Involvement of hydrogenases in the formation of highly catalytic Pd(0) nanoparticles by bioreduction of Pd(II) using Escherichia coli mutant strains

Escherichia coli produces at least three [NiFe] hydrogenases (Hyd-1, Hyd-2 and Hyd-3). Hyd-1 and Hyd-2 are membrane-bound respiratory isoenzymes with their catalytic subunits exposed to the periplasmic side of the membrane. Hyd-3 is part of the cytoplasmically oriented formate hydrogenlyase complex. In this work the involvement of each of these hydrogenases in Pd(II) reduction under acidic (pH 2.4) conditions was studied. While all three hydrogenases could contribute to Pd(II) reduction, the presence of either periplasmic hydrogenase (Hyd-1 or Hyd-2) was required to observe Pd(II) reduction rates comparable to the parent strain. An E. coli mutant strain genetically deprived of all hydrogenase activity showed negligible Pd(II) reduction. Electron microscopy suggested that the location of the resulting Pd(0) deposits was as expected from the subcellular localization of the particular hydrogenase involved in the reduction process. Membrane separation experiments established that Pd(II) reductase activity is membrane-bound and that hydrogenases are required to initiate Pd(II) reduction. The catalytic activity of the resulting Pd(0) nanoparticles in the reduction of Cr(VI) to Cr(III) varied according to the E. coli mutant strain used for the initial bioreduction of Pd(II). Optimum Cr(VI) reduction, comparable to that observed with a commercial Pd catalyst, was observed when the bio-Pd(0) catalytic particles were prepared from a strain containing an active Hyd-1. The results are discussed in the context of economic production of novel nanometallic catalysts.

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Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

Biochemistry and Cell Biology ◽

10.1139/o02-131 ◽

2002 ◽

Vol 80 (4) ◽

pp. 435-443 ◽

Cited By ~ 10

Author(s):

Damaraju Sambasivarao ◽

Raymond J Turner ◽

Peter T Bilous ◽

Richard A Rothery ◽

Gillian Shaw ◽

...

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Mutant Strain ◽

Physiological Function ◽

Signal Sequence ◽

Molybdenum Cofactor ◽

Wild Type ◽

Dmso Reductase ◽

E Coli ◽

Membrane Bound

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeBMoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

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Identification and partial characterization of an Escherichia coli mutant with altered hydrogenase activity

Canadian Journal of Biochemistry ◽

10.1139/o80-047 ◽

1980 ◽

Vol 58 (4) ◽

pp. 361-367 ◽

Cited By ~ 15

Author(s):

Bernard R. Glick ◽

Patrick Y. Wang ◽

Henry Schneider ◽

William G. Martin

Keyword(s):

Escherichia Coli ◽

Mutant Strain ◽

Filter Paper ◽

Specific Activity ◽

Anaerobic Growth ◽

Hydrogenase Activity ◽

Wild Type ◽

Coli Mutant ◽

In The Wild ◽

Membrane Bound

An Escherichia coli mutant strain with altered hydrogenase activity was isolated using a filter paper assay. This assay depends on the ability of hydrogenase-containing microorganisms to reduce methyl viologen impregnated in filter paper, producing purple-colored colonies in the presence of hydrogen. Membrane-bound and cytoplasmic hydrogenase activities of wild-type and mutant strains were compared by amperometric measurement of hydrogen production. The cytoplasmic activities of mutant and wild type were comparable. The membrane-bound activity was lower in the mutant than in the wild type. Upon addition of detergent to the membrane fraction the specific activity of the enzyme from the mutant strain increased so that it equalled that of the wild type. The mutant requires an exogenous electron acceptor for anaerobic growth providing evidence for the function of the hydrogenase in anaerobic growth.

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

Antioxidants ◽

10.3390/antiox10010013 ◽

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.

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

Journal of Food Protection ◽

10.4315/0362-028x-45.3.241 ◽

1982 ◽

Vol 45 (3) ◽

pp. 241-243 ◽

Cited By ~ 2

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.

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Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.184.13.3630-3639.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3630-3639 ◽

Cited By ~ 39

Author(s):

David E. Nelson ◽

Anindya S. Ghosh ◽

Avery L. Paulson ◽

Kevin D. Young

Keyword(s):

Escherichia Coli ◽

Site Directed Mutagenesis ◽

Outer Face ◽

Penicillin Binding Protein ◽

Membrane Anchor ◽

E Coli ◽

Membrane Bound ◽

Carboxy Terminal ◽

Membrane Anchoring ◽

Β Sheet

ABSTRACT Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar dd-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other dd-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the dd-carboxypeptidase enzymatic core.

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Modification by an R determinant for streptomycin of hissd phenotype in a mutant strain of Escherichia coli B

Genetics Research ◽

10.1017/s0016672300011721 ◽

1968 ◽

Vol 12 (2) ◽

pp. 109-116 ◽

Cited By ~ 1

Author(s):

A. M. Molina ◽

L. Calegari ◽

G. Conte

Keyword(s):

Escherichia Coli ◽

Cell Membrane ◽

Mutant Strain ◽

Minimal Medium ◽

Specific Requirement ◽

Resistance Level ◽

E Coli ◽

Level Characteristic ◽

Escherichia Coli B

When an R determinant for streptomycin is transferred into a conditionally streptomycin-dependent E. coli B mutant—which requires in minimal medium either histidine or streptomycin—the latter behaves like a histidineless strain. This phenotype modification shows that the repairing action of streptomycin is prevented. The specific requirement of the strain is not now replaced even by streptomycin concentrations up to 10000 µg/ml at which the conditionally streptomycin-dependent mutant could originally grow, and which are well beyond the resistance level characteristic of the R determinant itself. These data seem to suggest that a reduction in permeability of the cell membrane cannot be held responsible for the phenomenon observed.

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Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

Applied and Environmental Microbiology ◽

10.1128/aem.02545-09 ◽

2010 ◽

Vol 76 (7) ◽

pp. 2107-2114 ◽

Cited By ~ 35

Author(s):

Qingzhao Wang ◽

Mark S. Ou ◽

Y. Kim ◽

L. O. Ingram ◽

K. T. Shanmugam

Keyword(s):

Escherichia Coli ◽

Mutant Strain ◽

Pyruvate Dehydrogenase ◽

Metabolic Flux ◽

Anaerobic Growth ◽

Kinetic Properties ◽

Fermentation Products ◽

E Coli ◽

Pyruvate Node

ABSTRACT During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

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Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

Canadian Journal of Biochemistry ◽

10.1139/o79-101 ◽

1979 ◽

Vol 57 (6) ◽

pp. 813-821 ◽

Cited By ~ 49

Author(s):

Peter Dickie ◽

Joel H. Weiner

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Sodium Cholate ◽

Defined Medium ◽

Exchange Chromatography ◽

Fumarate Reductase ◽

Molar Ratios ◽

Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

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Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3448-3455.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3448-3455 ◽

Cited By ~ 84

Author(s):

JÃ¯Â¿Â½rg Rau ◽

Andreas Stolz

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Bacterial Species ◽

Azo Compounds ◽

Biochemical Tests ◽

Pronounced Increase ◽

E Coli ◽

Amino Terminal ◽

Azo Reductase ◽

Anaerobic Reduction

ABSTRACT Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.

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

Biochemical Journal ◽

10.1042/bj1900079 ◽

1980 ◽

Vol 190 (1) ◽

pp. 79-94 ◽

Cited By ~ 42

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]

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