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. 1991 Feb;10(2):467–476. doi: 10.1002/j.1460-2075.1991.tb07969.x

The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

J L Vizán 1, C Hernández-Chico 1, I del Castillo 1, F Moreno 1
PMCID: PMC452668  PMID: 1846808

Abstract

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baquero F., Bouanchaud D., Martinez-Perez M. C., Fernandez C. Microcin plasmids: a group of extrachromosomal elements coding for low-molecular-weight antibiotics in Escherichia coli. J Bacteriol. 1978 Aug;135(2):342–347. doi: 10.1128/jb.135.2.342-347.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown P. O., Peebles C. L., Cozzarelli N. R. A topoisomerase from Escherichia coli related to DNA gyrase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6110–6114. doi: 10.1073/pnas.76.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conrad S. E., Campbell J. L. Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids. Nucleic Acids Res. 1979 Jul 25;6(10):3289–3304. doi: 10.1093/nar/6.10.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cozzarelli N. R. DNA gyrase and the supercoiling of DNA. Science. 1980 Feb 29;207(4434):953–960. doi: 10.1126/science.6243420. [DOI] [PubMed] [Google Scholar]
  7. Davagnino J., Herrero M., Furlong D., Moreno F., Kolter R. The DNA replication inhibitor microcin B17 is a forty-three-amino-acid protein containing sixty percent glycine. Proteins. 1986 Nov;1(3):230–238. doi: 10.1002/prot.340010305. [DOI] [PubMed] [Google Scholar]
  8. Davis R., Vapnek D. In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency. J Bacteriol. 1976 Mar;125(3):1148–1155. doi: 10.1128/jb.125.3.1148-1155.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Diaz R., Staudenbauer W. L. Replication of the broad host range plasmid RSF1010 in cell-free extracts of Escherichia coli and Pseudomonas aeruginosa. Nucleic Acids Res. 1982 Aug 11;10(15):4687–4702. doi: 10.1093/nar/10.15.4687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drlica K., Franco R. J. Inhibitors of DNA topoisomerases. Biochemistry. 1988 Apr 5;27(7):2253–2259. doi: 10.1021/bi00407a001. [DOI] [PubMed] [Google Scholar]
  12. Franco R. J., Drlica K. DNA gyrase on the bacterial chromosome. Oxolinic acid-induced DNA cleavage in the dnaA-gyrB region. J Mol Biol. 1988 May 5;201(1):229–233. doi: 10.1016/0022-2836(88)90449-4. [DOI] [PubMed] [Google Scholar]
  13. Garrido M. C., Herrero M., Kolter R., Moreno F. The export of the DNA replication inhibitor Microcin B17 provides immunity for the host cell. EMBO J. 1988 Jun;7(6):1853–1862. doi: 10.1002/j.1460-2075.1988.tb03018.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  15. Gellert M., Fisher L. M., O'Dea M. H. DNA gyrase: purification and catalytic properties of a fragment of gyrase B protein. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6289–6293. doi: 10.1073/pnas.76.12.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Gellert M., O'Dea M. H., 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. doi: 10.1073/pnas.73.12.4474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Genilloud O., Moreno F., Kolter R. DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17. J Bacteriol. 1989 Feb;171(2):1126–1135. doi: 10.1128/jb.171.2.1126-1135.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gowrishankar J. Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli. J Bacteriol. 1989 Apr;171(4):1923–1931. doi: 10.1128/jb.171.4.1923-1931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Groisman E. A., Castilho B. A., Casadaban M. J. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1480–1483. doi: 10.1073/pnas.81.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herrero M., Moreno F. Microcin B17 blocks DNA replication and induces the SOS system in Escherichia coli. J Gen Microbiol. 1986 Feb;132(2):393–402. doi: 10.1099/00221287-132-2-393. [DOI] [PubMed] [Google Scholar]
  22. Higgins N. P., Peebles C. L., Sugino A., Cozzarelli N. R. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1773–1777. doi: 10.1073/pnas.75.4.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hillenbrand G., Staudenbauer W. L. Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication. Nucleic Acids Res. 1982 Feb 11;10(3):833–853. doi: 10.1093/nar/10.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang W. M. Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39. Nucleic Acids Res. 1986 Oct 10;14(19):7751–7765. doi: 10.1093/nar/14.19.7751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huang W. M. The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. Nucleic Acids Res. 1986 Sep 25;14(18):7379–7390. [PMC free article] [PubMed] [Google Scholar]
  26. Laviña M., Pugsley A. P., Moreno F. Identification, mapping, cloning and characterization of a gene (sbmA) required for microcin B17 action on Escherichia coli K12. J Gen Microbiol. 1986 Jun;132(6):1685–1693. doi: 10.1099/00221287-132-6-1685. [DOI] [PubMed] [Google Scholar]
  27. Mayo O., Hernández-Chico C., Moreno F. Microcin B17, a novel tool for preparation of maxicells: identification of polypeptides encoded by the IncFII minireplicon pMccB17. J Bacteriol. 1988 May;170(5):2414–2417. doi: 10.1128/jb.170.5.2414-2417.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Mizuuchi K., O'Dea M. H., Gellert M. DNA gyrase: subunit structure and ATPase activity of the purified enzyme. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5960–5963. doi: 10.1073/pnas.75.12.5960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morrison A., Higgins N. P., Cozzarelli N. R. Interaction between DNA gyrase and its cleavage site on DNA. J Biol Chem. 1980 Mar 10;255(5):2211–2219. [PubMed] [Google Scholar]
  31. Orr E., Fairweather N. F., Holland I. B., Pritchard R. H. Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12. Mol Gen Genet. 1979;177(1):103–112. doi: 10.1007/BF00267259. [DOI] [PubMed] [Google Scholar]
  32. Pruss G. J. DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. J Mol Biol. 1985 Sep 5;185(1):51–63. doi: 10.1016/0022-2836(85)90182-2. [DOI] [PubMed] [Google Scholar]
  33. Rose K. M. DNA topoisomerases as targets for chemotherapy. FASEB J. 1988 Jun;2(9):2474–2478. doi: 10.1096/fasebj.2.9.2836254. [DOI] [PubMed] [Google Scholar]
  34. San Millan J. L., Hernandez-Chico C., Pereda P., Moreno F. Cloning and mapping of the genetic determinants for microcin B17 production and immunity. J Bacteriol. 1985 Jul;163(1):275–281. doi: 10.1128/jb.163.1.275-281.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. San Millán J. L., Kolter R., Moreno F. Evidence that colicin X is microcin B17. J Bacteriol. 1987 Jun;169(6):2899–2901. doi: 10.1128/jb.169.6.2899-2901.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. San Millán J. L., Kolter R., Moreno F. Plasmid genes required for microcin B17 production. J Bacteriol. 1985 Sep;163(3):1016–1020. doi: 10.1128/jb.163.3.1016-1020.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Snyder M., Drlica K. DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J Mol Biol. 1979 Jun 25;131(2):287–302. doi: 10.1016/0022-2836(79)90077-9. [DOI] [PubMed] [Google Scholar]
  39. Sugino A., Higgins N. P., Brown P. O., Peebles C. L., Cozzarelli N. R. 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. doi: 10.1073/pnas.75.10.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sugino A., Peebles C. L., Kreuzer K. N., Cozzarelli N. R. 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. doi: 10.1073/pnas.74.11.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Tewey K. M., Rowe T. C., Yang L., Halligan B. D., Liu L. F. Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science. 1984 Oct 26;226(4673):466–468. doi: 10.1126/science.6093249. [DOI] [PubMed] [Google Scholar]
  43. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  44. Wang J. C. Recent studies of DNA topoisomerases. Biochim Biophys Acta. 1987 Jun 6;909(1):1–9. doi: 10.1016/0167-4781(87)90040-6. [DOI] [PubMed] [Google Scholar]
  45. Wyckoff E., Natalie D., Nolan J. M., Lee M., Hsieh T. Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. J Mol Biol. 1989 Jan 5;205(1):1–13. doi: 10.1016/0022-2836(89)90361-6. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Yang Y., Ames G. F. DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8850–8854. doi: 10.1073/pnas.85.23.8850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]

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