The Two-Component System RstA/RstB Regulates Expression of Multiple Efflux Pumps and Influences Anaerobic Nitrate Respiration in Pseudomonas fluorescens.

Di-Yin Li,Xu Yan,Yong-Xing He,Jian-Ting Han,Nannan Zhang,Zhiping Wang,Honghua Ge,Meng-Yuan Zhang,Zackery Bulman

doi:10.1128/msystems.00911-21

Di-Yin Li, Xu Yan + Show 7 more

Open Access

https://doi.org/10.1128/msystems.00911-21

Copy DOI

Abstract

ABSTRACTMultidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials. Expression of MDR efflux pumps can be either constitutive or transiently induced by various environmental signals, which are typically perceived by bacterial two-component systems (TCSs) and relayed to the bacterial nucleoid, where gene expression is modulated for niche adaptation. Here, we demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in Escherichia coli, confers resistance to multiple antibiotics in Pseudomonas fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. Moreover, we show that phosphorylation of the conserved Asp52 residue in RstA greatly enhances RstA-DNA interaction, and regulation of the multidrug resistance by RstA/RstB is dependent on the phosphorylation of the RstA Asp52 residue by RstB. Proteome analysis reveals RstA/RstB also positively regulates the efflux pump MexEF-OprN and enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. Our results suggest that, by coupling the expression of multiple efflux pumps and anaerobic nitrate respiration, RstA/RstB could play a role in defense against nitrosative stress caused by anaerobic nitrate respiration.IMPORTANCE Microenvironmental hypoxia typically increases bacterial multidrug resistance by elevating expression of multidrug efflux pumps, but the precise mechanism is currently not well understood. Here, we showed that the two-component system RstA/RstB not only positively regulated expression of several efflux pumps involved in multidrug resistance, but also promoted expression of enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis. These results suggested that, by upregulating expression of efflux pumps and pyoverdine biosynthesis-related enzymes, RstA/RstB could play a role in promoting bacterial tolerance to hypoxia by providing protection against nitrosative stress.

Highlights

Multidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials
High-throughput data sets of microbial fitness had been proved to be informative in predicting gene functions and recently genome-wide mutant fitness data from 32 diverse bacteria across a set of growth conditions were available from http://fit.genomics.lbl.gov/, prompting us to seek the possible genes related to antibiotic resistance
Function of the RstA/RstB Two-Component System the protein products encoded by the emhABC operon were characterized to constitute an efflux pump playing a critical role in antibiotic resistance [25], we reasoned that the strain lacking the resistance related genes should have high cofitness values with strains lacking the EmhABC efflux pump

Summary

Introduction

Multidrug resistance (MDR) efflux pumps are involved in bacterial intrinsic resistance to multiple antimicrobials. We demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in Escherichia coli, confers resistance to multiple antibiotics in Pseudomonas fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. We showed that the two-component system RstA/RstB positively regulated expression of several efflux pumps involved in multidrug resistance, and promoted expression of enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis These results suggested that, by upregulating expression of efflux pumps and pyoverdine biosynthesisrelated enzymes, RstA/RstB could play a role in promoting bacterial tolerance to hypoxia by providing protection against nitrosative stress. We demonstrate that RstA/RstB, a TCS previously shown to control acid-induced and biofilm-related genes in E. coli [27], confers resistance to multiple antibiotics in P. fluorescens by directly regulating the MDR efflux pumps EmhABC and MexCD-OprJ. We identify that RstA/RstB positively regulated the efflux pump MexEF-OprN and enzymes involved in anaerobic nitrate respiration and pyoverdine biosynthesis, and propose a role for RstA/RstB in the cellular defense against nitrosative stress

Methods

Results

Conclusion