title: Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda.
abstract: The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.
authors.affiliation: Pathology Division, National Fisheries Research and Development Institute, Busan 619-705, Korea.
authors.forename: Myoung Sug
authors.initials: MS
authors.lastname: Kim
authors.forename: Lyu Jin
authors.initials: LJ
authors.lastname: Jun
authors.forename: Soon Bum
authors.initials: SB
authors.lastname: Shin
authors.forename: Myoung Ae
authors.initials: MA
authors.lastname: Park
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Jung
authors.forename: Kwangil
authors.initials: K
authors.lastname: Kim
authors.forename: Kyung Ho
authors.initials: KH
authors.lastname: Moon
authors.forename: Hyun Do
authors.initials: HD
authors.lastname: Jeong
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D027081:DNA Gyrase / Q000235:genetics*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D020609:Edwardsiella tarda / Q000187:drug effects* / Q000235:genetics; D007723:Korea; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D015363:Quinolones / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D015363:Quinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase