Summary
A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycolinduced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4x107 transformants per μg of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.
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Bahl, C.P., Marians, K.J., Wu, R., Strawinsky, J., Narang, S.: A general method for inserting specific DNA sequences into cloning vehicles. Gene 1, 81–92 (1976)
Bernhard, K., SChrempf, H., Goebel, W.: Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J. Bact. 133, 897–903 (1978)
Bettinger, G.E., Young, F.E.: Transformation of Bacillus subtilis: Transforming ability of deoxyribunucleic acid in lysates of L-forms or protoplasts. J. Bact. 122, 987–993 (1975)
Bibb, M.J., Ward, J.M., Hopwood, D.A.: Transformation of plasmid DNA into Streptomyces at high frequency. Nature 274, 388–400 (1978)
Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Helling, R.B.: Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA. 70, 3240–3244 (1973)
Cohen, S.N., Chang, A.C.Y., Hsu, I.: Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA. 69, 2110–2114 (1972)
Dubnau, D.: Genetic transformation in Bacillus subtilis: A review with emphasis on the recombination mechanism. In: Microbiology-1976 (D. Schlessinger, es.), pp. 14–27. Wash, D.C.: American Society Microbiology, 1976
Duncan, C.H., Wilson, G.A., Young, F.E.: Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 75, 3664–3668 (1978)
Ehrlich, S.D.: Replication and expression of plasmids from Staphyloccus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 74, 1680–1682 (1977)
Ehrilich, S.D.: DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 75, 1433–1436 (1978)
Fodor, K., Alföldi, L.: Fusion of protoplasts of Bacillus megaterium. Proc. Natl. Acad. Sci. USA. 73, 2147–2150 (1976)
Gryczan, T.J., Contente, S., Dubnau, D.: Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bact. 134, 318–329 (1978)
Gryczan, T.J., Dubnau, D.: Construction and properties of chimeric plasmids in Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 75, 1428–1432 (1978)
Hinnen, A., Hicks, J.B., Fink, G.R.: Transformation of yeast. Proc. Natl. Acad. Sci. USA. 75, 1929–1933 (1978)
Landman, O.E., Ryter, A., Fréhel, C.: Gelatin-induced reversion of protoplasts of Bacillus subtilis to the bacillary form: Electronmicroscope and physical study. J. Bact. 96, 2154–2170 (1968)
Le Hégarat, J.-C., Anagnostopoulos, C.: Detection and characterization of naturally occurring plasmids in Bacillus subtilis. Mol. Gen. Genet. 157, 167–174 (1977)
Lovett, P.S., Bramucci, M.G.: Plasmid deoxyribonucleic acid in Bacillus subtilis and Bacillus pumilus. J. Bact. 124, 484–490 (1975)
Notani, N.K., Setlow, J.K.: Mechanisms of bacterial transformation and transfection, pp. 39–100. In: Progress in nucleic acid research and molecular biology (W.E. Cohn, ed.), Vol. 14. New York: Academic Press, Inc. 1974
Ostro, M.J., Giacomoni, D., Dray, S.: Incorporation of high molecular weight RNA into large artificial lipid vesicles. Biochem. Biophys. Res. Comm. 76, 836–842 (1977)
Schaeffer, P., Cami, G., Hotchkiss, R.D.: Fusion of bacterial protoplasts. Proc. Natl. Acad. Sci. USA. 73, 2151–2155 (1976)
Sharp, P.A., Sugden, B., Sambrok, J.: Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055–3063 (1973)
Tanaka, T., Kuroda, M., Sakaguchi, K.: Isolation and characterization of four plasmids from Bacillus subtilis. J. Bact. 129, 1487–1494 (1977)
Wyrick, P.B., Rogers, H.J.: Isolation and characterization of cell wall-defective variants of Bacillus subtilis and Bacillus licheniformis. J. Bact. 116, 456–465 (1973)
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Communicated by H.W. Boyer
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Chang, S., Cohen, S.N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molec. Gen. Genet. 168, 111–115 (1979). https://doi.org/10.1007/BF00267940
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DOI: https://doi.org/10.1007/BF00267940