Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli.

摘要

Isogenic mutants derived from quinolone-susceptible isolate WT by introducing gyrA (S83L, D87G) and parC (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 pgyrA and ptopA, respectively, transcriptionally fused to the reporter gene bla coding for TEM-1 beta-lactamase. The quotient (Qsc) of the beta-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 in gyrA had the strongest influence on quinolone resistance while leaving other parameters nearly unaffected. The gyrA double mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation. Phenotypic expression of the parC mutation (S80I) was dependent on the presence of at least one gyrA mutation. Expression of high-level fluoroquinolone resistance (ciprofloxacin MIC, > 4 micrograms/ml) required a combination of the gyrA double mutation and one parC mutation (S80I or E84K). Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of a parC mutation affected neither the doubling time nor the degree of supercoiling, while the presence of the gyrA D87G mutation was associated with a significant reduction in the degree of DNA supercoiling.