LfrR is a repressor that regulates expression of the efflux pump LfrA in Mycobacterium smegmatis
LfrR is a repressor that regulates expression of the efflux pump LfrA in Mycobacterium smegmatis
复制标题
DOI：
10.1128/aac.00656-06
复制
发表时间：
2006-12-01
影响因子：
4.9
通讯作者：
De Rossi, Edda
中科院分区：
文献类型：
--
作者：
Buroni, Silvia;Manina, Giulia;De Rossi, Edda
耻垢分枝杆菌的lfrA基因编码一个外排泵，介导对不同氟喹诺酮类药物、阳离子染料和蒽环类药物的耐药性。lfrR基因编码一种推定的抑制因子，位于lfrA上游，其缺失增加了lfrA的表达。在本研究中，逆转录pcr实验表明，这两个基因被组织为一个操纵子，并使用lacZ报告基因融合来鉴定lfrRA启动子区域。通过引物延伸定位转录起始位点，验证了lfrRA启动子的定位。此外，我们发现多药转运体LfrA的一些底物，如吖啶黄、溴化乙啶和罗丹明123，在可检测的转录水平上增强了LfrA的表达。LfrR蛋白从大肠杆菌中纯化出来，作为一个带有六组氨酸标签的融合蛋白，通过凝胶位移分析发现它特异性结合LfrR上游143bp的片段。此外，吖啶黄能够使LfrR与启动子分离，这表明该分子直接与LfrR相互作用，诱导lfrA表达。这些结果表明，LfrR抑制因子能够与不同的化合物结合，从而诱导LfrA多药物外排泵表达以响应这些化合物。总之，所有数据表明LfrA泵受到严格调节，抑制和诱导可以在对细胞有毒的临界底物浓度上下切换。
The lfrA gene of Mycobacterium smegmatis encodes an efflux pump which mediates resistance to different fluoroquinolones, cationic dyes, and anthracyclines. The deletion of the lfrR gene, coding for a putative repressor and localized upstream of lfrA, increased the lfrA expression. In this study, reverse transcription-PCR experiments showed that the two genes are organized as an operon, and lacZ reporter fusions were used to identify the lfrRA promoter region. The lfrRA promoter assignment was verified by mapping the transcription start site by primer extension. Furthermore, we found that some substrates of the multidrug transporter LfrA, e.g., acriflavine, ethidium bromide, and rhodamine 123, enhance lfrA expression at a detectable level of transcription. LfrR protein was purified from Escherichia coli as a fusion protein with a hexahistidine tag and found to bind specifically to a fragment 143 bp upstream of lfrR by gel shift analysis. Furthermore, acriflavine was able to cause the dissociation of the LfrR from the promoter, thus suggesting that this molecule interacts directly with LfrR, inducing lfrA expression. These results suggest that the LfrR repressor is able to bind to different compounds, which allows induction of LfrA multidrug efflux pump expression in response to these ones. Together, all data suggest that the LfrA pump is tightly regulated and that the repression and induction can be switched about a critical substrate concentration which is toxic for the cell.