Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action

  • Alejandra Bravo
  • Sabino Pacheco
  • Isabel Gómez
  • Blanca Garcia-Gómez
  • Janette Onofre
  • Mario Soberón
Chapter

Abstract

Bacillus thuringiensis (Bt) are Gram-positive bacteria that produce different insecticidal proteins, named Cry, Vip, and Cyt, during the sporulation phase of growth. Here we will describe each one of these classes of protein, their mechanism of action, and their three-dimensional structure if it is available. We will also describe the different strategies that have been used to find novel insecticidal genes that could be used in biological control of insect pests as well as the strategies to evolve known genes to produce proteins with improved toxicity against selected insect pests. These novel strategies include site-directed mutagenesis and domain swapping among different Cry toxins where novel hybrid proteins containing domains or loop regions from different Cry proteins were constructed, resulting in improved toxicity against selected insect pests. Finally we will describe high-throughput systems that have been used to evolve Cry toxins in vitro. Overall, Bt toxins represent one of the most successful strategies for the biocontrol of insect pests.

Keywords

Bacillus thuringiensis Cry toxins Vip toxins Cyt toxins Insect control 

References

  1. 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 69:5343–5353Google Scholar
  2. Arenas I, Bravo A, Soberón M, Gómez I (2010) Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin. J Biol Chem 285:12497–12503Google Scholar
  3. Bardan AH, Guzov VM, Huai Q, Kemp MM, Vishwanath P, Kain W, Nance AM, Evdokimov A, Moshiri F, Turner KH, Wang P, Malvar T, Liu DR (2016) Continous evolution of Bacillus thuringiensis toxins overcome insect resistance. Nature 553:58–63Google Scholar
  4. Baum JA, Chu CR, Rupar M, Brown GR, Donovan WP, Huesing JE, Ilagan O, Malvar TM, Pleau M, Walters M, Vaughn T (2004) Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte. Appl Environ Microbiol 70:4889–4898CrossRefPubMedPubMedCentralGoogle Scholar
  5. Baum JA, Suruku UR, Penn SR, Meyer SE, Subbarao S, Shi X, Flasinki S, Heck GR, Brown RS, Clark TL (2012) Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs. J Econ Entomol 105:616–624CrossRefPubMedGoogle Scholar
  6. Beard CE, Ranasinghe C, Akhurst RJ (2001) Screening for novel cry genes by hybridization. Lett Appl Microbiol 33:241–245Google Scholar
  7. Becker N (1997) Microbial control of mosquitoes: management of the upper Rhine mosquito population as model programme. Parasitol Today 13:485–487Google Scholar
  8. Bravo A (1997) Phylogenetic relationships of the Bacillus thuringiensis delta-endotoxin family proteins and their functional domains. J Bacteriol 179:2793–2801CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41:423–431CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bravo A, Gómez I, Porta H, García-Gómez BI, Rodriguez-Almazan C, Pardo L, Soberón M (2013) Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microb Biotechnol 6:17–26Google Scholar
  11. Burkeness EC, Dively G, Patton T, Morey AC, Hutchison WD (2010) Novel Vip3A Bacillus thuringiensis (Bt) maize approaches high-dose efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) under field conditions: implications for resistance management. GM Crops 1:337–343CrossRefGoogle Scholar
  12. Cantón PE, Reyes EZ, Ruiz de Escudero I, Bravo A, Soberón M (2011) Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism. Peptides 32:595–600CrossRefPubMedGoogle Scholar
  13. Carozzi NB, Kramer VC, Warren GW, Evola S, Koziel MG (1991) Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles. Appl Environ Microbiol 57:3057–3061PubMedPubMedCentralGoogle Scholar
  14. Cerón J, Ortíz A, Quintero R, Güereca L, Bravo A (1995) Specific PCR reaction primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain colletion. Appl Environ Microbiol 61:3826–3831Google Scholar
  15. Chen G, Shu C, LI Y, Song F, Guo Y, Li G, Zhang J (2014) Identification method of cry2 gene based on polymerase chain reaction-high throughput sequencing (PCR-HTS). Chin J Biol Cont 30:610–617Google Scholar
  16. Chougule NP, Li H, Liu H, Linz LB, Narva KE, Meade T, Bonning BC (2013) Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests. Proc Natl Acad Sci USA 110:8465–8470CrossRefPubMedPubMedCentralGoogle Scholar
  17. Chow E, Singh GJP, Gill SS (1989) Binding and aggregation of the 24 kDa toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers. Appl Environ Microbiol 55:2779–2788PubMedPubMedCentralGoogle Scholar
  18. Crickmore N, Bone EJ, Williams JA, Ellar DJ (1995) Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131:249–254Google Scholar
  19. Crickmore N, Baum J, Bravo A, Lereclus D, Narva K, Sampson K, Schnepf E, Sun M, Zeigler DR (2016) Bacillus thuringiensis toxin nomenclature http://www.btnomenclature.info/
  20. de Maagd RA, Weemen-Hendriks M, Stiekema W, Bosch D (2000) Domain III substitution in Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specific determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids. Appl Environ Microbiol 66:1559–1563CrossRefPubMedPubMedCentralGoogle Scholar
  21. de Maagd R, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199CrossRefPubMedGoogle Scholar
  22. de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 37:409–433CrossRefPubMedGoogle Scholar
  23. Dementiev A, Booard J, Sitatam A, Hey T, Kelker MS, Xu X, Hu Y, Vidal-Quist C, Chikwana V, Griffin S, McCaskill D, Wang NX, Hung S-C, Chan MK, Lee MM, Hughes J, Wegener A, Aroian RV, Narva KE, Berry C (2016) The pesticidal Cry6Aa toxin fom Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins. BMC Biol 14:71Google Scholar
  24. Eid J, Fehr A, GrayJ LK, Lyle J, Otto G, Peluso G, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J, Foquet M Gaertner A, Hardenbol P, Heiner Ch, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Mrphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Traves K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S (2009) Real-time DNA sequencing from single polymerase molecules. Science 323:133–138Google Scholar
  25. Fang J, Xu X, Wang P, Zhao JZ, Shelton AM, Cheng J, Feng M-G, Shen Z (2007) Characterization of chimeric Bacillus thuringiensis Vip3 toxins. Appl Environ Microbiol 73:956–961Google Scholar
  26. Girard F, Vachon V, Prefontaine G, Marceau L, Vincent LG, Ch SJ-L, Masson L, Laprade R (2008) Cysteine scanning mutagenesis of alpha 4 a putative pore forming helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa. Appl Environ Microbiol 74:2565–2572CrossRefPubMedPubMedCentralGoogle Scholar
  27. Gómez I, Sánchez J, Miranda R, Bravo A, Soberón M (2002) Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin. FEBS Lett 513:242–246CrossRefPubMedGoogle Scholar
  28. Gómez I, Arenas I, Benitez I, Miranda-Ríos J, Becerril B, Grande R, Almagro JC, Gómez I, Dean DH, Bravo A, Soberón 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 42:10482–10489Google Scholar
  29. Guillet P, Kurstak DC, Philippon B, Meyer R (1990) Use of Bacillus thuringiensis israelensis for onchocerciasis control in West Africa. In: de Barjac H, Sutherland DJ (eds) Bacterial control of mosquitoes and blackflies. Rutgers Univ Press, New Brunswick, pp 187–199CrossRefGoogle Scholar
  30. Han S, Craig JA, Putnam CD, Carozzi NB, Tainer JA (1999) Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex. Nat Struct Biol 6:932–936CrossRefPubMedGoogle Scholar
  31. Hofte H, Whiteley HR (1989) Insecticidal cristal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255PubMedPubMedCentralGoogle Scholar
  32. James C (2015) Global status of commercialized biotech/GM crops: 2015. ISAAA brief N° 51. ISAAA, IthacaGoogle Scholar
  33. Jiménez-Juárez N, Muñoz-Garay C, Gómez I, Saab-Rincon G, Damian-Alamazo JY, Gill SS, Soberón M, Bravo A (2007) Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non toxic to Manduca sexta larvae. J Biol Chem 282:21222–21229CrossRefPubMedGoogle Scholar
  34. Jucovic M, Walters FS, Warren GW, Palekar NV, Chen JS (2008) From enzyme to zymogen: engineering Vip2, an ADP-ribosyltransferase from Bacillus cereus, for conditional toxicity. Prot Eng Des Sel 21:631–638CrossRefGoogle Scholar
  35. Kalman S, Kiehne KL, Libs JL, Yamamoto T (1993) Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl Environ Microbiol 59:1131–1137Google Scholar
  36. Kuo WS, Chak KF (1996) Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl Environ Microbiol 62:1369–1377Google Scholar
  37. Lambert B, Peferoen M (1992) Insecticidal promise of Bacillus thuringiensis. Facts and mysteries about a successful biopesticide. BioScience 42:112–122CrossRefGoogle Scholar
  38. Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin. Appl Environ Microbiol 69:4648–4657CrossRefPubMedPubMedCentralGoogle Scholar
  39. Letowski J, Bravo A, Brousseau R, Masson L (2005) Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays. Appl Environ Microbiol 71:5391–5398Google Scholar
  40. Li H, Shu C, He X, Gao J, Liu R, Huang D (2012) Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis. Curr Microbiol 64:463–468Google Scholar
  41. López-Díaz JA, Cantón PE, Gill SS, Soberón M, Bravo A (2013) Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11Aa toxicity in Aedes aegypti larvae. Environ Microbiol 15:3030–3039PubMedPubMedCentralGoogle Scholar
  42. Mahon RJ, Downes SJ, James B (2012) Vip3A resistence alleles exist at high levels in Australian targets before release of cotton expressing this toxin. PLoS One 7:e39192CrossRefPubMedPubMedCentralGoogle Scholar
  43. Masson L, Schwab G, Mazza A, Brousseau R, Potvin L, Schwartz JLA (2004) Novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes. Biochemist 43:12349–12357CrossRefGoogle Scholar
  44. Muñóz-Garay C, Portugal L, Pardo-López L, Jiménez-Juárez N, Arenas I, Gómez I, Sánchez-López R, Arroyo R, Holzenburg A, Savva Ch G, Soberón M, Bravo A (2009) Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects. Biochim Biophys Acta Biomembr 1788:2229–2237CrossRefGoogle Scholar
  45. Neves MH, Berry C, Regis L (2014) Lysinibacillus sphaericus: toxins and mode of action, applications for mosquito control and resistance management. Adv Insect Physiol 47:89–176CrossRefGoogle Scholar
  46. Ohba M, Mizuki E, Uemori A (2009) Anticancer protein group from Bacillus thuringiensis. Anticancer Res 29:427–434PubMedGoogle Scholar
  47. Pacheco S, Gómez I, Arenas I, Saab-Rincon G, Rodríguez-Almazán C, Gill SS, Bravo A, Soberón M (2009) Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a “ping pong” binding mechanism with Manduca sexta aminopetidase-N and cadherin receptors. J Biol Chem 284:32750–32757CrossRefPubMedPubMedCentralGoogle Scholar
  48. Pardo-López L, Soberón M, Bravo A (2013) Bacillus thuringiensis insecticidal toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol Rev 37:3–22CrossRefPubMedGoogle Scholar
  49. Pérez C, Fernandez LE, Sun J, Folch JL, Gill SS, Soberón M, Bravo A (2005) Bacillus thuringiensis subsp. israeliensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. Proc Natl Acad Sci U S A 102:8303–18308CrossRefGoogle Scholar
  50. Pérez C, Muñoz-Garay C, Portugal L, Sánchez J, Gill SS, Soberón M, Bravo A (2007) Bacillus thuringiensis subsp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure. Cell Microbiol 9:2931–2937CrossRefPubMedPubMedCentralGoogle Scholar
  51. Prasifka PL, Rule DM, Storer NP, Nolting SP, Hendrix WH (2013) Evaluation of corn hybrids expressing Cry34Ab1/Cry35Ab1 and Cry3BbL against the western corn rootworm (Coleoptera: Chrysomelidae). J Econ Entomol 106:823–829CrossRefPubMedGoogle Scholar
  52. Promdonkoy B, Ellar DJ (2000) Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis. Biochem J 350:275–282CrossRefPubMedPubMedCentralGoogle Scholar
  53. Qaim M, Zilberman D (2003) Yield effects of genetically modified crops in developing countries. Science 299:900–902CrossRefPubMedGoogle Scholar
  54. Shu C, Zhang J, Chen G, Liang G, He K, Crickmore N, Huang D, Zhang J, Song F (2013) Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis. J Invertebr Pathol 114:31–33Google Scholar
  55. Soberón M, Pardo-López L, López I, Gómez I, Tabashnik B, Bravo A (2007) Engineering modified Bt toxins to counter insect resistance. Sciences 318:1640–1642CrossRefGoogle Scholar
  56. Soberón M, López-Díaz JA, Bravo A (2013) Cyt toxins produced by Bacillus thuringiensis: a protein fold conserved in several pathogenic microorganisms. Peptides 41:87–93CrossRefPubMedGoogle Scholar
  57. Walters FS, Stacy CM, Lee MK, Palekar N, Chen JS (2008) An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against western corn rootworm larvae. Appl Environ Microbiol 74:367–374CrossRefPubMedGoogle Scholar
  58. Wirth M, Georghiou GP, Federici BA (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 9:10536–10540CrossRefGoogle Scholar
  59. Wirth MC, Park HW, Walton WE, Federici BA (2005) Cyt of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus. Appl Environ Microbiol 71:185–189CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zavala LE, Pardo-López L, Cantón PE, Gómez I, Soberón 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 286:19109–11911CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Alejandra Bravo
    • 1
  • Sabino Pacheco
    • 1
  • Isabel Gómez
    • 1
  • Blanca Garcia-Gómez
    • 1
  • Janette Onofre
    • 1
  • Mario Soberón
    • 1
  1. 1.Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

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