荧光假单胞菌2P24中RstA蛋白的功能鉴定

Functional Identification of RstA in Pseudomonas fluorescens Strain 2P24

探究荧光假单胞菌2P24 中OmpR 家族转录因子RstA 的功能,明确其对EmhABC 外排泵的调控作用及机制。利用共适应分析预测RstA 的潜在功能;采用同源重组技术构建rstA、emhABC 基因缺失菌株ΔrstA 和ΔemhABC,检测野生型、ΔrstA、ΔemhABC 对多种抗生素的敏感性;通过qRT-PCR 和β-半乳糖苷酶实验检测emhABC 在野生型和ΔrstA 菌株中的转录、表达水平;表达纯化His-RstA 蛋白并经凝胶阻滞实验检测RstA 蛋白与emhABC 基因启动子区域的结合活性。结果显示,RstA 同EmhABC 存在共适应性,并预测RstA 与多种抗生素胁迫环境的适应相关;ΔrstA 和ΔemhABC 对多种抗生素耐受性下降;与野生株相比,突变株ΔrstA 中emhABC 的转录、表达水平均下降超过3 倍;成功表达纯化His-RstA 蛋白,经凝胶阻滞实验显示重组His-RstA 蛋白可与emhABC 基因启动子区域特异性结合。OmpR 家族转录因子RstA 通过结合在emhABC 上游启动子区域正向调控EmhABC 的表达,并影响荧光假单胞菌2P24 的多重耐药性。

This work is to identify the function of transcriptional regulator RstA from OmpR subfamily and clarify the role of RstA in the regulation of efflux pump EmhABC in Pseudomonas fluorescens strain 2P24. Cofitness data was obtained from Cofitness Browser to explore potential functions of RstA. The rstA and emhABC-deficient mutant strain ΔrstA and ΔemhABC were constructed by homologous recombination and minimum inhibitory concentration(MIC)values of wild-type strain,ΔrstA and ΔemhABC to several antibiotics were detected. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of emhABC in the wild-type strain and ΔrstA. Electrophoretic mobility shift assay(EMSA)of the purified His-RstA protein were employed to access the interaction between RstA and the emhABC promotor. As results,RstA presented similar fitness pattern with EmhABC,which was predicted to be responsible for adapting several antibiotics stress conditions,and the tolerances of ΔrstA and ΔemhABC to multiple antibiotics decreased. Comparing to the wild-type strain,ΔrstA showed 3-fold down-regulation to the transcription and expression of emhABC. Results of EMSA indicated that the recombinant purified protein His-RstA was able to bind to the promoter of emhABC specifically. Transcriptional regulator RstA directly activates the expression of efflux pump EmhABC by binding to the emhABC promotor and contributes to multi-antibiotic resistance in P. fluorescens strain 2P24 .