Novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein: enzymatic analysis of the mutant proteins.
Using the techniques of gap misrepair mutagenesis and site-directed mutagenesis, we have generated two novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein. DNA sequencing showed these mutations to be Ser-83----Ala and Gln-106----Arg. The mutant proteins were overproduced and purified, and their enzymatic properties were analyzed and compared with those of the wild-type enzyme. With ciprofloxacin and other quinolones, the inhibition of DNA supercoiling, relaxation, and decatenation and the induction of DNA cleavage were investigated for both wild-type and mutant enzymes. In each assay, the mutant enzymes were found to require approximately 10 times more drug to inhibit the reaction or induce cleavage than was the wild-type enzyme. However, the Ca2(+)-directed DNA cleavage reaction was indistinguishable for wild-type and mutant gyrases. We discuss models for the gyrase-mediated bactericidal effects of quinolone drugs.
Hallett
P
P
Department of Biochemistry, University of Leicester, United Kingdom.
Maxwell
A
A
D042462:4-Quinolones; D000890:Anti-Infective Agents / Q000494:pharmacology*; D004260:DNA Repair; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids; D011489:Protein Denaturation
D042462:4-Quinolones; D000890:Anti-Infective Agents; D004250:DNA Topoisomerases, Type II