The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification

ABSTRACT The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae. However, it remains unknown which ligands control the function of AcrR. T...

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Main Authors: Dana E. Harmon, Cristian Ruiz
Format: Article
Language:English
Published: American Society for Microbiology 2022-12-01
Series:mSphere
Subjects:
Online Access:https://journals.asm.org/doi/10.1128/msphere.00474-22
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author Dana E. Harmon
Cristian Ruiz
author_facet Dana E. Harmon
Cristian Ruiz
author_sort Dana E. Harmon
collection DOAJ
description ABSTRACT The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae. However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the acrAB promoter and in vivo gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli. In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli, namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both in vitro and in vivo, which prevents the binding of AcrR to the acrAB promoter and, ultimately, induces the expression of acrAB. Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. IMPORTANCE Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the Enterobacteriaceae family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli, and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator.
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spelling doaj.art-52897dcae2d74a59bd1c69daff1dd3232022-12-22T03:03:16ZengAmerican Society for MicrobiologymSphere2379-50422022-12-017610.1128/msphere.00474-22The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and DetoxificationDana E. Harmon0Cristian Ruiz1Department of Biology, California State University Northridge, Northridge, California, USADepartment of Biology, California State University Northridge, Northridge, California, USAABSTRACT The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae. However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the acrAB promoter and in vivo gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli. In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli, namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both in vitro and in vivo, which prevents the binding of AcrR to the acrAB promoter and, ultimately, induces the expression of acrAB. Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. IMPORTANCE Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the Enterobacteriaceae family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli, and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator.https://journals.asm.org/doi/10.1128/msphere.00474-22AcrRligandpolyaminesethidium bromidemultidrug efflux pumpAcrAB-TolC
spellingShingle Dana E. Harmon
Cristian Ruiz
The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
mSphere
AcrR
ligand
polyamines
ethidium bromide
multidrug efflux pump
AcrAB-TolC
title The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
title_full The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
title_fullStr The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
title_full_unstemmed The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
title_short The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification
title_sort multidrug efflux regulator acrr of escherichia coli responds to exogenous and endogenous ligands to regulate efflux and detoxification
topic AcrR
ligand
polyamines
ethidium bromide
multidrug efflux pump
AcrAB-TolC
url https://journals.asm.org/doi/10.1128/msphere.00474-22
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