Molecular Study and Partial Characterization of Iron-only Hydrogenase inDesulfovibrio fructosovorans

Casalot, Laurence; Hatchikian, Claude E.; Forget, Nicole; Philip, Pascale de; Dermoun, Zorah; Bélaı̈ch, Jean-Pierre; Rousset, Marc

doi:10.1006/anae.1997.0137

Cited by 28 publications

(14 citation statements)

References 35 publications

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“…Three different hydrogenases have been identified in this bacterium: [NiFe] and [Fe] hydrogenases (1,10,27), localized in the periplasm, and a heterotetrameric NADP-reducing [Fe] hydrogenase, localized in the cytoplasm (4,21,22). In the present work, we have combined physiological, genetic, and biochemical ap-proaches to determine, at the molecular level, the exact roles of these different hydrogenases in Tc(VII) reduction and precipitation.…”

Section: Technetium (mentioning

confidence: 99%

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase

Luca

Philip

Dermoun

et al. 2001

Appl Environ Microbiol

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107

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Resting cells of the sulfate-reducing bacterium Desulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfatereducing bacteria functions as a Tc(VII) reductase. The role of cytochrome c 3 in the mechanism is also discussed.

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Section: Technetium (mentioning

confidence: 99%

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase

Luca

Philip

Dermoun

et al. 2001

Appl Environ Microbiol

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107

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“…Three hydrogenases (periplasmic NiFe hydrogenase [23] and Fe hydrogenase [1] and a cytoplasmic NADP-reducing hydrogenase [18]) have been found in Desulfovibrio fructosivorans, together with a recently identified fourth hydrogenase (2), cytoplasmic membrane-bound NiFe hydrogenase (M. Rousset, unpublished data).…”

mentioning

confidence: 95%

Bioaccumulation of Palladium by Desulfovibrio fructosivorans Wild-Type and Hydrogenase-Deficient Strains

Mikheenko

Rousset

Dementin

et al. 2008

Appl Environ Microbiol

111

105

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“…In this species, three hydrogenases have been already characterized: a periplasmic [NiFe] hydrogenase which represents about 1% of the total proteins (8,20), a cytoplasmic NADPreducing hydrogenase (13), and a periplasmic [Fe] hydrogenase (4). In order to elucidate the relative importance of these various hydrogenases in the energy-generating metabolism of D. fructosovorans, deletions were first made by marker exchange mutagenesis of the genes encoding the [NiFe] hydrogenase (19) and the NADP-reducing hydrogenase (12).…”

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confidence: 99%

“…Methyl viologen reduction activity of the triple mutant. Hydrogenase activity in D. fructosovorans is usually determined by measurement of hydrogen-dependent methyl viologen reduction activity in soluble cellular extract obtained from cultures grown on a 30 mM fructose-50 mM sulfate medium (4,5,12,19). The presence of the hydrogenase activities in the D. fructosovorans strains was tested in native polyacrylamide gel electrophoresis (Fig.…”

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confidence: 99%

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

et al. 2002

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A strain devoid of the three hydrogenases characterized for Desulfovibrio fructosovorans was constructed using marker exchange mutagenesis. As expected, the H 2 -dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H ؉ -D 2 exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in D. fructosovorans.Molecular hydrogen plays an important role in the energygenerating metabolism of sulfate reducers belonging to the genus Desulfovibrio. Desulfovibrio species can alternatively utilize hydrogen as the sole source of electron and energy (2, 3) or can produce hydrogen when growing fermentatively on a suitable carbon source in the absence of sulfate as an electron acceptor (17). Furthermore, hydrogen is successively produced and consumed during the degradation of organic compounds in the presence of sulfate (7,22,23 [NiFeSe] on the basis of their metal contents), and the cellular location of hydrogenases vary considerably from one Desulfovibrio species to another (6,25). This diversity makes the role of these various hydrogenases difficult to determine.With the aim to study the role of hydrogenases in Desulfovibrio, we chose D. fructosovorans DSM 3604 (16) as a model. In this species, three hydrogenases have been already characterized: a periplasmic [NiFe] hydrogenase which represents about 1% of the total proteins (8, 20), a cytoplasmic NADPreducing hydrogenase (13), and a periplasmic [Fe] hydrogenase (4). In order to elucidate the relative importance of these various hydrogenases in the energy-generating metabolism of D. fructosovorans, deletions were first made by marker exchange mutagenesis of the genes encoding the [NiFe] hydrogenase (19) and the NADP-reducing hydrogenase (12). All mutants (single or double) showed significant growth on organic substrates as well as on medium containing H 2 as the sole energy source.Construction and molecular characterization of a triple mutant depleted of all three hydrogenases. In order to perform the marker exchange experiment, a 5-kb fragment containing the two structural genes (hydAB) coding for the [Fe] hydrogenase of D. fructosovorans (obtained by PCR amplification performed on genomic DNA by using oligonucleotides 1 [5Ј-AAACGGCGACGCCGTGGTCGGCAAGGTCAA-3Ј] and 2 [5Ј-CGATGTCGGTGCCCGGATATTT-3Ј]) was cloned in pMosblue-T-vector (Amersham) to give the recombinant plasmid pMBE9 (Fig. 1). A 1.3-kb fragment containing the gentamicin resistance gene (acc1) was obtained by PCR amplification performed on pML122 (10), using two oligonucleotides, one introducing a BspEI restriction site (in boldface) (Gm 1, 5Ј-TTAAATCCGGATGAAGGCACGAACCCAGTT-3Ј) and one located downstream from the BstEII restriction site (Gm 2, 5Ј-GACGCTTAGCACCTCTGATAGTT-3Ј). The amplification product was digested with BstEII and BspEI and cloned into pMBE9 with a deletion of the BstEII-BspEI fragment containing hydAB, to give pE9Gm (Fig. 1). This recombinant s...

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Molecular Study and Partial Characterization of Iron-only Hydrogenase inDesulfovibrio fructosovorans

Cited by 28 publications

References 35 publications

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase

Reduction of Technetium(VII) by Desulfovibrio fructosovorans Is Mediated by the Nickel-Iron Hydrogenase

Bioaccumulation of Palladium by Desulfovibrio fructosivorans Wild-Type and Hydrogenase-Deficient Strains

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

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