Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a
Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a
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DOI：
10.1128/jb.188.1.115-123.2006
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发表时间：
2006-01-01
影响因子：
3.2
通讯作者：
Foght, JM
中科院分区：
文献类型：
--
作者：
Hearn, EM;Gray, MR;Foght, JM
荧光假单胞菌cLP 6a中的EmhABC外排系统与属于耐药-增殖-分裂（RND）家族的多药和溶剂外排系统同源，并且负责多环芳烃转运、抗生素耐药性和甲苯外排。为了更好地理解RND外排泵中的底物转运，对EmhB泵进行突变分析。预测的EmhB三维结构的中央空腔内的氨基酸突变显示出对抗生素底物的选择性活性。与野生型相比，A384 P/A385 Y双突变体对罗丹明6 G的敏感性增加，与野生型相比，F386 A和N99 A单突变体对地喹铵的敏感性增加。同时，发现D101的羧酸侧链，位于中心空腔区域，对于多环芳烃运输和对EmhB的所有抗生素底物的抗性是必不可少的。位于周质孔结构域内的苯丙氨酸残基也被靶向诱变，并且F325 A和F281 A突变显著损害了所有EmhB底物的外排活性。外膜蛋白对接结构域中的一个突变（A206 S）增加了抗生素抗性和甲苯耐受性，证明了该结构域在转运活性中的重要作用。这些数据证明了中央腔和周质结构域在RND外排泵EmhB功能中的作用。
The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.