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Efficient transformation of Bacillus thuringiensis and B. cereus via electroporation: Transformation of acrystalliferous strains with a cloned delta-endotoxin gene

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Summary

Electroporation was used as a method to transform intact cells of Bacillus thuringiensis and B. cereus. With our optimized method a range of plasmid vectors could be transformed into strains of B. thuringiensis at frequencies of up to 107 transformants/μg DNA. This high frequency allows cloning experiments to be bone directly in B. thuringiensis. A bifunctional vector capable of replicating in Escherichia coli and in Bacillus spp. was constructed. The kurhd1 protoxin gene was cloned into this shuttle vector to produce plasmid pXI93, then transformed into B. thuringiensis HDl cryB and B. cereus 569K. The cloned protoxin gene was expressed in sporulating cultures of both strain HD1 cryB (pXI93) and 569K (pXI93), producing crystal protein active in biotests against larvae of Heliothis virescens. This demonstrates the usefulness of the electroporation method for the introduction of cloned toxin genes, in either their native or modified form, into a variety of host strains.

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References

  • Adang MJ, Staver MJ, Rocheleau TA, Leighton J, Barker RF, Thompson DV (1985) Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36:289–300

    Google Scholar 

  • Alikhanian SI, Ryabchenko NF, Bukanov NO, Sakanyan VA (1981) Transformation of Bacillus thuringiensis subsp. galleriae protoplasts by plasmids pBC16. J Bacteriol 146:7–9

    Google Scholar 

  • Aronson AI, Beckman W, Dunn P (1986) Bacillus thruingiensis and related insect pathogens. Microbiol Rev 50:1–24

    Google Scholar 

  • Bernhard K, Schrepf H, Goebel W (1978) Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol 133:897–903

    Google Scholar 

  • Bibb JJ, Ward JM, Hopwood DA (1978) Transformation of plasmid DNA into Streptomycetes at high frequency. Nature 274:398–400

    Google Scholar 

  • Brown BJ, Carlton BC (1980) Plasmid-mediated transformation in Bacillus megaterium. J Bacteriol 142:508–512

    Google Scholar 

  • Carlton BC, Gonzalez JM (1985a) The genetics and molecular biology of Bacillus thuringiensis. In: Dubnau DA (ed) The molecular biology of the bacilli, vol 2. Academic Press, New York, pp 211–249

    Google Scholar 

  • Carlton BC, Gonzalez JM (1985b) Plasmids and delta-endotoxin production in different subspecies of Bacillus thuringiensis. In: Hoch JA, Setlow P (eds) Molecular biology of microbial differentiation. Am Soc Microbiol, Washington, D.C., pp 246–252

    Google Scholar 

  • Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111–115

    Google Scholar 

  • Chassy BM, Flickinger JL (1987) Transformation of Lactobacillus casei by electroporation. FEMS Microbiol Lett 44:173–177

    Google Scholar 

  • Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Google Scholar 

  • Fiedler S, Wirth R (1988) Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem 170:38–44

    Google Scholar 

  • Fischer H-M, Lüthy P, Schweitzer S (1984) Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer. Arch Microbiol 139:213–217

    Google Scholar 

  • Garduno F, Thorne L, Walfield AM, Pollock TJ (1988) Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 54:277–279

    Google Scholar 

  • Geiser M, Schweitzer S, Grimm C (1986) The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HDl. Gene 48:109–118

    Google Scholar 

  • Gryczan T, Shivakumar AG, Dubnau D (1980) Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol 141:246–253

    Google Scholar 

  • Heierson A, Landen R, Lövgren A, Dalhammar G, Boman HG (1987) Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA. J Bacteriol 169:1147–1152

    Google Scholar 

  • Horinouchi S, Weisblum B (1982) Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150:815–825

    Google Scholar 

  • Huber-Lukac H (1982) Zur Struktur des delta-Endotoxins von Bacillus thuringiensis. PhD Thesis, Swiss Federal Institute of Technology, Zurich

    Google Scholar 

  • Klier A, Bourgouin C, Rapoport G (1983) Mating between Bacillus subtilis and Bacillus thuringiensis and transfer of cloned crystal genes. Mol Gen Genet 191:257–262

    Google Scholar 

  • Knutson JC, Yee D (1987) Electroporation: parameters affecting transfer of DNA into mammalian cells. Anal Biochem 164:44–52

    Google Scholar 

  • Kondo JK, McKay LL (1984) Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. Appl Environ Microbiol 48:252–259

    Google Scholar 

  • Mahler I, Halvorson HO (1980) Two erythromycin-resistance plasmids of diverse origin and their effect on sporulation in Bacillus subtilis. J Gen Microbiol 120:259–263

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Martin PA, Lohr JR, Dean DH (1981) Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid. J Bacteriol 145:980–983

    Google Scholar 

  • Miller JF, Dower WJ, Tompkins LS (1988) High-voltage electroporation of bacteria: Genetic transformation of Campylobacter jejeuni with plasmid DNA. Proc Natl Acad Sci USA 85:856–860

    Google Scholar 

  • Minnich SA, Aronson AJ (1984) Regulation of protoxin synthesis in Bacillus thuringiensis. J Bacteriol 158:447–454

    Google Scholar 

  • Obukowicz MG, Perlak FJ, Kusano-Kretzmer K, Mayer EJ, Bolten SL, Watrud LS (1986) Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads. J Bacteriol 168:982–989

    Google Scholar 

  • Polak J, Novick RP (1982) Closely related plasmids from Staphylococcus aureus and soil bacilli. Plasmid 7:152–162

    Google Scholar 

  • Powell IB, Achen MG, Hillier AJ, Davidson BE (1988) A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Appl Environ Microbiol 54:655–660

    Google Scholar 

  • Primrose SB, Ehrlich SD (1981) Isolation of plasmid deletion mutants and study of their instability. Plasmid 6:193–201

    Google Scholar 

  • Schall D (1986) Genübertragung zwischen Isolaten von, Bacillus thuringiensis durch Protoplastentransformation und-Fusion. PhD Thesis, Universität Tübingen

  • Schnepf HE, Whiteley HR (1981) Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci USA 78:2893–2897

    Google Scholar 

  • Schnepf HE, Wong HC, Whiteley HR (1985) The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem 260:6264–6272

    Google Scholar 

  • Shivakumar AG, Gundling GJ, Benson TA, Casuto D, Miller MF, Spear BB (1986) Vegetative expression of the δ-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis. J Bacteriol 166:194–204

    Google Scholar 

  • Shivarova N, Förster W, Jacob H-E, Grigorova R (1983) Microbiological implications of electric fields VII. Stimulation of plasmid transformation of Bacillus cereus protoplasts. Z Allgem Mikrobiol 23:595–599

    Google Scholar 

  • Stahly DP, Dingmann DW, Bulla LA, Aronson AI (1978) Possible origin and function of the parasporal crystals in Bacillus thuringiensis. Biochem Biophys Res Commun 84:581–588

    Google Scholar 

  • Taketo A (1988) DNA transfection of Escherichia coli by electroporation. Biochim Biophys Acta 494:318–324

    Google Scholar 

  • Vieira A, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Wabiko H, Raymond KC, Bulla LA (1986) Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis. DNA 5:305–314

    Google Scholar 

  • Wirth R, An FY, Clewell DB (1986) Highly efficient protoplast transformation system for Steptococcus faecalis and a new Escherichia coli-S faecalis shuttle vector. J Bacteriol 165:831–836

    Google Scholar 

  • Wong HC, Schnepf HE, Whiteley HR (1983) Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem 258:1960–1967

    Google Scholar 

  • Yousten AA, Rogoff MH (1969) Metabolism of Bacillus thuringiensis in relation to spore and crystal formation. J Bacteriol 100:1229–1236

    Google Scholar 

  • Yoshihama M, Higashiro K, Rao EA, Akedo M, Shanabruch WG, Follettie MT, Walker GC, Sinskey AJ (1985) Cloning vector system for Corynebacterium glutamicum. J Bacteriol 162:591–597

    Google Scholar 

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Authors and Affiliations

  1. Biotechnology Department, Ciba-Geigy Ltd., CH-4002, Basel, Switzerland

    Walter Schurter, Martin Geiser & Danièle Mathé

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  1. Walter Schurter
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  2. Martin Geiser
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  3. Danièle Mathé
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Communicated by H. Hennecke

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Schurter, W., Geiser, M. & Mathé, D. Efficient transformation of Bacillus thuringiensis and B. cereus via electroporation: Transformation of acrystalliferous strains with a cloned delta-endotoxin gene. Mol Gen Genet 218, 177–181 (1989). https://doi.org/10.1007/BF00330581

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  • DOI: https://doi.org/10.1007/BF00330581

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