Summary
Using plasmid pHV60, which contains a chloramphenicol resistance (Cmr) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cmr transformants had the mutant phenotype as expected. However, with a frequency of approximately 3×10-4 atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cmr marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.
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References
Biswal N, Kleinschmidt AK, Spatz HC, Trauntner TA (1967) Physical properties of the DNA of bacteriophage SP50. Mol Gen Genet 100:39–55
Bron S, Venema G (1972) Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterisation of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat Res 15:1–10
Burton K (1956) A study of the mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315–323
Davidoff-Abelson R, Dubnau D (1973) Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded deoxyribonucleic acid of donor origin. J Bacteriol 116:146–153
Dedonder RA, Lepesant JA, Lepesant-Keizalarova J, Billant A, Steinmetz M, Kunst F (1977) Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis. Appl Environ Microbiol 33:989–993
Fani R, Mastromei G, Polsinelli M, Venema G (1984) Isolation and characterization of Bacillus subtilis mutants altered in competence. J Bacteriol 157: 152–157
Henner DJ, Hoch JA (1980) The Bacillus subtilis chromosome. Microbiol Rev 44:57–82
Hoch FA, Barot M, Anagnostopoulos C (1967) Transformation and transduction in recombination-defective mutants of Bacillus subtilis. J Bacteriol 122: 25–33
Hohn B (1979) In vitro packaging of λ and cosmid DNA. Methods Enzymol 68:299–309
Ish-Horowicz D, Burke FJ (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2999
Janniere L, Niaudet B, Pierre E, Ehrlich SD (1985) Stable gene amplification in the chromosome of Bacillus subtilis Gene 40:47–55
Kok J, van Dijl JM, van der Vossen JMBM, Venema G (1985) Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol 50:94–101
Magnusson K, Hederstedt L, Rutberg L (1985) Cloning and expression in Escherichia coli of Sdh A, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex. J Bacteriol 162:1180–1185
Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Michel B, Niaudet B, Ehrlich SD (1983) Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids. Plasmid 10:1–10
Mulder JA, Venema G (1982) Isolation and partial characterization of Bacillus subtilis mutants impaired in DNA entry. J Bacteriol 150:260–268
Nester E, Lederberg J (1961) Linkage of genetic units of Bacillus subtilis in DNA transformation. Proc Natl Acad Sci USA 47:52–55
Niaudet B, Gose A, Ehrlich SD (1982) Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning. Gene 19:277–284
Piechowska M, Fox MS (1971) Fate of transforming deoxyribonucleate in Bacillus subtilis J Bacteriol 108:680–689
Rigby PWJ, Dieckman M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251
Smith H, de Vos W, Bron S (1983a) Transformation in Bacillus subtilis. Properties of DNA-binding-deficient mutants. J Bacteriol 153:12–20
Smith H, Wiersma K, Bron S, Venema G (1983b) Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein. J Bacteriol 156:101–108
Smith H, Wiersma K, Bron S, Venema G (1984) Transformation in Bacillus subtilis: a 75,000-dalton protein complex is involved in binding and entry of donor DNA. J Bacteriol 157:733–738
Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078
Venema G, Pritchard RH, Venema-Schroder T (1965) Fate of transforming deoxyribonucleic acid in Bacillus subtilis. J Bacteriol 89:1250–1255
Vermeulen CA, Venema G (1972) The effect of competence regime on competence, DNA absorption and integration of DNA in cultures of Bacillus subtilis. J Gen Microbiol 71:415–424
de Vos WM, Venema G (1982) Transformation of Bacillus subtilis competent cells: Identification of a protein involved in recombination. Mol Gen Genet 187:439–445
Youngman PJ, Perkins JB, Losick R (1983) Genetic transposition and insertional mutagenesis in Bacillus subtilis with the Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci USA 80:2305–2309
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Communicated by W. Gajewski
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Vosman, B., Kooistra, J., Olijve, J. et al. Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism. Molec Gen Genet 204, 524–531 (1986). https://doi.org/10.1007/BF00331035
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DOI: https://doi.org/10.1007/BF00331035