title: A single nucleotide in the promoter region modulates the expression of the β-lactamase OXA-61 in Campylobacter jejuni.
abstract: OBJECTIVE
  Despite prevalent β-lactam resistance in Campylobacter jejuni, an important zoonotic enteric pathogen, the molecular basis of β-lactamase-mediated β-lactam resistance is still largely unknown. In particular, some C. jejuni strains that carry β-lactamase gene blaOXA-61 (Cj0299) are still susceptible to β-lactams with undetected β-lactamase activity, suggesting blaOXA-61 is subjected to regulation. The objective of this study was to determine the regulatory mechanism of β-lactamase in C. jejuni.
  
  
  METHODS
  An ampicillin-resistant derivative of C. jejuni NCTC 11168 was subjected to whole genome sequencing and comparative genomics analysis. Complementary molecular experiments were further performed to examine the identified regulatory mechanism of blaOXA-61, which included complementation, promoter fusion assay, real-time RT-PCR, natural transformation using a defined PCR fragment, survey of clinical isolates and transcription start site mapping.
  
  
  RESULTS
  A single nucleotide mutation (G → T transversion) upstream of blaOXA-61 was identified in the ampicillin-resistant derivative of NCTC 11168. The role of the G → T point mutation in acquired β-lactam resistance through up-regulating the expression of blaOXA-61 was confirmed by multiple molecular approaches. The G → T transversion in the blaOXA-61 promoter was linked to high-level β-lactam resistance in C. jejuni isolates. Transcription start site mapping indicated that the G → T transversion restored the TATA box in the -10 region of blaOXA-61.
  
  
  CONCLUSIONS
  We demonstrated a novel genetic mechanism of β-lactamase regulation in C. jejuni in this study, which will provide insights into the regulation and evolution of β-lactam resistance in Campylobacter.
authors.affiliation: Department of Animal Science, The University of Tennessee, 2506 River Drive, Knoxville, TN 37996, USA.
authors.forename: Ximin
authors.initials: X
authors.lastname: Zeng
authors.forename: Samantha
authors.initials: S
authors.lastname: Brown
authors.forename: Barbara
authors.initials: B
authors.lastname: Gillespie
authors.forename: Jun
authors.initials: J
authors.lastname: Lin
mesh_terms: D000668:Ampicillin Resistance; D016123:Campylobacter jejuni / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D001618:beta-Lactamases
keywords: ampicillin resistance; comparative genomics; gene regulation