Summary
Competent B. subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA. Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg2+ ions in excess of the EDTA. At 30° and at 17° comparable numbers of transformants are produced from cells pre-locaded with DNA at 30° (after termination of uptake by the addition of DNA ase the samples were incubated at 37°). However, almost no transformants are produced when cells are exposed to DNA at 17°, although binding does take place then. Because DNA is taken up at 17° after having loaded the cells at 30°, whereas no uptake occurs after binding at 17°, it is suggested that binding of DNA to the cellular surface involves at least two steps.
In DNA re-extracted from cells at 17°, pre-loaded with DNA at 30°, little recombinant type activity is present, indicating that integration is blocked at 17°. However, physico-chemical analysis of the reextracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17°. This complex has a higher buoyant density than donor-recipient complexes formed at 30°.
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Arwert, F., Venema, G.: Evidence for a non-covalently bonded intermediate in recombination during transformation of Bacillus subtilis. In: Bacterial transformation (L. Archer, ed.), pp. 203–214. London: Academic Press 1973a
Arwert, F., Venema, G.: Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Molec. gen. Genet. 123, 185–198 (1973b)
Buitenwerf, J., Venema, G.: Transformation in Bacillus subtilis. Fate of transforming DNA in transformation-deficient mutants. Molec. gen. Genet. 151, 203–213 (1977)
Cahn, F.H., Fox, M.S.: Fractionation of transformable bacteria from competent cultures of Bacillus subtilis on Renografin gradients. J. Bact. 95, 867–875 (1968)
Davidoff-Abelson, R., Dubnau, D.: Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded deoxyribonucleic acid of donor origin. J. Bact. 116, 146–153 (1973a)
Davidoff-Abelson, R., Dubnau, D.: Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis. J. Bact. 116, 154–162 (1973b)
Dubnau, D., Cirigliano, C.: Fate of transforming DNA following uptake by competent Bacillus subtilis. VI. Non-covalent association of donor and recipient DNA. Molec. gen. Genet. 120, 101–106 (1973a)
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. molec. Biol. 56, 209–221 (1971)
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. Bact. 114, 273–286 (1973b)
Haest, C.W.M., Verkleij, A.J., De Gier, J., Scheek, R., Ververgaert, P.H.J., Van Deenen, L.L.M.: The effect of lipid phase transition on the architecture of bacterial membranes. Biochem. biophys. Acta (Amst.) 356, 17–26 (1974)
Joenje, H., Venema, G.: Different nuclease activities in competent and non-competent Bacillus subtilis. J. Bact. 122, 25–33 (1975)
Kooistra, J., Venema, G.: Effect of temperature on the fate of donor DNA in transformation of Haemophilus influenzae. In: Informative molecules in biological systems (L.G.H. Ledoux, ed.) Amsterdam: North-Holland Publishing Company 1971
Lacks, S.: Molecular fate of DNA in genetic transformation of Pneumococcus. J. molec. Biol. 5, 119–131 (1962)
Lacks, S., Greenberg, B.: Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae. J. molec. Biol. 101, 255–275 (1976)
Levine, J.S., Strauss, N.: Lag period characterizing the entry of transforming deoxyribonucleic acid into Bacillus subtilis. J. Bact. 89, 281–287 (1965)
Morrison, D.A.: Early intermediate state of transforming DNA during uptake by Bacillus subtilis. J. Bact. 108, 38–44 (1971)
Piechowska, M., Fox, M.S.: Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bact. 108, 680–689 (1971)
Schmid, R.W., Reilley, C.N.: New complexon for titration of calcium in the presence of magnesium. Anal. Chem. 29, 264–268 (1957)
Seto, H., Lopez, R., Tomasz, A.: Cell surface-located deoxyribonucleic acid receptors in transformable Pneumococci. J. Bact. 122, 1339–1350 (1975)
Seto, H., Tomasz, A.: Early stages in DNA binding and uptake during genetic transformation of Pneumococci. Proc. nat. Acad. Sci. (Wash.) 71, 1493–1498 (1974)
Seto, H., Tomasz, A.: Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of compotent Pneumococci. J. Bact. 126, 1113–1118 (1976)
Spizizen, J.: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. nat. Acad. Sci. (Wash.) 44, 1072–1078 (1958)
Venema, G., Pritchard, R.H., Venema-Schröder, T.: Fate of transforming deoxyribonucleic acid in Bacillus subtilis. J. Bact. 89, 1250–1255 (1965)
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Buitenwerf, J., Venema, G. Transformation in Bacillus subtilis: Biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells. Molec. Gen. Genet. 156, 145–155 (1977). https://doi.org/10.1007/BF00283487
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DOI: https://doi.org/10.1007/BF00283487