Impact ofgyrAandparCMutations on Quinolone Resistance, Doubling Time, and Supercoiling Degree ofEscherichia coli

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Abstract
Isogenic mutants derived from quinolone-susceptible isolate WT by introducinggyrA(S83L, D87G) andparC(S80I, E84K) mutations associated with quinolone resistance were characterized with respect to quinolone resistance, growth rate, and degree of global supercoiling. The latter was determined by use of a pair of reporter plasmids carrying supercoiling-dependent promoters pgyrAand ptopA, respectively, transcriptionally fused to the reporter geneblacoding for TEM-1 β-lactamase. The quotient (Qsc) of the β-lactamase specific activity determined for a mutant carrying either plasmid was taken as a measure of the degree of global supercoiling. These Qsc data were comparable to results obtained from the separation of topoisomers of plasmid pBR322 on chloroquine-containing agarose gels and indicate a reduced degree of negative supercoiling in resistant mutants relative to the parent, WT. The S83L mutation ingyrAhad the strongest influence on quinolone resistance while leaving other parameters nearly unaffected. ThegyrAdouble mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation. Phenotypic expression of theparCmutation (S80I) was dependent on the presence of at least onegyrAmutation. Expression of high-level fluoroquinolone resistance (ciprofloxacin MIC, >4 μg/ml) required a combination of thegyrAdouble mutation and oneparCmutation (S80I or E84K). Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of aparCmutation affected neither the doubling time nor the degree of supercoiling, while the presence of thegyrAD87G mutation was associated with a significant reduction in the degree of DNA supercoiling.