Functional Identification of RstA in Pseudomonas fluorescens Strain 2P24

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

Abstract

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

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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