Identification and Characterization of a Novel Multidrug Resistance Operon, mdtRP ( yusOP ), of Bacillus subtilis

Using comparative genome sequencing analysis, we identified a novel mutation inBacillus subtilis that confers a low level of resistance to fusidic acid. This mutation was located in themdtR (formerlyyusO ) gene, which encodes a MarR-type transcriptional regulator, and conferred a low level of resistance to several antibiotics, including novobiocin, streptomycin, and actinomycin D. Transformation experiments showed that thismdtR mutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream genemdtP (formerlyyusP ), which encodes a multidrug efflux transporter, is cotranscribed withmdtR as an operon. Disruption of themdtP gene completely abolished the multidrug resistance phenotype observed in themdtR mutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to themdtRP promoter, thus leading to repression of its transcription. Moreover, gel mobility shift analysis indicated that an Arg83 → Lys or Ala67 → Thr substitution in MdtR significantly reduces binding affinity to DNA, resulting in derepression ofmdtRP transcription. Low concentrations of fusidic acid induced the expression ofmdtP , although the level ofmdtP expression was much lower than that in themdtR disruptant. These findings indicate that the MdtR protein is a repressor of themdtRP operon and that the MdtP protein functions as a multidrug efflux transporter inB. subtilis .