title: Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator.
abstract: OBJECTIVE
  Constitutive overexpression of patAB has been observed in several unrelated fluoroquinolone-resistant laboratory mutants and clinical isolates; therefore, we sought to identify the cause of this overexpression.
  
  
  METHODS
  Constitutive patAB overexpression in two clinical isolates and a laboratory-selected mutant was investigated using a whole-genome transformation approach. To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter. Finally, mutation of the opposing base in the stem-loop structure was carried out.
  
  
  RESULTS
  We identified three novel mutations causing up-regulation of patAB. All three of these were located in the upstream region of patA and affected the same Rho-independent transcriptional terminator structure. Each mutation was predicted to destabilize the terminator stem-loop to a different degree, and there was a strong correlation between predicted terminator stability and patAB expression level. Using a GFP reporter of patA transcription, these terminator mutations led to increased transcription of a downstream gene. For one mutant sequence, terminator stability could be restored by mutation of the opposing base in the stem-loop structure, demonstrating that transcriptional suppression of patAB is mediated by the terminator stem-loop structure.
  
  
  CONCLUSIONS
  This study showed that a mutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance. Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials.
authors.affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Baylay
authors.affiliation: Antimicrobials Research Group, School of Immunity and Infection, Institute of Microbiology and Infection and College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK l.j.v.piddock@bham.ac.uk.
authors.forename: Laura J V
authors.initials: LJ
authors.lastname: Piddock
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009154:Mutation; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D061806:Transcription Termination, Genetic
chemical_list: D018528:ATP-Binding Cassette Transporters; D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones
keywords: antimicrobial resistance mechanisms; efflux; multidrug resistance; regulation