Summary
DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.
Similar content being viewed by others
References
Adachi T, Mizuuchi M, Robinson EA, Appella E, O'Dea MH, Gellert M, Mizuuchi K (1987) DNA sequence of the gyrB gene: application of a new sequencing strategy. Nucleic Acids Res 15:771–784
Bachmann BJ (1983) Linkage map of Escherichia coli K-12, Edition 7. Microbiol Rev 47:180–230
Bourguignon GJ, Levitt M, Sternglantz R (1973) Studies on the mechanism of action of nalidixic acid. Antimicrob Agents Chemother 4:479–486
Chou Py, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148
Cosloy SD, Oishi M (1973) The nature of the transformation process in Escherichia coli K12. Mol Gen Genet 124:1–10
Gellert M, Mizuuchi K, O'Dea MH, Ito T, Tomizawa J (1977) Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci USA 74:4772–4776
Hane MW, Wood TH (1969) Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J Bacteriol 99:238–241
Higgins NP, Cozzarelli NR (1982) The binding of DNA gyrase to DNA: analysis by retention by nitrocellulose filters. Nucleic Acids Res 10:6833–6847
Higgins NP, Peebles CL, Sugino A, Cozzarelli NR (1978) Purification of subunits of Escherichia coli DNA gyrase and reconstitution of subunits of enzymatic activity. Proc Natl Acad Sci USA 75:1773–1777
Hirai K, Aoyama H, Irikura T, Iyobe S, Mitsuhashi S (1986) Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob Agents Chemother 29:535–538
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Horowitz DS, Wang JC (1987) Mapping the active site tyrosine of Escherichia coli DNA gyrase. J Biol Chem 262:5339–5344
Hrebenda J, Heleszko H, Brzostek K, Bielecki J (1985) Mutation affecting resistance of Escherichia coli K12 to nalidixic acid. J Gen Microbiol 131:2285–2292
Huang WM (1986) The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. Nucleic Acids Res 14:7379–7390
Inoue S, Ohue T, Yamagishi J, Nakamura S, Shimizu M (1978) Mode of incomplete cross-resistance among pipemidic, piromidic, and nalidixic acids. Antimicrob Agents Chemother 14:240–245
Klevan L, Wang JC (1980) Deoxyribonucleic acid gyrase-deoxyribonucleic acid complex containing 140 base pairs of deoxyribonucleic acid and an α2β2 protein core. Biochemistry 19:5229–5234
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Lampe MF, Bott KF (1984) Cloning of the gyrA gene of Bacillus subtilis. Nucleic Acids Res 12:6307–6323
Lesher GY, Froelich EJ, Gruett MD, Bailey JH, Brundage RP (1962) 1,8-Naphthyridine derivatives. A new class of chemotherapeutic agents. J Med Pharm Chem 5:1063–1065
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Menzel R, Gellert M (1987) Fusions of the Escherichia coli gyrA and gyrB control regions to the galactokinase gene are inducible by coumermycin treatment. J Bacteriol 169:1272–1278
Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Mizuuchi K, O'Dea MH, Gellert M (1978) DNA gyrase: Subunit structure and ATPase activity of the purified enzyme. Proc Natl Acad Sci USA 75:5960–5963
Moriya S, Ogasawara N, Yoshikawa H (1985) Structure and function of the replication origin of the Bacillus subtilis chromosome. III. Nucleic Acids Res 13:2251–2265
Platz A, Sjöberg B-M (1980) Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region. J Bacteriol 143:561–568
Sanders CC, Sanders WE Jr, Goering RV, Werner V (1984) Selection of multiple antibiotic resistance by quinolones, β-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrob Agents Chemother 26:797–801
Sato K, Inoue Y, Fujii T, Aoyama H, Inoue M, Mitsuhashi S (1986) Purification and properties of DNA gyrase from a fluoroquinolone-resistant strain of Escherichia coli. Antimicrob Agents Chemother 30:777–780
Timmers K, Sternglantz R (1978) Ionization and divalent cation dissociation constants of nalidixic acid and oxolinic acid. Bioinorganic Chem 9:145–155
Wilkie NM, Clements JB, Boll W, Mantei N, Lonsdale D, Weissmann C (1979) Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus I. Nucleic Acids Res 7:859–877
Wolfson JS, Hooper DS (1985) The fluoroquinolones: Structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob Agents Chemother 28:581–586
Yamagishi J, Furutani Y, Inoue S, Ohue T, Nakamura S, Shimizu M (1981) New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity. J Bacteriol 148:450–458
Yamagishi J, Yoshida H, Yamayoshi M, Nakamura S (1986) Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet 204:367–373
Author information
Authors and Affiliations
Additional information
Communicated by M. Takanami
Rights and permissions
About this article
Cite this article
Yoshida, H., Kojima, T., Yamagishi, Ji. et al. Quinolone-resistant mutations of the gyrA gene of Escherichia coli . Mol Gen Genet 211, 1–7 (1988). https://doi.org/10.1007/BF00338386
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00338386