title: Molecular basis of non-mutational derepression of ramA in Klebsiella pneumoniae.
abstract: OBJECTIVE
  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.
authors.affiliation: Centre for Infection and Immunity, Health Sciences Building, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7AE, UK.
authors.forename: Shyamasree
authors.initials: S
authors.lastname: De Majumdar
authors.affiliation: Centre for Infection and Immunity, Health Sciences Building, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7AE, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Yu
authors.affiliation: School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, University Walk, Bristol BS18 1TD, UK.
authors.forename: James
authors.initials: J
authors.lastname: Spencer
authors.affiliation: Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.affiliation: Centre for Infection and Immunity, Health Sciences Building, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7AE, UK t.schneiders@qub.ac.uk.
authors.forename: Thamarai
authors.initials: T
authors.lastname: Schneiders
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D023061:Gene Order; D056426:Genetic Loci; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000235:genetics*; D008024:Ligands; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010640:Phenothiazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008024:Ligands; D010640:Phenothiazines; C095947:RamA protein, Klebsiella
keywords: Enterobacteriaceae; chlorpromazine; ligand interactions; repression; thioridazine