The project is directed toward the understanding of the mechanism of action and physiological role of the Bacillus subtilis multidrug efflux transporter Bmr. This membrane protein is able to transport a number of structurally unrelated bacteriostatic compounds and antibiotics out of bacteria. The homologs of this protein participate in the drug resistance phenomena in clinically important bacteria. In particular, the closest known structural and functional homolog of Bmr, the Staphylococcus protein NorA, is involved in resistance of S. aureus to norfloxacin and other fluoroquinolones. The mammalian membrane pump P-glycoprotein, although being structurally different from Bmr, effluxes a similar to Bmr spectrum of drugs. This protein is involved in resistance of tumor cells to chemotherapeutic agents. The mechanism of an unusually broad substrate specificity of P-glycoprotein and Bmr remains unknown. The bacterial origin of Bmr provides an opportunity to investigate this mechanism with a large variety of molecular genetic and biochemical methods. The proposed plan of experiments includes localization of the substrate-recognition site in the Bmr molecule and the study of the fine structure of this site by using site-directed mutagenesis. These experiments will address the question, whether all the substrates of Bmr bind to the same amino acid residues of the transporter molecule, or different drugs interact with different """"""""pockets"""""""" in the binding site. The mutagenesis studies will be comple- mented with the biochemical analysis of the drug transport process mediated by Bmr and its mutants. Other experiments will reveal the physiological role of Bmr and the nature of its cellular substrates. The bmr-containing operon will be sequenced and the homologs of the other genes of the operon will be identified in the sequence databases. Overexpression of Bmr leads to impairment of bacterial growth. The changes in metabolism and gene expression in the Bmr-overexpressing bacteria will be analyzed. Cellular genes reversing the Bmr-induced growth inhibition will be cloned and sequenced. Finally, a chromatographic analysis of substances effluxed by Bmr-overexpressing bacteria will be performed. The results of this study are expected to contribute to the understanding of both the theoretical and clinical aspects of the multidrug resistance phenomena in bacteria and tumor cells.
