Summary
The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kanr/Neor) and Bg/II digests of pTL12 (Tmpr, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.
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References
Aubert E, Farget F, Fouet A, Klier A, Rapoport G (1982) Use of a bifunctional cosmid for cloning large DNA fragments of B. subtilis. In: Ganesan AT, Chang S, Hoch JA (eds) Molecular cloning and gene regulation in Bacilli. Academic Press, New York, pp 11–24
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Dowling PC, Giorgi C, Roux L, Dethlefsen LA, Galantowicz ME, Blumberg BM, Kolakofsky D (1983) Molecular cloning of the 3′-proximal third of Sendai virus genome. Proc Natl Acad Sci USA 80:5213–5216
Dubnau D, Cirigliano C (1974) Genetic characterization of recombination-deficient mutants of Bacillus subtilis. J Bacteriol 117:488–493
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity—Addendum. Anal Biochem 137:266–267
Fujii M, Sakaguchi K (1980) A site-specific recE4-independent intramolecular recombination between Bacillus subtilis and Staphylococcus aureaus DNAs in hybrid plasmids. Gene 12:95–102
Grandi G, Mottes M, Scaramella V (1981) Specific pattern of instability of Escherichia coli hisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194. Plasmid 6:99–111
Gryczan TJ, Contente S, Dubnau D (1978) Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol 134:318–328
Hahn J, Dubnau D (1985) Analysis of plasmid deletional instability in Bacillus subtilis. J Bacteriol 162:1014–1023
Hofemeister J, Israeli-Reches M, Dubnau D (1983) Integration of plasmid pE194 at multiple sites on the Bacillus subtilis chromosome. Mol Gen Genet 189:58–68
Holtzclaw WD, Chapman LF (1975) Properties of some norvaline-resistant mutants of Bacillus subtilis. J Gen Microbiol 88:289–294
Keggins KM, Lovett PS, Duvall EJ (1978) Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc Natl Acad Sci USA 75:1423–1427
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning — a laboratory manual. Cold Spring Harbor Laboratory, NY
Novick RP, Iordanescu S, Surdeanu M, Edelman I (1981) Transduction-related cointegrate formation between staphylococcal plasmids: a new type of site-specific recombination. Plasmid 6:159–172
Novick RP, Projan SJ, Rosenblum W, Edelman I (1984) Staphylococcal plasmid cointegrates are formed by host and phage-mediated general rec systems that act on short regions of homology. Mol Gen Genet 195:374–377
Ostroff GR, Pene JJ (1984a) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis I. Construction and analysis of B. subtilis clone banks in Escherichia coli. Mol Gen Genet 193:299–305
Ostroff GR, Pene JJ (1984b) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis II. Transfer of sequences propagated in Escherichia coli to B. subtilis. Mol Gen Genet 193:306–311
Sato S, Hutchison III CA, Harris JI (1977) A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc Natl Acad Sci USA 74:542–546
Shishido K, Tanaka Y (1984) A restriction map of Bacillus subtilis tetracycline-resistance plasmid pNS1981. Plasmid 12:65–66
Shishido K, Noguchi N, Kim C, Ando T (1983) Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis. Plasmid 10:224–234
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Tanaka T, Kawano N (1980) Cloning vehicles for the homologous Bacillus subtilis host vector system. Gene 10:131–136
Uhlen M, Flock J-I, Philipson L (1981) RecE-independent deletions of recombinant plasmids in Bacillus subtilis. Plasmid 5:161–169
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Communicated by H. Böhme
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Temeyer, K.B., Chapman, L.F. Intermolecular recE4-independent recombination in Bacillus subtilis: Formation of plasmid pKBT1. Mol Gen Genet 210, 518–522 (1987). https://doi.org/10.1007/BF00327206
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DOI: https://doi.org/10.1007/BF00327206