Cry2A toxins from Bacillus thuringiensis expressed in insect cells are toxic to two lepidopteran insects

  • G. M. S. Lima
  • R. W. S. Aguiar
  • R. F. T. Corrêa
  • E. S. Martins
  • A. C. M. Gomes
  • T. Nagata
  • M. T. De-Souza
  • R. G. Monnerat
  • B. M. Ribeiro
Original Paper


The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects. The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC50 of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC50 of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus.


Baculovirus Cry2Aa Cry2Ab Bacillus thuringiensis Lepidopteran insects 



We are grateful to Embrapa Recursos Genéticos e Biotecnologia for supplying insect larvae. This work was supported by the following Brazilian agencies: PRONEX, FAPDF, CNPq, FINATEC and CAPES.


  1. Aguiar RWS, Martins ES, Valicente FH, Carneiro NP, Batista AC, Melatti VM et al (2006) A recombinant truncated Cry1Ca protein is toxic to lepidopteran insects and forms large cuboidal crystals in insect cells. Curr Microbiol 53:287–292. doi: 10.1007/s00284-005-0502-3 CrossRefGoogle Scholar
  2. Aronson AI (1994) Flexibility in protoxins composition of Bacillus thuringiensis. FEMS Microbiol Lett 117:21–28. doi: 10.1111/j.1574-6968.1994.tb06737.x CrossRefGoogle Scholar
  3. Aronson AI, Han E, McGaughey W, Johnson D (1991) The solubility if the inclusion proteins from Bacillus thuringiensis is depend upon protoxin composition and is a factoring toxicity to insects. Appl Environ Microbiol 57:981–986Google Scholar
  4. Baum JA, Malvar T (1995) Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol Microbiol 18:1–12. doi: 10.1111/j.1365-2958.1995.mmi_18010001.x CrossRefGoogle Scholar
  5. Bietlot HP, Vishnubhatla I, Carey PR, Pozsgay M, Kaplan H (1990) Characterization of the cysteine residues and disulphide linkages in the protein crystal of Bacillus thuringiensis. Biochem J 267:309–315Google Scholar
  6. Bravo A (2004) Familia de proteínas inseticidas de Bacillus thuringiensis. In: Bravo A, Ceron J (eds) Bacillus thuringiensis en el control biológico. Editorial Buena Semilla, Bogotá, pp 49–68Google Scholar
  7. Bravo A, Arrieta G, Benintende G, Real MD, Espinoza AM, Ibarra J et al (2001) Metodologias utilizadas en investigación sobre bactérias entomopatógenas. Unam, MéxicoGoogle Scholar
  8. Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435. doi: 10.1016/j.toxicon.2006.11.022 CrossRefGoogle Scholar
  9. Chang JH, Choi JY, Jin BR, Roh JY, Olszewski A, Seo SJ et al (2003) An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin. J Invertebr Pathol 84:30–37. doi: 10.1016/S0022-2011(03)00121-6 CrossRefGoogle Scholar
  10. Choma CT, Kaplan H (1990) Folding and unfolding of the protoxin from Bacillus thuringiensis: evidence that the toxic moiety is present in an active conformation. Biochemistry 29:10971–10977. doi: 10.1021/bi00501a015 CrossRefGoogle Scholar
  11. Crickmore N, Ellar DJ (1992) Involvement of possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. Mol Microbiol 6:1533–1537. doi: 10.1111/j.1365-2958.1992.tb00874.x CrossRefGoogle Scholar
  12. Crickmore N, Wheeler VC, Ellar DJ (1994) Use of an operon fusion to induce expression and crystallization of a Bacillus thuringiensis delta endotoxin enconded by a cryptic gene. Mol Gen Genet 242:365–378. doi: 10.1007/BF00280428 CrossRefGoogle Scholar
  13. Crickmore N, Bone EJ, Williams JA, Ellar DJ (1995) Contribution of the individual components of the delta-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131:249–254Google Scholar
  14. Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Rie J, Lereclus D et al (1998) Revision of the nomenclature of the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813Google Scholar
  15. Dankocsik C, Donovan WP, Jany CS (1990) Activation of a cryptical crystal protein gene of Bacillus thuringiensis subspecies kurstaki by gene fusion and determination of crystal protein insecticidal specificity. Mol Microbiol 4:2087–2094. doi: 10.1111/j.1365-2958.1990.tb00569.x CrossRefGoogle Scholar
  16. de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199. doi: 10.1016/S0168-9525(01)02237-5 CrossRefGoogle Scholar
  17. Donovan WP, Dankocsik CC, Gilbert MP, Gawron-Burke MC, Groat RG, Carlton BC (1988) Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki. J Biol Chem 263:561–567Google Scholar
  18. El-Bendary (2006) Bacillus thuringiensis and Bacillus sphaericus biopesticides production. J Basic Microbiol 46:158–170Google Scholar
  19. Estruch JJ, Carozzi NB, Desai N, Nicholas B, Duck NB, Warren GW, Koziel MG (1997) Transgenic plants: an emerging approach to pest control. Nat Biotecnol 15:137–141CrossRefGoogle Scholar
  20. Ferre J, van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Ann Rev Entomol 47:501–533CrossRefGoogle Scholar
  21. Finney DJ (1971) Probit analysis. Cambridge University Press, CambridgeGoogle Scholar
  22. Ge B, Bideshi D, Moar JW, Federici BA (1998) Diferential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis. FEMS Microbiol Lett 165:35–41CrossRefGoogle Scholar
  23. Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53:242–255Google Scholar
  24. Höfte H, de Greve H, Seurinck J, Jansens S, Mahillon J, Ampe C, Vandekerckhove J, Vanderbruggen H, van Montagu M, Zabeau M (1986) Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem 161:273–280CrossRefGoogle Scholar
  25. Iriarte J, Porcar M, Lecadet M, Caballero P (2000) Isolation and characterization of Bacillus thuringiensis strains from aquatic environments in Spain. Curr Microbiol 40:402–408CrossRefGoogle Scholar
  26. Jarvis DL (1997) Baculovirus expression vectors. In: Miller LK (ed) The baculovirus. Plenum, New York, pp 341–389Google Scholar
  27. Lereclus D, Bourgouin C, Lecadet MM, Klier A, Rapoport G (1989) Role, structure and molecular organization of the genes coding for the parasporal δ-endotoxins of Bacillus thuringiensis. In: Smith I, Slepecky RA, Setlow P (eds) Regulation of procaryotic development: structural and functional analysis of bacterial sporulation and germination. American Society for Microbiology, Washington, pp 255–276Google Scholar
  28. Lu A, Miller LK (1997) Regulation of baculoviruses late and very late expression. In: Miller LK (ed) The baculoviruses. Plenum Press, New York, pp 193–216Google Scholar
  29. Martens JW, Honéee G, Zuidema D, Van Lent JWM, Visser B, Vlak JM (1990) Insecticidal activity of a bacterial crystal protein expressed by a recombinant baculovirus in insect cells. Appl Environ Microbiol 56:2764–2770Google Scholar
  30. Martins ES, Aguiar RW, Martins NF, Melatti VM, Falcão R, Gomes AC, Ribeiro BM, Monnerat RG (2008) Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm(Spodoptera frugiperda). J Appl Microbiol 104:1363–1371CrossRefGoogle Scholar
  31. Merryweather AT, Weyer U, Harris MPG, Hirst M, Booth T, Possee D (1990) Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta-endotoxin. J Gen Virol 71:1535–1544CrossRefGoogle Scholar
  32. Moscardi F (1999) Assessment of the application of baculoviruses for control of Lepidoptera. Annu Rev Entomol 44:257–289CrossRefGoogle Scholar
  33. O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus expression vectors: a laboratory manual. Freeman, New YorkGoogle Scholar
  34. Pang Y, Frutos R, Federici BA (1992) Synthesis and toxicity of full length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells a baculovírus vector. J Gen Virol 73:89–101CrossRefGoogle Scholar
  35. Park H, Bideshi DK, Johnson JJ, Federici BA (1999) Diferential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence. FEMS Microbiol Lett 181:319–327CrossRefGoogle Scholar
  36. Pérez C, Muñoz-Garay C, Portugal LC, Sánchez J, Gill SS, Soberón M, Bravo AC (2007) Bacillus thuringiensis ssp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure. Cell Microbiol 9:2931–2937CrossRefGoogle Scholar
  37. Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29:303–329CrossRefGoogle Scholar
  38. Ribeiro BM, Crook NE (1993) Expression of full length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp. kurstaki in baculovírus and pathogenicity of the recombinant viruses. J Invertebr Pathol 62:121–130CrossRefGoogle Scholar
  39. Ribeiro BM, Crook NE (1998) Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis. Braz J Med Biol Res 31:763–769CrossRefGoogle Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, New YorkGoogle Scholar
  41. Sarfraz M (2004) Interaction between diamondback moth and Bacillus thuringiensis. Outlooks Pest Manag 15:167–171CrossRefGoogle Scholar
  42. Sasaki J, Asano S, Hashimoto N, Lay BW, Hastowo S, Bando H, Iizuka T (1997) Characterization of a cry2A gene cloned from an isolate of Bacillus thuringiensis serovar sotto. Curr Microbiol 35:1–8CrossRefGoogle Scholar
  43. 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 62:775–806Google Scholar
  44. Soberón M, Fernández LE, Pérez C, Gill SS, Bravo A (2007) Mode of action of mosquitocidal Bacillus thuringiensis toxins. Toxicon 49:597–600CrossRefGoogle Scholar
  45. Staples N, Ellar D, Crickmore N (2001) Cellular localization and characterization of the Bacillus thuringiensis Orf2 crystalization factor. Curr Microbiol 42:388–392CrossRefGoogle Scholar
  46. Szewczyk B, Hoyos-Carvajal L, Palusek M, Skrzecz I, Lobo de Souza M (2006) Baculoviruses- re-emerging biopesticides. Biotech Adv 24:143–160CrossRefGoogle Scholar
  47. Thomas WE, Ellar DJ (1983) Mechanism of action of Bacillus thuringiensis var israelensis delta-endotoxin. FEBS Lett 154:362–368CrossRefGoogle Scholar
  48. Vaeck M, Reynaerts A, Hofte A, Jansens S, De Beuckeleer M, Dean C, Zabeau M, Van Montagu M, Leemans J (1987) Transgenic plants protected from insect attack. Nature 328:33–37CrossRefGoogle Scholar
  49. Wang X, Ooi BG, Miller LK (1991) Baculovirus vectors for multiple gene expression and for occluded virus production. Gene 100:131–137CrossRefGoogle Scholar
  50. Widner WR, Whiteley HR (1989) Two highly related instecidal crystal proteins of Bacillus thuringiensis subsp kurstaki possess different host range specificities. J Bacteriol 171:965–974Google Scholar
  51. Wirth MC, Federici BA, Walton WE (2000) Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti. Appl Environ Microbiol 66:1093–1097CrossRefGoogle Scholar
  52. Wirth MC, Delécluse A, Walton WE (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 67:3280–3284CrossRefGoogle Scholar
  53. Xue JL, Cai QX, Zheng DS, Yuan ZM (2005) The synergistic between Cry1Aa and Cry1C from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera. Lett Appl Microbiol 40:460–465CrossRefGoogle Scholar
  54. Yamamoto T, McLaughlin RE (1981) Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. Kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem Biophys Res Commun 103:414–421CrossRefGoogle Scholar
  55. Zhang LL, Lin J, Lu OSL, Guan CY, Zhang QL, Guan Y, Zhang Y, Ji JT, Huang ZP, Guan X (2007) A novel Bacillus thuringiensis strain LLB6, isolated from bryophytes, and its new cry2Ac-type gene. Lett Appl Microbiol 44:301–307CrossRefGoogle Scholar
  56. Zhao JZ, Cao J, Li Y, Collins HL, Roush RT (2003) Earle, E.D., Shelton, A.M.: Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnol 21:1493–1497CrossRefGoogle Scholar
  57. Zhao JZ, Cao J, Collins HL, Bates SL, Roush RT, Earle ED, Shelton AM (2005) Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants. Proc Natl Acad Sci USA 102:8426–8430CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • G. M. S. Lima
    • 1
  • R. W. S. Aguiar
    • 1
  • R. F. T. Corrêa
    • 1
  • E. S. Martins
    • 1
    • 2
  • A. C. M. Gomes
    • 2
  • T. Nagata
    • 1
  • M. T. De-Souza
    • 1
  • R. G. Monnerat
    • 2
  • B. M. Ribeiro
    • 1
  1. 1.Department of Cell BiologyUniversity of BrasíliaBrasiliaBrazil
  2. 2.Embrapa – Recursos Genéticos e BiotecnologiaBrasiliaBrazil

Personalised recommendations