Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Carboxy-terminal half of Cry1C can help vegetative insecticidal protein to form inclusion bodies in the mother cell of Bacillus thuringiensis


Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth and has toxicity against a wide spectrum of lepidopteran insects. Unlike insecticidal crystal proteins, which are produced as parasporal crystal proteins within the cell during sporulation, VIP is secreted into the culture medium. Here, we show that Vip3Aa7 protein can be relocated into the mother cell of B. thuringiensis by altering its synthesis using cry1C promoters, combined with a cry1C transcription termination sequence at the 3′ region and a STAB-SD sequence from cry1C promoters at the 5′ region of the gene. Further, when the carboxy-terminal half of Cry1C was included in the construct, the synthesis of Vip3Aa7 markedly increased, and its expression was relocated into the mother cell in the form of inclusion bodies. The expression of Vip3Aa7 with higher yields in the form of inclusion bodies demonstrated here would facilitate the development of a suitable formulation for the application of this class of insecticidal protein in the field, and the described system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3


  1. Adams LF, Mathewes S, O’Hara P, Petersen A, Gurtler H (1994) Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var tenebrionis. Mol Microbiol 14:38–389

  2. Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:602–6032

  3. Agaisse H, Lereclus D (1996) STAB-SD: a Shine-Dalgarno sequence in the 5′ untranslated region is a determinant of mRNA stability. Mol Microbiol 20:633–643

  4. Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119

  5. Aronson AI (1993) The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol Microbiol 7:489–496

  6. Baum JA, Malvar T (1995) Regulation of insecticidal crystal protein production in Bacillus thuringiensis. Mol Microbiol 18:1–12

  7. Cai QL, Liu ZD, Sun M, Wei F, Yu ZN (2002) The analysis of Bacillus thuringiensis vegetative insecticical protein gene cloning and expression. J Bioeng 18:578–582

  8. Crickmore N, Ellar DJ (1992) Improvement of a possible chaperonin the efficient expression of a cloned CryIIA-endotoxin gene in Bacillus thuringiensis. Mol Microbiol 6:1533–1537

  9. de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming entomo pathogenic bacteria. Annu Rev Genet 37:409–433

  10. Du C, W Martin PA, Nickerson KW (1994) Comparison of disulfide contents and solubility at alkaline pH of insecticidal and noninsecticidal Bacillus thuringiensis protein crystals. Appl Environ Microbiol 60:3847–3853

  11. Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci U S A 93:5389–5394

  12. Ge AZ, Pfister RM, Dean DH (1990) Hyper expression of a Bacillus thuringiensis delta endotoxin gene in Escherichia coli: properties of the product. Gene 93:49–54

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

  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature 227:680–685

  15. Li L, Yang C, Liu ZD, Li F, Yu ZN (2000) Screening of acrystalliferous mutants from Bacillus thuringiensis and their transformation properties. Acta Microbiol Sin 40:85–90

  16. Lu SQ (1999) Construction of genetically engineered Bacillus thuringiensis strains with higher potency and wider spectrum against lepidopteran insects. Thesis for the degree of doctor of phidosophy, Huazhong Agricultural University

  17. Naresh A, Selvapandiyan A, Neema A, Bhatnagar RK (2003) Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis. Biochem Biophys Res Comun 310:158–162

  18. Park HW, Ge B, Bauer LS, Fedrici BA (1998) Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64:3932–3938

  19. Park HW, Bideshi DK, Federici BA (2000) Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield. Appl Environ Microbiol 66:4449–4455

  20. Rang C, Patricia G, Nathalie N, Jeroen VR, Roger F (2005) Novel Vip3-Related Protein from Bacillus thuringiensis. Appl Environ Microbiol 71:6276–6281

  21. Sambrook S, Fritsch EF, Mamiatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor University Press, Cold Spring Harbor

  22. Samir N, Elena MU, Mieke WH, Stefan D, Ivan M, de Maagd RA (2006) Carboxy-terminal effects on crystal formation and insecticidal properties of colorado potato beetle-active Bacillus thuringiensisd-endotoxins. Mol Biotechnol 32:185–196

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

  24. Shao ZZ, Liu ZD, Yu ZN (2001) Effects of the 20-kilodalton-helper protein on Cry1Ac production in Bacillus thuringiensis. Appl Environ Microbiol 67:5362–5369

  25. Strizhov N, Keler M, Mathur J, Koncz Kalman Z, Bosch D, Prudovsky E, Schell J, Sneh B, Koncz C, Zilberstein A (1996) A synthetic cry1C gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci U S A 93:15012–15017

  26. Wong HC, Chang S (1986) Identification of a positive retroregulator that stabilizes mRNAs in bacteria. Proc Natl Acad Sci U S A 83:3233–3237

  27. Yu ZN (1993) Production and application of bacillus thuringiensis preparations. Agricultural Publishing House, Beijing, pp 182–183

  28. Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects. Appl Environ Microbiol 63:532–536

  29. Zeng XH, Zhang HY, Yu ZN, Hu C (1998) Bioassay method of evaluating toxicity of Bacillus thuringiensis against larvae of Spodoptera exigua. Chin J Biol Control 14:172–175

  30. Zhu CG, Ruan LF, Peng DH, Yu ZN, Sun M (2006) Vegetative insecticidal protein enhancing the toxicity of Bacillus thuringiensis subsp kurstaki against Spodoptera exigua. Lett Appl Microbiol 42:109–114

Download references


This work was supported by grants from the National High Technology Research and Development Project of China (863 Program) (No.2006AA02Z174 and No. 2006AA03A243) and the National Basic Research Program of China (973 Program) (No.2003CB114201).

Author information

Correspondence to Ming Sun.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Song, R., Peng, D., Yu, Z. et al. Carboxy-terminal half of Cry1C can help vegetative insecticidal protein to form inclusion bodies in the mother cell of Bacillus thuringiensis . Appl Microbiol Biotechnol 80, 647–654 (2008).

Download citation


  • Vegetative insecticidal protein
  • Bacillus thuringiensis
  • Carboxy-terminal half
  • Relocation expression
  • Inclusion body