A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis
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.
KeywordsNon-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.
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.
This article does not contain any studies with human participants or animals performed by any of the authors
- 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. https://doi.org/10.1111/j.1365-2958.1994.tb00405.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1006/jipa.1995.1024 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1111/j.1574-6968.1989.tb03448.x Google Scholar
- Li CR, Du LX, Peng Q (2013) Construction of high-level expression vector for Bacillus thuringiensis. Microbiol China 40:350–361Google Scholar
- 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
- Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- 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
- Sambrook BJ, Russell DW (2015) Molecular cloning. In: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- 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
- 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. https://doi.org/10.1128/jb.178.2.424-434.1996 CrossRefPubMedPubMedCentralGoogle Scholar
- Weigel D, Glazebrook J (2010) Transmission electron microscopy (TEM) freeze substitution of plant tissues. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot4959
- 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. https://doi.org/10.1111/j.1574-6968.2007.01053.x CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1128/JB.00112-13 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1128/jb.175.9.2750-2753.1993 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1093/nar/26.5.1288 CrossRefPubMedPubMedCentralGoogle Scholar
- 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