Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 Jun;154(3):1077–1087. doi: 10.1128/jb.154.3.1077-1087.1983

Plasmid marker rescue transformation in Bacillus subtilis.

Y Weinrauch, D Dubnau
PMCID: PMC217577  PMID: 6406424

Abstract

We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome. This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis. Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid. Rescue transformation did not proceed by recircularization and replication of the donor DNA. Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA. The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA. Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products. Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges. Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation. We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination.

Full text

PDF
1077

Images in this article

1080Image
on p.1080

Selected References

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

  1. Bingham A. H., Atkinson T., Sciaky D., Roberts R. J. A specific endonuclease from Bacillus caldolyticus. Nucleic Acids Res. 1978 Oct;5(10):3457–3467. doi: 10.1093/nar/5.10.3457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bodmer W. F. Integration of deoxyribonuclease-treated DNA in bacillus subtilis transformation. J Gen Physiol. 1966 Jul;49(6):233–258. doi: 10.1085/jgp.49.6.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Contente S., Dubnau D. Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. Mol Gen Genet. 1979 Jan 2;167(3):251–258. doi: 10.1007/BF00267416. [DOI] [PubMed] [Google Scholar]
  4. Contente S., Dubnau D. Marker rescue transformation by linear plasmid DNA in Bacillus subtilis. Plasmid. 1979 Oct;2(4):555–571. doi: 10.1016/0147-619x(79)90054-4. [DOI] [PubMed] [Google Scholar]
  5. Darlington A. J., Bodmer W. F. Events occurring at the site of integration of a DNA molecule in Bacillus subtilis transformation. Genetics. 1968 Dec;60(4):681–684. doi: 10.1093/genetics/60.4.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubnau D., Cirigliano C. Fate of transforming DNA following uptake by competent Bacillus subtilis. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J Mol Biol. 1972 Feb 28;64(1):9–29. doi: 10.1016/0022-2836(72)90318-x. [DOI] [PubMed] [Google Scholar]
  7. Dubnau D., Cirigliano C. Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: size and distribution of the integrated donor segments. J Bacteriol. 1972 Aug;111(2):488–494. doi: 10.1128/jb.111.2.488-494.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dubnau D., Cirigliano C. Genetic characterization of recombination-deficient mutants of Bacillus subtilis. J Bacteriol. 1974 Feb;117(2):488–493. doi: 10.1128/jb.117.2.488-493.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
  10. Dubnau D., Davidoff-Abelson R., Scher B., Cirigliano C. Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: phenotypic characterization of radiation-sensitive recombination-deficient mutants. J Bacteriol. 1973 Apr;114(1):273–286. doi: 10.1128/jb.114.1.273-286.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gryczan T. J., Contente S., Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):318–329. doi: 10.1128/jb.134.1.318-329.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gryczan T. J., Grandi G., Hahn J., Grandi R., Dubnau D. Conformational alteration of mRNA structure and the posttranscriptional regulation of erythromycin-induced drug resistance. Nucleic Acids Res. 1980 Dec 20;8(24):6081–6097. doi: 10.1093/nar/8.24.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gryczan T., Contente S., Dubnau D. Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis. Mol Gen Genet. 1980 Feb;177(3):459–467. doi: 10.1007/BF00271485. [DOI] [PubMed] [Google Scholar]
  14. Gryczan T., Shivakumar A. G., Dubnau D. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol. 1980 Jan;141(1):246–253. doi: 10.1128/jb.141.1.246-253.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guild W. R., Cato A., Jr, Lacks S. Transformation and DNA size: two controlling parameters and the efficiency of the single strand intermediate. Cold Spring Harb Symp Quant Biol. 1968;33:643–645. doi: 10.1101/sqb.1968.033.01.072. [DOI] [PubMed] [Google Scholar]
  17. Hahn J., Grandi G., Gryczan T. J., Dubnau D. Translational attenuation of ermC: a deletion analysis. Mol Gen Genet. 1982;186(2):204–216. doi: 10.1007/BF00331851. [DOI] [PubMed] [Google Scholar]
  18. Hoch J. A., Barat M., Anagnostopoulos C. Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol. 1967 Jun;93(6):1925–1937. doi: 10.1128/jb.93.6.1925-1937.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horinouchi S., Weisblum B. Posttranscriptional modification of mRNA conformation: mechanism that regulates erythromycin-induced resistance. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7079–7083. doi: 10.1073/pnas.77.12.7079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hutchinson F., Hales H. B. Mechanism of the sensitization of bacterial transforming DNA to ultraviolet light by the incorporation of 5-bromouracil. J Mol Biol. 1970 May 28;50(1):59–69. doi: 10.1016/0022-2836(70)90103-8. [DOI] [PubMed] [Google Scholar]
  22. Imanaka T., Fujii M., Aiba S. Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids. J Bacteriol. 1981 Jun;146(3):1091–1097. doi: 10.1128/jb.146.3.1091-1097.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Imanaka T., Fujii M., Aramori I., Aiba S. Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol. 1982 Mar;149(3):824–830. doi: 10.1128/jb.149.3.824-830.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kühn S., Fritz H. J., Starlinger P. Close vicinity of IS1 integration sites in the leader sequence of the gal operon of E. coli. Mol Gen Genet. 1979 Jan 2;167(3):235–241. doi: 10.1007/BF00267414. [DOI] [PubMed] [Google Scholar]
  25. LOVELESS A., HOWARTH S. Mutation of bacteria at high levels of survival by ethyl methane sulphonate. Nature. 1959 Dec 5;184:1780–1782. doi: 10.1038/1841780a0. [DOI] [PubMed] [Google Scholar]
  26. Morrison D. A., Guild W. R. Activity of deoxyribonucleic acid fragments of defined size in Bacillus subtilis transformation. J Bacteriol. 1972 Oct;112(1):220–223. doi: 10.1128/jb.112.1.220-223.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Prozorov A. A., Bashkirov V. I., Lakomova N. M., Surikov N. N. Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis. Mol Gen Genet. 1982;185(2):363–364. doi: 10.1007/BF00330813. [DOI] [PubMed] [Google Scholar]
  29. Shivakumar A. G., Gryczan T. J., Kozlov Y. I., Dubnau D. Organization of the pE194 genome. Mol Gen Genet. 1980;179(2):241–252. doi: 10.1007/BF00425450. [DOI] [PubMed] [Google Scholar]
  30. Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Venema G. Bacterial transformation. Adv Microb Physiol. 1979;19:245–331. doi: 10.1016/s0065-2911(08)60200-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES