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. 1990 Jun;56(6):1710–1716. doi: 10.1128/aem.56.6.1710-1716.1990

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

M Trisrisook 1, S Pantuwatana 1, A Bhumiratana 1, W Panbangred 1
PMCID: PMC184498  PMID: 2200339

Abstract

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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

  1. 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]
  2. 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]
  3. Bourgouin C., Klier A., Rapoport G. Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis. Mol Gen Genet. 1986 Dec;205(3):390–397. doi: 10.1007/BF00338072. [DOI] [PubMed] [Google Scholar]
  4. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  5. González J. M., Jr, Carlton B. C. A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis. Plasmid. 1984 Jan;11(1):28–38. doi: 10.1016/0147-619x(84)90004-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Ibarra J. E., Federici B. A. Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1986 Feb;165(2):527–533. doi: 10.1128/jb.165.2.527-533.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. McDonald K. O., Burke W. F., Jr Plasmid transformation of Bacillus sphaericus 1593. J Gen Microbiol. 1984 Jan;130(1):203–208. doi: 10.1099/00221287-130-1-203. [DOI] [PubMed] [Google Scholar]
  10. McLean K. M., Whiteley H. R. Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1987 Mar;169(3):1017–1023. doi: 10.1128/jb.169.3.1017-1023.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  12. Myers P., Yousten A. A. Toxic activity of Bacillus sphaericus SSII-1 for mosquito larvae. Infect Immun. 1978 Mar;19(3):1047–1053. doi: 10.1128/iai.19.3.1047-1053.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Polak J., Novick R. P. Closely related plasmids from Staphylococcus aureus and soil bacilli. Plasmid. 1982 Mar;7(2):152–162. doi: 10.1016/0147-619x(82)90074-9. [DOI] [PubMed] [Google Scholar]
  14. Sekar V., Carlton B. C. Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis. Gene. 1985;33(2):151–158. doi: 10.1016/0378-1119(85)90089-7. [DOI] [PubMed] [Google Scholar]
  15. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  16. Thorne L., Garduno F., Thompson T., Decker D., Zounes M., Wild M., Walfield A. M., Pollock T. J. Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis". J Bacteriol. 1986 Jun;166(3):801–811. doi: 10.1128/jb.166.3.801-811.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Tyrell D. J., Bulla L. A., Jr, Andrews R. E., Jr, Kramer K. J., Davidson L. I., Nordin P. Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J Bacteriol. 1981 Feb;145(2):1052–1062. doi: 10.1128/jb.145.2.1052-1062.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Yamamoto T., Watkinson I. A., Kim L., Sage M. V., Stratton R., Akande N., Li Y., Ma D. P., Roe B. A. Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis. Gene. 1988 Jun 15;66(1):107–120. doi: 10.1016/0378-1119(88)90229-6. [DOI] [PubMed] [Google Scholar]
  23. Yousten A. A. Bacillus sphaericus: microbiological factors related to its potential as a mosquito larvicide. Adv Biotechnol Processes. 1984;3:315–343. [PubMed] [Google Scholar]

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