title: RamA, which controls expression of the MDR efflux pump AcrAB-TolC, is regulated by the Lon protease.
abstract: OBJECTIVE
  RamA regulates the AcrAB-TolC multidrug efflux system. Using Salmonella Typhimurium, we investigated the stability of RamA and its impact on antibiotic resistance.
  
  
  METHODS
  To detect RamA, we introduced ramA::3XFLAG::aph into plasmid pACYC184 and transformed this into Salmonella Typhimurium SL1344ramA::cat and lon::aph mutants. An N-terminus-deleted mutant [pACYC184ramA(Δ2-21)::3XFLAG::aph] in which the first 20 amino acids of RamA were deleted was also constructed. To determine the abundance and half-life of FLAG-tagged RamA, we induced RamA with chlorpromazine (50 mg/L) and carried out western blotting using anti-FLAG antibody. Susceptibility to antibiotics and phenotypic characterization of the lon mutant was also carried out.
  
  
  RESULTS
  We show that on removal of chlorpromazine, a known inducer of ramA, the abundance of RamA decreased to pre-induced levels. However, in cells lacking functional Lon, we found that the RamA protein was not degraded. We also demonstrated that the 21 amino acid residues of the RamA N-terminus are required for recognition by the Lon protease. Antimicrobial susceptibility and phenotypic tests showed that the lon mutant was more susceptible to fluoroquinolone antibiotics, was filamentous when observed by microscopy and grew poorly, but showed no difference in motility or the ability to form a biofilm. There was also no difference in the ability of the lon mutant to invade human intestinal cells (INT-407).
  
  
  CONCLUSIONS
  In summary, we show that the ATP-dependent Lon protease plays an important role in regulating the expression of RamA and therefore multidrug resistance via AcrAB-TolC in Salmonella Typhimurium.
authors.affiliation: Antimicrobials Research Group, School of Immunity and Infection and Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Vito
authors.initials: V
authors.lastname: Ricci
authors.forename: Jessica M A
authors.initials: JM
authors.lastname: Blair
authors.forename: Laura J V
authors.initials: LJ
authors.lastname: Piddock
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D015964:Gene Expression Regulation, Bacterial; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D049070:Protease La / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D049070:Protease La; D015534:Trans-Activators; C493251:AcrAB-TolC protein, Salmonella enterica; C531999:RamA protein, Salmonella typhimurium
keywords: Salmonella; proteolysis; transcription factors