Broad-host-range gyrase A gene probe.
The Escherichia coli gyrase A gene was cloned in the broad-host-range cosmid vector pLA2917. The resulting plasmid, pNJR3-2, conferred quinolone susceptibility on a gyrA mutant of E. coli. To analyze the expression of this E. coli gene in Pseudomonas aeruginosa, pNJR3-2 or pLA2917 was mobilized via conjugation into P. aeruginosa PAO2 and several well-characterized quinolone-resistant mutants of this strain. The vector pLA2917 did not significantly affect the quinolone susceptibilities of any of the P. aeruginosa strains. However, pNJR3-2 conferred wild-type quinolone susceptibility on P. aeruginosa cfxA (gyrA) mutants and intermediate quinolone susceptibility on cfxA-cfxB double mutants of P. aeruginosa. The quinolone susceptibility of P. aeruginosa PAO2 gyrA+ was unaffected by pNJR3-2. Also, pNJR3-2 had no significant effect on P. aeruginosa cfxB (permeability) mutants. These results demonstrate that the DNA gyrase A gene from E. coli is expressed in P. aeruginosa and confers dominant susceptibility on gyrA mutants. Thus, pNJR3-2 can be used to detect the quinolone resistance mutations that occur in the gyrase A gene of this organism. pNJR3-2 also appears to discriminate between mutations in gyrA and mutations which alter permeability. This gyrase A probe was used successfully in the analysis of quinolone resistance in clinical isolates of P. aeruginosa.
Robillard
N J
NJ
Pharmaceutical Division, Miles Inc., West Haven, Connecticut 06516.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005838:Genotype; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004250:DNA Topoisomerases, Type II