A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates

Multidrug transporters are membrane proteins which, by an unknown mechanism, recognize diverse toxic compounds and efflux them from cells. We found that two substrates of the Bacillus subtilis multidrug transporter Bmr, rhodamine 6G and tetraphenylphosphonium (TPP), enhance Bmr expression at the level of transcription. Gene knock-out experiments demonstrated that an open reading frame located immediately downstream of the bmr gene is required for this enhancement. The protein product of this open reading frame, BmrR, shows distinct sequence homology to several known bacterial transcription activator proteins, such as MerR and TipAL. Gel-mobility shift and DNase protection assays indicated that BmrR binds specifically, as a dimer, to the bmr gene promoter. Furthermore, the affinity of this binding was enhanced by rhodamine and TPP, thus suggesting that these structurally dissimilar molecules interact directly with BmrR. Indeed, we found that BmrR bound rhodamine 6G stoichiometrically, one rhodamine molecule/BmrR dimer, and that TPP competed with rhodamine for this binding. Our results indicate that the enhancement of Bmr expression by some of its substrates is due to the ability of the regulatory protein, BmrR, to bind structurally dissimilar compounds resulting in enhanced transcription of the transporter gene.