The QacR multidrug-binding repressor protein regulates the expression of the Staphylococcus aureus qacA gene, a multidrug resistance (MDR) locus that is prevalent in clinical isolates of this important human pathogen. In this paper we demonstrate that the range of structurally diverse compounds capable of inducing qacA transcription is significantly more varied than previously appreciated, particularly in relation to bivalent cations. For all of the newly identified inducing compounds, induction of qacA expression was correlated with a matching ability to dissociate QacR from operator DNA. Development of a ligand-binding assay based on intrinsic tryptophan fluorescence permitted dissociation constants to be determined for the majority of known QacR ligands, with values ranging from 0.2 to 82 µM. Highaffinity binding of a compound to QacR in vitro was not found to correlate very strongly with either its in vivo inducing abilities or its structure. The latter observation indicated that the QacR ligand-binding pocket appears to have evolved to accommodate a wide range of toxic hydrophobic cations, rather than a specific class of compound. Importantly, the antimicrobial ligands of QacR included several plant alkaloids that share structural similarities with synthetic MDR substrates. This is consistent with the suggestion that the qacA-qacR MDR locus was recently derived from genes that protect against natural antimicrobial compounds.

A series of Staphylococcus aureus-Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.

Mercury resistance regulator (MerR) is a 32 kD metalloregulatory protein that controls mercury resistance in Gram-negative bacteria by regulating transcription of the mer-operon. It is a member in the diverse MerR-family of transcriptional activators. In the presence of Hg (II) the regulator functions as an activator of the mer-genes while in the absence of Hg(II) it is a weak repressor.The monomer folds into three functional domains: a N-terminal DNA-binding domain, which is homologous among the MerR family proteins, a C-terminal Hg-binding domain, and an intervening region, which plays a role in active repression and in transcriptional activation. The DNA-binding domain contains a helix-turn-helix motif that binds to DNA between positions -35 and -10 on the operon. The Hg-binding domain has three conserved cysteine residues at positions C82, C117, and C126, which form the trigonal high-affinity Hg-binding site. Every dimer binds one Hg atom, but the mechanism of binding is still unknown. High affinity and selectivity of MerR for Hg has been utilized in biosensors and has been engineered for purification of mercury-contaminated drinking water [1,2].In the absence of Hg the inactive form of MerR causes a distortion of DNA in the middle of the mer-operator. In this inactive form the RNA polymerase binds adjacent to MerR in a closed complex, which has access only to the -35 site of the promoter, while the -10 site remains inaccessible. The distortion is caused by a 19 bp spacing between the -35 and -10 elements, which is unusually long compared to normal 17 bp in bacteria. Incoming Hg binds to MerR and causes unwinding and unkinking of DNA. The helix backbone is straightened out and unwound which brings the -35 and -10 promoter elements onto the same face of the DNA double helix. The reorientation of the -35 and -10 sequences allows them to interact with the RNA polymerase to form an open transcriptional complex and transcription is initiated [3].Our aims are to solve the crystal structure of MerR in active and inactive form and study the specific protein-DNA interactions, binding mechanisms and structural changes occurring upon ligand binding.[1] Brown, N.L., Stoyanov,