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Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae

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Abstract

In Enterococcus hirae, copper homeostasis is controlled by the cop operon, which encodes the copper-responsive repressor CopY, the copper chaperone CopZ, and two copper ATPases, CopA and CopB. The four genes are under control of CopY, which is a homodimeric zinc protein, [Zn(II)CopY]2. It acts as a copper-responsive repressor: when media copper is raised, CopY is released from the DNA, allowing transcription to proceed. This involves the conversion of [Zn(II)CopY]2 to [Cu(I)2CopY]2, which is no longer able to bind to the promoter. Binding analysis of [Zn(II)CopY]2 to orthologous promoters and to control DNA by surface plasmon resonance analysis defined the consensus sequence TACAnnTGTA as the repressor binding element, or “cop box”, of Gram-positive bacteria. Association and dissociation rates for the CopY–DNA interaction in the absence and presence of added copper were determined. The dissociation rate of [Zn(II)CopY]2 from the promoter was 7.3×10-6 s-1 and was increased to 5×10-5 s-1 in the presence of copper. This copper-induced change may be the underlying mechanism of copper induction. Induction of the cop operon was also assessed in vivo with a biosensor containing a lux reporter system under the control of the E. hirae cop promoter. Half-maximal induction of this biosensor was observed at 5 μM media copper, which delineates the ambient copper concentration to which the cop operon responds in vivo.

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Abbreviations

TCEP:

tris(2-carboxyethyl)phosphine

RU:

response units

TG buffer:

50 mM tris-SO4, pH 7.8, 5% (v/v) glycerol

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Acknowledgment

We thank Kristian Raaby Poulsen and Thomas Weber for valuable experimental help and Christopher Rensing for providing the E. coli CopA-knockout strain DW3110. This work was supported by grant 31-68075.02 from the Swiss National Foundation, and by the International Copper Association (M.S.) and the Deutsche Forschungsgemeinschaft DFG (G.M.).

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Authors and Affiliations

  1. Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 , Berne, Switzerland

    Reto Portmann, David Magnani, Jivko V. Stoyanov & Marc Solioz

  2. Department for Chemistry/Biochemistry, Free University of Berlin, Thielallee 63, 14195 , Berlin, Germany

    Ariane Schmechel & Gerd Multhaup

Authors
  1. Reto Portmann
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  2. David Magnani
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  3. Jivko V. Stoyanov
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  4. Ariane Schmechel
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  5. Gerd Multhaup
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  6. Marc Solioz
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Correspondence to Marc Solioz.

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Portmann, R., Magnani, D., Stoyanov, J.V. et al. Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae . J Biol Inorg Chem 9, 396–402 (2004). https://doi.org/10.1007/s00775-004-0536-1

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  • DOI: https://doi.org/10.1007/s00775-004-0536-1

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