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Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum

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Abstract

The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization. mop encodes a 69-amino-acid protein (M r 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis. Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop. This result was obtained independent of the availability of tungstate in the growth medium. mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein. On the basis of gel filtration, the native protein was a homohexamer of 48 kDa. The specificity of oxyanion binding was examined by protein mobility shift assay. Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop. Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility. Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding.

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Abbreviations

Mop :

Molybdate-binding protein

ABC transporter :

ATP-binding cassette transporter

RT-PCR :

Reverse transcription-polymerase chain reaction

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Acknowledgements

This work was supported by grants from the Land Sachsen-Anhalt and the Fonds der Chemischen Industrie. We thank Karl Peter Rücknagel for the N-terminal protein sequencing.

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Author notes

  1. Kathrin Fritsche

    Present address: Laboratory of Microbiology, Wageningen University, H. v. Suchtelenweg 4, 6703, CT Wageningen, The Netherlands

  2. Andreas Pich

    Present address: Institut für Pathologie, MH-Hannover, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany

Authors and Affiliations

  1. Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120, Halle, Germany

    Kathrin Makdessi, Kathrin Fritsche, Andreas Pich & Jan R. Andreesen

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  1. Kathrin Makdessi
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Correspondence to Jan R. Andreesen.

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Makdessi, K., Fritsche, K., Pich, A. et al. Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum . Arch Microbiol 181, 45–51 (2004). https://doi.org/10.1007/s00203-003-0624-6

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  • DOI: https://doi.org/10.1007/s00203-003-0624-6

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