Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(6):1837–1845. doi: 10.1128/jb.179.6.1837-1845.1997

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

F Auvray 1, M Coddeville 1, P Ritzenthaler 1, L Dupont 1
PMCID: PMC178904  PMID: 9068626

Abstract

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.

Full Text

The Full Text of this article is available as a PDF (878.4 KB).

Selected References

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

  1. Alegre M. T., Cournoyer B., Mesas J. M., Guérineau M., Normand P., Pernodet J. L. Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans. Appl Environ Microbiol. 1994 Dec;60(12):4279–4283. doi: 10.1128/aem.60.12.4279-4283.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Atlung T., Nielsen A., Rasmussen L. J., Nellemann L. J., Holm F. A versatile method for integration of genes and gene fusions into the lambda attachment site of Escherichia coli. Gene. 1991 Oct 30;107(1):11–17. doi: 10.1016/0378-1119(91)90291-i. [DOI] [PubMed] [Google Scholar]
  4. Bar-Nir D., Cohen A., Goedeke M. E. tDNA(ser) sequences are involved in the excision of Streptomyces griseus plasmid pSG1. Gene. 1992 Dec 1;122(1):71–76. doi: 10.1016/0378-1119(92)90033-l. [DOI] [PubMed] [Google Scholar]
  5. Barreiro V., Haggård-Ljungquist E. Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives. J Bacteriol. 1992 Jun;174(12):4086–4093. doi: 10.1128/jb.174.12.4086-4093.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown D. P., Idler K. B., Backer D. M., Donadio S., Katz L. Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans. Mol Gen Genet. 1994 Jan;242(2):185–193. doi: 10.1007/BF00391012. [DOI] [PubMed] [Google Scholar]
  7. Brown D. P., Idler K. B., Katz L. Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea. J Bacteriol. 1990 Apr;172(4):1877–1888. doi: 10.1128/jb.172.4.1877-1888.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Campbell A. M. Chromosomal insertion sites for phages and plasmids. J Bacteriol. 1992 Dec;174(23):7495–7499. doi: 10.1128/jb.174.23.7495-7499.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chopin A., Chopin M. C., Moillo-Batt A., Langella P. Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid. 1984 May;11(3):260–263. doi: 10.1016/0147-619x(84)90033-7. [DOI] [PubMed] [Google Scholar]
  10. Christiansen B., Brøndsted L., Vogensen F. K., Hammer K. A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1. J Bacteriol. 1996 Sep;178(17):5164–5173. doi: 10.1128/jb.178.17.5164-5173.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cluzel P. J., Veaux M., Rousseau M., Accolas J. P. Evidence for temperate bacteriophages in two strains of Lactobacillus bulgaricus. J Dairy Res. 1987 Aug;54(3):397–405. doi: 10.1017/s0022029900025577. [DOI] [PubMed] [Google Scholar]
  12. Dupont L., Boizet-Bonhoure B., Coddeville M., Auvray F., Ritzenthaler P. Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum. J Bacteriol. 1995 Feb;177(3):586–595. doi: 10.1128/jb.177.3.586-595.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gabriel K., Schmid H., Schmidt U., Rausch H. The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus. Nucleic Acids Res. 1995 Jan 11;23(1):58–63. doi: 10.1093/nar/23.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Griffin H. G., Gasson M. J. Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis. Mol Gen Genet. 1995 Jan 6;246(1):119–127. doi: 10.1007/BF00290140. [DOI] [PubMed] [Google Scholar]
  16. Hayashi T., Matsumoto H., Ohnishi M., Terawaki Y. Molecular analysis of a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa: structure of the attP-cos-ctx region and integration into the serine tRNA gene. Mol Microbiol. 1993 Mar;7(5):657–667. doi: 10.1111/j.1365-2958.1993.tb01157.x. [DOI] [PubMed] [Google Scholar]
  17. Hottinger H., Ohgi T., Zwahlen M. C., Dhamija S., Söll D. Allele-specific complementation of an Escherichia coli leuB mutation by a Lactobacillus bulgaricus tRNA gene. Gene. 1987;60(1):75–83. doi: 10.1016/0378-1119(87)90215-0. [DOI] [PubMed] [Google Scholar]
  18. Inouye S., Sunshine M. G., Six E. W., Inouye M. Retronphage phi R73: an E. coli phage that contains a retroelement and integrates into a tRNA gene. Science. 1991 May 17;252(5008):969–971. doi: 10.1126/science.1709758. [DOI] [PubMed] [Google Scholar]
  19. Jacob A. E., Hobbs S. J. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol. 1974 Feb;117(2):360–372. doi: 10.1128/jb.117.2.360-372.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Le Bourgeois P., Lautier M., van den Berghe L., Gasson M. J., Ritzenthaler P. Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion. J Bacteriol. 1995 May;177(10):2840–2850. doi: 10.1128/jb.177.10.2840-2850.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Le Marrec C., Moreau S., Loury S., Blanco C., Trautwetter A. Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70. J Bacteriol. 1996 Apr;178(7):1996–2004. doi: 10.1128/jb.178.7.1996-2004.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee C. Y., Buranen S. L., Ye Z. H. Construction of single-copy integration vectors for Staphylococcus aureus. Gene. 1991 Jul 15;103(1):101–105. doi: 10.1016/0378-1119(91)90399-v. [DOI] [PubMed] [Google Scholar]
  23. Lee M. H., Pascopella L., Jacobs W. R., Jr, Hatfull G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3111–3115. doi: 10.1073/pnas.88.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leer R. J., van Luijk N., Posno M., Pouwels P. H. Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet. 1992 Aug;234(2):265–274. doi: 10.1007/BF00283847. [DOI] [PubMed] [Google Scholar]
  25. Martin B., García P., Castanié M. P., Claverys J. P. The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol Microbiol. 1995 Jan;15(2):367–379. doi: 10.1111/j.1365-2958.1995.tb02250.x. [DOI] [PubMed] [Google Scholar]
  26. Martin B., Prats H., Claverys J. P. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product. Gene. 1985;34(2-3):293–303. doi: 10.1016/0378-1119(85)90138-6. [DOI] [PubMed] [Google Scholar]
  27. Martín C., Mazodier P., Mediola M. V., Gicquel B., Smokvina T., Thompson C. J., Davies J. Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol Microbiol. 1991 Oct;5(10):2499–2502. doi: 10.1111/j.1365-2958.1991.tb02095.x. [DOI] [PubMed] [Google Scholar]
  28. Mazodier P., Thompson C., Boccard F. The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes. Mol Gen Genet. 1990 Jul;222(2-3):431–434. doi: 10.1007/BF00633850. [DOI] [PubMed] [Google Scholar]
  29. Papp I., Dorgai L., Papp P., Jónás E., Olasz F., Orosz L. The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3' end of a putative proline tRNA gene. Mol Gen Genet. 1993 Aug;240(2):258–264. doi: 10.1007/BF00277064. [DOI] [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Porter R. D., Guild W. R. Characterization of some pneumococcal bacteriophages. J Virol. 1976 Aug;19(2):659–667. doi: 10.1128/jvi.19.2.659-667.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Powell Ian B., Achen Marc G., Hillier Alan J., Davidson Barrie E. A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation. Appl Environ Microbiol. 1988 Mar;54(3):655–660. doi: 10.1128/aem.54.3.655-660.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raya R. R., Fremaux C., De Antoni G. L., Klaenhammer T. R. Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites. J Bacteriol. 1992 Sep;174(17):5584–5592. doi: 10.1128/jb.174.17.5584-5592.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reiter W. D., Palm P., Yeats S. Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements. Nucleic Acids Res. 1989 Mar 11;17(5):1907–1914. doi: 10.1093/nar/17.5.1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ross W., Landy A. Patterns of lambda Int recognition in the regions of strand exchange. Cell. 1983 May;33(1):261–272. doi: 10.1016/0092-8674(83)90355-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Simon D., Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie. 1988 Apr;70(4):559–566. doi: 10.1016/0300-9084(88)90093-4. [DOI] [PubMed] [Google Scholar]
  38. Smokvina T., Mazodier P., Boccard F., Thompson C. J., Guérineau M. Construction of a series of pSAM2-based integrative vectors for use in actinomycetes. Gene. 1990 Sep 28;94(1):53–59. doi: 10.1016/0378-1119(90)90467-6. [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. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H., Hatfull G. F. New use of BCG for recombinant vaccines. Nature. 1991 Jun 6;351(6326):456–460. doi: 10.1038/351456a0. [DOI] [PubMed] [Google Scholar]
  41. Vrijbloed J. W., Madoń J., Dijkhuizen L. A plasmid from the methylotrophic actinomycete Amycolatopsis methanolica capable of site-specific integration. J Bacteriol. 1994 Nov;176(22):7087–7090. doi: 10.1128/jb.176.22.7087-7090.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van de Guchte M., Daly C., Fitzgerald G. F., Arendt E. K. Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363. Appl Environ Microbiol. 1994 Jul;60(7):2324–2329. doi: 10.1128/aem.60.7.2324-2329.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES