Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus

Authors

  • J. G. Shaw,

    1. Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
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  • M. J. Hamblin,

    1. Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
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  • D. J. Kelly

    Corresponding author
    1. Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
    • *For correspondence. Tel. (0742) 768555; ext. 4414; Fax (0742) 728697.

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Summary

A periplasmic binding protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5kb section sequenced. A complete open reading frame of 999bp was identified that encoded a 333-residue protein (DctP) with a molecular weight of 36 128 with a 26-residue amino-terminal signal peptide. The identity of this protein with the purified dicarboxylate-binding protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.

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