title: Constitutive SoxS expression in a fluoroquinolone-resistant strain with a truncated SoxR protein and identification of a new member of the marA-soxS-rob regulon, mdtG.
abstract: Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.
authors.affiliation: Department of Microbiology, Hospital Clínic, School of Medicine, University of Barcelona, Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Fàbrega
authors.forename: Robert G
authors.initials: RG
authors.lastname: Martin
authors.forename: Judah L
authors.initials: JL
authors.lastname: Rosner
authors.forename: M Mar
authors.initials: MM
authors.lastname: Tavio
authors.forename: Jordi
authors.initials: J
authors.lastname: Vila
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D024841:Fluoroquinolones / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D018085:Regulon / Q000235:genetics / Q000502:physiology*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D015534:Trans-Activators; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; C070725:SoxS protein, E coli