title: Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.
abstract: Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA-ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA-ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA-ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA-ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
authors.affiliation: Centre for Infection and Immunity, 4th Floor, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: R
authors.initials: R
authors.lastname: Rosenblum
authors.forename: E
authors.initials: E
authors.lastname: Khan
authors.forename: G
authors.initials: G
authors.lastname: Gonzalez
authors.forename: R
authors.initials: R
authors.lastname: Hasan
authors.forename: T
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; D001665:Binding Sites; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008911:Minocycline / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000078304:Tigecycline
chemical_list: C477884:AcrR protein, Klebsiella pneumoniae; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C095947:RamA protein, Klebsiella; D000078304:Tigecycline; D008911:Minocycline