The tcrB gene is part of the tcrYAZB operon conferring copper resistance in Enterococcus faecium and Enterococcus faecalis Henrik Hasman Correspondence Henrik Hasman hha@dfvf.dk Danish Institute for Food and Veterinary Research, Bu ¨ lowsvej 27, DK-1790 Copenhagen V, Denmark Received 11 April 2005 Revised 20 May 2005 Accepted 27 May 2005 The plasmid-localized tcrB (transferable copper-resistance gene B) gene from Enterococcus faecium was identified to be part of an operon called the tcrYAZB operon, which has a genetic organization similar to the copYZAB copper-homeostasis gene cluster from Enterococcus hirae. Putative promoter (P tcr )- and repressor-binding sites highly similar to the E. hirae cop-promoter region were identified upstream of the tcrYAZB genes. The P tcr promoter was cloned in both the absence and the presence of the proximal repressor-encoding tcrY gene into a promoter-probe vector. Induction of the promoter was shown in liquid growth medium containing increasing concentrations of copper sulphate. To determine the growth advantage conferred by the tcrYAZB genes in a copper environment, a tcr-deletion mutant was isolated, and its growth was compared with that of its copper-resistant ancestor (strain A17sv1) in sublethal concentrations of copper sulphate. A competition assay using these two isogenic strains showed that copper sulphate concentrations of 3 mmol l ”1 and above are sufficient to select for copper resistance. INTRODUCTION Copper is an essential trace metal to all living organisms, where it serves as a cofactor for a large number of enzymes. Therefore, all living cells have developed homeostatic mechanisms to ensure adequate levels of copper within the cell. One of the most studied bacterial copper homeo- stasis mechanisms is the copYZAB operon of the Gram- positive bacterium Enterococcus hirae, recently reviewed by Solioz & Stoyanov (2003). This operon encodes four pro- teins (CopY, CopZ, CopA and CopB) working in concert to maintain tolerable levels of copper inside the cell (Lu & Solioz, 2002). CopA and CopB are two membrane-localized CPx-type ATPases involved in Cu 2+ trafficking across the membrane. CPx-type ATPases (also called P-type ATPases) are soft-metal transporters, which all contain a CPC or CPH motif in their active site (Solioz & Vulpe, 1996). The CopA protein is probably an influx pump (Odermatt & Solioz, 1995; Wunderli-Ye & Solioz, 2001), while CopB is res- ponsible for copper efflux (Solioz & Odermatt, 1995). CopY is a transcriptional repressor of the copper-responsive pro- moter located upstream of the four genes (Strausak & Solioz, 1997). It affects the expression of the downstream genes through zinc-dependent binding to two regulatory operator sites overlapping the promoter (Strausak & Solioz, 1997). The central recognition site of the operator has been sug- gested to be the so-called cop box, which has the consensus sequence TACANNTGTA. This sequence is found in cop promoters from several different Gram-positive organisms, including Lactococcus lactis and Streptococcus mutans (Portmann et al., 2004), while a slightly modified sequence is present in Bacillus subtilis (TACGNNGGTA). When copper is in excess, copper ions replace the zinc atom embedded inside CopY, and DNA binding is abolished (Cobine et al., 1999). The fourth protein encoded by the copYZAB operon is CopZ, a copper chaperone responsible for Cu 2+ trafficking in the periplasm. Here, it transfers Cu 2+ to CopY (Cobine et al., 1999, 2002), and possibly also to CopB for transport to the exterior (Elam et al., 2002). Homeostatic mechanisms like the copYZAB system are rarely able to handle artificially elevated concentrations of copper. In response to toxic levels of copper, plasmid-borne copper resistance mechanisms are often employed. The pco system from Escherichia coli, and the cop system from Pseudo- monas syringae pv. tomato (Brown et al., 1995; Lee et al., 2002), are well-known model systems of transferable copper resistance in Gram-negative bacteria (Bender & Cooksey, 1986; Mellano & Cooksey, 1988). However, these systems do not involve CPx-type ATPases. The tcrB ( transferable copper- resistance gene B) gene is, to date, the only plasmid-encoded and transferable CPx-type copper ATPase gene described. The tcrB gene has been described in Enterococcus faecium (Hasman & Aarestrup, 2002) and Enterococcus faecalis (Aarestrup et al., 2002), where it confers copper resistance. Strains harbouring the tcrB gene have an MIC of 24 mmol l 21 for CuSO 4 , whereas strains lacking the tcrB gene have a MIC of 2–8 mmol l 21 (Hasman & Aarestrup, 0002-8109 G 2005 SGM Printed in Great Britain 3019 Microbiology (2005), 151, 3019–3025 DOI 10.1099/mic.0.28109-0