5; 20 mM MgCl2; 400 mM NaCl and 50% glycerol) for ArgR5aa and ArgR149 proteins. Protein concentrations were determined using Bradford assays or using QuBit fluorimetry, and we obtained 50% purity for ArgR5aa and 30% for ArgR149. For crosslinking analysis, the proteins were also expressed as histidine-tagged

fusion proteins by cloning the coding regions of all three genes into pQE31 (Qiagen Inc.) and purifying the overexpressed proteins according the manufacturer’s Y-27632 clinical trial instructions. The gel retardation assays were performed as described by Tian et al. (1992) and Villion & Szatmari (1998) using specific fragments labelled with digoxigenin by PCR. A 106-bp fragment containing the ARG box site was labelled with digoxygenin using 100 pmol of the following primers: ArgboxD (5′CTT GCG GAT CCG AGC TTC G) and ArgboxG (5′TTT CAG CCG AAT TCA GGG CTG) under the following conditions: 95 °C/30 s, 55 °C/30 s, 72 °C/30 s for 30 cycles, with a final extension at 72 °C/1 min. Reactions were carried out in 50-μL volumes using CP690550 Taq DNA polymerase (Qiagen Inc.) using 120 ng of pGS38 DNA as the template. The gel shift assay was performed with 2 ng labelled DNA,

1 μg of poly-dIdC in buffer containing 20 mM Tris-HCl pH 7.5; 10 mM MgCl2; 100 mM KCl; 10 mM CaCl2; 1 mM EDTA pH 8.8; 10% glycerol; and 5 mM l-arginine. The reactions were incubated for 30 min at 37 °C before electrophoresing on a 6% polyacrylamide gel. Five millimolar and 1 mM of l-arginine were added to the gel and the 0.5 × TBE running buffer, respectively. Gels were transferred onto Hybond-N+ (Amersham Biosciences) positively charged nylon membranes and UV cross-linked. Final detections were performed using CDP-Star (NEB), according to the standard digoxygenin detection methods, and followed by exposure to a Fujifilm SuperRX X-ray

film. N-terminal 6-histidine-tagged versions of ArgR, ArgR5aa and ArgR149 were purified using Ni-NTA affinity chromatography (Qiagen Inc.) as per the manufacturer’s instructions. Purified proteins were crosslinked 17-DMAG (Alvespimycin) HCl with various amounts of glutaraldehyde (Fisher) in 50 mM triethanolamine pH 8, containing 150 mM KCl and 1 mM l-arginine. Cross-linked complexes were analysed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie Brilliant Blue. In order to obtain ArgR mutants deficient in cer site-specific recombination, we used the technique of pentapeptide scanning mutagenesis, which inserts five amino acids into random regions of a DNA sequence. We isolated a series of ArgR mutants using an in vivo site-specific recombination assay in an argR− E. coli strain (DS956) containing plasmid pCS210. Mutants were screened for their inability to delete the cer-flanked lacZ gene of pCS210, which results in a blue phenotype in X-gal-containing media. These mutants were then screened for their ability to repress the argR∷lacZ fusion in strain EC146(λAZ-7).