Molecular and General Genetics MGG

, Volume 182, Issue 1, pp 99–105 | Cite as

Integration and excision of a plasmid in Bacillus subtilis

  • A. Galizzi
  • F. Scoffone
  • G. Milanesi
  • A. M. Albertini
Article

Summary

We have studied the behaviour in Bacillus subtilis of a plasmid (pPV21) carrying the thymidylate synthetase gene of phage ϕ3T (thyP3). The plasmid can transform efficiently the competent cells of all the strains tested. Polyethylene glycol (PEG)-mediated protoplast transformation is efficient only for recE, recD or recF mutants. When present in recombination proficient strains, the plasmid can be integrated into the chromosome, primarily at the thyA locus. This has been shown by genetic mapping and by blot-hybridization. A second less efficient site is at (or near to) the attachment site of phage ϕ3T. Excision of the plasmid restores the EcoRI restriction pattern of the parental DNA, although with the loss of the defective thyA endogenotic allele and the retention of the thyP exogenotic gene.

Keywords

Recombination Glycol Bacillus Genetic Mapping Bacillus Subtilis 

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References

  1. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  2. Campbell A, Berg D, Lederberg E, Starlinger P, Botstein D, Novick R, Szybalski W (1977) Nomenclature of transposable elements in prokaryotes. In: Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Laboratory, New York, pp 15–22Google Scholar
  3. Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111–115Google Scholar
  4. Cohen SN, Chang ACY, Boyer HW, Helling RB (1973) Construction of biologically functional bacterial plasmids “in vitro”. Proc Natl Acad Sci USA 70:3240–3244Google Scholar
  5. Denhardt DT (1966) A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23:641–643Google Scholar
  6. Duncan CH, Wilson GA, Young FE (1978) Mechanism of integrating foreign DNA during tranformation of Bacillus subtilis. Proc Natl Acad Sci USA 75:3664–3668Google Scholar
  7. Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1680–1682Google Scholar
  8. Ehrlich SD (1978) DNA cloning in Bacillus subtilis. Proc Natl Acad Sci USA 75:1433–1436Google Scholar
  9. Ehrlich SD, Bursztyn-Pettegrew H, Stroynowski I, Lederberg J (1976) Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli. Proc Natl Acad Sci USA 73:4145–4149Google Scholar
  10. Ehrlich SD, Jupp S, Niaudet B, Goze A (1978) Bacillus subtilis as a host for DNA cloning. In: Boyer HW, Nicosia S (eds) Genetic engineering. Elsevier-North Holland, Amsterdam, pp 25–32Google Scholar
  11. Gryczan TJ, Dubnau D (1978) Construction and properties of chimeric plasmids in Bacillus subtilis. Proc Natl Acad Sci USA 75:1428–1432Google Scholar
  12. Ganesan AT (1979) Genetic recombination during transformation in Bacillus subtilis: appearence of a deoxyribonucleic acid methylase. J Bacteriol 139:270–279Google Scholar
  13. Haldenwang WG, Banner CDB, Ollington JF, Losick R, Hoch JA, O'Connor MB, Sonensheim AL (1980) Mapping a cloned gene under sporulation control by insertion of a drug resistance marker into the Bacillus subtilis chromosome. J Bacteriol 142:90–98Google Scholar
  14. Henner DJ, Hoch JA (1980) The Bacillus subtilis chromosome. Microbiol Rev 44:57–82Google Scholar
  15. Hoch JA, Barat M, Anagnostopoulos C (1967) Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol 93:1925–1937Google Scholar
  16. Kelly RB, Cozzarelli NR, Deutschman NP, Lehman IR, Hornberg A (1970) Enzymatic synthesis of DNA XXXII: replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J Biol Chem 245:39–45Google Scholar
  17. Kreft J, Bernhard K, Goebel W (1978) Recombination plasmids capable of replication in B. subtilis and E. coli. Mol Gen Genet 162:59–67Google Scholar
  18. Mazza G, Galizzi A (1978) The genetics of DNA replication, repair and recombination in Bacillus subtilis. Microbiologica 1:111–135Google Scholar
  19. Mazza G, Marinone R, Ferrari E (1980) Plasmid transformation in Bacillus subtilis pHV15 cloning vector. Microbiologica 3:247–258Google Scholar
  20. Radloff R, Bauer W, Vinograd J (1967) A dye-buoyant density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci USA 57:1514–1521Google Scholar
  21. Rapoport G, Klier A, Billault A, Fargette F, Dedonder R (1979) Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Mol Gen Genet 176:239–245Google Scholar
  22. Romig WR (1962) Infection of Bacillus subtilis with phenol-extracted bacteriophages. Virology 16:452–459Google Scholar
  23. Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochem Biophys Acta 72:619–629Google Scholar
  24. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  25. Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078Google Scholar
  26. Strunl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039Google Scholar
  27. Zahler SA, Benjamin LG, Glatz BS, Winter PF, Goldstein BJ (1976) Genetic mapping of alsA, alsR, thyA, kauA and citD markers in Bacillus subtilis In: Schlessinger (ed) Microbiology 1976. American Society for Microbiology, Washington, pp 35–43Google Scholar
  28. Williams MT, Young FE (1977) Temperate Bacillus subtilis bacteriophage ϕ3T: chromosomal attachment site and comparison with temperate bacteriophages ϕ105 and SPO2. J Virol 21:522–529Google Scholar
  29. Yamagishi H, Takahashi I (1968) Transducing particles of PBS-1. Virology 36:639–645Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • A. Galizzi
    • 1
  • F. Scoffone
    • 2
  • G. Milanesi
    • 2
  • A. M. Albertini
    • 1
  1. 1.Istituto di GeneticaUniversità degli StudiPaviaItaly
  2. 2.Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R.PaviaItaly

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