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PHYSIOLOGY AND METABOLISM

Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in Geobacter sulfurreducens and Engineering of Fumarate Reduction in Geobacter metallireducens

Jessica E. Butler, Richard H. Glaven, Abraham Esteve-Núñez, Cinthia Núñez, Evgenya S. Shelobolina, Daniel R. Bond, Derek R. Lovley
Jessica E. Butler
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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  • For correspondence: jbutler@microbio.umass.edu
Richard H. Glaven
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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Abraham Esteve-Núñez
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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Cinthia Núñez
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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Evgenya S. Shelobolina
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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Daniel R. Bond
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
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DOI: 10.1128/JB.188.2.450-455.2006
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  • FIG. 1.
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    FIG. 1.

    Structure and mutagenesis of the fumarate reductase/succinate dehydrogenase operon of G. sulfurreducens. (A) The operon structure of the genes, GSU1176 (frdC), GSU1177 (frdA), and GSU1178 (frdB), showing the location (black bar) of the gene disruption with the kanamycin resistance cassette in the mutant strain. The homologous genes in G. metallireducens are organized and spaced identically, with 85%, 93%, and 91% amino acid sequence identity between the C, A, and B subunits, respectively. (B) Transcription of the operon in G. sulfurreducens grown with fumarate as electron acceptor, analyzed by Northern blot (10 μg RNA/lane) using a fragment of frdA as a probe. The expected size of a transcript of frdCAB is 3.3 kb and that of frdAB is 2.6 kb.

  • FIG. 2.
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    FIG. 2.

    Phylogenetic tree of representative catalytic subunits from putative B-type, heterotrimeric enzymes which were most similar to G. sulfurreducens. Sequences were aligned using Clustal X (38), and distances and branching order were determined using the neighbor-joining method (32). A total of 1,000 replicates were used for bootstrap analysis. The clade containing the W. succinogenes fumarate reductase is indicated by FrdA and the clade containing the B. subtilis succinate dehydrogenase is indicated by SdhA. Geobacter species subunits are shown in bold.

  • FIG. 3.
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    FIG. 3.

    Growth of wild-type and frdA-deficient G. sulfurreducens strains with excess acetate as the electron donor and Fe(III) as the electron acceptor. (A) Cell growth and Fe(III) reduction of the wild type with 20 mM fumarate supplementation and with no fumarate supplementation. (B) Cell growth and Fe(III) reduction of the frdA-deficient strain with 20 mM fumarate supplementation and with no fumarate supplementation. Experiments were run in parallel with inocula of ca. 6 × 106 late-log-phase cells adapted to the appropriate medium. Data are means ± standard deviations of triplicate cultures.

  • FIG. 4.
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    FIG. 4.

    Metabolism of organic acids in wild-type and frdA-deficient G. sulfurreducens strains grown with excess acetate as the electron donor and Fe(III) as the electron acceptor. Data are means of analyses by high-pressure liquid chromatography of the fumarate supplemented cultures shown in Fig. 3. (A) Metabolism of acetate, fumarate, succinate, and malate in the wild-type strain. (B) Metabolism of acetate, fumarate, succinate, and malate in the frdA-deficient mutant strain. Samples were taken from the triplicate cultures at the same time as those from Fig. 3.

  • FIG. 5.
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    FIG. 5.

    A model for succinate production during growth with fumarate supplementation in the wild-type and frdA-deficient G. sulfurreducens strains. (A) In the wild-type strain, succinate can be produced by both the terminal fumarate reductase, FrdCAB, and by the TCA cycle reactions. Succinate can be exchanged with fumarate from external media, obviating the need for a succinate dehydrogenase. (B) In the frdA-deficient mutant strain, neither a terminal fumarate reductase nor a succinate dehydrogenase is present, so succinate is produced only by the TCA cycle reactions and must be exchanged with external fumarate to allow continued acetate oxidation.

  • FIG. 6.
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    FIG. 6.

    Growth of G. metallireducens and G. sulfurreducens with excess acetate as the electron donor and fumarate as the sole electron acceptor. Shown are wild-type G. metallireducens and strains of G. metallireducens and G. sulfurreducens in which dcuB, the putative fumarate transporter from G. sulfurreducens, is being constitutively expressed in trans. Inocula were 2.5% late-log-phase cells adapted to the appropriate medium, with the strains carrying pRGdcuB grown in the presence of 275 μM spectinomycin. Data are means ± standard deviations of triplicate cultures.

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Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in Geobacter sulfurreducens and Engineering of Fumarate Reduction in Geobacter metallireducens
Jessica E. Butler, Richard H. Glaven, Abraham Esteve-Núñez, Cinthia Núñez, Evgenya S. Shelobolina, Daniel R. Bond, Derek R. Lovley
Journal of Bacteriology Dec 2005, 188 (2) 450-455; DOI: 10.1128/JB.188.2.450-455.2006

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Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in Geobacter sulfurreducens and Engineering of Fumarate Reduction in Geobacter metallireducens
Jessica E. Butler, Richard H. Glaven, Abraham Esteve-Núñez, Cinthia Núñez, Evgenya S. Shelobolina, Daniel R. Bond, Derek R. Lovley
Journal of Bacteriology Dec 2005, 188 (2) 450-455; DOI: 10.1128/JB.188.2.450-455.2006
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KEYWORDS

Fumarates
Geobacter
Succinate Dehydrogenase
Succinic Acid

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