Summary
Recombinant plasmid DNA cloned in E. coli via the bifunctional vector pDH5060 suffered deletions when returned to B. subtilis. However, DNA preparations of identical chimeras containing homologous or heterologous sequences stably transformed B. subtilis at high efficiency when isolated from B. subtilis. The vector pDH5060, however, was not affected and could be stably shuttled between E. coli and B. subtilis at high frequency. These problems affected the transfer of clone pools and individual chimeras, irrespective of the restriction or recombination phenotype of B. subtilis recipients. Deleted chimeras lost at least one end of cloned inserts, and in most cases, flanking plasmid sequences. Single plasmid forms (intact or deleted) were isolated from several hundred individual Cmr-transformants this suggests that events leading to deletion of chimeric plasmid DNA occur during transformation by restriction of unmodified insert sequences propagated in the intermediate host, E. coli. This conclusion is discussed with regard to the mechanism of plasmid transformation in B. subtilis.
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References
Arrand JR, Myers PA, Roberts RJ (1978) A new restriction endonuclease from Streptomyces albus G. J Mol Biol 118:127–135
Bensi G, Igleasias A, Canosi U, Trautner TA (1981) Plasmid transformation in Bacillus subtilis. The significance of partial homology between plasmid and recipient cell DNAs. Mol Gen Genet 184:400–404
Bernhard K, Schrempf H, Goebel W (1978) Bacteriocin and antibiotic resistance in Bacillus cereus and Bacillus subtilis. J Bacteriol 133:897–903
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Bron S, Murray K, Trautner TA (1975) Restriction modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R. Mol Gen Genet 143:13–23
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 S (ed) Microbial drug resistance. University Park Press, Baltimore, Maryland, pp 23–32
Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1620–1682
Ehrlich SD, Jupp S, Niadet B, Goze A (1978) In: Boyer HW and Nicosia S (eds) Genetic engineering. Elsevier/North-Holland, Amsterdam, pp 25–32
Fouet A, Klier A, Rapoport G (1982) Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis. Mol Gen 186:399–404
Goebel W, Kreft J, Bernhard K, Schremph H, Weidinger G (1978) In: Boyer HW, Nicosia S (eds) Genetic engineering. Elsevier/North-Holland, Amsterdam, pp 47–58
Grandi G, Mottes M, Sgaramelli 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
Gryzcan TJ, Contente S, Dubnau D (1978) Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol 134:318–329
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
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Klein RD, Selsing E, Wells RD (1980) A rapid microscal technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91
Kreft J, Bernhard K, Goebel W (1978) Recombinant plasmids capable of replication in B. subtilis and E. coli. Mol Gen Genet 162:59–67
Marcina FL, Jones KR, Wood PH (1980) Chimeric streptococcal plasmids and their use as molecular cloning vehicles in Streptococcus sanguis (Challis). J Bacteriol 143:1425–1435
Michel B, Palla E, Niaudet B, Ehrlich SD (1980) DNA cloning in Bacillus subtilis: III Efficiency of random-segment cloning and insertional inactivation vectors. Gene 12:147–154
O'Farrell Ph, Kutter E, Nakanishi M (1980) A restriction map of the bacteriophage T4 genome. Mol Gen Genet 179:421–435
Ostroff GR, Pène JJ (1984) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: construction and analysis of B. subtilis clone banks in Escherichia coli. Mol Gen Genet 193:299–305
Ostroff GR, Pène JJ (1983) Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. III. Isolation of a spontaneous mutant of B. subtilis with enhanced transformability for Escherichia coli propagated chimeric plasmid DNA. J Bacteriol 156:934–936
Proverez AA, Belova TS, Surikov NN (1980) Transformation and transduction of Bacillus subtilis strains with the BsuR restriction-modification system by means of modified and unmodified DNA of pUB110 plasmid. Mol Gen Genet 180:135–138
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 176:239–245
Scher B, Dubnau D (1976) Purification and properties of a manganese-stimulated endonuclease from Bacillus subtilis. J Bacteriol 126:429–438
Shibato T, Ando T (1974) Host controlled modification and restriction in Bacillus subtilis. Mol Gen Genet 131:275–280
Shibato T, Ikawa S, Kim C, Ando T (1976) Site-specific deoxyribonucleases in Bacillus subtilis and other Bacillus strains. J Bacteriol 128:473–476
Tanaka T (1979) Restriction of plasmid-mediated transformation in Bacillus subtilis 168. Mol Gen Genet 175:235–237
Uhlén M, Flock JI, Philipson L (1981) RecE independent deletions of recombinant plasmids in Bacillus subtilis. Plasmid 6:161–169
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Communicated by G.A. O'Donovan
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Ostroff, G.R., Pène, J.J. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis . Molec Gen Genet 193, 306–311 (1984). https://doi.org/10.1007/BF00330685
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DOI: https://doi.org/10.1007/BF00330685