CLONING AND NUCLEOTIDE-SEQUENCE OF PSEUDOMONAS-AERUGINOSA DNA GYRASE GYRA GENE FROM STRAIN PAO1 AND QUINOLONE-RESISTANT CLINICAL ISOLATES

The Pseudomonas aerginosa DNA gyrase gyrA gene was cloned and sequence d from strain PAO1. An open reading frame of 2,769 bp was found; it co ded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with E scherichia coli GyrA and 54% identity with Bacillus subtilis GyrA, alt hough conserved regions were present throughout the sequences, particu larly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally compleme nt the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. col i or P. aeruginosa with mutationally altered gyrA genes caused a rever sion to wild-type quinolone susceptibility, indicating that the intrin sic susceptibility of the PAO1 GyrA to quinolones is comparable to tha t of the E. coli enzyme. PCR was used to amplify 360 bp of P. aerugino sa gyrA encompassing the so-called quinolone resistance-determining re gion from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates teste d; these mutations caused the following alterations in the sequence of GyrA: Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 t o Ile. The resistance mechanisms in the other six isolates are unknown . The results of the study suggested that mechanisms other than a muta tional alteration in gyrA are the most common mechanism of ciprofloxac in resistance in P. aeruginosa from the lungs of patients with cystic fibrosis.