QUINOLONE RESISTANCE LOCUS NFXD OF ESCHERICHIA-COLI IS A MUTANT ALLELE OF THE PARE GENE ENCODING A SUBUNIT OF TOPOISOMERASE-IV

The locus nfxD, which contributes to high-level quinolone resistance i n Escherichia coli KF111b (gyrA(r) nfxB nfxD), is only expressed in th e presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (g yrA(r) nfxD). DNA sequence analysis of DH161 revealed no changes in th e topoisomerase n: parC quinolone resistance-determining region but di d identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-Length cloned parE(+) partially compleme nted the resistance phenotype in KF111b and DH161, but did not complem ent the resistance phenotype in strain KF130 (gyrA(r)). No complementa tion was seen with cloned, truncated parE(+). To confirm these finding s, gyrA(r) was first outcrossed from KF130 into E. coli W3110parE10 [p arE temperature sensitive(Ts)] and KL16. The transduced strains KL16 a nd W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfl oxacin resistance in the parE(Ts) background twofold at 30 degrees C a nd fourfold at 42 degrees C compared to those for cloned parE from KL1 6. The same experiment with a non-Ts background revealed a twofold inc rease in the norfloxacin MIC at both 30 and 42 degrees C, These data i dentify the nfxD conditional resistance locus as a mutant allele of pa rE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norflox acin in E. coli.