Abstract
Using the techniques of gap misrepair mutagenesis and site-directed mutagenesis, we have generated two novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein. DNA sequencing showed these mutations to be Ser-83----Ala and Gln-106----Arg. The mutant proteins were overproduced and purified, and their enzymatic properties were analyzed and compared with those of the wild-type enzyme. With ciprofloxacin and other quinolones, the inhibition of DNA supercoiling, relaxation, and decatenation and the induction of DNA cleavage were investigated for both wild-type and mutant enzymes. In each assay, the mutant enzymes were found to require approximately 10 times more drug to inhibit the reaction or induce cleavage than was the wild-type enzyme. However, the Ca2(+)-directed DNA cleavage reaction was indistinguishable for wild-type and mutant gyrases. We discuss models for the gyrase-mediated bactericidal effects of quinolone drugs.
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- Adachi T., Mizuuchi M., Robinson E. A., Appella E., O'Dea M. H., Gellert M., Mizuuchi K. DNA sequence of the E. coli gyrB gene: application of a new sequencing strategy. Nucleic Acids Res. 1987 Jan 26;15(2):771–784. doi: 10.1093/nar/15.2.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barrett J. F., Sutcliffe J. A., Gootz T. D. In vitro assays used to measure the activity of topoisomerases. Antimicrob Agents Chemother. 1990 Jan;34(1):1–7. doi: 10.1128/aac.34.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Cullen M. E., Wyke A. W., Kuroda R., Fisher L. M. 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. doi: 10.1128/aac.33.6.886. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drlica K., Engle E. C., Manes S. H. DNA gyrase on the bacterial chromosome: possibility of two levels of action. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6879–6883. doi: 10.1073/pnas.77.11.6879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
- Gellert M., Mizuuchi K., O'Dea M. H., Itoh T., Tomizawa J. I. 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. doi: 10.1073/pnas.74.11.4772. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hallett P., Grimshaw A. J., Wigley D. B., Maxwell A. Cloning of the DNA gyrase genes under tac promoter control: overproduction of the gyrase A and B proteins. Gene. 1990 Sep 1;93(1):139–142. doi: 10.1016/0378-1119(90)90148-k. [DOI] [PubMed] [Google Scholar]
- Hallett P., Mehlert A., Maxwell A. Escherichia coli cells resistant to the DNA gyrase inhibitor, ciprofloxacin, overproduce a 60 kD protein homologous to GroEL. Mol Microbiol. 1990 Mar;4(3):345–353. doi: 10.1111/j.1365-2958.1990.tb00602.x. [DOI] [PubMed] [Google Scholar]
- Hane M. W., Wood T. H. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J Bacteriol. 1969 Jul;99(1):238–241. doi: 10.1128/jb.99.1.238-241.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horowitz D. S., Wang J. C. Mapping the active site tyrosine of Escherichia coli DNA gyrase. J Biol Chem. 1987 Apr 15;262(11):5339–5344. [PubMed] [Google Scholar]
- Hussain K., Elliott E. J., Salmond G. P. The parD- mutant of Escherichia coli also carries a gyrAam mutation. The complete sequence of gyrA. Mol Microbiol. 1987 Nov;1(3):259–273. doi: 10.1111/j.1365-2958.1987.tb01932.x. [DOI] [PubMed] [Google Scholar]
- Klevan L., Wang J. C. Deoxyribonucleic acid gyrase-deoxyribonucleic acid complex containing 140 base pairs of deoxyribonucleic acid and an alpha 2 beta 2 protein core. Biochemistry. 1980 Nov 11;19(23):5229–5234. doi: 10.1021/bi00564a012. [DOI] [PubMed] [Google Scholar]
- Kreuzer K. N., Cozzarelli N. R. Escherichia coli mutants thermosensitive for deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic acid replication, transcription, and bacteriophage growth. J Bacteriol. 1979 Nov;140(2):424–435. doi: 10.1128/jb.140.2.424-435.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krueger S., Zaccai G., Wlodawer A., Langowski J., O'Dea M., Maxwell A., Gellert M. Neutron and light-scattering studies of DNA gyrase and its complex with DNA. J Mol Biol. 1990 Jan 5;211(1):211–220. doi: 10.1016/0022-2836(90)90021-D. [DOI] [PubMed] [Google Scholar]
- LESHER G. Y., FROELICH E. J., GRUETT M. D., BAILEY J. H., BRUNDAGE R. P. 1,8-NAPHTHYRIDINE DERIVATIVES. A NEW CLASS OF CHEMOTHERAPEUTIC AGENTS. J Med Pharm Chem. 1962 Sep;91:1063–1065. doi: 10.1021/jm01240a021. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Liu L. F. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem. 1989;58:351–375. doi: 10.1146/annurev.bi.58.070189.002031. [DOI] [PubMed] [Google Scholar]
- Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxwell A., Gellert M. Mechanistic aspects of DNA topoisomerases. Adv Protein Chem. 1986;38:69–107. doi: 10.1016/s0065-3233(08)60526-4. [DOI] [PubMed] [Google Scholar]
- Mizuuchi K., Fisher L. M., O'Dea M. H., Gellert M. DNA gyrase action involves the introduction of transient double-strand breaks into DNA. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1847–1851. doi: 10.1073/pnas.77.4.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mizuuchi K., Mizuuchi M., O'Dea M. H., Gellert M. Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins. J Biol Chem. 1984 Jul 25;259(14):9199–9201. [PubMed] [Google Scholar]
- Nakamura S., Nakamura M., Kojima T., Yoshida H. gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli. Antimicrob Agents Chemother. 1989 Feb;33(2):254–255. doi: 10.1128/aac.33.2.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nelson E. M., Tewey K. M., Liu L. F. Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1361–1365. doi: 10.1073/pnas.81.5.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neu H. C. Clinical utility of DNA gyrase inhibitors. Pharmacol Ther. 1989;41(1-2):207–221. doi: 10.1016/0163-7258(89)90107-1. [DOI] [PubMed] [Google Scholar]
- Reece R. J., Dauter Z., Wilson K. S., Maxwell A., Wigley D. B. Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein. J Mol Biol. 1990 Oct 20;215(4):493–495. doi: 10.1016/S0022-2836(05)80162-7. [DOI] [PubMed] [Google Scholar]
- Reece R. J., Maxwell A. Tryptic fragments of the Escherichia coli DNA gyrase A protein. J Biol Chem. 1989 Nov 25;264(33):19648–19653. [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shen L. L., Kohlbrenner W. E., Weigl D., Baranowski J. Mechanism of quinolone inhibition of DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes. J Biol Chem. 1989 Feb 15;264(5):2973–2978. [PubMed] [Google Scholar]
- Shen L. L., Mitscher L. A., Sharma P. N., O'Donnell T. J., Chu D. W., Cooper C. S., Rosen T., Pernet A. G. Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug--DNA binding model. Biochemistry. 1989 May 2;28(9):3886–3894. doi: 10.1021/bi00435a039. [DOI] [PubMed] [Google Scholar]
- Shen L. L., Pernet A. G. Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA. Proc Natl Acad Sci U S A. 1985 Jan;82(2):307–311. doi: 10.1073/pnas.82.2.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shortle D., Grisafi P., Benkovic S. J., Botstein D. Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1588–1592. doi: 10.1073/pnas.79.5.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steck T. R., Drlica K. Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation. Cell. 1984 Apr;36(4):1081–1088. doi: 10.1016/0092-8674(84)90058-8. [DOI] [PubMed] [Google Scholar]
- Swanberg S. L., Wang J. C. Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase. J Mol Biol. 1987 Oct 20;197(4):729–736. doi: 10.1016/0022-2836(87)90479-7. [DOI] [PubMed] [Google Scholar]
- Walton L., Elwell L. P. In vitro cleavable-complex assay to monitor antimicrobial potency of quinolones. Antimicrob Agents Chemother. 1988 Jul;32(7):1086–1089. doi: 10.1128/aac.32.7.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
- Wolfson J. S., Hooper D. C. The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob Agents Chemother. 1985 Oct;28(4):581–586. doi: 10.1128/aac.28.4.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1128/jb.148.2.450-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamagishi J., Yoshida H., Yamayoshi M., Nakamura S. Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet. 1986 Sep;204(3):367–373. doi: 10.1007/BF00331012. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1128/aac.34.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yoshida H., Kojima T., Yamagishi J., Nakamura S. Quinolone-resistant mutations of the gyrA gene of Escherichia coli. Mol Gen Genet. 1988 Jan;211(1):1–7. doi: 10.1007/BF00338386. [DOI] [PubMed] [Google Scholar]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
- Zweerink M. M., Edison A. Inhibition of Micrococcus luteus DNA gyrase by norfloxacin and 10 other quinolone carboxylic acids. Antimicrob Agents Chemother. 1986 Apr;29(4):598–601. doi: 10.1128/aac.29.4.598. [DOI] [PMC free article] [PubMed] [Google Scholar]