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. 1989 Jul 1;261(1):99–105. doi: 10.1042/bj2610099

Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

J Carroll 1, J Li 1, D J Ellar 1
PMCID: PMC1138787  PMID: 2549968

Abstract

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

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Selected References

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

  1. Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. 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. 1985;36(3):289–300. doi: 10.1016/0378-1119(85)90184-2. [DOI] [PubMed] [Google Scholar]
  2. Aronson A. I., Beckman W., Dunn P. Bacillus thuringiensis and related insect pathogens. Microbiol Rev. 1986 Mar;50(1):1–24. doi: 10.1128/mr.50.1.1-24.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bechtel D. B., Bulla L. A., Jr Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. J Bacteriol. 1976 Sep;127(3):1472–1481. doi: 10.1128/jb.127.3.1472-1481.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chungjatupornchai W., Höfte H., Seurinck J., Angsuthanasombat C., Vaeck M. Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera. Eur J Biochem. 1988 Apr 5;173(1):9–16. doi: 10.1111/j.1432-1033.1988.tb13960.x. [DOI] [PubMed] [Google Scholar]
  5. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem. 1986 Dec 1;161(2):273–280. doi: 10.1111/j.1432-1033.1986.tb10443.x. [DOI] [PubMed] [Google Scholar]
  9. Knowles B. H., Thomas W. E., Ellar D. J. Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action. FEBS Lett. 1984 Mar 26;168(2):197–202. doi: 10.1016/0014-5793(84)80245-8. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  12. Li J., Henderson R., Carroll J., Ellar D. X-ray analysis of the crystalline parasporal inclusion in Bacillus thuringiensis var. tenebrionis. J Mol Biol. 1988 Feb 5;199(3):543–544. doi: 10.1016/0022-2836(88)90625-0. [DOI] [PubMed] [Google Scholar]
  13. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  14. Northrop J., Slepecky R. A. Sporulation mutations induced by heat in Bacillus subtilis. Science. 1967 Feb 17;155(3764):838–839. doi: 10.1126/science.155.3764.838. [DOI] [PubMed] [Google Scholar]
  15. Schnepf H. E., Whiteley H. R. Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene. J Biol Chem. 1985 May 25;260(10):6273–6280. [PubMed] [Google Scholar]
  16. Schnepf H. E., Wong H. C., Whiteley H. R. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol Chem. 1985 May 25;260(10):6264–6272. [PubMed] [Google Scholar]
  17. 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]
  18. Stewart G. S., Ellar D. J. Precursor processing during the maturation of a spore-coat protein in Bacillus megaterium KM. Biochem J. 1983 Feb 15;210(2):411–417. doi: 10.1042/bj2100411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Tungpradubkul S., Settasatien C., Panyim S. The complete nucleotide sequence of a 130 kDa mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. israelensis. Nucleic Acids Res. 1988 Feb 25;16(4):1637–1638. doi: 10.1093/nar/16.4.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ward E. S., Ellar D. J. Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli. J Mol Biol. 1986 Sep 5;191(1):1–11. doi: 10.1016/0022-2836(86)90417-1. [DOI] [PubMed] [Google Scholar]
  24. Ward E. S., Ellar D. J. Nucleotide sequence of a Bacillus thuringiensis var. israelensis gene encoding a 130 kDa delta-endotoxin. Nucleic Acids Res. 1987 Sep 11;15(17):7195–7195. doi: 10.1093/nar/15.17.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]

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