Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Dec;61(12):4230–4235. doi: 10.1128/aem.61.12.4230-4235.1995

Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein.

A Delécluse 1, M L Rosso 1, A Ragni 1
PMCID: PMC167734  PMID: 8534090

Abstract

A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.

Full Text

The Full Text of this article is available as a PDF (307.4 KB).

Selected References

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

  1. Adams L. F., Visick J. E., Whiteley H. R. A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli. J Bacteriol. 1989 Jan;171(1):521–530. doi: 10.1128/jb.171.1.521-530.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angsuthanasombat C., Crickmore N., Ellar D. J. Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):63–68. doi: 10.1016/0378-1097(92)90584-b. [DOI] [PubMed] [Google Scholar]
  3. Arantes O., Lereclus D. Construction of cloning vectors for Bacillus thuringiensis. Gene. 1991 Dec 1;108(1):115–119. doi: 10.1016/0378-1119(91)90495-w. [DOI] [PubMed] [Google Scholar]
  4. Barberis-Maino L., Berger-Bächi B., Weber H., Beck W. D., Kayser F. H. IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris. Gene. 1987;59(1):107–113. doi: 10.1016/0378-1119(87)90271-x. [DOI] [PubMed] [Google Scholar]
  5. Bietlot H. P., Vishnubhatla I., Carey P. R., Pozsgay M., Kaplan H. Characterization of the cysteine residues and disulphide linkages in the protein crystal of Bacillus thuringiensis. Biochem J. 1990 Apr 15;267(2):309–315. doi: 10.1042/bj2670309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bourgouin C., Delécluse A., Ribier J., Klier A., Rapoport G. A Bacillus thuringiensis subsp. israelensis gene encoding a 125-kilodalton larvicidal polypeptide is associated with inverted repeat sequences. J Bacteriol. 1988 Aug;170(8):3575–3583. doi: 10.1128/jb.170.8.3575-3583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang C., Yu Y. M., Dai S. M., Law S. K., Gill S. S. High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes. Appl Environ Microbiol. 1993 Mar;59(3):815–821. doi: 10.1128/aem.59.3.815-821.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dai S. M., Gill S. S. In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases. Insect Biochem Mol Biol. 1993 Mar;23(2):273–283. doi: 10.1016/0965-1748(93)90008-g. [DOI] [PubMed] [Google Scholar]
  10. Delecluse A., Bourgouin C., Klier A., Rapoport G. Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis. Plasmid. 1989 Jan;21(1):71–78. doi: 10.1016/0147-619x(89)90088-7. [DOI] [PubMed] [Google Scholar]
  11. Delécluse A., Charles J. F., Klier A., Rapoport G. Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity. J Bacteriol. 1991 Jun;173(11):3374–3381. doi: 10.1128/jb.173.11.3374-3381.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Delécluse A., Poncet S., Klier A., Rapoport G. Expression of cryIVA and cryIVB Genes, Independently or in Combination, in a Crystal-Negative Strain of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol. 1993 Nov;59(11):3922–3927. doi: 10.1128/aem.59.11.3922-3927.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dervyn E., Poncet S., Klier A., Rapoport G. Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1995 May;177(9):2283–2291. doi: 10.1128/jb.177.9.2283-2291.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Koni P. A., Ellar D. J. Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin. J Mol Biol. 1993 Jan 20;229(2):319–327. doi: 10.1006/jmbi.1993.1037. [DOI] [PubMed] [Google Scholar]
  17. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lereclus D., Arantès O., Chaufaux J., Lecadet M. Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett. 1989 Jul 15;51(1):211–217. doi: 10.1016/0378-1097(89)90511-9. [DOI] [PubMed] [Google Scholar]
  19. Martin C., Timm J., Rauzier J., Gomez-Lus R., Davies J., Gicquel B. Transposition of an antibiotic resistance element in mycobacteria. Nature. 1990 Jun 21;345(6277):739–743. doi: 10.1038/345739a0. [DOI] [PubMed] [Google Scholar]
  20. Mollet B., Iida S., Shepherd J., Arber W. Nucleotide sequence of IS26, a new prokaryotic mobile genetic element. Nucleic Acids Res. 1983 Sep 24;11(18):6319–6330. doi: 10.1093/nar/11.18.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nicolas L., Nielsen-Leroux C., Charles J. F., Delécluse A. Respective role of the 42- and 51-kDa components of the Bacillus sphaericus toxin overexpressed in Bacillus thuringiensis. FEMS Microbiol Lett. 1993 Feb 1;106(3):275–280. doi: 10.1111/j.1574-6968.1993.tb05976.x. [DOI] [PubMed] [Google Scholar]
  22. Polzin K. M., Shimizu-Kadota M. Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J Bacteriol. 1987 Dec;169(12):5481–5488. doi: 10.1128/jb.169.12.5481-5488.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Poncet S., Anello G., Delécluse A., Klier A., Rapoport G. Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol. 1993 Nov;59(11):3928–3930. doi: 10.1128/aem.59.11.3928-3930.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Porter A. G., Davidson E. W., Liu J. W. Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbiol Rev. 1993 Dec;57(4):838–861. doi: 10.1128/mr.57.4.838-861.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  28. Wu D., Federici B. A. Improved production of the insecticidal CryIVD protein in Bacillus thuringiensis using cryIA(c) promoters to express the gene for an associated 20-kDa protein. Appl Microbiol Biotechnol. 1995 Jan;42(5):697–702. doi: 10.1007/BF00171947. [DOI] [PubMed] [Google Scholar]
  29. Wu D., Johnson J. J., Federici B. A. Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis. Mol Microbiol. 1994 Sep;13(6):965–972. doi: 10.1111/j.1365-2958.1994.tb00488.x. [DOI] [PubMed] [Google Scholar]
  30. de Barjac H., Lecadet M. M. Dosage biochimique de l'exotoxine thermostable de B. thuringiensis d'après l'inhibition d'ARN-polymérases bactériennes. C R Acad Sci Hebd Seances Acad Sci D. 1976 Jun 21;282(23):2119–2122. [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES