scholarly journals Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

1977 ◽  
Vol 164 (1) ◽  
pp. 199-211 ◽  
Author(s):  
Robert W. Jones ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Respiratory Enzymes ◽  
Radical Species ◽  
Benzyl Viologen ◽  
E Coli ◽  
Nitrite Reductase Activity

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.

scholarly journals The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli

1980 ◽  
Vol 190 (1) ◽  
pp. 79-94 ◽  
Author(s):  
Robert W. Jones ◽  
Alan Lamont ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Proton Translocation ◽  
Deficient Mutant ◽  
Benzyl Viologen ◽  
Low Concentrations ◽  
Nitrite Reductase Activity

Low concentrations (1–50μm) of ubiquinol1 were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol1 drove an outward translocation of protons with a corrected →H+/2e− stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309–323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol1 was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA−menA−), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol1. Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ+) to the oxidized species (DQ++) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV+) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV++. In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol1 are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate→H+/2e− stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol1 (QH2), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed→H+/2e− stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]

scholarly journals Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

2002 ◽  
Vol 184 (5) ◽  
pp. 1314-1323 ◽  
Author(s):  
Valley Stewart ◽  
Yiran Lu ◽  
Andrew J. Darwin
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Null Alleles ◽  
E Coli ◽  
Periplasmic Nitrate Reductase ◽  
Nitrate Concentrations ◽  
K 12

ABSTRACT Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

scholarly journals Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase

1979 ◽  
Vol 184 (1) ◽  
pp. 45-50 ◽  
Author(s):  
E Cadenas ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Selection Method ◽  
Separation Procedure ◽  
Physical Separation ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93–101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).

scholarly journals Effect of ruminal administration ofEscherichia coliwild type or a genetically modified strain with enhanced high nitrite reductase activity on methane emission and nitrate toxicity in nitrate-infused sheep

2005 ◽  
Vol 94 (5) ◽  
pp. 691-697 ◽  
Author(s):  
C. Sar ◽  
B. Mwenya ◽  
B. Pen ◽  
K. Takaura ◽  
R. Morikawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Body Weight ◽  
Single Dose ◽  
Methane Emission ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Wild Type ◽  
E Coli ◽  
Reduction Activity ◽  
Nitrite Reductase Activity

The effects of two kinds ofEscherichia coli(E. coli) strain, wild-typeE. coliW3110 andE. colinir-Ptac, which has enhanced NO2reduction activity, on oral CH4emission and NO3toxicity in NO3-treated sheep were assessed in a respiratory hood system in a 4×6 Youden square design. NO3(1·3g NaNO3/kg0·75body weight) and/orE. colistrains were delivered into the rumen through a fistula as a single dose 30min after the morning meal.Escherichia colicells were inoculated for sheep to provide an initialE. colicell density of optical density at 660nm of 2, which corresponded to 2×1010cells/ml. The six treatments consisted of saline,E. coliW3110,E. colinir-Ptac, NO3, NO3plusE. coliW3110, and NO3plusE. colinir-Ptac. CH4emission from sheep was reduced by the inoculation ofE. coliW3110 orE. colinir-Ptac by 6% and 12%, respectively. NO3markedly inhibited CH4emission from sheep. Compared with sheep given NO3alone, the inoculation ofE. coliW3110 to NO3-infused sheep lessened ruminal and plasma toxic NO2accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO2accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation ofE. colinir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation ofE. coliW3110 orE. colinir-Ptac. The inoculation ofE. coliW3110 may abate NO3toxicity when NO3is used to inhibit CH4emission from ruminants.

Functional Analysis of the Escherichia coli Molybdopterin Cofactor Biosynthesis Protein MoeA by Site-Directed Mutagenesis

2002 ◽  
Vol 383 (2) ◽  
pp. 319-323 ◽  
Author(s):  
C. Sandu ◽  
R. Brandsch
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Sitedirected Mutagenesis ◽  
Cofactor Biosynthesis

AbstractFive moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Momolybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

scholarly journals In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Antioxidants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 13
Author(s):  
Elena Forte ◽  
Sergey A. Siletsky ◽  
Vitaliy B. Borisov
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Dissociation Constants ◽  
Reductase Activity ◽  
Ammonia Concentration ◽  
High Concentration ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cytochrome Bo3 ◽  
Oxygen Reductase

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives1

1982 ◽  
Vol 45 (3) ◽  
pp. 241-243 ◽  
Author(s):  
M. KRUK ◽  
J. S. LEE
Keyword(s):  
Escherichia Coli ◽  
Benzoic Acid ◽  
Sorbic Acid ◽  
Reductase Activity ◽  
Resting Cells ◽  
E Coli

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes II. Changes in the Cell Envelope of Escherichia coli

1970 ◽  
Vol 1 (3) ◽  
pp. 311-318
Author(s):  
D. Friedberg ◽  
I. Friedberg ◽  
M. Shilo
Keyword(s):  
Escherichia Coli ◽  
Polymorphonuclear Leukocytes ◽  
Cytoplasmic Membrane ◽  
Morphological Changes ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intact Cells ◽  
Lysosomal Fraction ◽  
E Coli ◽  
Absorbing Material

Interaction of lysosomal fraction with Escherichia coli caused damage to the cell envelope of these intact cells and to the cytoplasmic membrane of E. coli spheroplasts. The damage to the cytoplasmic membrane was manifested in the release of 260-nm absorbing material and β-galactosidase from the spheroplasts, and by increased permeability of cryptic cells to O -nitrophenyl-β- d -galactopyranoside; damage to the cell wall was measured by release of alkaline phosphatase. Microscope observation showed morphological changes in the cell envelope.

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