摘要
目的评价金葡菌多药耐药主动外排蛋白QacAα-螺旋13位于细胞膜侧的氨基酸对于底物转运的重要性。方法用半胱氨酸定点诱变的方法突变E407,S408,M409,Y410,D411和L412,测序确定突变后,测定最低抑菌浓度(MIC)和进行运输试验比较突变前后细菌转运功能的变化。结果MIC分析表明突变株E407,Y410和D411耐药性显著降低,运输实验结果与MIC结果一致,提示这3株突变株失去对底物的转运能力。结论QacA蛋白α-螺旋13上的3个氨基酸可能与底物的结合和转运功能密切相关。
Objective To determine the importance of amino acid residues in and flanking cytoplasmic end in α-helix 13 of multidrug exporter QacA from Staphylococcus aureus. Methods Site-directed mutagenesis was used to mutate six residues including FA07, S408, M409, Y410, D411 and L412 into cysteine. After complete sequencing to make sure the mutation, minimum inhibitory concentration （MIC） analyses and the fluorimetric transport assay were performed to assess the resistant profiles and the transport capability of strains expressing these mutants. Results MIC analysis suggested that mutants E407C, Y410C and D411C lost all the resistance to the detected substrates. And the results of fluorimetric transport assay were in good agreement with those of MIC analysis, which means E407C, Y410C, and D411C did not confer transport to the tested substrates. Conclusion Residues FA07, Y410 and D411 were functionally important residues, probably involved in the substrate binding and translocation of QacA.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2009年第7期429-433,共5页
Chinese Journal of Antibiotics
基金
国家自然科学基金青年基金(编号:30400373)
重庆市自然科学基金重点项目及重庆市自然科学基金面上项目赞助
