AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening

Jon W. Weeks, Teresa Celaya-Kolb, Sara Pecora, Rajeev Misra

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

In Escherichia coli, the TolC-AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four-residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC-mediated phage or colicin infection. However, despite the null-like drug sensitivity phenotype, chemical cross-linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA-mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB-dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state.

Original languageEnglish (US)
Pages (from-to)1468-1483
Number of pages16
JournalMolecular Microbiology
Volume75
Issue number6
DOIs
StatePublished - 2010

Fingerprint

Drug Hypersensitivity
Mutant Proteins
Anti-Bacterial Agents
Phenotype
Genetic Suppression
Colicins
Substrate Specificity
Bacteriophages
Hypersensitivity
Escherichia coli
Infection
Pharmaceutical Preparations
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Microbiology
  • Medicine(all)

Cite this

AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening. / Weeks, Jon W.; Celaya-Kolb, Teresa; Pecora, Sara; Misra, Rajeev.

In: Molecular Microbiology, Vol. 75, No. 6, 2010, p. 1468-1483.

Research output: Contribution to journalArticle

@article{fd9de4723130437987d08c34a8f9bce7,
title = "AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening",
abstract = "In Escherichia coli, the TolC-AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four-residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC-mediated phage or colicin infection. However, despite the null-like drug sensitivity phenotype, chemical cross-linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA-mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB-dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state.",
author = "Weeks, {Jon W.} and Teresa Celaya-Kolb and Sara Pecora and Rajeev Misra",
year = "2010",
doi = "10.1111/j.1365-2958.2010.07068.x",
language = "English (US)",
volume = "75",
pages = "1468--1483",
journal = "Molecular Microbiology",
issn = "0950-382X",
publisher = "Wiley-Blackwell",
number = "6",

}

TY - JOUR

T1 - AcrA suppressor alterations reverse the drug hypersensitivity phenotype of a TolC mutant by inducing TolC aperture opening

AU - Weeks, Jon W.

AU - Celaya-Kolb, Teresa

AU - Pecora, Sara

AU - Misra, Rajeev

PY - 2010

Y1 - 2010

N2 - In Escherichia coli, the TolC-AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four-residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC-mediated phage or colicin infection. However, despite the null-like drug sensitivity phenotype, chemical cross-linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA-mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB-dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state.

AB - In Escherichia coli, the TolC-AcrAB complex forms a major antibiotic efflux system with broad substrate specificity. During the complex assembly, the periplasmic helices and bottom turns of TolC are thought to interact with a hairpin helix of AcrA and hairpin loops of AcrB respectively. In the present study we show that a four-residue substitution in TolC's turn 1, which connects outer helices 3 and 4 proximal to TolC's periplasmic aperture, confers antibiotic hypersensitivity, without affecting TolC-mediated phage or colicin infection. However, despite the null-like drug sensitivity phenotype, chemical cross-linking analysis revealed no apparent defects in the ability of the mutant TolC protein to physically interact with AcrA and AcrB. A role for TolC turn 1 residues in the functional assembly of the tripartite efflux pump complex was uncovered through isolating suppressor mutations of the mutant TolC protein that mapped within acrA and by utilizing a labile AcrA protein. The data showed that AcrA-mediated suppression of antibiotic sensitivity was achieved by dilating the TolC aperture/channel in an AcrB-dependent manner. The results underscore the importance of the periplasmic turn 1 of TolC in the functional assembly of the tripartite efflux complex and AcrA in transitioning TolC from its closed to open state.

UR - http://www.scopus.com/inward/record.url?scp=77949360141&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77949360141&partnerID=8YFLogxK

U2 - 10.1111/j.1365-2958.2010.07068.x

DO - 10.1111/j.1365-2958.2010.07068.x

M3 - Article

VL - 75

SP - 1468

EP - 1483

JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

IS - 6

ER -