Applied Microbiology and Biotechnology

, Volume 102, Issue 8, pp 3687–3699 | Cite as

A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis

  • Xin Zhang
  • Tantan Gao
  • Qi Peng
  • Lai Song
  • Jie Zhang
  • Yunrong Chai
  • Dongmei Sun
  • Fuping Song
Applied genetics and molecular biotechnology


Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σE and σK. Here, we report a novel σE-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.


Non-cry gene promoter P5014 cry1Ac Bacillus thuringiensis 



We thank F He (CAAS) and X Tian (CAAS) for participating in some of the work, S Huang (CAAS) for providing technical support in preparing the AFM pictures, and X Chen (CAAS) and Y Xiao (CAAS) for some experimental performances.

Author contributions

XZ, JZ, DS, and FS designed the experiments. XZ performed the experiments. TG, QP, and FS analyzed the results. LS analyzed the RNA-Seq data. TG, YC, and FS wrote the manuscript.


This study was funded by the National Natural Science Foundation of China (No. 31530095 and No. 31300085).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors

Supplementary material

253_2018_8836_MOESM1_ESM.pdf (266 kb)
Supplementary Table S1 (PDF 266 kb)


  1. Agaisse H, Lereclus D (1994) Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis. Mol Microbiol 13:97–107. CrossRefPubMedGoogle Scholar
  2. Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Arantes O, Lereclus D (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119. CrossRefPubMedGoogle Scholar
  4. Boydston JA, Yue L, Kearney JF, Turnbough CLJ (2006) The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. J Bacteriol 188:7440–7448. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bradley D, Harkey MA, Kim MK, Biever KD, Bauer LS (1995) The insecticidal CryIB crystal protein of Bacillus thuringiensis spp. thuringiensis has dual specificity to Coleopteran and Lepidopteran larvae. J Invertebr Pathol 65:162–173. CrossRefPubMedGoogle Scholar
  6. Bravo A, Agaisse H, Salamitou S, Lereclus D (1996) Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis. Mol Gen Genet 250:734–741. PubMedGoogle Scholar
  7. Bravo A, Likivivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol Biol 41:423–431. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brizzard BL, Schnepf HE, Kronstad JW (1991) Expression of the cryIB crystal protein gene of Bacillus thuringiensis. Mol Gen Genet 231:59–64. CrossRefPubMedGoogle Scholar
  9. Brown KL (1993) Transcriptional regulation of the Bacillus thuringiensis subsp. thompsoni crystal protein gene operon. J Bacteriol 175:7951–7957. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Brown KL, Whiteley HR (1988) Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc Natl Acad Sci U S A 85:4166–4170. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Brown KL, Whiteley HR (1990) Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J Bacteriol 172:6682–6688. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chen J, Hou K, Qin P, Liu H, Yi B, Yang W, Wu W (2014) RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes. BMC Genomics 15:571. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dervyn E, Poncet S, Klier A, Rapoport G (1995) Transcriptional regulation of the cryIVD gene operon from Bacillus thuringiensis subsp. israelensis. J Bacteriol 177:2283–2291. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Desouza MT, Lecadet MM, Lereclus D (1993) Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription. J Bacteriol 175:2952–2960. CrossRefGoogle Scholar
  15. Du C, Nickerson KW (1996) Bacillus thuringiensis HD-73 spores have surface-localized Cry1Ac toxin: physiological and pathogenic consequences. Appl Environ Microbiol 62:3722–3726PubMedPubMedCentralGoogle Scholar
  16. Du L, Wei J, Han L, Chen Z, Zhang J, Song F, Huang D (2011) Characterization of Bacillus thuringiensis sigK disruption mutant and its influence on activation of cry3A promoter. Wei Sheng Wu Xue Bao 51:1177–1184PubMedGoogle Scholar
  17. Du L, Qiu L, Peng Q, Lereclus D, Zhang J, Song F, Huang D (2012) Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor. Appl Environ Microbiol 78:4164–4168. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gerwick BC, Sparks TC (2014) Natural products for pest control: an analysis of their role, value and future. Pest Manag Sci 70:1169–1185. CrossRefPubMedGoogle Scholar
  19. He X, Sun Z, He K, Guo S (2017) Biopolymer microencapsulations of Bacillus thuringiensis crystal preparations for increased stability and resistance to environmental stress. App Microbiol Biotechnol 101:2779–2789. CrossRefGoogle Scholar
  20. Huang D, Zhang J, Song F, Lang Z (2007) Microbial control and biotechnology research on Bacillus thuringiensis in China. J Invertebr Pathol 95:175–180. CrossRefPubMedGoogle Scholar
  21. Kroos L, Zhang B, Ichikawa H, Yu YT (1999) Control of sigma factor activity during Bacillus subtilis sporulation. Mol Microbiol 31:1285–1294. CrossRefPubMedGoogle Scholar
  22. Lacey LA, Grzywacz D, Shapiro-Ilan DI, Frutos R, Brownbridge M, Goettel MS (2015) Insect pathogens as biological control agents: back to the future. J Invertebr Pathol 132:1–41. CrossRefPubMedGoogle Scholar
  23. Lereclus D, Arantes O, Chaufaux J, Lecadet M (1989) Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60:211–217. Google Scholar
  24. Li CR, Du LX, Peng Q (2013) Construction of high-level expression vector for Bacillus thuringiensis. Microbiol China 40:350–361Google Scholar
  25. Liu R, Yu G, Zou W, Du T (2008) Improvements in technique of madding ultrathin section for transmission electron microscope. Jiangxi For Sci Technol 41–43Google Scholar
  26. Macaluso A, Mettus AM (1991) Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA. J Bacteriol 173:1353–1356. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  28. Ni D, Xu P, Gallagher S (2016) Immunoblotting and immunodetection. In: Coligan JE (ed) Current protocols in immunology, Wiley, Hoboken, pp 8.10.11–18.10.36Google Scholar
  29. Peng Q, Wang G, Liu G, Zhang J, Song F (2015) Identification of metabolism pathways directly regulated by sigma54 factor in Bacillus thuringiensis. Front Microbiol 6:407. PubMedPubMedCentralGoogle Scholar
  30. Sambrook BJ, Russell DW (2015) Molecular cloning. In: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  31. Sanchis V, Gohar M, Chaufaux J, Arantes O, Meier A, Agaisse H, Cayley J, Lereclus D (1999) Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance. Appl Environ Microbiol 65:4032–4039PubMedPubMedCentralGoogle Scholar
  32. Saxild HH, Andersen L, Hammer K (1996) Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein. J Bacteriol 178:424–434. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A 54:704–711. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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 62:775–806PubMedPubMedCentralGoogle Scholar
  35. Shu C, Yu H, Wang R, Fen S, Su X, Huang D, Zhang J, Song F (2009) Characterization of two novel cry8 genes from Bacilllus thuringiensis strain BT185. Curr Microbiol 58:389–392. CrossRefPubMedGoogle Scholar
  36. Skerlova J, Fabry M, Hubalek M, Otwinowski Z, Rezacova P (2014) Structure of the effector-binding domain of deoxyribonucleoside regulator DeoR from Bacillus subtilis. FEBS J 281:4280–4292. CrossRefPubMedGoogle Scholar
  37. Wang G, Zhang J, Song F, Wu J, Feng S, Huang D (2006) Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests. Appl Microbiol Biotechnol 72:924–930. CrossRefPubMedGoogle Scholar
  38. Weigel D, Glazebrook J (2010) Transmission electron microscopy (TEM) freeze substitution of plant tissues. Cold Spring Harb Protoc.
  39. Wong HC, Schnepf HE, Whiteley HR (1983) Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem 258:1960–1967PubMedGoogle Scholar
  40. Xue J, Liang G, Crickmore N, Li H, He K, Song F, Feng X, Huang D, Zhang J (2008) Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects. FEMS Microbiol Lett 280:95–101. CrossRefPubMedGoogle Scholar
  41. Yang H, Wang P, Peng Q, Rong R, Liu C, Lereclus D, Zhang J, Song F, Huang D (2012) Weak transcription of the cry1Ac gene in nonsporulating Bacillus thuringiensis cells. Appl Environ Microbiol 78:6466–6474. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yang J, Peng Q, Chen Z, Deng C, Shu C, Zhang J, Huang D, Song F (2013) Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis. J Bacteriol 195:2887–2897. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Yoshisue H, Fukada T, Yoshida K, Sen K, Kurosawa S, Sakai H, Komano T (1993) Transcriptional regulation of Bacillus thuringiensis subsp. israelensis mosquito larvicidal crystal protein gene cryIVA. J Bacteriol 175:2750–2753. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zeng XM, Saxild HH, Switzer RL (2000) Purification and characterization of the DeoR repressor of Bacillus subtilis. J Bacteriol 182:1916–1922. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Zhang JB, Schairer HU, Schnetter W, Lereclus D, Agaisse H (1998) Bacillus popilliae cry18Aa operon is transcribed by sigma(E) and sigma(K) forms of RNA polymerase from a single initiation site. Nucleic Acids Res 26:1288–1293. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Zheng Q, Wang G, Zhang Z, Qu N, Zhang Q, Peng Q, Zhang J, Gao J, Song F (2014) Expression of cry1Ac gene directed by PexsY promoter of the exsY gene encoding component protein of exosporium basal layer in Bacillus thuringiensis. Acta Microbiol Sin 54:1138–1145Google Scholar
  47. Zhou C, Zheng Q, Peng Q, Du L, Shu C, Zhang J, Song F (2014) Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant. Appl Microbiol Biotechnol 98:7901–7909. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Life Science and TechnologyHeilongjiang Bayi Agricultural UniversityDaqingChina
  2. 2.State Key Laboratory for Biology of Plant Diseases and Insect PestsInstitute of Plant Protection, Chinese Academy of Agricultural SciencesBeijingChina
  3. 3.CAS Key Laboratory of Genome Sciences and InformationBeijing Institute of Genomics, Chinese Academy of SciencesBeijingChina
  4. 4.Department of BiologyNortheastern UniversityBostonUSA

Personalised recommendations