Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene.
Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene.
复制标题
MepR（金黄色葡萄球菌 MepA 多药外排泵基因的抑制子）中替代突变的功能后果。
DOI：
10.1128/jb.00565-13
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发表时间：
2013
影响因子：
3.2
通讯作者：
Kaatz,GlennW
中科院分区：
文献类型：
--
作者：
Schindler,BryanD;Seo,SusanM;Jacinto,PaulineL;Kumaraswami,Muthiah;Birukou,Ivan;Brennan,RichardG;Kaatz,GlennW
编码金黄色葡萄球菌MepA多药外排蛋白的MepA的表达被MarR同源MepR抑制。MepR二聚体与meprandmepa上游的操作符结合方式不同，在mepaoperator处的亲和力最大。MepR替代突变可能导致mepa过表达，A103V在临床菌株中最常见。利用alacZreporter基因测定法对MepR和其他MepR替换的功能后果进行评估，结果显示，Q18P、F27L、G97E和A103V替换存在时，抑制因子活性显著降低。敏感性和外流试验普遍支持报告数据，电泳迁移率转移试验（emsa）证实meprandmepaa操作子的MepR F27L和A103V的亲和力受损。其中一个突变蛋白含有两个取代位点（T94P和T132M）；T132M弥补了T94P的功能缺陷，挽救了A103V的功能缺陷，但没有挽救F27L的功能缺陷，使其成为一个有限范围的抑制因子。另一个具有10个取代的导数的功能受到最小的影响，这可能是一个涉及几个残基之间相互作用的抑制的极端例子。通过在载脂蛋白mepr上模拟突变，确定了观察到的功能效应的结构相关性。F27L和A103V可能通过DNA识别螺旋的重新定位影响蛋白质-DNA相互作用。MepR替代突变的负面功能后果可能是由于结构可塑性的干扰，螺旋排列的改变，蛋白质同源DNA亲和力的降低，或者可能是MepR原体的关联。结构测定将进一步深入了解这些和其他影响MepR功能的突变的后果，特别是T132M抑制基因。
The expression ofmepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream ofmepRandmepA, with affinity being greatest at themepAoperator. MepR substitution mutations may result inmepAoverexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using alacZreporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for themepRandmepAoperators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.