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Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli.
Mol Microbiol. 2004 Nov; 54(3):620-31.MM

Abstract

Genetic data have suggested that TolC, AcrA and AcrB constitute a major antibiotic efflux system in Escherichia coli. Through reversion analysis of an unstable and antibiotic-sensitive TolC mutant (TolCP246R,S350C), we isolated extragenic suppressors that mapped within the acrRAB loci. DNA sequence analysis revealed that 18 isolates contained 10 different missense mutations within the acrA gene, whereas a single isolate had a missense mutation within the acrR gene, which codes for the acrAB repressor. Besides reversing the hypersensitivity phenotype of TolCP246R,S350C, AcrA and AcrR alterations elevated the mutant TolC protein level, thus indicating that the mechanism of suppression involves the stabilization of an unstable mutant TolC protein. Eight of the 10 AcrA alterations were clustered in the 202-265 region of the mature protein, whereas the other two suppressors affected residues 30 and 146. Based on the recently solved crystal structure of MexA, an AcrA counterpart from Pseudomonas aeruginosa, the regions encompassing residues 30 and 202-265 constitute the alpha+beta-domain of AcrA (MexA), whereas that of 146 form the alpha-domain. The data suggest that residues of these two AcrA domains either directly or indirectly influence interactions with TolC. Curiously, the stability of three mutant AcrA proteins, bearing an L222Q, L222R or P265R substitution, became dependent on the presence of either wild-type or mutant TolC. This dependence of the mutant AcrA proteins on TolC further supported the notion of a direct physical interaction between these two proteins. Because a mutation in acrR or acrAB expression from a multicopy plasmid also suppressed the TolCP246R,S350C defects, it indicated that wild-type AcrA when produced in high levels presumably establishes similar interactions with the mutant TolC protein as do the suppressor forms of AcrA produced from the chromosomal copy. The AcrA-mediated suppression of mutant TolC phenotypes and the stabilization of mutant TolC protein were dependent on AcrB, reflecting the existence of a functional complex between TolC and AcrAB in vivo.

Authors+Show Affiliations

Microbiology Graduate Program, School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15491355

Citation

Gerken, Henri, and Rajeev Misra. "Genetic Evidence for Functional Interactions Between TolC and AcrA Proteins of a Major Antibiotic Efflux Pump of Escherichia Coli." Molecular Microbiology, vol. 54, no. 3, 2004, pp. 620-31.
Gerken H, Misra R. Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. Mol Microbiol. 2004;54(3):620-31.
Gerken, H., & Misra, R. (2004). Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. Molecular Microbiology, 54(3), 620-31.
Gerken H, Misra R. Genetic Evidence for Functional Interactions Between TolC and AcrA Proteins of a Major Antibiotic Efflux Pump of Escherichia Coli. Mol Microbiol. 2004;54(3):620-31. PubMed PMID: 15491355.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli. AU - Gerken,Henri, AU - Misra,Rajeev, PY - 2004/10/20/pubmed PY - 2005/7/7/medline PY - 2004/10/20/entrez SP - 620 EP - 31 JF - Molecular microbiology JO - Mol. Microbiol. VL - 54 IS - 3 N2 - Genetic data have suggested that TolC, AcrA and AcrB constitute a major antibiotic efflux system in Escherichia coli. Through reversion analysis of an unstable and antibiotic-sensitive TolC mutant (TolCP246R,S350C), we isolated extragenic suppressors that mapped within the acrRAB loci. DNA sequence analysis revealed that 18 isolates contained 10 different missense mutations within the acrA gene, whereas a single isolate had a missense mutation within the acrR gene, which codes for the acrAB repressor. Besides reversing the hypersensitivity phenotype of TolCP246R,S350C, AcrA and AcrR alterations elevated the mutant TolC protein level, thus indicating that the mechanism of suppression involves the stabilization of an unstable mutant TolC protein. Eight of the 10 AcrA alterations were clustered in the 202-265 region of the mature protein, whereas the other two suppressors affected residues 30 and 146. Based on the recently solved crystal structure of MexA, an AcrA counterpart from Pseudomonas aeruginosa, the regions encompassing residues 30 and 202-265 constitute the alpha+beta-domain of AcrA (MexA), whereas that of 146 form the alpha-domain. The data suggest that residues of these two AcrA domains either directly or indirectly influence interactions with TolC. Curiously, the stability of three mutant AcrA proteins, bearing an L222Q, L222R or P265R substitution, became dependent on the presence of either wild-type or mutant TolC. This dependence of the mutant AcrA proteins on TolC further supported the notion of a direct physical interaction between these two proteins. Because a mutation in acrR or acrAB expression from a multicopy plasmid also suppressed the TolCP246R,S350C defects, it indicated that wild-type AcrA when produced in high levels presumably establishes similar interactions with the mutant TolC protein as do the suppressor forms of AcrA produced from the chromosomal copy. The AcrA-mediated suppression of mutant TolC phenotypes and the stabilization of mutant TolC protein were dependent on AcrB, reflecting the existence of a functional complex between TolC and AcrAB in vivo. SN - 0950-382X UR - https://www.unboundmedicine.com/medline/citation/15491355/Genetic_evidence_for_functional_interactions_between_TolC_and_AcrA_proteins_of_a_major_antibiotic_efflux_pump_of_Escherichia_coli_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0950-382X&date=2004&volume=54&issue=3&spage=620 DB - PRIME DP - Unbound Medicine ER -