Effect of ionophores on denitrification inFlexibacter canadensis

1995 ◽  
Vol 41 (3) ◽  
pp. 227-234 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Donald F. Niven
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Proton Motive Force ◽  
Nitrate Transport ◽  
Nitric Oxide Reductase ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Nitrite Production ◽  
Carbonyl Cyanide ◽  
No Consumption

Denitrification by Flexibacter canadensis was investigated by measuring the production and (or) consumption of nitrite, nitric oxide (NO), and nitrous oxide (N2O) under anaerobic conditions. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2,4-dinitrophenol, and nigericin, but not valinomycin-K+inhibited the production of nitrite and N2O from nitrate by intact cells. However, CCCP, FCCP, 2,4-dinitrophenol, nigericin, and valinomycin-K+did not affect nitrite production from nitrate by cell-free extracts. These results suggest that nitrate transport was dependent on the transmembrane pH gradient but not on the membrane potential. CCCP, FCCP, and nigericin but not 2,4-dinitrophenol and valinomycin-K+caused NO accumulation during the reduction of nitrite, and also inhibited NO consumption and N2O production from nitrite by intact cells. These results preclude an explanation for NO accumulation based on the collapse of the proton motive force by ionophores, and imply that CCCP, FCCP, and nigericin perhaps dissociated a nitrite reductase–nitric oxide reductase complex, and (or) inhibited nitric oxide reductase specifically. 2,4-Dinitrophenol and CCCP did not inhibit the reduction of N2O to dinitrogen. Addition of ≤ 1.16 μM dissolved NO did not affect the production of nitrite from nitrate, or the disappearance of nitrite or N2O. The rate of NO consumption was linear with concentrations of dissolved NO up to 67 nM. Above 67 nM NO, NO consumption was inhibited, suggesting that NO is toxic to nitric oxide reductase.Key words: ionophores, denitrification, nitric oxide, Flexibacter canadensis.

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.

scholarly journals Nitrosomonas europaea Expresses a Nitric Oxide Reductase during Nitrification

2004 ◽  
Vol 186 (13) ◽  
pp. 4417-4421 ◽  
Author(s):  
Hubertus J. E. Beaumont ◽  
Bas van Schooten ◽  
Sylvia I. Lens ◽  
Hans V. Westerhoff ◽  
Rob J. M. van Spanning
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Gene Cluster ◽  
Nitrosomonas Europaea ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Aerobic Growth ◽  
No Consumption ◽  
Dependent Activity ◽  
Alternative Site

ABSTRACT In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.

scholarly journals Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp. Strain FA1

2003 ◽  
Vol 69 (9) ◽  
pp. 5216-5221 ◽  
Author(s):  
Bharat Bhushan ◽  
Louise Paquet ◽  
Jim C. Spain ◽  
Jalal Hawari
Keyword(s):  
Nitrous Oxide ◽  
Enzyme Preparation ◽  
Electron Transfer Reaction ◽  
Garden Soil ◽  
Resting Cells ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Cell Biomass ◽  
Membrane Enzyme ◽  
Enzyme Catalyzed Reactions

ABSTRACT The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h−1 mg of cell biomass−1 and 11.5 ± 0.4 nmol h−1 mg of protein−1, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO2 −), 1.5 molecules of nitrous oxide (N2O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.

scholarly journals Analysis of multiple haloarchaeal genomes suggests that the quinone-dependent respiratory nitric oxide reductase is an important source of nitrous oxide in hypersaline environments

2017 ◽  
Vol 9 (6) ◽  
pp. 788-796 ◽  
Author(s):  
Javier Torregrosa-Crespo ◽  
Pedro González-Torres ◽  
Vanesa Bautista ◽  
Julia M. Esclapez ◽  
Carmen Pire ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Nitric Oxide Reductase ◽  
Hypersaline Environments

Production and consumption of nitric oxide by denitrifyingFlexibacter canadensis

1995 ◽  
Vol 41 (7) ◽  
pp. 585-591 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Yiu-Kwok Chan
Keyword(s):  
Nitric Oxide ◽  
Nitrite Reductase ◽  
Chelating Agent ◽  
No Production ◽  
Production And Consumption ◽  
Carbonyl Cyanide ◽  
No Consumption ◽  
High Concentrations ◽  
Ph Range ◽  
Triton X

Production and consumption of nitric oxide (NO) by Flexibacter canadensis cells under anaerobic conditions was investigated using a chemiluminescence NO analyzer. Net NO production from nitrite in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was pH dependent, increased in the pH range from 4.5 to 6.5, and sharply decreased at pH >6.5. CCCP inhibited NO consumption but only at pH values ≤6.5. This can explain why CCCP stimulation of NO production depends on the pH. Denitrification of nitrite at high concentrations (≥5 mM) also resulted in net NO accumulation. Diethyldithiocarbamate, a copper chelating agent, prevented not only net production of NO during the reduction of nitrite in the presence of CCCP, but also production of nitrous oxide (N2O) from nitrite in the presence of C2H2. This suggests that F. canadensis may possess a copper-type nitrite reductase. However, cytochrome cd1- and copper-containing nitrite reductase DNA probes from Pseudomonas species did not hybridize with the total DNA of F. canadensis, indicating that the nitrite reductase of F. canadensis may possess unique properties. In addition to diethyldithiocarbamate, sulfide, carbon monoxide, azide, cyanide, hydroxylamine and Triton X-100 prevented net NO production from nitrite in the presence of CCCP, and also inhibited NO consumption. C2H2, an inhibitor of N2O reductase, did not affect NO production or consumption.Key words: nitrite reductase, nitric oxide (NO), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flexibacter canadensis.

scholarly journals Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

2015 ◽  
Vol 82 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Brian J. Vaccaro ◽  
Michael P. Thorgersen ◽  
W. Andrew Lancaster ◽  
Morgan N. Price ◽  
Kelly M. Wetmore ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Electron Acceptor ◽  
Pseudomonas Stutzeri ◽  
Basic Function ◽  
Nitric Oxide Reductase ◽  
Content Type ◽  
Nitric Oxide Release ◽  
Peripheral Proteins ◽  
Cofactor Biosynthesis

ABSTRACTEnzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.

scholarly journals Diverse effects of nitric oxide reductase NorV on Aeromonas hydrophila virulence-associated traits under aerobic and anaerobic conditions

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Jin Liu ◽  
Yuhao Dong ◽  
Nannan Wang ◽  
Shuiyan Ma ◽  
Chengping Lu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Aeromonas Hydrophila ◽  
Dependent Manner ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Significant Difference ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

Abstract NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection.

scholarly journals Structural basis for nitrous oxide generation by bacterial nitric oxide reductases

2012 ◽  
Vol 367 (1593) ◽  
pp. 1195-1203 ◽  
Author(s):  
Yoshitsugu Shiro ◽  
Hiroshi Sugimoto ◽  
Takehiko Tosha ◽  
Shingo Nagano ◽  
Tomoya Hino
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Structural Basis ◽  
Nitric Oxide Reductase ◽  
Respiratory Enzymes ◽  
Functional Differences ◽  
Cytochrome Oxidases ◽  
Structural Differences ◽  
Nitric Oxide Reduction ◽  
Copper Oxidase

The crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.

scholarly journals Two Conserved Glutamates in the Bacterial Nitric Oxide Reductase Are Essential for Activity but Not Assembly of the Enzyme

2001 ◽  
Vol 183 (1) ◽  
pp. 189-199 ◽  
Author(s):  
Gareth Butland ◽  
Stephen Spiro ◽  
Nicholas J. Watmough ◽  
David J. Richardson
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Transmembrane Helix ◽  
Spectroscopic Characterization ◽  
Nitric Oxide Reductase ◽  
Intact Cells ◽  
Heterologous System ◽  
Proton Uptake ◽  
Heme Copper Oxidases

ABSTRACT The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an FeB–heme-Fe dinuclear catalytic center rather than a CuB–heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues inParacoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex inEscherichia coli. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.

Sign in / Sign up

Export Citation Format

Share Document