.) P.Beauv. were performed. The study was conducted in two localities: heavy contaminated waste tip Skalny located in Upper Silesia, and Blonia City Park in Bielsko-Biala which place was chosen as a control area. For both species high activity of the enzyme was detected. The nitrate reductase activity varied between 99 to 9093 nmol per g dry mass per hour for B. rutabulum and 265 to 5135 nmol per g d.m. per hour of nitrite synthesized for A. undulatum respectively on Skalny waste tip. In the control area the results varied between 747 to 1077 for B. rutabulum and 171 to 518 nmol per g d.m. per hour of nitrite synthesized for A. undulatum, respectively. The differences were statistically significant only between the two species but not between habitats probably due to high dispersion and small amount of replications. The levels of nitrate and nitrite in stream water in both areas were also measured. In the Skalny waste tip there were high and reached 1.66 mg · dm-3 of nitrite and 65 mg · dm-3 of nitrate, respectively. In the control area these amounts were lower and reach zero level for nitrite and 4.5 mg · dm-3 of nitrate, respectively.

O2 regulation of denitrification in Flexibacter canadensis

1994 ◽  
Vol 40 (11) ◽  
pp. 916-921 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Donald F. Niven
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Reductase Activity ◽  
Inhibitory Effect ◽  
Nitrate Transport ◽  
Intact Cells ◽  
Benzyl Viologen ◽  
Whole Cells ◽  
Nitrate And Nitrite

We studied the sensitivity to oxygen of the reductases involved in denitrification by whole cells and membrane fractions of Flexibacter canadensis. All of the nitrate reductase activity was found in the membrane fraction, suggesting that the nitrate reductase of F. canadensis is largely or entirely a membrane-bound enzyme. Methyl viologen and benzyl viologen were good electron donors to nitrate reductase in both whole cells and membrane fractions, whereas glucose and glycerol were effective in whole cells but, as expected, not in membrane fractions. Oxygen, generated by means of H2O2 plus catalase, inhibited the production of nitrite from nitrate by intact cells but not by membrane fractions, suggesting that O2 exerts its inhibitory effect at the level of nitrate transport rather than nitrate reduction. In intact cells, the rates of nitric oxide accumulation during reduction of nitrite in the presence of 20 μM carbonyl cyanide m-chlorophenylhydrazone, and consumption of nitric oxide and nitrous oxide, decreased as the concentration of H2O2 was increased. The concentrations of H2O2 giving 50% inhibition of reduction of nitrate and nitrite were 0.34 and 0.12 mM, respectively. In contrast, the rates of nitric oxide and nitrous oxide consumption were inhibited by only 36 and 32% at a concentration of H2O2 of 3.99 mM. These results indicate that the reduction of both nitric oxide and nitrous oxide is relatively tolerant to oxygen, and that nitrite reductase is much more sensitive to oxygen than the other reductases.Key words: nitrate reductase, nitrate transport, denitrification, O2 inhibition, Flexibacter canadensis.

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