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Volume 141, Issue 3

protoxin: location of toxic border and requirement of non-toxic domain for high-level production of active toxin Free

Abstract

Summary

Insecticidal crystal proteins, or protoxins, of are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination codons were introduced from the 3′-end of the DNA sequence encoding the toxic domain of the 1155-residue gene product. The mutated and the intact genes were placed under the control of the inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae. Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin. Furthermore, the non-toxic protein seemed to be unstable inthe extracts. To investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both and . The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous . Upon induction of PrecA in , and upon sporulation in , there was a large accumulation of gene products which formed inclusion bodies. The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive. Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble. Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.

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Keyword(s): Bacillus thuringiensis subsp , berliner 1715 , crystal protein , deletion analysis and insecticide
© Society for General Microbiology 1995
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1995-03-01
2024-04-20
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References

  1. Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. 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]
  2. Aronson A. I., Beckman W., Dunn P. 1986; Bacillus thuringiensis and related insect pathogens. Microbiol Rev 50:1–24
     [Google Scholar]
  3. Aronson A. I., Han E.-S., Mcgaughey W., Johnson D. 1991; The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects. Appl Environ Microbiol 57:981–986
     [Google Scholar]
  4. Bulla L. A. Jr, Kramer K. J., Davidson L. I. 1977; Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis . J Bacteriol 130:375–383
     [Google Scholar]
  5. Bulla L. A. Jr, Bechtel D. B., Kramer K. J., Shethna Y. I., Aronson A. I., Fitz-James P. C. 1980; Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis . CRC Crit Rev Microbiol 8:147–204
     [Google Scholar]
  6. Chamberlain J. P. 1979; Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem 98:132–135
     [Google Scholar]
  7. Chung C. T., Miller R. H. 1988; A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res 16:3580
     [Google Scholar]
  8. Ge A. Z., Shivarova N. I., Dean D. H. 1989; Location of the Bombyx mori specificity domain on a Bacillus thuringiensis δ-endotoxin protein. Proc Natl Acad Sci USA 86:4037–4041
     [Google Scholar]
  9. Goodman N. S., Gottfried R. J., Rogoff M. H. 1967; Biphasic system for separation of spores crystals of Bacillus thuringiensis . J Bacteriol 94:485
     [Google Scholar]
  10. Haider M. Z., Ellar D. J. 1989; Functional mapping of an entomocidal δ-endotoxin. Single amino acid changes produced by sitrected mutagenesis influence toxicity and specificity of the protein. J mol Biol 208:183–194
     [Google Scholar]
  11. Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. 1989; A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17:6545–6551
     [Google Scholar]
  12. Höfte H., De Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., Van Montagu M., Zebeau M., Vaeck M. 1986; Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem 161:273–280
     [Google Scholar]
  13. Höfte H., Whiteley H. R. 1989; Insecticidal crystal proteins of Bacillus thuringiensis . Microbiol Rev 53:242–255
     [Google Scholar]
  14. 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]
  15. Klier A., Fargette F., Ribier J., Rapoport G. 1982; Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715 . EMBO J 1:791–799
     [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
     [Google Scholar]
  17. Li J., Carroll J. , Ellar D. J. 1991; Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. Nature 353:815–821
     [Google Scholar]
  18. Little J. W., Mount D. W. 1982; The SOS regulatory system of Escherichia coli . Cell 29:11–22
     [Google Scholar]
  19. Lüthy P., Ebersold H. R. 1981 Bacillus thuringiensis δ-endotoxin: histopathology and molecular mode of action. In Pathogenesis of Invertebrate Microbial Diseases pp 235–267 Edited by Davidson E. W. Totowa, NJ: Allenheld, Osmun & Co;
     [Google Scholar]
  20. Nakamura K., Oshie K., Shimizu M., Takada Y., Oeda K., Ohkawa H. 1990; Construction of chimeric insecticidal proteins between the 130-kDa and 135-kDa proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship. Agric Biol Chem 54:715–724
     [Google Scholar]
  21. Pao-lntara M., Angsuthanasombat C., Panyim S. 1988; The mosquito larvicidal activity of 130 delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment. Biochem Biophys Res Commun 153:294–300
     [Google Scholar]
  22. Peterson G. L. 1977; A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356
     [Google Scholar]
  23. Rapoport G., Klier A., Billault A., Fargette F., Dedonder R. 1979; Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Mol & Gen Genet 176:239–245
     [Google Scholar]
  24. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  25. Sancar A., Hack A. M., Rupp W. D. 1977; Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
     [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
     [Google Scholar]
  27. Schnepf H. E., Whiteley H. R. 1985; Delineation of a toxin-encoding segment o Bacillus thuringiensis crystal protein gene. J Biol Chem 260:6273–6280
     [Google Scholar]
  28. Schnepf H. E., Tomczak K., Ortega J. P., Whiteley H. R. 1990; Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis . J Biol Chem 265:20923–20930
     [Google Scholar]
  29. Schurter W., Geiser M., Mathé D. 1989; 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
     [Google Scholar]
  30. Sharpe E. S., Nickerson K. W., Bulla L. A. Jr, Aronson J. N. 1975; Separation of spores and parasporal crystals of Bacillus thuringiensis in gradients of certain X-ray contrasting agents. Appl Microbiol 30:1052–1053
     [Google Scholar]
  31. Shirakawa M., Tsurimoto T., Matsubara K. 1984; Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli . Gene 28:127–132
     [Google Scholar]
  32. Stahly D. P., Dingman D. W., Bulla L. A. Jr, Aronson A. I. 1978; Possible origin and function of the parasporal crystals in Bacillus thuringiensis . Biochem Biophys Res Commun 84:581–588
     [Google Scholar]
  33. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived syster insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
     [Google Scholar]
  34. Wabiko H., Held G. A., Bulla L. A. Jr 1985; Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity. Appl Environ Microbiol 49:706–708
     [Google Scholar]
  35. Wabiko H., Raymond K. C., Bulla L. A. Jr. 1986; Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis. DNA 5:305–314
     [Google Scholar]
  36. Whiteley H. R., Schnepf H. E. 1986; The molecular biology of parasporal crystal body formation in Bacillus thuringiensis . Annu Rev Microbiol 40:549–576
     [Google Scholar]
  37. Widner W. R., Whiteley H. R. 1990; Location of the dipteran specificity regin a lepidopteran-dipteran crystal protein from Bacillus thuringiensis . J Bacteriol 172:2826–2832
     [Google Scholar]
  38. Wong H. C., Chang S. 1986; Identification of a positive retroregulator that stabilizes mRNAs in bacteria. Proc Natl Acad Sci USA 83:3233–3237
     [Google Scholar]
  39. Wong H. C., Schnepf H. E., Whiteley H. R. 1983; Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem 258:1960–1967
     [Google Scholar]
  40. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103–119
     [Google Scholar]
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Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin
Microbiology 141, 629 (1995); https://doi.org/10.1099/13500872-141-3-629
/content/journal/micro/10.1099/13500872-141-3-629
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