MULTIDRUG-RESISTANCE PROTEINS QACA AND QACB FROM STAPHYLOCOCCUS-AUREUS - MEMBRANE TOPOLOGY AND IDENTIFICATION OF RESIDUES INVOLVED IN SUBSTRATE-SPECIFICITY

The closely related multidrug efflux pumps QacA and QacB, from the bac terial pathogen Staphylococcus aureus, both confer resistance to vario us 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 nu cleotide substitutions, Random hydroxylamine mutagenesis of qacB was u ndertaken, selecting for variants that conferred increased resistance to divalent cations, Both QacA and the QacB mutants capable of conferr ing resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in th ese positions, Site-directed mutagenesis of qacA confirmed the importa nce of an acidic residue within this region of QacA in conferring resi stance to divalent cations. Membrane topological analysis using alkali ne phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments, Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane seg ments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.