Summary
Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tcr gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec+ recipients. Loss of sequences from hybrid plasmids was not prevented in a r - m - recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.
Similar content being viewed by others
References
Bochner BR, Huang HC, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933
Bolivar F, Backman K (1979) Plasmids of Escherichia coli as clonning vectors. Methods Enzymol 68:245–267
Bott KF, Wilson FE, Stewart GC (1981) Characterization of Bacillus subtilis rRNA genes. In: Levinson HS, Sonenshein AL, Tipper DJ (eds) Sporulation and germination. Am Soc Microbiol. Washington DC, pp 119–122
Boyer HW, Roulland-Dussoix (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472
Canosi U, Morelli G, Trautner TA (1978) The relationship between molecular structure and transformation efficiency of some Staphylococcus aureus plasmids isolated from Bacillus subtilis. Mol Gen Genet 166:259–267
Clewell DB, Yagi Y, Tomich P (1979) In: Mitsuhashi (ed) Microbiol resistance. University Park Press, Baltimore, Maryland, pp 23–32
Contente S, Dubnau D (1979) Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. Mol Gen Genet 167:251–258
Dedonder RA, Lepesant JA, Lepesant-Kejzlarova J, Billault A, Steinmetz M, Kunst F (1977) Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol 33:989–993
Dubnau D, Davidoff-Abelson R, Scher B, Cirigliano C (1973) Fate of transforming DNA after uptake by competent Bacillus subtilis: phenotypic characterization of radiation sensitive recombination-deficient mutants. J Bacteriol 114:273–286
Ducan CH, Wilson GA, Young FE (1978) Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proc Natl Acad Sci USA 75:3664–3668
Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1620–1682
Ehrlich SD (1978) DNA cloning in Bacillus subtilis. Proc Natl Acad Sci USA 75:1433–1436
Erickson RJ (1976) Industrial applications of the Bacilli: a review and prospectus. In: Schlessinger D (ed) Microbiology-1976. Am Soc Microbiol, Washington DC, pp 406–419
Ferrari FA, Lang D, Ferrari E, Hoch J (1982) Molecular cloning of the spOB sporulation locus in bacteriophage lambda. J Bacteriol 152:809–814
Gray O, Chang S (1981) Molecular cloning and expression of Bacillus licheniformis β-lactamase gene in Escherichia coli and Bacillus subtilis. J Bacteriol 145:422–428
Greene PJ, Betlach MC, Goodman HM, Boyer HW (1974) The EcoRI restriction endonuclease. In: Wickner RB (ed) Methods in molecular biology series, DNA replication and biosynthesis; vol 7. Marcel Dekker, Inc, New York, pp 87–111
Gryzcan TJ, Contente S, Dubnau D (1978) Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol 134:318–323
Gryzcan TJ, Contente S, Dubnau D (1980) Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis. Mol Gen Genet 177:459–467
Haldenwang WG, Banner CDB, Ollington JF, Losick R, Hoch JA, O'Connor MB, Sonensheim AL (1980) Mapping of a cloned gene under sporulation control by insertion of a drug resistance marker into the Bacillus subtilis chromosome. J Bacteriol 142:90–98
Hemphill HE, Whiteley HR (1975) Bacteriophages of Bacillus subtilis. Bacteriol Rev 39:257–315
Henner DJ, Hoch JA (1980) The Bacillus subtilis chromosome. Microbiol Rev 44:57–82
Hoch JA (1978) Developmental genetics at the beginning of a new era. In: Chambliss G, Vary JC (eds) Spores VIII. Am Soc Microbiol, Washington DC, pp 119–121
Hughes RC (1970) Autolysis of isolated cell walls of Bacillus licheniformis NTC 6346 and Bacillus subtilis Marburg strain 168. Preparation of products and characterization of the mucopeptide fragments. Biochem J 119:849–860
Hutchinson KW, Halvorson HO (1980) Cloning of randomly cleaved DNA fragments from a 105 lysogen of Bacillus subtilis: identification of prophage containing clones. Gene 8:267–278
Kawamura F, Saito H, Ikeda Y (1979) A method for construction of specialized transducing phage P11 of Bacillus subtilis. Gene 5:87–91
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
Kreft J, Bernhard K, Goebel W (1978) Recombinant plasmids capable of replication in B. subtilis and E. coli. Mol Gen Genet 162:59–67
Kupersztoch YM, Helinski DR (1973) A catenated DNA molecule as an intermediate in the replication of the resistance transfer factor R6K in Escherichia coli. Biochem Biophys Res Comm 54:1451–1459
Lederberg EM, Cohen SN (1974) Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol 199:1072–1074
Lovett PS, Bramucci MG (1976) Plasmid deoxyribonucleic acid in Bacillus subtilis and Bacillus pumilis. In: Schlesinger D (ed) Microbiology 1976. Amer Soc for Micro, Washington DC, pp 388–393
Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1112
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acids from micro-organisms. J Mol Biol 3:208–218
Nester EW, Schafer M, Lederberg J (1963) Gene linkage in DNA transfer: A cluster of genes concerned with aromatic biosynthesis in Bacillus subtilis. Genetics 48:529–551
Radloff R, Bauer W, Vinograd J (1967) A dye-buoyant density method for the detection and isolation of closed circular duplex DNA; the closed circular DNA in HeLa cells. Proc Natl Acad Sci USA 57:1514–1521
Rapoport G, Klier A, Billault F, Fargette F, Dedonder R (1979) Construction of a colony bank of Escherichia coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis. Mol Gen Genet 176:236–245
Shibato T, Ando T (1974) Host controlled modification and restriction in Bacillus subtilis. Mol Gen Genet 131:275–280
Shure M, Vinograd J (1976) The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method. Cell 8:215–226
Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078
Struhl K, Cameron JR, Davis RW (1976) Functional genetic expression of eucaryotic DNA in Escherichia coli. Proc Natl Acad Sci USA 73:1471–1475
Tanaka T (1979) Restriction of plasmid-mediated transformation in Bacillus subtilis 168. Mol Gen Genet 175:235–237
Tanaka T, Kawano N (1980) Cloning vehicles for the homologous Bacillus subtilis host-vector system. Gene 10:131–136
Uhlen M, Flock JI, Phillipson L (1981) RecE independent deletions of recombinant plasmids in Bacillus subtilis. Plasmid 6:161–169
Author information
Authors and Affiliations
Additional information
Communicated by G.A. O'Donovan
Rights and permissions
About this article
Cite this article
Ostroff, G.R., Pène, J.J. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis . Molec Gen Genet 193, 299–305 (1984). https://doi.org/10.1007/BF00330684
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330684