Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(8):2731–2739. doi: 10.1128/jb.179.8.2731-2739.1997

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

G D Tribble 1, A C Parker 1, C J Smith 1
PMCID: PMC179024  PMID: 9098073

Abstract

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.

Full Text

The Full Text of this article is available as a PDF (740.8 KB).

Selected References

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

  1. 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]
  2. Caparon M. G., Scott J. R. Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism. Cell. 1989 Dec 22;59(6):1027–1034. doi: 10.1016/0092-8674(89)90759-9. [DOI] [PubMed] [Google Scholar]
  3. Clewell D. B., Flannagan S. E., Ike Y., Jones J. M., Gawron-Burke C. Sequence analysis of termini of conjugative transposon Tn916. J Bacteriol. 1988 Jul;170(7):3046–3052. doi: 10.1128/jb.170.7.3046-3052.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clewell D. B., Flannagan S. E., Jaworski D. D. Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol. 1995 Jun;3(6):229–236. doi: 10.1016/s0966-842x(00)88930-1. [DOI] [PubMed] [Google Scholar]
  5. Collins F. S., Weissman S. M. Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6812–6816. doi: 10.1073/pnas.81.21.6812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Currier T. C., Nester E. W. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem. 1976 Dec;76(2):431–441. doi: 10.1016/0003-2697(76)90338-9. [DOI] [PubMed] [Google Scholar]
  7. DeShazer D., Wood G. E., Friedman R. L. Boiling eliminates artifact banding when sequencing double-stranded templates. Biotechniques. 1994 Aug;17(2):288–290. [PubMed] [Google Scholar]
  8. 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]
  9. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Hecht D. W., Malamy M. H. Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis. J Bacteriol. 1989 Jul;171(7):3603–3608. doi: 10.1128/jb.171.7.3603-3608.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hecht D. W., Thompson J. S., Malamy M. H. Characterization of the termini and transposition products of Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5340–5344. doi: 10.1073/pnas.86.14.5340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  14. Li L. Y., Shoemaker N. B., Salyers A. A. Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes. J Bacteriol. 1995 Sep;177(17):4992–4999. doi: 10.1128/jb.177.17.4992-4999.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li L. Y., Shoemaker N. B., Wang G. R., Cole S. P., Hashimoto M. K., Wang J., Salyers A. A. The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette. J Bacteriol. 1995 Jul;177(14):3940–3945. doi: 10.1128/jb.177.14.3940-3945.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lu F., Churchward G. Conjugative transposition: Tn916 integrase contains two independent DNA binding domains that recognize different DNA sequences. EMBO J. 1994 Apr 1;13(7):1541–1548. doi: 10.1002/j.1460-2075.1994.tb06416.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lu F., Churchward G. Tn916 target DNA sequences bind the C-terminal domain of integrase protein with different affinities that correlate with transposon insertion frequency. J Bacteriol. 1995 Apr;177(8):1938–1946. doi: 10.1128/jb.177.8.1938-1946.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mays T. D., Smith C. J., Welch R. A., Delfini C., Macrina F. L. Novel antibiotic resistance transfer in Bacteroides. Antimicrob Agents Chemother. 1982 Jan;21(1):110–118. doi: 10.1128/aac.21.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nunes-Düby S. E., Azaro M. A., Landy A. Swapping DNA strands and sensing homology without branch migration in lambda site-specific recombination. Curr Biol. 1995 Feb 1;5(2):139–148. doi: 10.1016/s0960-9822(95)00035-2. [DOI] [PubMed] [Google Scholar]
  20. Ochman H., Ayala F. J., Hartl D. L. Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Methods Enzymol. 1993;218:309–321. doi: 10.1016/0076-6879(93)18023-6. [DOI] [PubMed] [Google Scholar]
  21. Parker A. C., Smith C. J. Genetic and biochemical analysis of a novel Ambler class A beta-lactamase responsible for cefoxitin resistance in Bacteroides species. Antimicrob Agents Chemother. 1993 May;37(5):1028–1036. doi: 10.1128/aac.37.5.1028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Privitera G., Dublanchet A., Sebald M. Transfer of multiple antibiotic resistance between subspecies of Bacteroides fragilis. J Infect Dis. 1979 Jan;139(1):97–101. doi: 10.1093/infdis/139.1.97. [DOI] [PubMed] [Google Scholar]
  23. Rasmussen B. A., Bush K., Tally F. P. Antimicrobial resistance in Bacteroides. Clin Infect Dis. 1993 Jun;16 (Suppl 4):S390–S400. doi: 10.1093/clinids/16.supplement_4.s390. [DOI] [PubMed] [Google Scholar]
  24. Sadowski P. D. Site-specific genetic recombination: hops, flips, and flops. FASEB J. 1993 Jun;7(9):760–767. doi: 10.1096/fasebj.7.9.8392474. [DOI] [PubMed] [Google Scholar]
  25. Salyers A. A., Shoemaker N. B., Li L. Y. In the driver's seat: the Bacteroides conjugative transposons and the elements they mobilize. J Bacteriol. 1995 Oct;177(20):5727–5731. doi: 10.1128/jb.177.20.5727-5731.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scott J. R., Churchward G. G. Conjugative transposition. Annu Rev Microbiol. 1995;49:367–397. doi: 10.1146/annurev.mi.49.100195.002055. [DOI] [PubMed] [Google Scholar]
  27. Shoemaker N. B., Barber R. D., Salyers A. A. Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector. J Bacteriol. 1989 Mar;171(3):1294–1302. doi: 10.1128/jb.171.3.1294-1302.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shoemaker N. B., Salyers A. A. A cryptic 65-kilobase-pair transposonlike element isolated from Bacteroides uniformis has homology with Bacteroides conjugal tetracycline resistance elements. J Bacteriol. 1990 Apr;172(4):1694–1702. doi: 10.1128/jb.172.4.1694-1702.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shoemaker N. B., Wang G. R., Salyers A. A. The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family. J Bacteriol. 1996 Jun;178(12):3594–3600. doi: 10.1128/jb.178.12.3594-3600.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shoemaker N. B., Wang G. R., Stevens A. M., Salyers A. A. Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element. J Bacteriol. 1993 Oct;175(20):6578–6587. doi: 10.1128/jb.175.20.6578-6587.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith C. J., Callihan D. R. Analysis of rRNA restriction fragment length polymorphisms from Bacteroides spp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals. J Clin Microbiol. 1992 Apr;30(4):806–812. doi: 10.1128/jcm.30.4.806-812.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith C. J. Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region. J Bacteriol. 1985 Mar;161(3):1069–1073. doi: 10.1128/jb.161.3.1069-1073.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith C. J., Parker A. C. A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids. Mol Microbiol. 1996 May;20(4):741–750. doi: 10.1111/j.1365-2958.1996.tb02513.x. [DOI] [PubMed] [Google Scholar]
  34. Smith C. J., Parker A. C. Identification of a circular intermediate in the transfer and transposition of Tn4555, a mobilizable transposon from Bacteroides spp. J Bacteriol. 1993 May;175(9):2682–2691. doi: 10.1128/jb.175.9.2682-2691.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith C. J., Parker A., Rogers M. B. Plasmid transformation of Bacteroides spp. by electroporation. Plasmid. 1990 Sep;24(2):100–109. doi: 10.1016/0147-619x(90)90012-2. [DOI] [PubMed] [Google Scholar]
  36. Smith C. J., Rollins L. A., Parker A. C. Nucleotide sequence determination and genetic analysis of the Bacteroides plasmid, pBI143. Plasmid. 1995 Nov;34(3):211–222. doi: 10.1006/plas.1995.0007. [DOI] [PubMed] [Google Scholar]
  37. Smith C. J., Spiegel H. Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp. J Bacteriol. 1987 Aug;169(8):3450–3457. doi: 10.1128/jb.169.8.3450-3457.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  39. Stark W. M., Boocock M. R., Sherratt D. J. Catalysis by site-specific recombinases. Trends Genet. 1992 Dec;8(12):432–439. [PubMed] [Google Scholar]
  40. Stevens A. M., Shoemaker N. B., Li L. Y., Salyers A. A. Tetracycline regulation of genes on Bacteroides conjugative transposons. J Bacteriol. 1993 Oct;175(19):6134–6141. doi: 10.1128/jb.175.19.6134-6141.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Valentine P. J., Shoemaker N. B., Salyers A. A. Mobilization of Bacteroides plasmids by Bacteroides conjugal elements. J Bacteriol. 1988 Mar;170(3):1319–1324. doi: 10.1128/jb.170.3.1319-1324.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES