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. 1968 Mar;95(3):876–885. doi: 10.1128/jb.95.3.876-885.1968

Purification of Competent Cells in the Bacillus subtilis Transformation System

Charles Hadden 1, E W Nester 2
PMCID: PMC252105  PMID: 4966831

Abstract

Transformed cells have been separated from nontransformed cells by centrifugation on a density gradient of Renografin-76. Separation was achieved both on a linear gradient and on a discontinuous gradient. Under optimal conditions, all of the cells in one band (median density, 1.110 g/ml) were transformants, whereas virtually all cells in the other (median density, 1.131) were nontransformants. In some instances, recentrifugation of the transformant band further enriched the transformant population. The transformed population can also be enriched by zonal centrifugation in a linear gradient of Ficoll. However, this technique is far less efficient than centrifugation in Renografin-76. Since the density of competent cells is identical to that of transformants, we conclude that the low density is a property of competent cells. The significance of this low density to the physiology of competent cells is discussed.

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Selected References

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

  1. BODMER W. F., GANESAN A. T. BIOCHEMICAL AND GENETIC STUDIES OF INTEGRATION AND RECOMBINATION IN BACILLUS SUBTILIS TRANSFORMATION. Genetics. 1964 Oct;50:717–738. doi: 10.1093/genetics/50.4.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bott K. F., Wilson G. A. Development of competence in the Bacillus subtilis transformation system. J Bacteriol. 1967 Sep;94(3):562–570. doi: 10.1128/jb.94.3.562-570.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. GOODGAL S. H., HERRIOTT R. M. Studies on transformations of Hemophilus influenzae. I. Competence. J Gen Physiol. 1961 Jul;44:1201–1227. doi: 10.1085/jgp.44.6.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HOLTER H., MOLLER K. M. A substance for aqueous density gradients. Exp Cell Res. 1958 Dec;15(3):631–632. doi: 10.1016/0014-4827(58)90116-2. [DOI] [PubMed] [Google Scholar]
  6. MacDonald R. E., Turnock G., Forchhammer J. The synthesis and function of ribosomes in a new mutant of Escherichia coli. Proc Natl Acad Sci U S A. 1967 Jan;57(1):141–147. doi: 10.1073/pnas.57.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Manor H., Haselkorn R. Size fractionation of exponentially growing Escherichia coli. Nature. 1967 Jun 3;214(5092):983–986. doi: 10.1038/214983a0. [DOI] [PubMed] [Google Scholar]
  8. McCarthy C., Nester E. W. Macromolecular synthesis in newly transformed cells of Bacillus subtilis. J Bacteriol. 1967 Jul;94(1):131–140. doi: 10.1128/jb.94.1.131-140.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. NESTER E. W., STOCKER B. A. BIOSYNTHETIC LATENCY IN EARLY STAGES OF DEOXYRIBONUCLEIC ACIDTRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1963 Oct;86:785–796. doi: 10.1128/jb.86.4.785-796.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nester E W, Schafer M, Lederberg J. Gene Linkage in DNA Transfer: A Cluster of Genes Concerned with Aromatic Biosynthesis in Bacillus Subtilis. Genetics. 1963 Apr;48(4):529–551. doi: 10.1093/genetics/48.4.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pollard E. C., Grady L. J. CsCl density gradient centrifugation studies of intact bacterial cells. Biophys J. 1967 Mar;7(2):205–213. doi: 10.1016/S0006-3495(67)86584-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. REILLY B. E., SPIZIZEN J. BACTERIOPHAGE DEOXYRIBONUCLEATE INFECTION OF COMPETENT BACILLUS SUBTILIS. J Bacteriol. 1965 Mar;89:782–790. doi: 10.1128/jb.89.3.782-790.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. SCHAEFFER P. Existence d'une compétition entre molécules d'acides désoxyribonucléiques pour la pénétration dans les bactéries transformables. C R Hebd Seances Acad Sci. 1957 Jul 8;245(2):230–231. [PubMed] [Google Scholar]
  14. STOCKER B. A. TRANSFORMATION OF BACILLUS SUBTILIS TO MOTILITY AND PROTOTROPHY: MICROMANIPULATIVE ISOLATION OF BACTERIA OF TRANSFORMED PHENOTYPE. J Bacteriol. 1963 Oct;86:797–804. doi: 10.1128/jb.86.4.797-804.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tamir H., Gilvarg C. Density gradient centrifugation for the separation of sporulating forms of bacteria. J Biol Chem. 1966 Mar 10;241(5):1085–1090. [PubMed] [Google Scholar]
  16. WADE H. E. Variation in the phosphorus content of Escherichia coli during cultivation. J Gen Microbiol. 1952 Aug;7(1-2):24–30. doi: 10.1099/00221287-7-1-2-24. [DOI] [PubMed] [Google Scholar]
  17. Wolstenholme D. R., Vermeulen C. A., Venema G. Evidence for the involvement of membranous bodies in the processes leading to genetic transformation in Bacillus subtilis. J Bacteriol. 1966 Oct;92(4):1111–1121. doi: 10.1128/jb.92.4.1111-1121.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. YOUNG F. E., SPIZIZEN J. INCORPORATION OF DEOXYRIBONUCLEIC ACID IN THE BACILLUS SUBTILIS TRANSFORMATION SYSTEM. J Bacteriol. 1963 Sep;86:392–400. doi: 10.1128/jb.86.3.392-400.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Young F. E. Competence in Bacillus subtilis transformation system. Nature. 1967 Feb 25;213(5078):773–775. doi: 10.1038/213773a0. [DOI] [PubMed] [Google Scholar]

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