Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity

I. T. Paulsen, M. H. Brown, T. G. Littlejohn, B. A. Mitchell, R. A. Skurray

Research output: Contribution to journalArticle

220 Citations (Scopus)

Abstract

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and β-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Original languageEnglish
Pages (from-to)3630-3635
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number8
DOIs
Publication statusPublished - 16 Apr 1996
Externally publishedYes

Keywords

  • drug efflux pumps
  • major facilitator superfamily
  • protein topology

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