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Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuni

Authors

  • Edward Guccione,

    1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
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    • These authors contributed equally to this work.

  • Andrew Hitchcock,

    1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
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    • These authors contributed equally to this work.

  • Stephen J. Hall,

    1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
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    • Present address: Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

  • Francis Mulholland,

    1. Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK.
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  • Neil Shearer,

    1. Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK.
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  • Arnoud H. M. Van Vliet,

    1. Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK.
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  • David J. Kelly

    Corresponding author
    1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
      *E-mail d.kelly@sheffield.ac.uk; Tel. (+44) 114 222 4414; Fax (+44) 114 272 8697.
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*E-mail d.kelly@sheffield.ac.uk; Tel. (+44) 114 222 4414; Fax (+44) 114 272 8697.

Summary

Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some ε-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni-specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment.

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