Skip to main content
SpringerLink
Log in
Book cover

Bacillus thuringiensis Biotechnology pp 93–113Cite as

Protein Engineering of Bacillus thuringiensis δ-Endotoxins

  • Chapter
  • First Online:

Abstract

Protein engineering of insecticidal Bt δ-endotoxins is a powerful tool for designing novel Cry toxins with altered properties, including changing the toxin’s specificity. By following some elementary rules governing the structure/function relationship, it has been possible to create new toxins with modified properties including increased toxicity and binding affinity, enhanced ion-transport activity, and changes in insect specificity. These methods have also produced valuable information and have led to an improved understanding of the mode of action of these important biopesticides. The results discussed in this chapter derive from rational molecular design where protein structure is modified by incorporating single or multiple amino acid substitutions aimed at modifying specific protein functions. In this review, we analyze several protein modifications that have been successfully used for creating stable, functional proteins with minimal structural alterations. The understanding and proper use of protein engineering approaches may help in implementing appropriate pest management strategies by improving the efficacy of these toxins against insect pests.

This is a preview of subscription content, log in via an institution.

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   219.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   279.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

References

  • Abdullah MA, Alzate O, Mohammad M, McNall RJ, Adang MJ, Dean DH (2003) Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering. Appl Environ Microbiol 69(9):5343–5353

    Article  CAS  Google Scholar 

  • Ahmad W, Ellar DJ (1990) Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein. FEMS Microbiol Lett 56(1–2):97–104

    Article  CAS  Google Scholar 

  • Alcantara EP, Alzate O, Lee MK, Curtiss A, Dean DH (2001) Role of a-helix 7 of Bacillus thuringiensis Cry1Ab δ-endotoxin in membrane insertion, structural stability, and ion channel activity. Biochemistry 40(8):2540–2547

    Article  CAS  Google Scholar 

  • Altenbach C, Marti T, Khorana HG, Hubbell WL (1990) Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants. Science 248(4959):1088–1092

    Article  CAS  Google Scholar 

  • Altenbach C, Greenhalgh DA, Khorana HG, Hubbell WL (1994) A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci U S A 91(5):1667–1671

    Article  CAS  Google Scholar 

  • Alzate O, You T, Claybon M, Osorio C, Curtiss A, Dean DH (2006) Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa δ-endotoxin on ion-channel formation in biological membranes. Biochemistry 45(45):13597–13605

    Article  CAS  Google Scholar 

  • Alzate O, Hemann CF, Osorio C, Hille R, Dean DH (2009) Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes. BMC Biochem 10:25

    Article  Google Scholar 

  • Alzate O, Osorio C, Florez AM, Dean DH (2010) Participation of valine 171 in α-Helix 5 of Bacillus thuringiensis Cry1Ab δ-endotoxin in translocation of toxin into Lymantria dispar midgut membranes. Appl Environ Microbiol 76(23):7878–7880

    Article  CAS  Google Scholar 

  • Arenas I, Bravo A, Soberon M, Gomez I (2010) Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin. J Biol Chem 285(17):12497–12503

    Article  CAS  Google Scholar 

  • Arnold S, Curtiss A, Dean DH, Alzate O (2001) The role of a proline-induced broken-helix motif in α-helix 2 of Bacillus thuringiensis δ-endotoxins. FEBS Lett 490(1–2):70–74

    Article  CAS  Google Scholar 

  • Aronson A (1995) The protoxin composition of Bacillus thuringiensis insecticidal inclusions affects solubility and toxicity. Appl Environ Microbiol 61(11):4057–4060

    CAS  Google Scholar 

  • Aronson A (2000) Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis Bacillus thuringiensisδ-endotoxin. Appl Environ Microbiol 66(10):4568–4570

    Article  CAS  Google Scholar 

  • Atherton NM (1993) Principles of electron spin resonance. Ellis Horwood, Prentice Hall, London

    Google Scholar 

  • Audtho M, Valaitis AP, Alzate O, Dean DH (1999) Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 δ-endotoxin by protein engineering. Appl Environ Microbiol 65(10):4601–4605

    CAS  Google Scholar 

  • Berova N, Nakanishi K, Woody R (2000) Circular dichroism: principles and applications, 2nd edn. Wiley-VCH, New York

    Google Scholar 

  • Bietlot HP, Vishnubhatla I, Carey PR, Pozsgay M, Kaplan H (1990) Characterization of the cysteine residues and disulfide linkages in the protein crystal of Bacillus thuringiensis. Biochem J 267:309–315

    CAS  Google Scholar 

  • Boonserm P, Davis P, Ellar DJ, Li J (2005) Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 348(2):363–382

    Article  CAS  Google Scholar 

  • Boonserm P, Mo M, Angsuthanasombat C, Lescar J (2006) Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution. J Bacteriol 188(9):3391–3401

    Article  CAS  Google Scholar 

  • Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill SS, Soberon M (2004) Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta 1667(1):38–46

    Article  CAS  Google Scholar 

  • Burton SL, Ellar DJ, Li J, Derbyshire DJ (1999) N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognized by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. J Mol Biol 287:1011–1022

    Article  CAS  Google Scholar 

  • Carroll J, Wolfersberger MG, Ellar DJ (1997) The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism. J Cell Sci 110:3099–3104

    CAS  Google Scholar 

  • Chandra A, Ghosh P, Mandaokar AD, Bera AK, Sharma RP, Das S, Kumar PA (1999) Amino acid substitution in α-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to HelicoverpaarmigeraHubner. FEBS Lett 458(2):175–179

    Article  CAS  Google Scholar 

  • Chen XJ, Curtiss A, Alcantara E, Dean DH (1995) Mutations in domain I of Bacillus thuringiensisδ-endotoxin CryIAbreduce the irreversible binding of toxin to Manduca sexta brush border membrane vesicles. J Biol Chem 270(11):6412–6419

    Article  CAS  Google Scholar 

  • Choma CT, Surewicz WK, Carey PR, Pozsgay M, Raynor T, Kaplan H (1990) Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis: structural implications. Eur J Biochem 189:523–527

    Article  CAS  Google Scholar 

  • Coux F, Vachon V, Rang C, Moozar K, Masson L, Royer M, Bes M, Rivest S, Brousseau R, Schwartz JL, Laprade R, Frutos R (2001) Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac. J Biol Chem 276(38):35546–35551

    Article  CAS  Google Scholar 

  • Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):807–813

    CAS  Google Scholar 

  • Crickmore N, Zeigler DR, Schnepf E, Van Rie J, Lereclus D, Baum J, Bravo A, Dean DH (2011) Bacillus thuringiensis toxin nomenclature. http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

    Google Scholar 

  • de Maagd RA, Kwa MS, van der Klei H, Yamamoto T, Schipper B, Vlak JM, Stiekema WJ, Bosch D (1996) Domain III substitution in Bacillus thuringiensis δ-endotoxin CryIA(b) results in superior toxicity for Spodopteraexigua and altered membrane protein recognition. Appl Environ Microbiol 62(5):1537–1543

    CAS  Google Scholar 

  • de Maagd RA, Weemen-Hendriks M, Stiekema W, Bosch D (2000) Bacillus thuringiensis δ-endotoxin Cry1C domain III can function as a specificity determinant for Spodopteraexigua in different, but not all, Cry1-Cry1C hybrids. Appl Environ Microbiol 66(4):1559–1563

    Article  Google Scholar 

  • Fortier M, Vachon V, Frutos R, Schwartz JL, Laprade R (2007) Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins. Appl Environ Microbiol 73(19):6208–6213

    Article  CAS  Google Scholar 

  • Gatehouse JA (2011) Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects. Curr Protein Pept Sci. (Epub ahead of print)

    Google Scholar 

  • Gazit E, Shai Y (1993) Structural and functional characterization of the alpha 5 segment of Bacillus thuringiensis δ-endotoxin. Biochemistry 32(13):3429–3436

    Article  CAS  Google Scholar 

  • Gazit E, La Rocca P, Sansom MS, Shai Y (1998) The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis δ-endotoxin are consistent with an “umbrella-like” structure of the pore. Proc Natl Acad Sci U S A 95(21):12289–12294

    Article  CAS  Google Scholar 

  • Ge AZ, Shivarova NI, Dean DH (1989) Location of the Bombyxmori specificity domain on a Bacillus thuringiensis δ-endotoxin protein. Proc Natl Acad Sci U S A 86(11):4037–4041

    Article  CAS  Google Scholar 

  • Ge AZ, Rivers D, Milne R, Dean DH (1991) Functional domains of Bacillus thuringiensis insecticidal crystal proteins: refinement of Heliothisvirescens and Trichoplusianispecificity domains on CryIA(c). J Biol Chem 266:17954–17958

    CAS  Google Scholar 

  • Girard F, Vachon V, Prefontaine G, Marceau L, Su Y, Larouche G, Vincent C, Schwartz JL, Masson L, Laprade R (2008) Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa. Appl Environ Microbiol 74(9):2565–2572

    Article  CAS  Google Scholar 

  • Gomez I, Sanchez J, Miranda R, Bravo A, Soberon M (2002) Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin. FEBS Lett 513(2–3):242–246

    Article  CAS  Google Scholar 

  • Gomez I, Dean DH, Bravo A, Soberon M (2003) Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin. Biochemistry 42(35):10482–10489

    Article  CAS  Google Scholar 

  • Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890

    Article  CAS  Google Scholar 

  • Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz JL, Brousseau R, Cygler M (1995) Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation. J Mol Biol 254(3):447–464

    Article  CAS  Google Scholar 

  • Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255

    Google Scholar 

  • Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol 7(9):735–739

    Article  CAS  Google Scholar 

  • Hubbell WL, Altenbach C, Hubbell CM, Khorana HG (2003) Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. Adv Protein Chem 63:243–290

    Article  CAS  Google Scholar 

  • Hussain SR, Aronson AI, Dean DH (1996) Substitution of residues on the proximal side of Cry1A Bacillus thuringiensis δ-endotoxins affects irreversible binding to Manduca sexta midgut membrane. Biochem Biophys Res Commun 226(1):8–14

    Article  CAS  Google Scholar 

  • Hussain SA, Flórez AM, Dean DH, Alzate O (2010) Preferential protection of domains II and III of Bacillus thuringiensis Cry1Aa toxin by brush border membrane vesicles. Rev Colomb Biotechnol 12(2):14–26

    CAS  Google Scholar 

  • Jimenez-Juarez N, Munoz-Garay C, Gomez I, Saab-Rincon G, Damian-Almazo JY, Gill SS, Soberon M, Bravo A (2007) Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae. J Biol Chem 282(29):21222–21229

    Article  CAS  Google Scholar 

  • Jimenez-Juarez N, Munoz-Garay C, Gomez I, Gill SS, Soberon M, Bravo A (2008) The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides 29(2):318–323

    Article  CAS  Google Scholar 

  • Kaur S (2000) Molecular approaches towards development of novel Bacillus thuringiensis biopesticides. World J Microbiol Biotechnol 16:781–793

    Article  CAS  Google Scholar 

  • Kirouac M, Vachon V, Quievy D, Schwartz JL, Laprade R (2006) Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles. Appl Environ Microbiol 72(1):506–515

    Article  CAS  Google Scholar 

  • Kumar AS, Aronson AI (1999) Analysis of mutations in the pore-forming region essential for insecticidal activity of a Bacillus thuringiensis δ-endotoxin. J Bacteriol 181(19):6103–6107

    CAS  Google Scholar 

  • Lebel G, Vachon V, Prefontaine G, Girard F, Masson L, Juteau M, Bah A, Larouche G, Vincent C, Laprade R, Schwartz JL (2009) Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles. Appl Environ Microbiol 75(12):3842–3850

    Article  CAS  Google Scholar 

  • Lee MK, Young BA, Dean DH (1995) Domain III exchanges of Bacillus thuringiensis CryIA toxins affect binding to different gypsy moth midgut receptors. Biochem Biophys Res Commun 216:306–312

    Article  CAS  Google Scholar 

  • Li J, Carroll J, Ellar DJ (1991) Crystal structure of insecticidal d-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. Nature 353:815–821

    Article  CAS  Google Scholar 

  • Li J, Derbyshire DJ, Promdonkoy B, Ellar DJ (2001) Structural implications for the transformation of the Bacillus thuringiensis δ-endotoxins from water-soluble to membrane-inserted forms. Biochem Soc Trans 29(Pt 4):571–577

    Article  CAS  Google Scholar 

  • Lightwood DJ, Ellar DJ, Jarrett P (2000) Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac δ-endotoxin. Appl Environ Microbiol 66(12):5174–5181

    Article  CAS  Google Scholar 

  • Likitvivatanavong S, Chen J, Evans AM, Bravo A, Soberon M, Gill SS (2011) Multiple receptors as targets of cry toxins in mosquitoes. J Agric Food Chem 59(7):2829–2838

    Article  CAS  Google Scholar 

  • Liu XS, Dean DH (2006) Redesigning Bacillus thuringiensis Cry1Aa toxin into a mosquito toxin. Protein Eng Des Sel 19(3):107–111

    Article  Google Scholar 

  • Lorence A, Darszon A, Diaz C, Lievano A, Quintero R, Bravo A (1995) Delta-endotoxins induce cation channels in Spodopterafrugiperda brush border membranes in suspension and in planar lipid bilayers. FEBS Lett 360(3):217–222

    Article  CAS  Google Scholar 

  • Masson L, Tabashnik BE, Liu YB, Brousseau R, Schwartz JL (1999) Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel. J Biol Chem 274(45):31996–32000

    Article  CAS  Google Scholar 

  • Munoz-Garay C, Sanchez J, Darszon A, de Maagd RA, Bakker P, Soberon M, Bravo A (2006) Permeability changes of Manduca sexta midgut brush border membranes induced by oligomeric structures of different Cry toxins. J Membr Biol 212(1):61–68

    Article  CAS  Google Scholar 

  • Munoz-Garay C, Rodriguez-Almazan C, Aguilar JN, Portugal L, Gomez I, Saab-Rincon G, Soberon M, Bravo A (2009) Oligomerization of Cry11Aa from Bacillus thuringiensis has an important role in toxicity against Aedesaegypti. Appl Environ Microbiol 75(23):7548–7550

    Article  CAS  Google Scholar 

  • Nair MS, Dean DH (2008) All domains of Cry1A toxins insert into insect brush border membranes. J Biol Chem 283(39):26324–26331

    Article  CAS  Google Scholar 

  • Pacheco S, Gomez I, Arenas I, Saab-Rincon G, Rodriguez-Almazan C, Gill SS, Bravo A, Soberon M (2009) Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a “ping pong” binding mechanism with Manduca sextaaminopeptidase-N and cadherin receptors. J Biol Chem 284(47):32750–32757

    Article  CAS  Google Scholar 

  • Pardo-Lopez L, Gomez I, Rausell C, Sanchez J, Soberon M, Bravo A (2006) Structural changes of the Cry1Ac oligomeric pre-pore from Bacillus thuringiensis induced by N-Acetylgalactosamine facilitates toxin membrane insertion. Biochemistry 45(34):10329–10336

    Article  CAS  Google Scholar 

  • Pigott CR, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev 71(2):255–281

    Article  CAS  Google Scholar 

  • Pozsgay M, Fast P, Kaplan H, Carey PR (1987) The effect of sunlight on the protein crystals from Bacillus thuringiensis subsp. kurstaki HD1 and NRD12, a Raman spectroscopy study. J Invertebr Pathol 50:246–253

    Article  CAS  Google Scholar 

  • Pusztai M, Fast P, Gringorten L, Kaplan H, Lessard T, Carey PR (1991) The mechanism of sunlight-mediated inactivation of Bacillus thuringiensis crystals. Biochem J 273(Pt 1):43–47

    CAS  Google Scholar 

  • Rajamohan F, Dean DH (1996) Molecular biology of bacteria on biological control of insects. In: Gunasekaran M, Weber DJ (eds) Molecular biology of the biological control of pests and diseases of plants. CRC Press, Boca Raton, pp 105–122

    Google Scholar 

  • Rajamohan F, Alcantara E, Lee MK, Chen XJ, Curtiss A, Dean DH (1995) Single amino acid changes in domain II of Bacillus thuringiensis CryIAb δ-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles. J Bacteriol 177(9):2276–2282

    CAS  Google Scholar 

  • Rajamohan F, Alzate O, Cotrill JA, Curtiss A, Dean DH (1996a) Protein engineering of Bacillus thuringiensis δ-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae. Proc Natl Acad Sci U S A 93(25):14338–14343

    Article  CAS  Google Scholar 

  • Rajamohan F, Cotrill JA, Gould F, Dean DH (1996b) Role of domain II, loop 2 residues of Bacillus thuringiensis CryIAb δ-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothisvirescens. J Biol Chem 271(5):2390–2396

    Article  CAS  Google Scholar 

  • Rausell C, Munoz-Garay C, Miranda-CassoLuengo R, Gomez I, Rudino-Pinera E, Soberon M, Bravo A (2004a) Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate. Biochemistry 43(1):166–174

    Article  CAS  Google Scholar 

  • Rausell C, Pardo-Lopez L, Sanchez J, Munoz-Garay C, Morera C, Soberon M, Bravo A (2004b) Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel. J Biol Chem 279(53):55168–55175

    Article  CAS  Google Scholar 

  • Rodriguez-Almazan C, Zavala LE, Munoz-Garay C, Jimenez-Juarez N, Pacheco S, Masson L, Soberon M, Bravo A (2009) Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity. PLoS One 4(5):e5545

    Article  Google Scholar 

  • Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P (2011) Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol J 9(3):283–300

    Article  CAS  Google Scholar 

  • Schnepf HE (1995) Bacillus thuringiensis toxins: regulation, activities and structural diversity. Curr Opin Biotechnol 6:305–312

    Article  CAS  Google Scholar 

  • Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):775–806

    CAS  Google Scholar 

  • Schwartz JL, Juteau M, Grochulski P, Cygler M, Prefontaine G, Brousseau R, Masson L (1997a) Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering. FEBS Lett 410(2–3):397–402

    Article  CAS  Google Scholar 

  • Schwartz JL, Lu YJ, Sohnlein P, Brousseau R, Laprade R, Masson L, Adang MJ (1997b) Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors. FEBS Lett 412:270–276

    Article  CAS  Google Scholar 

  • Schwartz JL, Potvin L, Chen XJ, Brousseau R, Laprade R, Dean DH (1997c) Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin. Appl Environ Microbiol 63(10):3978–3984

    CAS  Google Scholar 

  • Shan S, Zhang Y, Ding X, Hu S, Sun Y, Yu Z, Liu S, Zhu Z, Xia L (2010) A Cry1Ac toxin variant generated by directed evolution has enhanced toxicity against Lepidopteran insects. Curr Microbiol 62(2):358–365

    Article  Google Scholar 

  • Soberon M, Pardo-Lopez L, Lopez I, Gomez I, Tabashnik BE, Bravo A (2007) Engineering modified Bt toxins to counter insect resistance. Science 318(5856):1640–1642

    Article  CAS  Google Scholar 

  • Tigue NJ, Jacoby J, Ellar DJ (2001) The α-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins. Appl Environ Microbiol 67(12):5715–5720

    Article  CAS  Google Scholar 

  • Vachon V, Prefontaine G, Rang C, Coux F, Juteau M, Schwartz JL, Brousseau R, Frutos R, Laprade R, Masson L (2004) Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities. Appl Environ Microbiol 70(10):6123–6130

    Article  CAS  Google Scholar 

  • van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystal proteins. J Invertebr Pathol 101(1):1–16

    Article  Google Scholar 

  • Wolfersberger MG (1990) The toxicity of two Bacillus thuringiensis δ-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins. Experientia 46(5):475–477

    Article  CAS  Google Scholar 

  • Wolfersberger MG, Spaeth DD (1987) Activity of spore-crystal preparations from twenty serotypes of Bacillus thuringiensis toward Manduca sexta larvae in vivo and in vitro. Z Angew Entomol 103:138–141

    Google Scholar 

  • Wolfersberger M, Lüthy P, Maurer A, Parenti P, Sacchi FV, Giordana B, Hanozet GM (1987) Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pierisbrassicae). Comp Biochem Physiol 86A:301–308

    Article  CAS  Google Scholar 

  • Wu D, Aronson AI (1992) Localized mutagenesis defines regions of the Bacillus thuringiensis δ-endotoxin involved in toxicity and specificity. J Biol Chem 267(4):2311–2317

    CAS  Google Scholar 

  • Zavala LE, Pardo-Lopez L, Canton PE, Gomez I, Soberon M, Bravo A (2011) Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent after insertion of part of domain I into the membrane. J Biol Chem 286:19109–19117

    Article  CAS  Google Scholar 

  • Zhang X, Candas M, Griko NB, Taussig R, Bulla LA Jr (2006) A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc Natl Acad Sci U S A 103(26):9897–9902

    Article  CAS  Google Scholar 

  • Zhuang M, Oltean DI, Gomez I, Pullikuth AK, Soberon M, Bravo A, Gill SS (2002) Heliothisvirescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation. J Biol Chem 277(16):13863–13872

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Drs. Rehan Hussain (Columbus Children’s Hospital) and Carol Parker (University of Victoria Proteome Center) for careful review of the manuscript, and to Mr. Diego Velez for his contribution to Figs. 5.1–5.3. Special thanks to Dr. Donald H. Dean from the Entomology Department, The Ohio State University, for his support and encouragement.

Author information

Authors and Affiliations

  1. Laboratorio de Biología Molecular y Biotecnología, Facultad de Medicina, Universidad de Santander, Bucaramanga, Colombia

    Dr. Alvaro M. Florez

  2. Systems Proteomics Center, Program in Molecular Biology and Biotechnology, School of Medicine, University of North Carolina, 438A Taylor Hall, CB# 7090, Mason Farm Road, 27599, Chapel Hill, NC, USA

    Dr. Cristina Osorio & Dr. Oscar Alzate

  3. Department of Cell and Developmental Biology, School of Medicine, University of North Carolina, 438A Taylor Hall, CB# 7090, Mason Farm Road, 27599, Chapel Hill, NC, USA

    Dr. Oscar Alzate

Authors
  1. Dr. Alvaro M. Florez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dr. Cristina Osorio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dr. Oscar Alzate
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Oscar Alzate .

Editor information

Editors and Affiliations

  1. Autónoma de Puebla, Facultad de Ciencias Químicas, Benemérita Universidad, Blv. 14 sur y Av. San Claudio, Puebla, 72570, Mexico

    Estibaliz Sansinenea

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media B.V.

About this chapter

Cite this chapter

Florez, A.M., Osorio, C., Alzate, O. (2012). Protein Engineering of Bacillus thuringiensis δ-Endotoxins. In: Sansinenea, E. (eds) Bacillus thuringiensis Biotechnology. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-3021-2_5

Download citation

Publish with us

Policies and ethics

Search

Navigation