Skip to main content
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1993 Jun;37(6):1297–1304. doi: 10.1128/aac.37.6.1297

A novel dihydrofolate reductase cassette inserted in an integron borne on a Tn21-like element.

E Heikkilä 1, M Skurnik 1, L Sundström 1, P Huovinen 1
PMCID: PMC187956  PMID: 8392309

Abstract

In this study, a 498-bp dhfrXII gene coding for trimethoprim resistance was found inserted in a cassette-like manner in the recombinationally active locus, the integron, borne on a transposon Tn21-like element. The dhfrXII cassette is distinct from those cassettes earlier observed in integrons and was found here upstream of two similarly inserted cassettes. The second one carried the new unidentified orfF, which is 85% identical to the orfD cassette in R46. The third cassette contained the aadA2 gene mediating spectinomycin resistance. The plasmid carrying this Tn21-like element was originally isolated from a trimethoprim-resistant urinary tract pathogen, Escherichia coli, from Turku City Hospital, Turku, Finland. By colony hybridization and polymerase chain reaction, this group of three cassettes, including dhfrXII, was detected in four additional E. coli strains of similar origin and in four Shigella strains isolated in Finland but originating from Asia. The dihydrofolate reductase produced from dhfrXII showed an unusual drug resistance in that 50% of the enzymatic activity remained at a trimethoprim concentration of 1 mM.

Full text

PDF

Selected References

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

  1. Amyes S. G., Smith J. T. R-factor trimethoprim resistance mechanism: an insusceptible target site. Biochem Biophys Res Commun. 1974 May 20;58(2):412–418. doi: 10.1016/0006-291x(74)90380-5. [DOI] [PubMed] [Google Scholar]
  2. Amyes S. G., Smith J. T. The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388. Eur J Biochem. 1976 Jan 15;61(2):597–603. doi: 10.1111/j.1432-1033.1976.tb10055.x. [DOI] [PubMed] [Google Scholar]
  3. Argos P., Landy A., Abremski K., Egan J. B., Haggard-Ljungquist E., Hoess R. H., Kahn M. L., Kalionis B., Narayana S. V., Pierson L. S., 3rd The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 1986 Feb;5(2):433–440. doi: 10.1002/j.1460-2075.1986.tb04229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barth P. T., Datta N., Hedges R. W., Grinter N. J. Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J Bacteriol. 1976 Mar;125(3):800–810. doi: 10.1128/jb.125.3.800-810.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Brisson N., Hohn T. Nucleotide sequence of the dihydrofolate-reductase gene borne by the plasmid R67 and conferring methotrexate resistance. Gene. 1984 May;28(2):271–274. doi: 10.1016/0378-1119(84)90266-x. [DOI] [PubMed] [Google Scholar]
  8. Brown N. L., Misra T. K., Winnie J. N., Schmidt A., Seiff M., Silver S. The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol Gen Genet. 1986 Jan;202(1):143–151. doi: 10.1007/BF00330531. [DOI] [PubMed] [Google Scholar]
  9. Cameron F. H., Groot Obbink D. J., Ackerman V. P., Hall R. M. Nucleotide sequence of the AAD(2'') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388. Nucleic Acids Res. 1986 Nov 11;14(21):8625–8635. doi: 10.1093/nar/14.21.8625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collis C. M., Hall R. M. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol. 1992 Mar;174(5):1574–1585. doi: 10.1128/jb.174.5.1574-1585.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Diver W. P., Grinsted J., Fritzinger D. C., Brown N. L., Altenbuchner J., Rogowsky P., Schmitt R. DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721. Mol Gen Genet. 1983;191(2):189–193. doi: 10.1007/BF00334812. [DOI] [PubMed] [Google Scholar]
  13. Fleming M. P., Datta N., Grüneberg R. N. Trimethoprim resistance determined by R factors. Br Med J. 1972 Mar 18;1(5802):726–728. doi: 10.1136/bmj.1.5802.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Flensburg J., Steen R. Nucleotide sequence analysis of the trimethoprim resistant dihydrofolate reductase encoded by R plasmid R751. Nucleic Acids Res. 1986 Jul 25;14(14):5933–5933. doi: 10.1093/nar/14.14.5933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fling M. E., Elwell L. P. Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases. J Bacteriol. 1980 Feb;141(2):779–785. doi: 10.1128/jb.141.2.779-785.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fling M. E., Kope J., Richards C. Characterization of plasmid pAZ1 and the type III dihydrofolate reductase gene. Plasmid. 1988 Jan;19(1):30–38. doi: 10.1016/0147-619x(88)90060-1. [DOI] [PubMed] [Google Scholar]
  17. Fling M. E., Kopf J., Richards C. Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase. Nucleic Acids Res. 1985 Oct 11;13(19):7095–7106. doi: 10.1093/nar/13.19.7095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fling M. E., Richards C. The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7. Nucleic Acids Res. 1983 Aug 11;11(15):5147–5158. doi: 10.1093/nar/11.15.5147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grinsted J., de la Cruz F., Schmitt R. The Tn21 subgroup of bacterial transposable elements. Plasmid. 1990 Nov;24(3):163–189. doi: 10.1016/0147-619x(90)90001-s. [DOI] [PubMed] [Google Scholar]
  20. Hall R. M., Brookes D. E., Stokes H. W. Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol. 1991 Aug;5(8):1941–1959. doi: 10.1111/j.1365-2958.1991.tb00817.x. [DOI] [PubMed] [Google Scholar]
  21. Hall R. M., Vockler C. The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. Nucleic Acids Res. 1987 Sep 25;15(18):7491–7501. doi: 10.1093/nar/15.18.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harr R., Fällman P., Häggström M., Wahlström L., Gustafsson P. GENEUS, a computer system for DNA and protein sequence analysis containing an information retrieval system for the EMBL data library. Nucleic Acids Res. 1986 Jan 10;14(1):273–284. doi: 10.1093/nar/14.1.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heikkilä E., Siitonen A., Jahkola M., Fling M., Sundström L., Huovinen P. Increase of trimethoprim resistance among Shigella species, 1975-1988: analysis of resistance mechanisms. J Infect Dis. 1990 Jun;161(6):1242–1248. doi: 10.1093/infdis/161.6.1242. [DOI] [PubMed] [Google Scholar]
  24. Heikkilä E., Sundström L., Huovinen P. Trimethoprim resistance in Escherichia coli isolates from a geriatric unit. Antimicrob Agents Chemother. 1990 Oct;34(10):2013–2015. doi: 10.1128/aac.34.10.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huovinen P., Mattila T., Kiminki O., Pulkkinen L., Huovinen S., Koskela M., Sunila R., Toivanen P. Emergence of trimethoprim resistance in fecal flora. Antimicrob Agents Chemother. 1985 Aug;28(2):354–356. doi: 10.1128/aac.28.2.354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huovinen P. Trimethoprim resistance. Antimicrob Agents Chemother. 1987 Oct;31(10):1451–1456. doi: 10.1128/aac.31.10.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hyde D. R., Tu C. P. tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution. Cell. 1985 Sep;42(2):629–638. doi: 10.1016/0092-8674(85)90120-5. [DOI] [PubMed] [Google Scholar]
  28. Jansson C., Sköld O. Appearance of a new trimethoprim resistance gene, dhfrIX, in Escherichia coli from swine. Antimicrob Agents Chemother. 1991 Sep;35(9):1891–1899. doi: 10.1128/aac.35.9.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Martin C., Timm J., Rauzier J., Gomez-Lus R., Davies J., Gicquel B. Transposition of an antibiotic resistance element in mycobacteria. Nature. 1990 Jun 21;345(6277):739–743. doi: 10.1038/345739a0. [DOI] [PubMed] [Google Scholar]
  30. Martinez E., de la Cruz F. Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J. 1990 Apr;9(4):1275–1281. doi: 10.1002/j.1460-2075.1990.tb08236.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martinez E., de la Cruz F. Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet. 1988 Feb;211(2):320–325. doi: 10.1007/BF00330610. [DOI] [PubMed] [Google Scholar]
  32. Matthews D. A., Bolin J. T., Burridge J. M., Filman D. J., Volz K. W., Kaufman B. T., Beddell C. R., Champness J. N., Stammers D. K., Kraut J. Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim. J Biol Chem. 1985 Jan 10;260(1):381–391. [PubMed] [Google Scholar]
  33. Nucifora G., Chu L., Silver S., Misra T. K. Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358. J Bacteriol. 1989 Aug;171(8):4241–4247. doi: 10.1128/jb.171.8.4241-4247.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nücken E. J., Henschke R. B., Schmidt F. R. Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603. J Gen Microbiol. 1989 Apr;135(4):761–765. doi: 10.1099/00221287-135-4-761. [DOI] [PubMed] [Google Scholar]
  35. Ouellette M., Bissonnette L., Roy P. H. Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7378–7382. doi: 10.1073/pnas.84.21.7378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ouellette M., Roy P. H. Homology of ORFs from Tn2603 and from R46 to site-specific recombinases. Nucleic Acids Res. 1987 Dec 10;15(23):10055–10055. doi: 10.1093/nar/15.23.10055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parsons Y., Hall R. M., Stokes H. W. A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100. Antimicrob Agents Chemother. 1991 Nov;35(11):2436–2439. doi: 10.1128/aac.35.11.2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pattishall K. H., Acar J., Burchall J. J., Goldstein F. W., Harvey R. J. Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J Biol Chem. 1977 Apr 10;252(7):2319–2323. [PubMed] [Google Scholar]
  39. Paulsen I. T., Littlejohn T. G., Rådström P., Sundström L., Sköld O., Swedberg G., Skurray R. A. The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob Agents Chemother. 1993 Apr;37(4):761–768. doi: 10.1128/aac.37.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Saunders S. E., Burke J. F. Rapid isolation of miniprep DNA for double strand sequencing. Nucleic Acids Res. 1990 Aug 25;18(16):4948–4948. doi: 10.1093/nar/18.16.4948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Simonsen C. C., Chen E. Y., Levinson A. D. Identification of the type I trimethoprim-resistant dihydrofolate reductase specified by the Escherichia coli R-plasmid R483: comparison with procaryotic and eucaryotic dihydrofolate reductases. J Bacteriol. 1983 Sep;155(3):1001–1008. doi: 10.1128/jb.155.3.1001-1008.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Singh K. V., Reves R. R., Pickering L. K., Murray B. E. Identification by DNA sequence analysis of a new plasmid-encoded trimethoprim resistance gene in fecal Escherichia coli isolates from children in day-care centers. Antimicrob Agents Chemother. 1992 Aug;36(8):1720–1726. doi: 10.1128/aac.36.8.1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sköld O., Widh A. A new dihydrofolate reductase with low trimethoprim sensitivity induced by an R factor mediating high resistance to trimethoprim. J Biol Chem. 1974 Jul 10;249(13):4324–4325. [PubMed] [Google Scholar]
  46. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stokes H. W., Hall R. M. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol. 1989 Dec;3(12):1669–1683. doi: 10.1111/j.1365-2958.1989.tb00153.x. [DOI] [PubMed] [Google Scholar]
  48. Stone D., Smith S. L. The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. J Biol Chem. 1979 Nov 10;254(21):10857–10861. [PubMed] [Google Scholar]
  49. Sundström L., Roy P. H., Sköld O. Site-specific insertion of three structural gene cassettes in transposon Tn7. J Bacteriol. 1991 May;173(9):3025–3028. doi: 10.1128/jb.173.9.3025-3028.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sundström L., Rådström P., Swedberg G., Sköld O. Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol Gen Genet. 1988 Aug;213(2-3):191–201. doi: 10.1007/BF00339581. [DOI] [PubMed] [Google Scholar]
  51. Sundström L., Sköld O. The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings. Antimicrob Agents Chemother. 1990 Apr;34(4):642–650. doi: 10.1128/aac.34.4.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sundström L., Swedberg G., Sköld O. Characterization of transposon Tn5086, carrying the site-specifically inserted gene dhfrVII mediating trimethoprim resistance. J Bacteriol. 1993 Mar;175(6):1796–1805. doi: 10.1128/jb.175.6.1796-1805.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sundström L., Vinayagamoorthy T., Sköld O. Novel type of plasmid-borne resistance to trimethoprim. Antimicrob Agents Chemother. 1987 Jan;31(1):60–66. doi: 10.1128/aac.31.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Swift G., McCarthy B. J., Heffron F. DNA sequence of a plasmid-encoded dihydrofolate reductase. Mol Gen Genet. 1981;181(4):441–447. doi: 10.1007/BF00428733. [DOI] [PubMed] [Google Scholar]
  55. Ward E., Grinsted J. The nucleotide sequence of the tnpA gene of Tn21. Nucleic Acids Res. 1987 Feb 25;15(4):1799–1806. doi: 10.1093/nar/15.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wiedemann B., Meyer J. F., Zühlsdorf M. T. Insertions of resistance genes into Tn21-like transposons. J Antimicrob Chemother. 1986 Oct;18 (Suppl 100):85–92. doi: 10.1093/jac/18.supplement_c.85. [DOI] [PubMed] [Google Scholar]
  57. Wohlleben W., Arnold W., Bissonnette L., Pelletier A., Tanguay A., Roy P. H., Gamboa G. C., Barry G. F., Aubert E., Davies J. On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette. Mol Gen Genet. 1989 Jun;217(2-3):202–208. doi: 10.1007/BF02464882. [DOI] [PubMed] [Google Scholar]
  58. Wylie B. A., Amyes S. G., Young H. K., Koornhof H. J. Identification of a novel plasmid-encoded dihydrofolate reductase mediating high-level resistance to trimethoprim. J Antimicrob Chemother. 1988 Oct;22(4):429–435. doi: 10.1093/jac/22.4.429. [DOI] [PubMed] [Google Scholar]
  59. Wylie B. A., Koornhof H. J. Nucleotide sequence of dihydrofolate reductase type VI. J Med Microbiol. 1991 Oct;35(4):214–218. doi: 10.1099/00222615-35-4-214. [DOI] [PubMed] [Google Scholar]
  60. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES