The qepA Gene is Dependent on Upstream Sequences to Reduce Susceptibility to Ciprofloxacin

• The plasmid-borne qepA gene encodes a quinolone efflux pump that confers resistance to ciprofloxacin in clinical isolates of Escherichia coli. According to published data qepA is transcribed from a promoter 31 bp upstream of the coding sequence. We asked whether ciprofloxacin MIC associated with qepA in E. coli would vary as a function of mRNA expression level. To our surprise we found that the annotated qepA coding sequence was not sufficient to increase MIC. We decided to investigate the role played by surrounding sequences in determining resistance to ciprofloxacin. The qepA region was engineered onto the E. coli chromosome and mutations were generated to test the significance of surrounding sequences for expression of resistance. MIC was measured by broth microdilution. A 3.2 kb fragment from a resistance plasmid, including the annotated qepA coding sequence (1.5 kb), generated an 8-fold ciprofloxacin MIC increase when recombined into the E. coli chromosome. Deletion analysis revealed that the MIC increase was dependent on sequences upstream of qepA, annotated as ∆int1/groEL and ∆dfr2. Combining sequence analysis and mutagenesis, we identified a promoter within the ∆int1/groEL sequence, required for expression of resistance. The predicted transcript included two open reading frames (orf1 261 bp, orf2 189 bp) upstream of qepA. Deletion analysis revealed the essentiality of orf2 for the MIC increase. In conclusion, we have identified a new promoter for qepA, provided evidence that expression of the qepA coding sequence is not sufficient for resistance, and that resistance is influenced by sequences upstream of the qepA coding sequence. Details of the resistance mechanism remain to be elucidated.

Ämnesord

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

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