New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cour) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nalr) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA (Nalr) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA (Nalr) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.