Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Sep;174(17):5584–5592. doi: 10.1128/jb.174.17.5584-5592.1992

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.

R R Raya 1, C Fremaux 1, G L De Antoni 1, T R Klaenhammer 1
PMCID: PMC206502  PMID: 1512192

Abstract

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.

Full text

PDF
5584

Images in this article

5586Image
on p.5586
5588Image
on p.5588
5589Image
on p.5589

Selected References

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

  1. Astumian J. H., Waldman A. S., Scocca J. J. Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli. J Bacteriol. 1989 Mar;171(3):1747–1750. doi: 10.1128/jb.171.3.1747-1750.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  3. Barksdale L., Arden S. B. Persisting bacteriophage infections, lysogeny, and phage conversions. Annu Rev Microbiol. 1974;28(0):265–299. doi: 10.1146/annurev.mi.28.100174.001405. [DOI] [PubMed] [Google Scholar]
  4. Brown D. P., Chiang S. J., Tuan J. S., Katz L. Site-specific integration in Saccharopolyspora erythraea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101. J Bacteriol. 1988 May;170(5):2287–2295. doi: 10.1128/jb.170.5.2287-2295.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davidson B. E., Powell I. B., Hillier A. J. Temperate bacteriophages and lysogeny in lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):79–90. doi: 10.1111/j.1574-6968.1990.tb04880.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Hagerman P. J. Sequence-directed curvature of DNA. Annu Rev Biochem. 1990;59:755–781. doi: 10.1146/annurev.bi.59.070190.003543. [DOI] [PubMed] [Google Scholar]
  8. Hsu P. L., Ross W., Landy A. The lambda phage att site: functional limits and interaction with Int protein. Nature. 1980 May 8;285(5760):85–91. doi: 10.1038/285085a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kleeman E. G., Klaenhammer T. R. Adherence of Lactobacillus species to human fetal intestinal cells. J Dairy Sci. 1982 Nov;65(11):2063–2069. doi: 10.3168/jds.S0022-0302(82)82462-4. [DOI] [PubMed] [Google Scholar]
  10. Kuhstoss S., Richardson M. A., Rao R. N. Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2. J Bacteriol. 1989 Jan;171(1):16–23. doi: 10.1128/jb.171.1.16-23.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lee C. Y., Buranen S. L. Extent of the DNA sequence required in integration of staphylococcal bacteriophage L54a. J Bacteriol. 1989 Mar;171(3):1652–1657. doi: 10.1128/jb.171.3.1652-1657.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee C. Y., Iandolo J. J. Integration of staphylococcal phage L54a occurs by site-specific recombination: structural analysis of the attachment sites. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5474–5478. doi: 10.1073/pnas.83.15.5474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee C. Y., Iandolo J. J. Structural analysis of staphylococcal bacteriophage phi 11 attachment sites. J Bacteriol. 1988 May;170(5):2409–2411. doi: 10.1128/jb.170.5.2409-2411.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leong J. M., Nunes-Düby S., Lesser C. F., Youderian P., Susskind M. M., Landy A. The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor. J Biol Chem. 1985 Apr 10;260(7):4468–4477. [PubMed] [Google Scholar]
  15. Luchansky J. B., Kleeman E. G., Raya R. R., Klaenhammer T. R. Genetic transfer systems for delivery of plasmid deoxyribonucleic acid to Lactobacillus acidophilus ADH: conjugation, electroporation, and transduction. J Dairy Sci. 1989 Jun;72(6):1408–1417. doi: 10.3168/jds.S0022-0302(89)79248-1. [DOI] [PubMed] [Google Scholar]
  16. Madon J., Moretti P., Hütter R. Site-specific integration and excision of pMEA100 in Nocardia mediterranei. Mol Gen Genet. 1987 Sep;209(2):257–264. doi: 10.1007/BF00329651. [DOI] [PubMed] [Google Scholar]
  17. Omer C. A., Cohen S. N. Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site. Mol Gen Genet. 1984;196(3):429–438. doi: 10.1007/BF00436190. [DOI] [PubMed] [Google Scholar]
  18. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  19. Raya R. R., Kleeman E. G., Luchansky J. B., Klaenhammer T. R. Characterization of the temperate bacteriophage phi adh and plasmid transduction in Lactobacillus acidophilus ADH. Appl Environ Microbiol. 1989 Sep;55(9):2206–2213. doi: 10.1128/aem.55.9.2206-2213.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Romero D. A., Klaenhammer T. R. IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1992 Feb;58(2):699–702. doi: 10.1128/aem.58.2.699-702.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Scheirlinck T., Mahillon J., Joos H., Dhaese P., Michiels F. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl Environ Microbiol. 1989 Sep;55(9):2130–2137. doi: 10.1128/aem.55.9.2130-2137.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sechaud L., Cluzel P. J., Rousseau M., Baumgartner A., Accolas J. P. Bacteriophages of lactobacilli. Biochimie. 1988 Mar;70(3):401–410. doi: 10.1016/0300-9084(88)90214-3. [DOI] [PubMed] [Google Scholar]
  24. Shimizu-Kadota M., Tsuchida N. Physical mapping of the virion and the prophage DNAs of a temperate Lactobacillus phage phi FSW. J Gen Microbiol. 1984 Feb;130(2):423–430. doi: 10.1099/00221287-130-2-423. [DOI] [PubMed] [Google Scholar]
  25. Shyamala V., Ames G. F. Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene. 1989 Dec 7;84(1):1–8. doi: 10.1016/0378-1119(89)90132-7. [DOI] [PubMed] [Google Scholar]
  26. Sosio M., Madoń J., Hütter R. Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans. Mol Gen Genet. 1989 Jul;218(1):169–176. doi: 10.1007/BF00330580. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Travers A. A. DNA conformation and protein binding. Annu Rev Biochem. 1989;58:427–452. doi: 10.1146/annurev.bi.58.070189.002235. [DOI] [PubMed] [Google Scholar]
  29. Waldman A. S., Goodman S. D., Scocca J. J. Nucleotide sequences and properties of the sites involved in lysogenic insertion of the bacteriophage HP1c1 genome into the Haemophilus influenzae chromosome. J Bacteriol. 1987 Jan;169(1):238–246. doi: 10.1128/jb.169.1.238-246.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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 Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES