Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA
Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA
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DOI：
10.1111/j.1574-6968.2010.02143.x
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发表时间：
2010-12-01
影响因子：
2.1
通讯作者：
Saluz, Hans-Peter
中科院分区：
文献类型：
--
作者：
Kruegel, Hans;Licht, Andreas;Saluz, Hans-Peter
两个独立的cervimycin C（CmC）的枯草芽孢杆菌耐药克隆进行了鉴定，每个携带两个突变的基因间区域之前的ABC转运蛋白基因bmrA。在双突变体中，实时PCR显示bmrA mRNA的量增加，稳定性增加。因此，膜蛋白的分离在64 kDa处产生对应于BmrA的强条带。分析表明，一个突变优化了-35盒序列，赋予对3 μ M CmC的抗性，而单独的+6突变没有影响，但增加了携带-35突变的菌株在5 μ M CmC下生长的潜力。转录融合揭示了双突变体的bmrA启动子活性升高。电泳迁移率变动分析（EMSA）证实，与野生型相比，该突变体的RNA聚合酶的结合亲和力高30倍，这种效果是由于bmrA启动子的-35盒改变。体外转录实验证实了EMSA的结果。在肝素存在下的EMSA表明突变不影响开放复合物的形成和/或稳定性。半衰期测量表明+6突变使bmrA mRNA稳定约2倍。总的来说，我们发现ABC转运蛋白通过启动子区两个突变的累积效应赋予抗生素耐药性。
Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 mu M CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 mu M CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA approximate to 2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.