The RstA/RstB Two-component System Activates Efflux Pumps And Influences Multi-drug Resistance In Pseudomonas Fluorescens

【Objective】This work is to identify the function of a two-component system RstA/RstB and investigate the molecular mechanism of RstA/RstB on multidrug resistance in Pseudomonas fluorescens strain 2P24.【Methods】Cofitness analysis was performed to explore possible functions of the two-component system RstA/RstB and its correlation with other proteins.Mutant strains,including genes deletion of rstA,rstBsensor,rstBkinase,emhABC,cmeAB,mfs and singel point mutations of D52 A,D52E,were constructed by homologous recombination and minimum inhibitory concentration 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 target genes,emhABC and cmeAB.Protein expression and purification in vitro included Mbp-RstA,Mbp-D52 A,Mbp-D52 E and Mbp-tag.Electrophoretic mobility shift assay(EMSA)and DNase I footprinting assay were employed to access the interaction activities and binding domains between RstA and target promotors.In vitro phosphorylation and protein cross-linking were performed by using high-energy phosphorylation reagents and cross-linking reagents.Exploring the biological processes in which RstA may be involved by using label-free quantitative proteomic analysis and bioinformatics analysis.【Results】As results,RstA/RstB has similar fitness pattern with efflux pumps EmhABC and CmeAB.As a two-component system of Pseudomonas fluorescens 2p24,RstA/RstB has not yet been identified and we first confirmed that RstA/RstB can modulate the efflux pumps’ expression and further modify multiple-antibiotic resistance.We identified that phosphorylation on site D52 of RstA induced the dimerization and gain of function.Also,the catalytic activity of the RstB kinase domain is necessary.Intriguingly,we found out that RstA possibly functioning as a global regulator involved in many biological processes,especially responding to many kinds of stresses,such as heat damage resistance,anaerobic survival,secondary metabolites production,biofilm formation and harmful substances efflux.【 Conclusion 】 The RstA/RstB two-component system is an important sensory-response regulation system of Pseudomonas fluorescens 2P24,which regulates the expression of multiple efflux pumps through complex mechanisms and affects the multidrug resistance.As a global regulator,RstA plays an important role in many physiology processes,which is critical for the adaptation and survival of bacteria in complex environments.This study provides new clues for the study of multi-drug resistance of Pseudomonas and provides a theoretical basis for the development of new antibiotics or antibiotic adjuvants.