Identification and Characterization of a Novel Multidrug Resistance Operon, mdtRP (yusOP), of Bacillus subtilis
Identification and Characterization of a Novel Multidrug Resistance Operon, mdtRP (yusOP), of Bacillus subtilis
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DOI：
10.1128/jb.00151-09
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发表时间：
2009-05-01
影响因子：
3.2
通讯作者：
Ochi, Kozo
中科院分区：
文献类型：
--
作者：
Kim, Ji-Yun;Inaoka, Takashi;Ochi, Kozo
使用比较基因组测序分析，我们确定了一种新的突变枯草芽孢杆菌，赋予低水平的耐夫西地酸。这种突变位于mdtR（以前的yusO）基因中，该基因编码MarR型转录调节因子，并赋予对几种抗生素（包括新生霉素、链霉素和放线菌素D）的低水平抗性。转化实验表明，mdtR突变是多药耐药的原因。北方印迹分析显示，下游基因mdtP（以前的yusP），它编码的多药外排转运蛋白，与mdtR作为操纵子共转录。mdtP基因的破坏完全消除了在mdtR突变体中观察到的多药耐药表型。DNA酶I足迹法和引物延伸分析表明，MdtR蛋白直接结合mdtRP启动子，从而导致其转录的抑制。此外，凝胶迁移率变动分析表明，Arg 83-> Lys或Ala 67-> Thr取代MdtR显著降低与DNA的结合亲和力，导致mdtRP转录的去抑制。低浓度的夫西地酸诱导mdtP的表达，尽管mdtP的表达水平远低于mdtR破坏剂。这些发现表明MdtR蛋白是mdtRP操纵子的阻遏物，并且MdtP蛋白在B中作为多药外排转运蛋白发挥功能。枯草芽孢杆菌
Using comparative genome sequencing analysis, we identified a novel mutation in Bacillus subtilis that confers a low level of resistance to fusidic acid. This mutation was located in the mdtR ( formerly yusO) 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 this mdtR mutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream gene mdtP ( formerly yusP), which encodes a multidrug efflux transporter, is cotranscribed with mdtR as an operon. Disruption of the mdtP gene completely abolished the multidrug resistance phenotype observed in the mdtR mutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to the mdtRP 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 of mdtRP transcription. Low concentrations of fusidic acid induced the expression of mdtP, although the level of mdtP expression was much lower than that in the mdtR disruptant. These findings indicate that the MdtR protein is a repressor of the mdtRP operon and that the MdtP protein functions as a multidrug efflux transporter in B. subtilis.