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. 1997 Aug;63(8):3254–3260. doi: 10.1128/aem.63.8.3254-3260.1997

Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. jegathesan.

H Cheong 1, S S Gill 1
PMCID: PMC168624  PMID: 9251213

Abstract

A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B. thuringiensis subsp. jegathasan was cloned employing a limited-growth PCR screening method with forward and reverse oligonucleotide primers designed from N-terminal amino acid sequences of native and trypsin-cleaved protein, respectively. The expressed protein showed little cross-reactivity to the antibody raised against the Cyt1Aa protein. Unlike Cyt1Aa and Cyt2Aa expression, there was little or no visible crystal inclusion formation under microscopic observation. The amino acid sequence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, respectively. The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions between alpha-helices and beta-sheets and one in the loop region between beta-sheets 5 and 6. beta-Blocks 4 to 7 are also conserved, not only structurally but also among the amino acids in the hydrophobic faces. Mosquitocidal activity assays indicated that the Cyt2Bb toxin had less toxicity than Cyt1Aa and had about 600-times-lower toxicity than the wild-type whole toxin crystal. However, both the Cyt2Bb and the Cyt1Aa toxin showed comparable levels of hemolytic activity.

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Selected References

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  1. 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]
  2. Armstrong J. L., Rohrmann G. F., Beaudreau G. S. Delta endotoxin of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1985 Jan;161(1):39–46. doi: 10.1128/jb.161.1.39-46.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Chow E., Singh G. J., Gill S. S. Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers. Appl Environ Microbiol. 1989 Nov;55(11):2779–2788. doi: 10.1128/aem.55.11.2779-2788.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Georghiou G. P., Wirth M. C. Influence of Exposure to Single versus Multiple Toxins of Bacillus thuringiensis subsp. israelensis on Development of Resistance in the Mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl Environ Microbiol. 1997 Mar;63(3):1095–1101. doi: 10.1128/aem.63.3.1095-1101.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gill S. S., Cowles E. A., Pietrantonio P. V. The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol. 1992;37:615–636. doi: 10.1146/annurev.en.37.010192.003151. [DOI] [PubMed] [Google Scholar]
  9. Gill S. S., Hornung J. M. Cytolytic activity of Bacillus thuringiensis proteins to insect and mammalian cell lines. J Invertebr Pathol. 1987 Jul;50(1):16–25. doi: 10.1016/0022-2011(87)90140-6. [DOI] [PubMed] [Google Scholar]
  10. Gill S. S., Hornung J. M., Ibarra J. E., Singh G. J., Federici B. A. Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins. Appl Environ Microbiol. 1987 Jun;53(6):1251–1256. doi: 10.1128/aem.53.6.1251-1256.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gill S. S., Singh G. J., Hornung J. M. Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins. Infect Immun. 1987 May;55(5):1300–1308. doi: 10.1128/iai.55.5.1300-1308.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Held G. A., Kawanishi C. Y., Huang Y. S. Characterization of the parasporal inclusion of Bacillus thuringiensis subsp. kyushuensis. J Bacteriol. 1990 Jan;172(1):481–483. doi: 10.1128/jb.172.1.481-483.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Kawalek M. D., Benjamin S., Lee H. L., Gill S. S. Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol. 1995 Aug;61(8):2965–2969. doi: 10.1128/aem.61.8.2965-2969.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Knowles B. H., Blatt M. R., Tester M., Horsnell J. M., Carroll J., Menestrina G., Ellar D. J. A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Lett. 1989 Feb 27;244(2):259–262. doi: 10.1016/0014-5793(89)80540-x. [DOI] [PubMed] [Google Scholar]
  16. Knowles B. H., White P. J., Nicholls C. N., Ellar D. J. A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis. Proc Biol Sci. 1992 Apr 22;248(1321):1–7. doi: 10.1098/rspb.1992.0035. [DOI] [PubMed] [Google Scholar]
  17. Koni P. A., Ellar D. J. Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins. Microbiology. 1994 Aug;140(Pt 8):1869–1880. doi: 10.1099/13500872-140-8-1869. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Li J., Koni P. A., Ellar D. J. Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J Mol Biol. 1996 Mar 22;257(1):129–152. doi: 10.1006/jmbi.1996.0152. [DOI] [PubMed] [Google Scholar]
  20. Maddrell S. H., Lane N. J., Harrison J. B., Overton J. A., Moreton R. B. The initial stages in the action of an insecticidal delta-endotoxin of Bacillus thuringiensis var. israelensis on the epithelial cells of the malpighian tubules of the insect, Rhodnius prolixus. J Cell Sci. 1988 May;90(Pt 1):131–144. doi: 10.1242/jcs.90.1.131. [DOI] [PubMed] [Google Scholar]
  21. Ragni A., Thiéry I., Delécluse A. Characterization of six highly mosquitocidal Bacillus thuringiensis strains that do not belong to H-14 serotype. Curr Microbiol. 1996 Jan;32(1):48–54. doi: 10.1007/s002849900009. [DOI] [PubMed] [Google Scholar]
  22. Ross L. S., Gill S. S. Limited growth PCR screening of a plasmid library. Biotechniques. 1996 Sep;21(3):382-4, 386. doi: 10.2144/96213bm08. [DOI] [PubMed] [Google Scholar]
  23. Slatin S. L., Abrams C. K., English L. Delta-endotoxins form cation-selective channels in planar lipid bilayers. Biochem Biophys Res Commun. 1990 Jun 15;169(2):765–772. doi: 10.1016/0006-291x(90)90397-6. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Thomas W. E., Ellar D. J. Mechanism of action of Bacillus thuringiensis var israelensis insecticidal delta-endotoxin. FEBS Lett. 1983 Apr 18;154(2):362–368. doi: 10.1016/0014-5793(83)80183-5. [DOI] [PubMed] [Google Scholar]
  26. Von Tersch M. A., Slatin S. L., Kulesza C. A., English L. H. Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide. Appl Environ Microbiol. 1994 Oct;60(10):3711–3717. doi: 10.1128/aem.60.10.3711-3717.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Ward E. S., Ellar D. J., Chilcott C. N. Single amino acid changes in the Bacillus thuringiensis var. israelensis delta-endotoxin affect the toxicity and expression of the protein. J Mol Biol. 1988 Aug 5;202(3):527–535. doi: 10.1016/0022-2836(88)90283-5. [DOI] [PubMed] [Google Scholar]
  30. Wu D., Federici B. A. A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol. 1993 Aug;175(16):5276–5280. doi: 10.1128/jb.175.16.5276-5280.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Yu Y. M., Ohba M., Gill S. S. Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins. Appl Environ Microbiol. 1991 Apr;57(4):1075–1081. doi: 10.1128/aem.57.4.1075-1081.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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