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Journal of Bacteriology, July 2003, p. 3804-3812, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3804-3812.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of the Copper-Transporting Efflux System CusCFBA of Escherichia coli

Sylvia Franke,1 Gregor Grass,1,2 Christopher Rensing,2 and Dietrich H. Nies1*

Molekulare Mikrobiologie, Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle, Germany,1 Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 857312

Received 23 January 2003/ Accepted 21 April 2003

The cus determinant of Escherichia coli encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux. CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance. Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance. Substitutions for several other methionine residues of this protein did not have any effect. The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein. CusF bound one copper per polypeptide. The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm. Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis. CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay. In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapartite resistance system that involves the novel periplasmic copper-binding protein CusF. These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex.

* Corresponding author. Mailing address: Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06099 Halle, Germany. Phone: (49)-345-5526352. Fax: (49)-345-5527010. E-mail: d.nies{at}mikrobiologie.uni-halle.de.

Journal of Bacteriology, July 2003, p. 3804-3812, Vol. 185, No. 13
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.13.3804-3812.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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