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Molecular and General Genetics MGG

, Volume 235, Issue 1, pp 113–121 | Cite as

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum β-lactamase TEM-3

  • Claude Mabilat
  • Joaõ Lourençao-Vital
  • Sylvie Goussard
  • Patrice Courvalin
Article

Summary

The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum β-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or −2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was, in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.

Key words

Amplification mapping Integron TEM Tn1 Tn21 

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References

  1. Arthur A, Nimmo ER, Hettle SJH, Sheratt DJ (1984) Transposition and transposition immunity of transposon Tn3 derivatives having different ends. EMBO J 3:1723–1729Google Scholar
  2. Cameron FH, Obbink DJG, Ackerman VP, Hall RM (1986) 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 14:8625–8635Google Scholar
  3. Chanal CM, Sirot DL, Petit A, Labia R, Morand A, Sirot JL, Cluzel RA (1989) Multiplicity of TEM-derived β-lactamases from Klebsiella pneumoniae strains isolated at the same hospital and relationships between the responsible plasmids (1989) Antimicrob Agents Chemother 33:1915–1920Google Scholar
  4. Chen ST, Clowes RC (1987) Variations between the nucleotide sequence of Tn1, Tn2, and Tn3 and expression of β-lactamase in Pseudomonas aeruginosa and Escherichia coli. J Bacteriol 169:913–916Google Scholar
  5. Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol 108:1244–1249Google Scholar
  6. DeGraaf J, Elwell LP, Falkow S (1976) Molecular nature of two β-lactamase specifying plasmids isolated from Haemophilus influenzae type b. J Bacteriol 126:439–446Google Scholar
  7. De La Cruz F, Grinsted J (1982) Genetic and molecular characterization of Tn21, a multiresistance transposon from R100.1. J Bacteriol 151:222–228Google Scholar
  8. Dessen P, Fondrat C, Valencien C, Mugnier C (1990) BISANCE: A French service for access to biomolecular sequence databases. Comput Appl Biosci 6:355–356Google Scholar
  9. Diver WP, Grinsted J, Fritzinger DC, Brown NL, Altenbuchner J, Rogowsky P, Schmitt R (1983) DNA sequences of and complementation by the tnpR genes of Tn21, Tn501, and Tn1721. Mol Gen Genet 191:189–193Google Scholar
  10. Gerlach BA, Wiedemann B (1985) Tn3 as the molecular basis of ampicillin resistance in E. coli — An epidemiological survey. Zentralb Bakteriol Hyg 260:139–150Google Scholar
  11. Griffin HG, Foster TJ, Falkiner FR, Carr ME, Coleman CC (1985) Molecular analysis of multiple-resistance plasmids transferred from gram-negative bacteria isolated in a urological unit. Antimicrob Agents Chemother 28:413–418Google Scholar
  12. Grinsted J, De La Cruz F, Schmitt R (1990) The Tn21 subgroup of bacterial transposable elements. Plasmid 24:163–189Google Scholar
  13. Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E (1979) DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition. Cell 18:1153–1163Google Scholar
  14. Hollingshead SK, Vapnek D (1985) Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenyltransferase. Plasmid 13:17–30Google Scholar
  15. Hopkins JD, O'Brien TF, Syvanen M (1988) Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae. Plasmid 20:163–166Google Scholar
  16. Hyde DR, Tu CPD (1985) tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate. Cell 42:629–638Google Scholar
  17. Kitzis MD, Billot-Klein D, Goldstein FW, Williamson R, Tran Van Nhieu G, Carlet J, Acar JF, Gutmann L (1988) Dissemination of the novel plasmid-mediated β-lactamase CTX-1, which confers resistance to broad-spectrum cephalosporins, and its inhibition by β-lactamase inhibitors. Antimicrob Agents Chemother 32:9–14Google Scholar
  18. Kontomichalou P, Mitani M, Clowes RC (1970) Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under relaxed or stringent control. J Bacteriol 104:34–44Google Scholar
  19. Kopecko DJ, Brevet J, Cohen SN (1976) Involvement of multiple translocating DNA segments and recombination hot-spots in the structural evolution of bacterial plasmids. J Mol Biol 108:333–360Google Scholar
  20. Labigne-Roussel A, Witchitz J, Courvalin P (1982) Modular evolution of disseminated Inc7-M plasmids encoding gentamicin resistance. Plasmid 8:215–231Google Scholar
  21. Labigne-Roussel A, Briaux-Gerbaud S, Courvalin P (1983) Tn1525, a kanamycin R determinant flanked by two direct copies of IS15. Mol Gen Genet 189:90–101Google Scholar
  22. Lett MC, Benett PM, Vidon DJM (1985) Characterization of Tn3926 a new mercury-resistance transposon from Yersinia enterocolitica. Gene 40:79–91Google Scholar
  23. Mabilat C, Courvalin P (1990) Development of “oligotyping” for characterization and molecular epidemiology of TEM β-lactamases in Enterobacteriaceae. Antimicrob Agents Chemother 34:2210–2216Google Scholar
  24. Mabilat C, Goussard S, Sougakoff W, Spencer R, Courvalin P (1990) Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum β-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae. Plasmid 23:27–34Google Scholar
  25. Medeiros AA (1984) β-lactamases. Br Med Bull 40:18–27Google Scholar
  26. Mercier J, Lachapelle J, Couture F, Lafond M, Vézina G, Boissinot M, Levesque RC (1990) Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related β-lacta-mase transposons. J Bacteriol 172:3745–3757Google Scholar
  27. Nobuta K, Tolmasky ME, Crosa LM, Crosa JH (1988) Sequencing and expression of the 6′-N-acetyltransferase gene of the transposon Tn1331 from Klebsiella pneumoniae. J Bacteriol 170:3769–3773Google Scholar
  28. Nucifora G, Chu L, Silver S, Misra TK (1989) Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358. J Bacteriol 171:4241–4247Google Scholar
  29. Ouellette M, Bissonnette L, Roy PH (1987) Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 β-lactamase gene. Proc Natl Acad Sci USA 84:7372–7378Google Scholar
  30. Paul GC, Gerbaud G, Buré A, Philippon AM, Pangon B, Courvalin P (1989) TEM-4, a new plasmid mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli. Antimicrob Agents Chemother 33:1958–1963Google Scholar
  31. Petit A, Sirot DL, Chanal CM, Sirot JL, Labia R, Gerbaud G, Cluzel RA (1988) Novel plasmid-mediated β-lactamase in clinical isolates of Klebsiella pneumoniae more resistant to ceftazidime than to other broad-spectrum cephalosporins. Antimicrob Agents Chemother 32:626–630Google Scholar
  32. Petit A, Gerbaud G, Sirot D, Courvalin P, Sirot J (1990) Molecular epidemiology of TEM-3 (CTX-1) β-lactamase. Antimicrob Agents Chemother 34:219–224Google Scholar
  33. Roberts M, Elwell LP, Falkow S (1977) Molecular characterization of two β-lactamase specifying plasmids isolated from Neisseria gonorrhoeae. J Bacteriol 131:557–563Google Scholar
  34. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  35. Schmidt F, Klopfer-Kaul I (1984) Evolutionary relationship between Tn21-like elements and pBP201, a plasmid from Klebsiella pneumoniae mediating resistance to gentamicin and eight other drugs. Mol Gen Genet 197:109–119Google Scholar
  36. Schmidt F, Nücken EJ, Henschke RB (1989) Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons. Mol Microbiol 3:1545–1555Google Scholar
  37. Sirot D, Sirot J, Labia R, Morand P, Courvalin P, Darfeuille-Michaud A, Perroux R, Cluzel R (1987) Transferable resistance to third generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-1, a novel β-lactamase. J Antimicrob Chemother 20:323–334Google Scholar
  38. Sirot D, De Champs C, Chanal C, Labia R, Darfeuille-Michaud A, Perroux R, Sirot J (1991) Translocation of antibiotic resistance determinants including an extended-spectrum β-lactamase between conjugative plasmids of Klebsiella pneumoniae and Escherichia coli. Antimicrob Agents Chemother 35:1576–1581Google Scholar
  39. Smith CA, Thomas CM (1987) Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncPβ sub-group are closely related. Mol Gen Genet 206:419–427Google Scholar
  40. Smith KT, Long CM, Bowman B, Manos MM (1990) Using cosolvents to enhance PCR amplification. Amplifications 5:16–17Google Scholar
  41. Sougakoff W, Goussard S, Courvalin P (1988) The TEM-3 β-lactamase, which hydrolyses broad-spectrum cephalosporins, is derived from the TEM-2 penicillinase by two amino acid substitutions. FEMS Microbiol Lett 56:343–348Google Scholar
  42. Stokes HW, Hall RM (1989) A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol 3:1669–1683Google Scholar
  43. Sundström L, Radström P, Swedberg G, Sköld O (1988) Site-specific recombination promotes linkage between trimethoprim and sulfonamide resistance genes. Sequence characterization of dhfrV and sull and a recombination-active locus on Tn21. Mol Gen Genet 213:191–201Google Scholar
  44. Tait RC, Rempel H, Rodriguez RL, Kado CI (1985) The aminoglycoside resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene. Gene 36:97–104Google Scholar
  45. Tanaka M, Yamamoto T, Sawai T (1983) Evolution of complex resistance transposons from an ancestral mercury transposon. J Bacteriol 153:1432–1438Google Scholar
  46. Tolmasky ME (1990) Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331. Plasmid 24:218–226Google Scholar
  47. Tran Van Nhieu G, Collatz E (1987) Primary structure of an aminoglycoside 6′-N-acetyltransferase, AAC(6′)-4, fused in vivo with the signal peptide of the Tn3-encoded β-lactamase. J Bacteriol 169:5708–5714Google Scholar
  48. Trieu-Cuot P, Courvalin P (1984) Nucleotide sequence of the transposable element IS15. Gene 30:113–120Google Scholar
  49. Turner AK, Grinsted J (1989) DNA sequence of the transposase gene of the class II transposon, Tn3926. Nucleic Acids Res 17:1757Google Scholar
  50. Ward JM, Grinsted J (1987) The nucleotide sequence of the tnpA gene of Tn21. Nucleic Acids Res 15:1799–1806Google Scholar
  51. Wohlleben W, Arnold W, Bissonnette L, Pelletier A, Tanguay A, Roy PH, Gamboa GC, Barry GF, Aubert E, Davies J, Kagan SA (1989) 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 217:202–208Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Claude Mabilat
    • 1
  • Joaõ Lourençao-Vital
    • 1
  • Sylvie Goussard
    • 1
  • Patrice Courvalin
    • 1
  1. 1.Unité des Agents Antibactériens, 28 rue du Dr. RouxInstitut PasteurParis Cedex 15

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