荧光假单胞菌2P24中RstA蛋白的功能鉴定

Objective] This work is to identify the function of transcriptional regulator RstA from OmpR subfamily and investigate the role of RstA in the regulation of efflux pump EmhABC in Pseudomonas fluorescens strain 2P24. [Methods] Cofitness data was obtained from Cofitness Browser to explore possible functions of RstA. The mutant strain ΔrstA and ΔemhABC were constructed by homologous recombination and minimum inhibitory concentration (MIC) values of several antibiotics were detected among this strains. Quantitative real-time PCR assay and β-galactosidase assay were performed to detect the transcriptional levels of emhABC in the wild-type strain 2P24 and ΔrstA. Electrophoretic mobility shift assay（EMSA）were employed to access the interaction between RstA and the emhABC promotor.[Results] As results, RstA has similar fitness pattern with EmhABC, which are responsible to adapt several antibiotics stress conditions. Comparing to the wild-type strain 2P24, ΔrstA and ΔemhABC show higher susceptibility to various antibiotics and the deletion of rstA performed about 3-fold down-regulation to the transcription of emhABC. Results of EMSA indicated that the purified protein RstA is able to bind to the promoter of emhABC specifically. [Conclusion] Transcriptional regulator RstA directly activates the expression of efflux pump EmhABC by binding to the emhABC promotor and contributes to multi-antibiotic resistance in Pseudomonas fluorescens strain 2P24.