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Journal of Bacteriology, December 2001, p. 7102-7109, Vol. 183, No. 24
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.24.7102-7109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The Staphylococcal QacR Multidrug Regulator Binds a Correctly Spaced Operator as a Pair of Dimers

Steve Grkovic,1 Melissa H. Brown,1 Maria A. Schumacher,2 Richard G. Brennan,2 and Ronald A. Skurray1,*

School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia,1 and Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-30982

Received 7 June 2001/Accepted 20 September 2001

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.

* Corresponding author. Mailing address: School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, New South Wales 2006, Australia. Phone: 61-2-9351-2376. Fax: 61-2-9351-4771. E-mail: skurray{at}bio.usyd.edu.au.

Journal of Bacteriology, December 2001, p. 7102-7109, Vol. 183, No. 24
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.24.7102-7109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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