title: NorA plasmid resistance to fluoroquinolones: role of copy number and norA frameshift mutations.
abstract: Staphylococcus aureus NorA protein is a transmembrane multidrug efflux pump that confers low-level resistance to hydrophilic fluoroquinolones. The norA gene promoter is active in Escherichia coli HB101. We have examined the genetic basis of norA-mediated resistance in E. coli by introducing a wild-type norA gene into HB101 in plasmid pCL1921, pBR322, or pUC18 exhibiting copy numbers that spanned a 22-fold range. Increased ciprofloxacin resistance correlated with norA transcript levels seen by Northern (RNA) analysis. Thus, contrary to some reports, a wild-type norA gene confers fluoroquinolone resistance in E. coli in a copy-number-dependent fashion and does not require mutational activation. Interestingly, a multicopy pUC19norA derivative gave transformants exhibiting a range of resistance phenotypes. The norA gene of one transformant carried a single base deletion (ATACAAT to AACAAT; the deleted base is underlined) in the putative--10 Pribnow box resulting in a promoter down-regulatory mutation; a second plasmid had acquired a frameshift producing a null mutation at codon 112. These mutations override the dual resistance-growth-inhibitory phenotype of high-copy-number norA plasmids. The results have implications for using the standard E. coli HB101 system to assess NorA function and potentially for plasmid-borne transmission of norA-mediated drug resistance.
authors.affiliation: Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, United Kingdom.
authors.forename: L
authors.initials: L
authors.lastname: Sun
authors.forename: S
authors.initials: S
authors.lastname: Sreedharan
authors.forename: K
authors.initials: K
authors.lastname: Plummer
authors.forename: L M
authors.initials: LM
authors.lastname: Fisher
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D024841:Fluoroquinolones; D016368:Frameshift Mutation; D005784:Gene Amplification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D024841:Fluoroquinolones