• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. Aug 1996; 40(8): 1881–1888.
PMCID: PMC163434

Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus.


Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.

Full Text

The Full Text of this article is available as a PDF (295K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ali JA, Jackson AP, Howells AJ, Maxwell A. The 43-kilodalton N-terminal fragment of the DNA gyrase B protein hydrolyzes ATP and binds coumarin drugs. Biochemistry. 1993 Mar 16;32(10):2717–2724. [PubMed]
  • Belland RJ, Morrison SG, Ison C, Huang WM. Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol Microbiol. 1994 Oct;14(2):371–380. [PubMed]
  • Brockbank SM, Barth PT. Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus. J Bacteriol. 1993 Jun;175(11):3269–3277. [PMC free article] [PubMed]
  • Contreras A, Maxwell A. gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase. Mol Microbiol. 1992 Jun;6(12):1617–1624. [PubMed]
  • Cullen ME, Wyke AW, Kuroda R, Fisher LM. Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones. Antimicrob Agents Chemother. 1989 Jun;33(6):886–894. [PMC free article] [PubMed]
  • Domagala JM. Structure-activity and structure-side-effect relationships for the quinolone antibacterials. J Antimicrob Chemother. 1994 Apr;33(4):685–706. [PubMed]
  • Ferrero L, Cameron B, Crouzet J. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrob Agents Chemother. 1995 Jul;39(7):1554–1558. [PMC free article] [PubMed]
  • Ferrero L, Cameron B, Manse B, Lagneaux D, Crouzet J, Famechon A, Blanche F. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones. Mol Microbiol. 1994 Aug;13(4):641–653. [PubMed]
  • Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. [PubMed]
  • Gellert M, Mizuuchi K, O'Dea MH, Itoh T, Tomizawa JI. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4772–4776. [PMC free article] [PubMed]
  • Gellert M, O'Dea MH, Itoh T, Tomizawa J. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4474–4478. [PMC free article] [PubMed]
  • Goswitz JJ, Willard KE, Fasching CE, Peterson LR. Detection of gyrA gene mutations associated with ciprofloxacin resistance in methicillin-resistant Staphylococcus aureus: analysis by polymerase chain reaction and automated direct DNA sequencing. Antimicrob Agents Chemother. 1992 May;36(5):1166–1169. [PMC free article] [PubMed]
  • Hallett P, Maxwell A. Novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein: enzymatic analysis of the mutant proteins. Antimicrob Agents Chemother. 1991 Feb;35(2):335–340. [PMC free article] [PubMed]
  • Hooper DC, Wolfson JS. Fluoroquinolone antimicrobial agents. N Engl J Med. 1991 Feb 7;324(6):384–394. [PubMed]
  • Hopewell R, Oram M, Briesewitz R, Fisher LM. DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance. J Bacteriol. 1990 Jun;172(6):3481–3484. [PMC free article] [PubMed]
  • Hori S, Ohshita Y, Utsui Y, Hiramatsu K. Sequential acquisition of norfloxacin and ofloxacin resistance by methicillin-resistant and -susceptible Staphylococcus aureus. Antimicrob Agents Chemother. 1993 Nov;37(11):2278–2284. [PMC free article] [PubMed]
  • Horowitz DS, Wang JC. Mapping the active site tyrosine of Escherichia coli DNA gyrase. J Biol Chem. 1987 Apr 15;262(11):5339–5344. [PubMed]
  • Hoshino K, Kitamura A, Morrissey I, Sato K, Kato J, Ikeda H. Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition. Antimicrob Agents Chemother. 1994 Nov;38(11):2623–2627. [PMC free article] [PubMed]
  • Ito H, Yoshida H, Bogaki-Shonai M, Niga T, Hattori H, Nakamura S. Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus. Antimicrob Agents Chemother. 1994 Sep;38(9):2014–2023. [PMC free article] [PubMed]
  • Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H. New topoisomerase essential for chromosome segregation in E. coli. Cell. 1990 Oct 19;63(2):393–404. [PubMed]
  • Kato J, Suzuki H, Ikeda H. Purification and characterization of DNA topoisomerase IV in Escherichia coli. J Biol Chem. 1992 Dec 25;267(36):25676–25684. [PubMed]
  • Khodursky AB, Zechiedrich EL, Cozzarelli NR. Topoisomerase IV is a target of quinolones in Escherichia coli. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11801–11805. [PMC free article] [PubMed]
  • Korten V, Huang WM, Murray BE. Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis. Antimicrob Agents Chemother. 1994 Sep;38(9):2091–2094. [PMC free article] [PubMed]
  • Kreiswirth BN, Löfdahl S, Betley MJ, O'Reilly M, Schlievert PM, Bergdoll MS, Novick RP. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature. 1983 Oct 20;305(5936):709–712. [PubMed]
  • Lewis RJ, Singh OM, Smith CV, Maxwell A, Skarzynski T, Wonacott AJ, Wigley DB. Crystallization of inhibitor complexes of an N-terminal 24 kDa fragment of the DNA gyrase B protein. J Mol Biol. 1994 Aug 5;241(1):128–130. [PubMed]
  • Li XZ, Livermore DM, Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrob Agents Chemother. 1994 Aug;38(8):1732–1741. [PMC free article] [PubMed]
  • Margerrison EE, Hopewell R, Fisher LM. Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins. J Bacteriol. 1992 Mar;174(5):1596–1603. [PMC free article] [PubMed]
  • Maxwell A. The interaction between coumarin drugs and DNA gyrase. Mol Microbiol. 1993 Aug;9(4):681–686. [PubMed]
  • Neidhardt FC, Bloch PL, Smith DF. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. [PMC free article] [PubMed]
  • Neyfakh AA, Borsch CM, Kaatz GW. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrob Agents Chemother. 1993 Jan;37(1):128–129. [PMC free article] [PubMed]
  • Ng EY, Trucksis M, Hooper DC. Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother. 1994 Jun;38(6):1345–1355. [PMC free article] [PubMed]
  • Ohshita Y, Hiramatsu K, Yokota T. A point mutation in norA gene is responsible for quinolone resistance in Staphylococcus aureus. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1028–1034. [PubMed]
  • Oram M, Fisher LM. 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrob Agents Chemother. 1991 Feb;35(2):387–389. [PMC free article] [PubMed]
  • Pattee PA, Neveln DS. Transformation analysis of three linkage groups in Staphylococcus aureus. J Bacteriol. 1975 Oct;124(1):201–211. [PMC free article] [PubMed]
  • Peng H, Marians KJ. Escherichia coli topoisomerase IV. Purification, characterization, subunit structure, and subunit interactions. J Biol Chem. 1993 Nov 15;268(32):24481–24490. [PubMed]
  • Peng H, Marians KJ. Decatenation activity of topoisomerase IV during oriC and pBR322 DNA replication in vitro. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8571–8575. [PMC free article] [PubMed]
  • Peterson LR, Willard KE, Sinn LM, Fasching CE, Gerding DN. GyrA sequence analysis of Staphylococcus aureus and methicillin-resistant S. aureus strains selected, in vitro, for high-level ciprofloxacin resistance. Diagn Microbiol Infect Dis. 1993 Aug-Sep;17(2):97–101. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Soussy CJ, Wolfson JS, Ng EY, Hooper DC. Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus. Antimicrob Agents Chemother. 1993 Dec;37(12):2588–2592. [PMC free article] [PubMed]
  • Sreedharan S, Oram M, Jensen B, Peterson LR, Fisher LM. DNA gyrase gyrA mutations in ciprofloxacin-resistant strains of Staphylococcus aureus: close similarity with quinolone resistance mutations in Escherichia coli. J Bacteriol. 1990 Dec;172(12):7260–7262. [PMC free article] [PubMed]
  • Sreedharan S, Peterson LR, Fisher LM. Ciprofloxacin resistance in coagulase-positive and -negative staphylococci: role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother. 1991 Oct;35(10):2151–2154. [PMC free article] [PubMed]
  • Stahl ML, Pattee PA. Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA. J Bacteriol. 1983 Apr;154(1):406–412. [PMC free article] [PubMed]
  • Sugino A, Bott KF. Bacillus subtilis deoxyribonucleic acid gyrase. J Bacteriol. 1980 Mar;141(3):1331–1339. [PMC free article] [PubMed]
  • Sugino A, Higgins NP, Brown PO, Peebles CL, Cozzarelli NR. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4838–4842. [PMC free article] [PubMed]
  • Sugino A, Peebles CL, Kreuzer KN, Cozzarelli NR. Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4767–4771. [PMC free article] [PubMed]
  • Suto MJ, Domagala JM, Roland GE, Mailloux GB, Cohen MA. Fluoroquinolones: relationships between structural variations, mammalian cell cytotoxicity, and antimicrobial activity. J Med Chem. 1992 Dec 11;35(25):4745–4750. [PubMed]
  • Trucksis M, Hooper DC, Wolfson JS. Emerging resistance to fluoroquinolones in staphylococci: an alert. Ann Intern Med. 1991 Mar 1;114(5):424–426. [PubMed]
  • Trucksis M, Wolfson JS, Hooper DC. A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus. J Bacteriol. 1991 Sep;173(18):5854–5860. [PMC free article] [PubMed]
  • Tse YC, Kirkegaard K, Wang JC. Covalent bonds between protein and DNA. Formation of phosphotyrosine linkage between certain DNA topoisomerases and DNA. J Biol Chem. 1980 Jun 25;255(12):5560–5565. [PubMed]
  • Wang JC. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. [PubMed]
  • Wang JC. Recent studies of DNA topoisomerases. Biochim Biophys Acta. 1987 Jun 6;909(1):1–9. [PubMed]
  • Willmott CJ, Maxwell A. A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex. Antimicrob Agents Chemother. 1993 Jan;37(1):126–127. [PMC free article] [PubMed]
  • Yamagishi J, Furutani Y, Inoue S, Ohue T, Nakamura S, Shimizu M. New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity. J Bacteriol. 1981 Nov;148(2):450–458. [PMC free article] [PubMed]
  • Yoshida H, Bogaki M, Nakamura M, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob Agents Chemother. 1990 Jun;34(6):1271–1272. [PMC free article] [PubMed]
  • Yoshida H, Bogaki M, Nakamura M, Yamanaka LM, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli. Antimicrob Agents Chemother. 1991 Aug;35(8):1647–1650. [PMC free article] [PubMed]
  • Yoshida H, Bogaki M, Nakamura S, Ubukata K, Konno M. Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol. 1990 Dec;172(12):6942–6949. [PMC free article] [PubMed]
  • Yoshida H, Nakamura M, Bogaki M, Nakamura S. Proportion of DNA gyrase mutants among quinolone-resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1990 Jun;34(6):1273–1275. [PMC free article] [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...