Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine

Abstract

The Bacillus subtilis multidrug transporter Bmr effluxes structurally diverse toxic compounds out of bacterial cells. Antihypertensive alkaloid reserpine reverses Bmr-mediated multidrug resistance by inhibiting drug transport. We have obtained a mutant of the bmr gene that provides a normal level of multidrug resistance, which can, however, only be reversed by very high concentrations of reserpine. Reduction of Bmr sensitivity to reserpine has been caused by the substitution of Leu for Val286 in the Bmr molecule. This mutation also led to a dramatic decrease of [3H]reserpine binding to membrane vesicles prepared from the Bmr-overexpressing bacteria. Leucine is larger than valine by one methylene group. Substitution of Val286 with a smaller residue, glycine, had an opposite effect. It led to increased sensitivity of Bmr to reserpine and increased affinity of reserpine binding to the membranes prepared from Bmr-overexpressing bacteria. Neither of the mutations significantly changed the sensitivity of Bmr to rescinnamine, a structural analog of reserpine. The results suggest that Val286 is involved in the formation of the reserpine-binding site of the Bmr molecule.