荧光假单胞菌2P24中RstA蛋白的功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2019-0097

生物技术通报 ›› 2019, Vol. 35 ›› Issue (6): 107-113.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0097

荧光假单胞菌2P24中RstA蛋白的功能鉴定

李谛音¹, 何永兴², 韩建庭², 李坤¹, 王志平¹, 李妙慧³

1. 兰州大学第二医院泌尿外科甘肃省泌尿系疾病研究重点实验室,兰州730030;
2. 兰州大学生命科学学院细胞活动与逆境适应教育部重点实验室,兰州730000;
3. 四川大学华西临床医学院,成都610041

收稿日期:2019-01-25 出版日期:2019-06-26 发布日期:2019-07-08
作者简介:李谛音,女,硕士研究生,研究方向：泌尿系感染,微生物转录调控机制;E-mail：lity16@lzu.edu.cn
基金资助:
国家自然科学基金项目（31770535,31300616）,甘肃省自然科学基金项目（17JR5RA208）

Functional Identification of RstA in Pseudomonas fluorescens Strain 2P24

LI Di-yin¹, HE Yong-xing², HAN Jian-ting², LI Kun¹, WANG Zhi-ping¹, LI Miao-hui³

1. Institute of Urology,Lanzhou University Second Hospital,Key Laboratory of Gansu Province for Urological Diseases,Lanzhou 730030;
2. Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations,School of Life Sciences,Lanzhou University,Lanzhou 730000;
3. West China School of Medicine,Sichuan University,Chengdu 610041

Received:2019-01-25 Published:2019-06-26 Online:2019-07-08

摘要/Abstract

摘要： 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。

关键词: 荧光假单胞菌2P24, RstA蛋白, 外排泵, 多重耐药性

Abstract: 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,ΔrstAand 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 .

Key words: Pseudomonas fluorescens strain 2P24, RstA protein, efflux pump, multi-drug resistance

李谛音, 何永兴, 韩建庭, 李坤, 王志平, 李妙慧. 荧光假单胞菌2P24中RstA蛋白的功能鉴定[J]. 生物技术通报, 2019, 35(6): 107-113.

LI Di-yin, HE Yong-xing, HAN Jian-ting, LI Kun, WANG Zhi-ping, LI Miao-hui. Functional Identification of RstA in Pseudomonas fluorescens Strain 2P24[J]. Biotechnology Bulletin, 2019, 35(6): 107-113.

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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

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