Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1990 Aug 15;270(1):133–136. doi: 10.1042/bj2700133

The construction of Bacillus thuringiensis strains expressing novel entomocidal delta-endotoxin combinations.

N Crickmore 1, C Nicholls 1, D J Earp 1, T C Hodgman 1, D J Ellar 1
PMCID: PMC1131688  PMID: 2168699

Abstract

Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahmad W., Ellar D. J. Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein. FEMS Microbiol Lett. 1990 Mar 1;56(1-2):97–104. doi: 10.1016/0378-1097(90)90132-a. [DOI] [PubMed] [Google Scholar]
  2. Angsuthanasombat C., Chungjatupornchai W., Kertbundit S., Luxananil P., Settasatian C., Wilairat P., Panyim S. Cloning and expression of 130-kd mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli. Mol Gen Genet. 1987 Jul;208(3):384–389. doi: 10.1007/BF00328128. [DOI] [PubMed] [Google Scholar]
  3. Ballester S., Lopez P., Espinosa M., Alonso J. C., Lacks S. A. Plasmid structural instability associated with pC194 replication functions. J Bacteriol. 1989 May;171(5):2271–2277. doi: 10.1128/jb.171.5.2271-2277.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banner C. D., Moran C. P., Jr, Losick R. Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis. J Mol Biol. 1983 Aug 5;168(2):351–365. doi: 10.1016/s0022-2836(83)80023-0. [DOI] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bone E. J., Ellar D. J. Transformation of Bacillus thuringiensis by electroporation. FEMS Microbiol Lett. 1989 Apr;49(2-3):171–177. doi: 10.1016/0378-1097(89)90033-5. [DOI] [PubMed] [Google Scholar]
  7. Brown K. L., Whiteley H. R. Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4166–4170. doi: 10.1073/pnas.85.12.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bulla L. A., Jr, Bechtel D. B., Kramer K. J., Shethna Y. I., Aronson A. I., Fitz-James P. C. Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis. Crit Rev Microbiol. 1980;8(2):147–204. doi: 10.3109/10408418009081124. [DOI] [PubMed] [Google Scholar]
  9. Carroll J., Li J., Ellar D. J. Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis. Biochem J. 1989 Jul 1;261(1):99–105. doi: 10.1042/bj2610099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chilcott C. N., Ellar D. J. Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro. J Gen Microbiol. 1988 Sep;134(9):2551–2558. doi: 10.1099/00221287-134-9-2551. [DOI] [PubMed] [Google Scholar]
  11. Donovan W. P., Dankocsik C., Gilbert M. P. Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1988 Oct;170(10):4732–4738. doi: 10.1128/jb.170.10.4732-4738.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer H. M., Lüthy P., Schweitzer S. Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer. Arch Microbiol. 1984 Oct;139(2-3):213–217. doi: 10.1007/BF00402002. [DOI] [PubMed] [Google Scholar]
  13. González J. M., Jr, Brown B. J., Carlton B. C. Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6951–6955. doi: 10.1073/pnas.79.22.6951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. González J. M., Jr, Carlton B. C. Patterns of plasmid DNA in crystalliferous and acrystalliferous strains of Bacillus thuringiensis. Plasmid. 1980 Jan;3(1):92–98. doi: 10.1016/s0147-619x(80)90038-4. [DOI] [PubMed] [Google Scholar]
  15. Haider M. Z., Ellar D. J. Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin. Biochem J. 1987 Nov 15;248(1):197–201. doi: 10.1042/bj2480197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haider M. Z., Ward E. S., Ellar D. J. Cloning and heterologous expression of an insecticidal delta-endotoxin gene from Bacillus thuringiensis var. aizawai IC1 toxic to both lepidoptera and diptera. Gene. 1987;52(2-3):285–290. doi: 10.1016/0378-1119(87)90055-2. [DOI] [PubMed] [Google Scholar]
  17. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  18. Heierson A., Landén R., Lövgren A., Dalhammar G., Boman H. G. Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA. J Bacteriol. 1987 Mar;169(3):1147–1152. doi: 10.1128/jb.169.3.1147-1152.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herrnstadt C., Gilroy T. E., Sobieski D. A., Bennett B. D., Gaertner F. H. Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego. Gene. 1987;57(1):37–46. doi: 10.1016/0378-1119(87)90174-0. [DOI] [PubMed] [Google Scholar]
  20. Höfte H., Seurinck J., Van Houtven A., Vaeck M. Nucleotide sequence of a gene encoding an insecticidal protein of Bacillus thuringiensis var. tenebrionis toxic against Coleoptera. Nucleic Acids Res. 1987 Sep 11;15(17):7183–7183. doi: 10.1093/nar/15.17.7183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martin P. A., Lohr J. R., Dean D. H. Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid. J Bacteriol. 1981 Feb;145(2):980–983. doi: 10.1128/jb.145.2.980-983.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Peeters B. P., de Boer J. H., Bron S., Venema G. Structural plasmid instability in Bacillus subtilis: effect of direct and inverted repeats. Mol Gen Genet. 1988 Jun;212(3):450–458. doi: 10.1007/BF00330849. [DOI] [PubMed] [Google Scholar]
  24. Peijnenburg A. C., Bron S., Venema G. Plasmid deletion formation in Bacillus subtilis. Plasmid. 1988 Jul;20(1):23–32. doi: 10.1016/0147-619x(88)90004-2. [DOI] [PubMed] [Google Scholar]
  25. Sekar V., Thompson D. V., Maroney M. J., Bookland R. G., Adang M. J. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7036–7040. doi: 10.1073/pnas.84.20.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shibano Y., Yamagata A., Nakamura N., Iizuka T., Sugisaki H., Takanami M. Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein. Gene. 1985;34(2-3):243–251. doi: 10.1016/0378-1119(85)90133-7. [DOI] [PubMed] [Google Scholar]
  27. Stewart G. S., Johnstone K., Hagelberg E., Ellar D. J. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J. 1981 Jul 15;198(1):101–106. doi: 10.1042/bj1980101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomas W. E., Ellar D. J. Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J Cell Sci. 1983 Mar;60:181–197. doi: 10.1242/jcs.60.1.181. [DOI] [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Waalwijk C., Dullemans A. M., van Workum M. E., Visser B. Molecular cloning and the nucleotide sequence of the Mr 28 000 crystal protein gene of Bacillus thuringiensis subsp. israelensis. Nucleic Acids Res. 1985 Nov 25;13(22):8207–8217. doi: 10.1093/nar/13.22.8207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ward E. S., Ellar D. J. Cloning and expression of two homologous genes of Bacillus thuringiensis subsp. israelensis which encode 130-kilodalton mosquitocidal proteins. J Bacteriol. 1988 Feb;170(2):727–735. doi: 10.1128/jb.170.2.727-735.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ward E. S., Ellar D. J., Todd J. A. Cloning and expression in Escherichia coli of the insecticidal delta-endotoxin gene of Bacillus thuringiensis var. israelensis. FEBS Lett. 1984 Oct 1;175(2):377–382. doi: 10.1016/0014-5793(84)80772-3. [DOI] [PubMed] [Google Scholar]
  33. Ward E. S., Ridley A. R., Ellar D. J., Todd J. A. Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J Mol Biol. 1986 Sep 5;191(1):13–22. doi: 10.1016/0022-2836(86)90418-3. [DOI] [PubMed] [Google Scholar]
  34. Yamamoto T., McLaughlin R. E. Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. Kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem Biophys Res Commun. 1981 Nov 30;103(2):414–421. doi: 10.1016/0006-291x(81)90468-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES