Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Sep;175(18):5806–5813. doi: 10.1128/jb.175.18.5806-5813.1993

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

P Langella 1, Y Le Loir 1, S D Ehrlich 1, A Gruss 1
PMCID: PMC206659  PMID: 8376328

Abstract

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.

Full text

PDF
5806

Images in this article

Selected References

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

  1. Behnke D., Gilmore M. S. Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301. Mol Gen Genet. 1981;184(1):115–120. doi: 10.1007/BF00271206. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Biswas I., Gruss A., Ehrlich S. D., Maguin E. High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol. 1993 Jun;175(11):3628–3635. doi: 10.1128/jb.175.11.3628-3635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bougueleret L., Bieth G., Horodniceanu T. Conjugative R plasmids in group C and G streptococci. J Bacteriol. 1981 Feb;145(2):1102–1105. doi: 10.1128/jb.145.2.1102-1105.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brantl S., Behnke D., Alonso J. C. Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035. Nucleic Acids Res. 1990 Aug 25;18(16):4783–4790. doi: 10.1093/nar/18.16.4783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brantl S., Behnke D. Copy number control of the streptococcal plasmid pIP501 occurs at three levels. Nucleic Acids Res. 1992 Feb 11;20(3):395–400. doi: 10.1093/nar/20.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bron S., Holsappel S., Venema G., Peeters B. P. Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates. Mol Gen Genet. 1991 Apr;226(1-2):88–96. doi: 10.1007/BF00273591. [DOI] [PubMed] [Google Scholar]
  8. Bruand C., Ehrlich S. D., Jannière L. Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1. EMBO J. 1991 Aug;10(8):2171–2177. doi: 10.1002/j.1460-2075.1991.tb07752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buu-Hoï A., Bieth G., Horaud T. Broad host range of streptococcal macrolide resistance plasmids. Antimicrob Agents Chemother. 1984 Feb;25(2):289–291. doi: 10.1128/aac.25.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Clewell D. B. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol Rev. 1981 Sep;45(3):409–436. doi: 10.1128/mr.45.3.409-436.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clewell D. B., Yagi Y., Dunny G. M., Schultz S. K. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J Bacteriol. 1974 Jan;117(1):283–289. doi: 10.1128/jb.117.1.283-289.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Engel H. W., Soedirman N., Rost J. A., van Leeuwen W. J., van Embden J. D. Transferability of macrolide, lincomycin, and streptogramin resistances between group A, B, and D streptococci, Streptococcus pneumoniae, and Staphylococcus aureus. J Bacteriol. 1980 May;142(2):407–413. doi: 10.1128/jb.142.2.407-413.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Evans R. P., Jr, Macrina F. L. Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives. J Bacteriol. 1983 Jun;154(3):1347–1355. doi: 10.1128/jb.154.3.1347-1355.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gennaro M. L., Kornblum J., Novick R. P. A site-specific recombination function in Staphylococcus aureus plasmids. J Bacteriol. 1987 Jun;169(6):2601–2610. doi: 10.1128/jb.169.6.2601-2610.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gibson E. M., Chace N. M., London S. B., London J. Transfer of plasmid-mediated antibiotic resistance from streptococci to lactobacilli. J Bacteriol. 1979 Jan;137(1):614–619. doi: 10.1128/jb.137.1.614-619.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gonzalez C. F., Kunka B. S. Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501. Appl Environ Microbiol. 1983 Jul;46(1):81–89. doi: 10.1128/aem.46.1.81-89.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gruss A., Ehrlich S. D. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. doi: 10.1128/mr.53.2.231-241.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holo H., Nes I. F. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Appl Environ Microbiol. 1989 Dec;55(12):3119–3123. doi: 10.1128/aem.55.12.3119-3123.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horaud T., Le Bouguenec C., Pepper K. Molecular genetics of resistance to macrolides, lincosamides and streptogramin B (MLS) in streptococci. J Antimicrob Chemother. 1985 Jul;16 (Suppl A):111–135. doi: 10.1093/jac/16.suppl_a.111. [DOI] [PubMed] [Google Scholar]
  22. Horodniceanu T., Bouanchaud D. H., Bieth G., Chabbert Y. A. R plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother. 1976 Nov;10(5):795–801. doi: 10.1128/aac.10.5.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krah E. R., 3rd, Macrina F. L. Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501. J Bacteriol. 1989 Nov;171(11):6005–6012. doi: 10.1128/jb.171.11.6005-6012.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krah E. R., 3rd, Macrina F. L. Identification of a region that influences host range of the streptococcal conjugative plasmid pIP501. Plasmid. 1991 Jan;25(1):64–69. doi: 10.1016/0147-619x(91)90007-j. [DOI] [PubMed] [Google Scholar]
  25. Langella P., Chopin A. Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. FEMS Microbiol Lett. 1989 Jul 15;51(1):149–152. doi: 10.1016/0378-1097(89)90498-9. [DOI] [PubMed] [Google Scholar]
  26. Le Chatelier E., Ehrlich S. D., Jannière L. Biochemical and genetic analysis of the unidirectional theta replication of the S. agalactiae plasmid pIP501. Plasmid. 1993 Jan;29(1):50–56. doi: 10.1006/plas.1993.1006. [DOI] [PubMed] [Google Scholar]
  27. Maguin E., Duwat P., Hege T., Ehrlich D., Gruss A. New thermosensitive plasmid for gram-positive bacteria. J Bacteriol. 1992 Sep;174(17):5633–5638. doi: 10.1128/jb.174.17.5633-5638.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mercenier A., Chassy B. M. Strategies for the development of bacterial transformation systems. Biochimie. 1988 Apr;70(4):503–517. doi: 10.1016/0300-9084(88)90086-7. [DOI] [PubMed] [Google Scholar]
  29. Projan S. J., Carleton S., Novick R. P. Determination of plasmid copy number by fluorescence densitometry. Plasmid. 1983 Mar;9(2):182–190. doi: 10.1016/0147-619x(83)90019-7. [DOI] [PubMed] [Google Scholar]
  30. Pucci M. J., Monteschio M. E., Kemker C. L. Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp. Appl Environ Microbiol. 1988 Feb;54(2):281–287. doi: 10.1128/aem.54.2.281-287.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Romero D. A., Slos P., Robert C., Castellino I., Mercenier A. Conjugative mobilization as an alternative vector delivery system for lactic streptococci. Appl Environ Microbiol. 1987 Oct;53(10):2405–2413. doi: 10.1128/aem.53.10.2405-2413.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schaberg D. R., Clewell D. B., Glatzer L. Conjugative transfer of R-plasmids from Streptococcus faecalis to Staphylococcus aureus. Antimicrob Agents Chemother. 1982 Aug;22(2):204–207. doi: 10.1128/aac.22.2.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Smith M. D., Clewell D. B. Return of Streptococcus faecalis DNA cloned in Escherichia coli to its original host via transformation of Streptococcus sanguis followed by conjugative mobilization. J Bacteriol. 1984 Dec;160(3):1109–1114. doi: 10.1128/jb.160.3.1109-1114.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Swinfield T. J., Jannière L., Ehrlich S. D., Minton N. P. Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis. Plasmid. 1991 Nov;26(3):209–221. doi: 10.1016/0147-619x(91)90044-w. [DOI] [PubMed] [Google Scholar]
  37. Swinfield T. J., Oultram J. D., Thompson D. E., Brehm J. K., Minton N. P. Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1. Gene. 1990 Mar 1;87(1):79–90. [PubMed] [Google Scholar]
  38. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. West C. A., Warner P. J. Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacillus plantarum. Appl Environ Microbiol. 1985 Nov;50(5):1319–1321. doi: 10.1128/aem.50.5.1319-1321.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES