The IcaR and TcaR proteins were expressed in E. coli BL21 (DE3) and purified as already described [17, 40] . The MarR homologous gene, SAR2349, was amplified directly from the S. aureus MRSA252 genome by polymerase chain reaction (PCR) and subsequently cloned into expression vector pET-32. This construct was transferred into E. coli of Arctic Express TM (DE3) RIL strain. The His 6 -tagged wild-type protein was over-expressed in Difco Luria-Bertani (LB) broth containing 50 mg/l ampicillin to an optical density at 600 nm of 0.5-0.6 and then induced with 0.5 mM IPTG (isopropyl-b-D-thiogalactopyranoside). Cells were grown for 2 days at 16uC. The cells were then harvested by centrifugation at 12,000 g for 30 min and disrupted by Constant Cell Disruption System (CONSTANT SYSTEM Ltd, UK) with lysis buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 20 mM imidazole. The homogenate was centrifuged at 27,000g for 30 min and the cell-free extract was loaded onto a Ni 2+ -NTA column, which had been previously equilibrated with lysis buffer. The column was washed with lysis buffer, and the His 6 -tagged SAR2349 was subsequently eluted by a linear gradient of imidazole from 10 mM to 500 mM. His-tagged SAR2349 eluted was dialyzed twice against 5 liters of buffer (20 mM Tris-HCl, pH 8.0, and 500 mM NaCl) and then subjected to Thrombin digestion to remove the tag. The mixture was then passed through another Ni 2+ -NTA column, and subsequently untagged SAR2349 protein was dialyzed twice against 3 liters of buffer (20 mM Tris-HCl, pH 8.0) and then passed through a Q-Sepharose anion-exchange column for further purification, and subsequently SAR2349 was eluted by a linear gradient of 10 mM to 500 mM NaCl-containing buffer and then dialyzed twice against 5 liters of buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2 mM DTT) for storage. The purified SAR2349 protein was finally concentrated by 3 kDa cut-off size membrane of Amicon ultra-15 centrifugal filter units (Millipore, MA, USA) for storage at -80uC.