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. 1996 Sep;40(9):2054–2061. doi: 10.1128/aac.40.9.2054

Sequence analysis, purification, and study of inhibition by 4-quinolones of the DNA gyrase from Mycobacterium smegmatis.

V Revel-Viravau 1, Q C Truong 1, N Moreau 1, V Jarlier 1, W Sougakoff 1
PMCID: PMC163472  PMID: 8878580

Abstract

We determined the nucleotide sequence of a 6-kb DNA region harboring the recF, orf192, gyrB, and gyrA genes from Mycobacterium smegmatis mc(2)155. The amino acid sequences deduced from gyrA and gyrB displayed 89 and 86% identity, respectively, with the DNA gyrase from Mycobacterium tuberculosis, and 67 and 65% identity, respectively, with that from Streptomyces coelicolor. An open reading frame encoding the C-terminal region of the M. smegmatis RecF polypeptide was found upstream from gyrB and was 57% identical to the open reading frame encoding the C-terminal region of the S. coelicolor RecF protein. The gene orf192 was identified between recF and gyrB and was 39% identical to orf191 found in S. coelicolor in the recF-gyrB region. The M. smegmatis DNA gyrase, which was purified by affinity chromatography on novobiocin-Sepharose, consisted of two polypeptides with apparent molecular masses of 98 and 80 kDa. Determination of the N-terminal amino acid sequence of the B subunit confirmed GTG as the start codon in gyrB. Analysis of the supercoiling activity of the enzyme indicated that the M. smegmatis DNA gyrase was characterized by a specific activity equivalent to that of the Escherichia coli DNA gyrase. Inhibition of this activity by 4-quinolones was investigated by determining the 50% inhibitory concentrations (IC50S) of nalidixic acid, ofloxacin, and ciprofloxacin. The results indicated that the inhibitory activities of these drugs against the M. smegmatis DNA gyrase were markedly lower than those previously reported for the E. coli DNA gyrase. The results also suggested that the higher levels of activity of ofloxacin and ciprofloxacin against M. smegmatis (MICs, 0.5 to 1 microgram/ml), in contrast to that of nalidixic acid (MIC, 256 micrograms/ml), could be related to the higher inhibitory activities of fluoroquinolones against the DNA gyrase from this species (IC50S, 7 to 14 micrograms/ml) compared with that of nalidixic acid (IC50, 1,400 micrograms/ml).

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Selected References

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  1. Adachi T., Mizuuchi K., Menzel R., Gellert M. DNA sequence and transcription of the region upstream of the E. coli gyrB gene. Nucleic Acids Res. 1984 Aug 24;12(16):6389–6395. doi: 10.1093/nar/12.16.6389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Alangaden G. J., Manavathu E. K., Vakulenko S. B., Zvonok N. M., Lerner S. A. Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrob Agents Chemother. 1995 Aug;39(8):1700–1703. doi: 10.1128/aac.39.8.1700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bazile S., Moreau N., Bouzard D., Essiz M. Relationships among antibacterial activity, inhibition of DNA gyrase, and intracellular accumulation of 11 fluoroquinolones. Antimicrob Agents Chemother. 1992 Dec;36(12):2622–2627. doi: 10.1128/aac.36.12.2622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belland R. J., Morrison S. G., Ison C., Huang W. M. Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol Microbiol. 1994 Oct;14(2):371–380. doi: 10.1111/j.1365-2958.1994.tb01297.x. [DOI] [PubMed] [Google Scholar]
  6. Brockbank S. M., Barth P. T. Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus. J Bacteriol. 1993 Jun;175(11):3269–3277. doi: 10.1128/jb.175.11.3269-3277.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calcutt M. J. Gene organization in the dnaA-gyrA region of the Streptomyces coelicolor chromosome. Gene. 1994 Dec 30;151(1-2):23–28. doi: 10.1016/0378-1119(94)90628-9. [DOI] [PubMed] [Google Scholar]
  8. Cambau E., Sougakoff W., Besson M., Truffot-Pernot C., Grosset J., Jarlier V. Selection of a gyrA mutant of Mycobacterium tuberculosis resistant to fluoroquinolones during treatment with ofloxacin. J Infect Dis. 1994 Aug;170(2):479–483. doi: 10.1093/infdis/170.2.479. [DOI] [PubMed] [Google Scholar]
  9. Cambau E., Sougakoff W., Jarlier V. Amplification and nucleotide sequence of the quinolone resistance-determining region in the gyrA gene of mycobacteria. FEMS Microbiol Lett. 1994 Feb 1;116(1):49–54. doi: 10.1111/j.1574-6968.1994.tb06674.x. [DOI] [PubMed] [Google Scholar]
  10. Colman S. D., Hu P. C., Bott K. F. Mycoplasma pneumoniae DNA gyrase genes. Mol Microbiol. 1990 Jul;4(7):1129–1134. doi: 10.1111/j.1365-2958.1990.tb00687.x. [DOI] [PubMed] [Google Scholar]
  11. Dimri G. P., Das H. K. Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae. Nucleic Acids Res. 1990 Jan 11;18(1):151–156. doi: 10.1093/nar/18.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dugaiczyk A., Goold R., diSibio G., Myers R. M. Improved sequencing of cosmids using new primers and linearized DNA. Nucleic Acids Res. 1992 Dec 11;20(23):6421–6422. doi: 10.1093/nar/20.23.6421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  14. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  15. Fujita M. Q., Yoshikawa H., Ogasawara N. Structure of the dnaA region of Pseudomonas putida: conservation among three bacteria, Bacillus subtilis, Escherichia coli and P. putida. Mol Gen Genet. 1989 Feb;215(3):381–387. doi: 10.1007/BF00427033. [DOI] [PubMed] [Google Scholar]
  16. Gaal T., Barkei J., Dickson R. R., deBoer H. A., deHaseth P. L., Alavi H., Gourse R. L. Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants. J Bacteriol. 1989 Sep;171(9):4852–4861. doi: 10.1128/jb.171.9.4852-4861.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Galtier N., Gouy M. Molecular phylogeny of Eubacteria: a new multiple tree analysis method applied to 15 sequence data sets questions the monophyly of gram-positive bacteria. Res Microbiol. 1994 Sep;145(7):531–541. doi: 10.1016/0923-2508(94)90030-2. [DOI] [PubMed] [Google Scholar]
  18. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Guillemin I., Cambau E., Jarlier V. Sequences of conserved region in the A subunit of DNA gyrase from nine species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones. Antimicrob Agents Chemother. 1995 Sep;39(9):2145–2149. doi: 10.1128/aac.39.9.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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. doi: 10.1128/aac.35.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hooper D. C., Wolfson J. S. Mode of action of the new quinolones: new data. Eur J Clin Microbiol Infect Dis. 1991 Apr;10(4):223–231. doi: 10.1007/BF01966994. [DOI] [PubMed] [Google Scholar]
  23. 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. doi: 10.1128/aac.38.9.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kureishi A., Diver J. M., Beckthold B., Schollaardt T., Bryan L. E. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates. Antimicrob Agents Chemother. 1994 Sep;38(9):1944–1952. doi: 10.1128/aac.38.9.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  26. Madhusudan K., Nagaraja V. Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli. Microbiology. 1995 Dec;141(Pt 12):3029–3037. doi: 10.1099/13500872-141-12-3029. [DOI] [PubMed] [Google Scholar]
  27. Madhusudan K., Ramesh V., Nagaraja V. Molecular cloning of gyrA and gyrB genes of Mycobacterium tuberculosis: analysis of nucleotide sequence. Biochem Mol Biol Int. 1994 Jul;33(4):651–660. [PubMed] [Google Scholar]
  28. Margerrison E. E., Hopewell R., Fisher L. M. 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. doi: 10.1128/jb.174.5.1596-1603.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moore R. A., Beckthold B., Wong S., Kureishi A., Bryan L. E. Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori. Antimicrob Agents Chemother. 1995 Jan;39(1):107–111. doi: 10.1128/aac.39.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moreau N. J., Robaux H., Baron L., Tabary X. Inhibitory effects of quinolones on pro- and eucaryotic DNA topoisomerases I and II. Antimicrob Agents Chemother. 1990 Oct;34(10):1955–1960. doi: 10.1128/aac.34.10.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moriya S., Ogasawara N., Yoshikawa H. Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res. 1985 Apr 11;13(7):2251–2265. doi: 10.1093/nar/13.7.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Muñoz R., Bustamante M., de la Campa A. G. Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance. J Bacteriol. 1995 Jul;177(14):4166–4170. doi: 10.1128/jb.177.14.4166-4170.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Orr E., Staudenbauer W. L. Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity. J Bacteriol. 1982 Jul;151(1):524–527. doi: 10.1128/jb.151.1.524-527.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Revel V., Cambau E., Jarlier V., Sougakoff W. Characterization of mutations in Mycobacterium smegmatis involved in resistance to fluoroquinolones. Antimicrob Agents Chemother. 1994 Sep;38(9):1991–1996. doi: 10.1128/aac.38.9.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sandler S. J., Chackerian B., Li J. T., Clark A. J. Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop. Nucleic Acids Res. 1992 Feb 25;20(4):839–845. doi: 10.1093/nar/20.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staudenbauer W. L., Orr E. DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties. Nucleic Acids Res. 1981 Aug 11;9(15):3589–3603. doi: 10.1093/nar/9.15.3589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stein D. C., Danaher R. J., Cook T. M. Characterization of a gyrB mutation responsible for low-level nalidixic acid resistance in Neisseria gonorrhoeae. Antimicrob Agents Chemother. 1991 Apr;35(4):622–626. doi: 10.1128/aac.35.4.622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sutcliffe J. A., Gootz T. D., Barrett J. F. Biochemical characteristics and physiological significance of major DNA topoisomerases. Antimicrob Agents Chemother. 1989 Dec;33(12):2027–2033. doi: 10.1128/aac.33.12.2027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Takiff H. E., Salazar L., Guerrero C., Philipp W., Huang W. M., Kreiswirth B., Cole S. T., Jacobs W. R., Jr, Telenti A. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother. 1994 Apr;38(4):773–780. doi: 10.1128/aac.38.4.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thiara A. S., Cundliffe E. Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin. EMBO J. 1988 Jul;7(7):2255–2259. doi: 10.1002/j.1460-2075.1988.tb03065.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thiara A. S., Cundliffe E. Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides. Mol Microbiol. 1993 May;8(3):495–506. doi: 10.1111/j.1365-2958.1993.tb01593.x. [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. DNA topoisomerases: why so many? J Biol Chem. 1991 Apr 15;266(11):6659–6662. [PubMed] [Google Scholar]
  45. Wang Y., Huang W. M., Taylor D. E. Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations. Antimicrob Agents Chemother. 1993 Mar;37(3):457–463. doi: 10.1128/aac.37.3.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Yoshida H., Bogaki M., Nakamura M., Yamanaka L. M., Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli. Antimicrob Agents Chemother. 1991 Aug;35(8):1647–1650. doi: 10.1128/aac.35.8.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Soolingen D., Hermans P. W., de Haas P. E., Soll D. R., van Embden J. D. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 1991 Nov;29(11):2578–2586. doi: 10.1128/jcm.29.11.2578-2586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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