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. 1997 Feb;63(2):468–473. doi: 10.1128/aem.63.2.468-473.1997

Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain.

I Thiery 1, A Delécluse 1, M C Tamayo 1, S Orduz 1
PMCID: PMC168337  PMID: 9023925

Abstract

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.

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Selected References

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  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. 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]
  5. 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]
  6. 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]
  7. Brown K. L., Whiteley H. R. Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J Bacteriol. 1990 Dec;172(12):6682–6688. doi: 10.1128/jb.172.12.6682-6688.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chilcott C. N., Ellar D. J. Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro. J Gen Microbiol. 1988 Sep;134(9):2551–2558. doi: 10.1099/00221287-134-9-2551. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Delécluse A., Bourgouin C., Klier A., Rapoport G. Specificity of action on mosquito larvae of Bacillus thuringiensis israelensis toxins encoded by two different genes. Mol Gen Genet. 1988 Sep;214(1):42–47. doi: 10.1007/BF00340177. [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. Drobniewski F. A., Ellar D. J. Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J Bacteriol. 1989 Jun;171(6):3060–3067. doi: 10.1128/jb.171.6.3060-3067.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Earp D. J., Ellar D. J. Bacillus thuringiensis var. morrisoni strain PG14: nucleotide sequence of a gene encoding a 27kDa crystal protein. Nucleic Acids Res. 1987 Apr 24;15(8):3619–3619. doi: 10.1093/nar/15.8.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Orduz S., Rojas W., Correa M. M., Montoya A. E., de Barjac H. A new serotype of Bacillus thuringiensis from Colombia toxic to mosquito larvae. J Invertebr Pathol. 1992 Jan;59(1):99–103. doi: 10.1016/0022-2011(92)90118-n. [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. 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]
  23. 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]
  24. 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]
  25. Visick J. E., Whiteley H. R. Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli. J Bacteriol. 1991 Mar;173(5):1748–1756. doi: 10.1128/jb.173.5.1748-1756.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  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. 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]
  31. 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]

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