Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the
divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation
products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken.
Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in
vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in
response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein
was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer,
whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of
inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as
dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator
half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual
characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does
not appear to require the prior self-assembly of QacR into tetramers