1887

Abstract

The larvicidal activity of subsp. against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new -like coding sequence named . In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a -trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in . Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of of Cyt1Ca in recombinant cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28981-0
2006-09-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/9/2651.html?itemId=/content/journal/micro/10.1099/mic.0.28981-0&mimeType=html&fmt=ahah

References

  1. Al-Yahyaee, S. A. & Ellar, D. J. ( 1995; ). Cell targeting of a pore-forming toxin, CytA delta-endotoxin from Bacillus thuringiensis subspecies israelensis, by conjugating CytA with anti-Thy 1 monoclonal antibodies and insulin. Bioconj Chem 7, 451–460.
    [Google Scholar]
  2. Arantes, O. & Lereclus, D. ( 1991; ). Construction of cloning vectors for Bacillus thuringiensis. Gene 108, 115–119.[CrossRef]
    [Google Scholar]
  3. Baneyx, F. ( 1999; ). Recombinant protein expression in E. coli. Curr Opin Biotechnol 10, 411–421.[CrossRef]
    [Google Scholar]
  4. Ben-Dov, E., Boussiba, S. & Zaritsky, A. ( 1995; ). Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis subsp. israelensis. J Bacteriol 177, 2851–2857.
    [Google Scholar]
  5. Ben-Dov, E., Einav, M., Peleg, N., Boussiba, S. & Zaritsky, A. ( 1996; ). Restriction map of the 125-kilobase of Bacillus thuringiensis subsp. israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae. Appl Environ Microbiol 62, 3140–3145.
    [Google Scholar]
  6. Berry, C., O'Neil, S., Ben-Dov, E. & 7 other authors ( 2002; ). Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68, 5082–5095.[CrossRef]
    [Google Scholar]
  7. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  8. Cheong, H. & Gill, S. S. ( 1997; ). Cloning and characterization of a cytolytic and mosquitocidal δ-endotoxin from Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 63, 3254–3260.
    [Google Scholar]
  9. Crickmore, N., Bone, E. J., Williams, J. A. & Ellar, D. J. ( 1995; ). Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131, 249–254.
    [Google Scholar]
  10. Delécluse, A., Juárez-Pérez, V. & Berry, C. ( 2000; ). Vector-active toxins: structure and diversity. In Entomopathogenic Bacteria: from Laboratory to Field Application, pp. 101–125. Edited by J.-F. Charles, A. Delécluse & C. Nielsen-leRoux. Dordrecht: Kluwer.
  11. Douek, J., Einav, M. & Zaritsky, A. ( 1992; ). Sensitivity to plating of Escherichia coli cells expressing the cytA gene from Bacillus thuringiensis var. israelensis. Mol Gen Genet 232, 162–165.[CrossRef]
    [Google Scholar]
  12. Du, J., Knowles, B. H., Li, J. & Ellar, D. J. ( 1999; ). Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer. Biochem J 338, 185–193.[CrossRef]
    [Google Scholar]
  13. Gazit, E., Burshtein, N., Ellar, D. J., Sawyer, T. & Shai, Y. ( 1997; ). Bacillus thuringiensis cytolytic toxin associates specifically with its synthetic helices A and C in the membrane bound state. Implications for the assembly of oligomeric transmembrane pores. Biochemistry 36, 15546–15554.[CrossRef]
    [Google Scholar]
  14. Goldberg, L. H. & Margalit, J. ( 1977; ). A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitatus, Aedes aegypti and Culex pipiens. Mosq News 37, 355–358.
    [Google Scholar]
  15. Guerchicoff, A., Ugalde, R. A. & Rubinstein, C. P. ( 1997; ). Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 63, 2716–2721.
    [Google Scholar]
  16. Hofte, H. & Whiteley, H. R. ( 1989; ). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53, 242–255.
    [Google Scholar]
  17. Itsko, M., Manasherob, R. & Zaritsky, A. ( 2005; ). Partial restoration of anti-bacterial activity of a cryptic ORF (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by site-directed mutagenesis. J Bacteriol 187, 6379–6385.[CrossRef]
    [Google Scholar]
  18. Juárez-Pérez, V., Guerchicoff, A., Rubinstein, C. & Delécluse, A. ( 2002; ). Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin. Appl Environ Microbiol 68, 1228–1231.[CrossRef]
    [Google Scholar]
  19. Khasdan, V., Ben-Dov, E., Manasherob, R., Boussiba, S. & Zaritsky, A. ( 2001; ). Toxicity and synergism in transgenic Escherichia coli expressing four genes from Bacillus thuringiensis subsp. israelensis. Environ Microbiol 3, 798–806.[CrossRef]
    [Google Scholar]
  20. Koni, P. A. & Ellar, D. J. ( 1993; ). Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin. J Mol Biol 229, 319–327.[CrossRef]
    [Google Scholar]
  21. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–685.
    [Google Scholar]
  22. Li, J., Koni, P. A. & Ellar, D. J. ( 1996; ). Structure of the mosquitocidal δ-endotoxin CytB from Bacillus thuringiensis subsp. kyushuensis and implications for membrane pore formation. J Mol Biol 257, 129–152.[CrossRef]
    [Google Scholar]
  23. Manasherob, R., Zaritsky, A., Ben-Dov, E., Saxena, D., Barak, Z. & Einav, M. ( 2001; ). Effect of accessory proteins P19 and P20 on cytolytic activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli. Curr Microbiol 43, 355–364.[CrossRef]
    [Google Scholar]
  24. Manasherob, R., Zaritsky, A., Metzler, Y., Ben-Dov, E., Itsko, M. & Fishov, I. ( 2003; ). Compaction of the Escherichia coli nucleoid caused by Cyt1Aa. Microbiology 149, 3553–3564.[CrossRef]
    [Google Scholar]
  25. Margalith, Y. & Ben-Dov, E. ( 2000; ). Biological control by Bacillus thuringiensis subsp. israelensis. In Insect Pest Management: Techniques for Environmental Protection, pp. 243–301. Edited by J. E. Rechcigl & N. A. Rechcigl. Boca Raton, FL: CRC Press.
  26. Nisnevitch, M., Cohen, S., Ben-Dov, E., Zaritsky, A., Soffer, Y. & Cahan, R. ( 2006; ). Cyt2Ba of Bacillus thuringiensis israelensis activation by putative endogenous protease. Biochem Biophys Res Commun 344, 99–105.[CrossRef]
    [Google Scholar]
  27. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. & Dean, D. H. ( 1998; ). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 775–806.
    [Google Scholar]
  28. Stein, C., Jones, G. W., Chalmers, T. & Berry, C. ( 2006; ). Transcriptional analysis of the toxin-coding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 72, 1771–1776.[CrossRef]
    [Google Scholar]
  29. Stewart, G. S., Johnstone, K., Hagelberg, E. & Ellar, D. J. ( 1981; ). Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J 198, 101–106.
    [Google Scholar]
  30. Thiery, I., Delecluse, A., Tamayo, M. C. & Orduz, S. ( 1997; ). Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain. Appl Environ Microbiol 63, 468–473.
    [Google Scholar]
  31. Thomas, W. E. & Ellar, D. J. ( 1983; ). Bacillus thuringiensis var. israelensis crystal δ-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J Cell Sci 60, 181–197.
    [Google Scholar]
  32. Waalwijk, C., Dullemans, A. M., van Workum, M. E. S. & Visser, B. ( 1985; ). Molecular cloning and the nucleotide sequence of the Mr 28 000 crystal protein gene of Bacillus thuringiensis subsp. israelensis. Nucleic Acids Res 13, 8207–8217.[CrossRef]
    [Google Scholar]
  33. Wirth, M. C., Georghiou, G. P. & Federici, B. A. ( 1997; ). CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc Natl Acad Sci U S A 94, 10536–10540.[CrossRef]
    [Google Scholar]
  34. Wirth, M. C., Delecluse, A. & Walton, W. E. ( 2001; ). Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis synergize Bacillus sphaericus against Aedes aegypti and resistant Culex quinquefasciatus (Diptera: Culicidae). Appl Environ Microbiol 67, 3280–3284.[CrossRef]
    [Google Scholar]
  35. Wirth, M. C., Park, H. W., Walton, W. E. & Federici, B. A. ( 2005; ). Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus. Appl Environ Microbiol 71, 185–189.[CrossRef]
    [Google Scholar]
  36. Wu, D. & Federici, B. A. ( 1993; ). A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol 175, 5276–5280.
    [Google Scholar]
  37. Wu, D., Johnson, J. J. & Federici, B. A. ( 1994; ). Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis. Mol Microbiol 13, 965–972.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28981-0
Loading
/content/journal/micro/10.1099/mic.0.28981-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error