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. 1989 Feb;171(2):965–974. doi: 10.1128/jb.171.2.965-974.1989

Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.

W R Widner 1, H R Whiteley 1
PMCID: PMC209689  PMID: 2914879

Abstract

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryB1 and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryB1 gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryB1 is the distal gene of an operon which is comprised of three open reading frames (designated orf1, orf2, and cryB1). The proteins encoded by cryB1 and orf2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orf1 or cryB2 gene products are present in cuboidal crystals. The protein encoded by orf2 has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orf1 and orf2 are not toxic to either test insect.

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Selected References

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

  1. Anderson S. Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic Acids Res. 1981 Jul 10;9(13):3015–3027. doi: 10.1093/nar/9.13.3015. [DOI] [PMC free article] [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. Bott K. F., Wilson G. A. Development of competence in the Bacillus subtilis transformation system. J Bacteriol. 1967 Sep;94(3):562–570. doi: 10.1128/jb.94.3.562-570.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. Chak K. F., Ellar D. J. Cloning and expression in Escherichia coli of an insecticidal crystal protein gene from Bacillus thuringiensis var. aizawai HD-133. J Gen Microbiol. 1987 Oct;133(10):2921–2931. doi: 10.1099/00221287-133-10-2921. [DOI] [PubMed] [Google Scholar]
  7. Dame J. B., Williams J. L., McCutchan T. F., Weber J. L., Wirtz R. A., Hockmeyer W. T., Maloy W. L., Haynes J. D., Schneider I., Roberts D. Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science. 1984 Aug 10;225(4662):593–599. doi: 10.1126/science.6204383. [DOI] [PubMed] [Google Scholar]
  8. Donovan W. P., Dankocsik C. C., Gilbert M. P., Gawron-Burke M. C., Groat R. G., Carlton B. C. Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki. J Biol Chem. 1988 Jan 5;263(1):561–567. [PubMed] [Google Scholar]
  9. Duvall E. J., Williams D. M., Mongkolsuk S., Lovett P. S. Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis. J Bacteriol. 1984 Jun;158(3):784–790. doi: 10.1128/jb.158.3.784-790.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ferguson B., Jones N., Richter J., Rosenberg M. Adenovirus E1a gene product expressed at high levels in Escherichia coli is functional. Science. 1984 Jun 22;224(4655):1343–1346. doi: 10.1126/science.6374895. [DOI] [PubMed] [Google Scholar]
  11. Haider M. Z., Knowles B. H., Ellar D. J. Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. Eur J Biochem. 1986 May 2;156(3):531–540. doi: 10.1111/j.1432-1033.1986.tb09612.x. [DOI] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Herrnstadt C., Gilroy T. E., Sobieski D. A., Bennett B. D., Gaertner F. H. Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego. Gene. 1987;57(1):37–46. doi: 10.1016/0378-1119(87)90174-0. [DOI] [PubMed] [Google Scholar]
  14. Knowles B. H., Ellar D. J. Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin. J Cell Sci. 1986 Jul;83:89–101. doi: 10.1242/jcs.83.1.89. [DOI] [PubMed] [Google Scholar]
  15. Kronstad J. W., Schnepf H. E., Whiteley H. R. Diversity of locations for Bacillus thuringiensis crystal protein genes. J Bacteriol. 1983 Apr;154(1):419–428. doi: 10.1128/jb.154.1.419-428.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kronstad J. W., Whiteley H. R. Three classes of homologous Bacillus thuringiensis crystal-protein genes. Gene. 1986;43(1-2):29–40. doi: 10.1016/0378-1119(86)90005-3. [DOI] [PubMed] [Google Scholar]
  17. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mowatt M. R., Clayton C. E. Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol Cell Biol. 1987 Aug;7(8):2838–2844. doi: 10.1128/mcb.7.8.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Schnepf H. E., Whiteley H. R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci U S A. 1981 May;78(5):2893–2897. doi: 10.1073/pnas.78.5.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Sucov H. M., Benson S., Robinson J. J., Britten R. J., Wilt F., Davidson E. H. A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. Structure of the gene and derived sequence of the protein. Dev Biol. 1987 Apr;120(2):507–519. doi: 10.1016/0012-1606(87)90254-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Ward E. S., Ridley A. R., Ellar D. J., Todd J. A. Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J Mol Biol. 1986 Sep 5;191(1):13–22. doi: 10.1016/0022-2836(86)90418-3. [DOI] [PubMed] [Google Scholar]
  33. Whiteley H. R., Schnepf H. E. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Annu Rev Microbiol. 1986;40:549–576. doi: 10.1146/annurev.mi.40.100186.003001. [DOI] [PubMed] [Google Scholar]
  34. Wong H. C., Schnepf H. E., Whiteley H. R. Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem. 1983 Feb 10;258(3):1960–1967. [PubMed] [Google Scholar]
  35. Yamamoto T., McLaughlin R. E. Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. Kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem Biophys Res Commun. 1981 Nov 30;103(2):414–421. doi: 10.1016/0006-291x(81)90468-x. [DOI] [PubMed] [Google Scholar]

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