1887

Abstract

The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the λ integrase family and second, in the presence of this integrase, a 56 bp fragment is sufficient for efficient recombination with the chromosomal site in the host subsp. MG1363. In the present work, this site was analysed and a 43 bp region was found to be the smallest fragment able to participate fully in recombination. studies showed that the TP901-1 integrase binds this 43 bp fragment, the 56 bp and a larger fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of sites, obtained by recombination between an site containing a mutation in this TC dinucleotide and a wild-type site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for sites with a decreased ability to participate in recombination, two bases located outside the core region of were shown to be involved in the binding of the TP901-1 integrase.

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2001-08-01
2020-01-20
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References

  1. Azaro M. A., Landy A.. 1997; The isomeric preference of Holliday junctions influences resolution bias by lambda integrase. EMBO J16:3744–3755[CrossRef]
    [Google Scholar]
  2. Bannam T. L., Crellin P. K., Rood J. I.. 1995; Molecular genetics of the chloramphenicol resistance transposon Tn 4451 from Clostridium perfringens : the TnpX site-specific recombinase excises a circular transposon molecule. Mol Microbiol16:535–551[CrossRef]
    [Google Scholar]
  3. Bonekamp F., Clemmensen K., Karlström O., Jensen K. F.. 1984; Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli : an example of operon polarity control through the coupling of translation to transcription. EMBO J3:2857–2861
    [Google Scholar]
  4. Breüner A., Brøndsted L., Hammer K.. 1999; Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1. J Bacteriol181:7291–7297
    [Google Scholar]
  5. Brøndsted L., Hammer K.. 1999; Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis . Appl Environ Microbiol65:752–758
    [Google Scholar]
  6. Campbell A. M.. 1992; Chromosomal insertion sites for phages and plasmids. J Bacteriol174:7495–7499
    [Google Scholar]
  7. Carrasco C. D., Ramaswamy K. S., Ramasubramanian T. S., Golden J. W.. 1994; Anabaena xisF gene encodes a developmentally regulated site-specific recombinase. Genes Dev8:74–83[CrossRef]
    [Google Scholar]
  8. Christiansen B.. 1995; Site-specific integration of the lactococcal temperate phage TP901-1. PhD thesis Technical University of Denmark;
  9. Christiansen B., Johnsen M. G., Stenby E., Vogensen F. K., Hammer K.. 1994; Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration. J Bacteriol176:1069–1076
    [Google Scholar]
  10. Christiansen B., Brøndsted L., Vogensen F. K., Hammer K.. 1996; A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1. J Bacteriol178:5164–5173
    [Google Scholar]
  11. Chung Y. S., Dubnau D.. 1998; All seven comG open reading frames are required for DNA binding during transformation of Bacillus subtilis . J Bacteriol180:41–45
    [Google Scholar]
  12. Crellin P. K., Rood J. I.. 1997; The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn 4451 . J Bacteriol179:5148–5156
    [Google Scholar]
  13. Gasson M. J.. 1983; Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol154:1–9
    [Google Scholar]
  14. Gopaul D. N., Van Duyne G. D.. 1999; Structure and mechanism in site-specific recombination. Curr Opinion Struct Biol9:14–20[CrossRef]
    [Google Scholar]
  15. Groth A. C., Olivares E. C., Thyagarajan B., Calos M. P.. 2000; A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA97:5995–6000[CrossRef]
    [Google Scholar]
  16. Guo F., Gopaul N., Van Duyne G. D.. 1999; Asymmetric DNA bending in the Cre- loxP site-specific recombination synapse. Proc Natl Acad Sci USA96:7143–7148[CrossRef]
    [Google Scholar]
  17. Hayes F., Daly C., Fitzgerald G. F.. 1990; Identification of the minimal replicon of Lactococcus lactis subsp. lactis UC317 plasmid pCI305. Appl Environ Microbiol56:202–209
    [Google Scholar]
  18. Holo H., Nes I.. 1989; High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol55:3119–3123
    [Google Scholar]
  19. Israelsen H., Madsen S. M., Vrang A., Hansen E. B., Johansen E.. 1995; Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn 917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol61:2540–2547
    [Google Scholar]
  20. Kuhstoss S., Rao R. N.. 1991; Analysis of the integration function of the streptomycete bacteriophage ϕC31. J Mol Biol222:897–908[CrossRef]
    [Google Scholar]
  21. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  22. Lunsford R. D., Roble A. G.. 1997; comYA , a gene similar to comGA of Bacillus subtilis , is essential for competence-factor-dependent DNA transformation in Streptococcus gordonii . J Bacteriol179:3122–3126
    [Google Scholar]
  23. Lutz R., Bujard H.. 1997; Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res25:1203–1210[CrossRef]
    [Google Scholar]
  24. Matsuura M., Noguchi T., Yamaguchi D., Aida T., Asayama M., Takahashi H., Shirai M.. 1996; The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome. J Bacteriol178:3374–3376
    [Google Scholar]
  25. Mertens G., Klippel A., Fuss H., Kahmann R., Blöcker H., Frank R.. 1988; Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin. EMBO J7:1219–1227
    [Google Scholar]
  26. Miller J. H.. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Mizuuchi M., Mizuuchi K.. 1985; The extent of DNA sequence required for a functional bacterial attachment site of phage λ. Nucleic Acids Res13:1193–1208[CrossRef]
    [Google Scholar]
  28. Popham D. L., Stragier P.. 1992; Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the σK-encoding gene. Proc Natl Acad Sci USA89:5991–5995[CrossRef]
    [Google Scholar]
  29. Richet E., Abcarian P., Nash H. A.. 1988; Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex. Cell52:9–17[CrossRef]
    [Google Scholar]
  30. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  31. Sanger F., Nicklen S., Coulson A. R.. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA74:5463–5467[CrossRef]
    [Google Scholar]
  32. Sato T., Samori Y., Kobayashi Y.. 1990; The cisA cistron of Bacillus subtilis sporulation gene spoIVC encodes a protein homologous to a site-specific recombinase. J Bacteriol172:1092–1098
    [Google Scholar]
  33. Stark W. M., Boocock M. R., Sherrat D. J.. 1989; Site-specific recombination by Tn 3 resolvase. Trends Genet5:304–309[CrossRef]
    [Google Scholar]
  34. Terzaghi B. E., Sandine W. E.. 1975; Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol29:807–813
    [Google Scholar]
  35. Thorpe H. M., Smith M. C. M.. 1998; In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA95:5505–5510[CrossRef]
    [Google Scholar]
  36. Thorpe H. M., Wilson S. E., Smith M. C. M.. 2000; Control of directionality in the site-specific recombination system of the Streptomyces phage ϕC31. Mol Microbiol38:232–241[CrossRef]
    [Google Scholar]
  37. Wang H., Roberts A. P., Lyras D., Rood J. I., Wilks M., Mullany P.. 2000; Characterization of the ends and target sites of the novel conjugative transposon Tn 5397 from Clostridium difficile : excision and circularization is mediated by the large resolvase, TndX. J Bacteriol182:3775–3783[CrossRef]
    [Google Scholar]
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