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Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae

De Majumdar, Shyamasree, Yu, Jing, Spencer, James, Tikhonova, Irina G. and Schneiders, Thamarai (2014) Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae. Journal of Antimicrobial Chemotherapy, 69 (10). pp. 2681-2689. [Journal article]

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URL: http://dx.doi.org/10.1093/jac/dku203

DOI: 10.1093/jac/dku203

Abstract

OBJECTIVES:The ram locus, consisting of the romA-ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression.METHODS:Quantitative real-time PCR analyses were performed to determine expression levels of the romA-ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure.RESULTS:Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA-ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR-romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63.CONCLUSIONS:These data demonstrate that phenothiazines can modulate loci linked to the microbe-drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.

Item Type:Journal article
Keywords:repression, Enterobacteriaceae, ligand interactions, chlorpromazine, thioridazine
Faculties and Schools:Faculty of Life and Health Sciences > School of Biomedical Sciences
Faculty of Life and Health Sciences
Research Institutes and Groups:Biomedical Sciences Research Institute > Genomic Medicine
Biomedical Sciences Research Institute
ID Code:38123
Deposited By: Dr Shyamasree De Majumdar
Deposited On:13 Jun 2017 09:26
Last Modified:13 Jun 2017 09:26

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