Abstract
Six strains of Bacillus thuringiensis previously selected as highly toxic against Manduca sexta and Plutella xylostella were analyzed by PCR screening in order to identify the cry genes active on Lepidoptera. According to their gene content and insecticidal potency, these strains were cultured and aliquots taken at different pre- and post-sporulation times. Total RNA was extracted and used as template in RT-PCR analyses directed to identify mRNAs of the previously identified cry genes. Results showed transcription of genes cry1A, cry1E, cry1I, and cry2 even before the onset of sporulation. However, this early transcription did not lead to an appreciable parasporal protein synthesis until t5–t9, as deduced from SDS-PAGE profiles. As for cry1I gene, the corresponding protein was not detected, as expected, but cry1I mRNAs were present at least until t5. Interestingly, strains expressing four cry genes from the end of the log phase onwards exhibited kinetics characterized by a very long transition phase, whereas the strain expressing only one cry gene showed a very short transition phase. Strains expressing three genes showed an intermediate profile. These results indicate that the transcription of B. thuringiensis cry1 and cry2 genes in natural strains can start several hours before massive crystal synthesis occurs and that this translation is probably competing with transcriptional regulators required for the sporulation onset.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Agaisse H, Lereclus D (1994) Expression in Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spoOA mutant. J Bacteriol 176:4734–4741
Agaisse H, Lereclus D (1995) How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177:6027–6032
Crickmore N, Zeigler DR, Feitelson J, Schnepf HE, Van Rie J, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813
de Hoon MJL, Eichenberger P, Vitkup D (2010) Hierarchical evolution of the bacterial sporulation network. Curr Biol 20:R735–R745
De Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evoled specific toxins to colonize the insect world. Trends Genet 17:193–199
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 37:409–433
Federici BA (2005) Insecticidal bacteria. An overwhelming success for invertebrate pathology. J Invertebr Pathol 89:30–38
García-Gómez BI, Sánchez J, Martínez de Castro DL, Ibarra JE, Bravo A, Soberón M (2013) Efficient production of Bacillus thuringiensis Cry1AMod toxins under regulation of cry3Aa promoter and single cysteine mutations in the protoxin region. Appl Environ Microbiol 79:6969–6973
Gleave AP, Williams R, Hedges RJ (1993) Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki. Appl Environ Microbiol 59:1683–1687
Gong Y, Li M, Xu D, Wang H, He J, Wu D, Chen D, Qiu N, Bao Q, Sun M, Yu Z (2012) Comparative proteomic analysis revealed metabolic changes and the translational regulation of Cry protein synthesis in Bacillus thuringiensis. J Proteom 75:1235–1246
Ibarra JE, Federici BA (1987) An alternative bioassay employing neonate larvae for determining the toxicity of suspended particles to mosquitoes. J Am Mosq Control Assoc 3:187–192
Juárez-Pérez VM, Ferrandis MD, Frutos R (1997) PCR-based approach for the detection of novel Bacillus thuringiensis genes. Appl Environ Microbiol 63:2997–3002
Kostichka K, Warren GW, Mullins M, Mullins AD, Palekar NV, Craig JA, Koziel MG, Estruch JJ (1996) Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase. J Bacteriol 178:2141–2144
Lereclus D, Agaisse H, Grandvalet C, Salamitou S, Gominet M (2000) Regulation of toxin and virulence gene transcription in Bacillus thuringiensis. Int J Med Microbiol 290:295–299
Park H-W, Ge B, Bauer LS, Federici 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
Pérez-García G, Basurto-Ríos R, Ibarra JE (2010) Potential effect of a putative σH-driven promoter on the over expression of the Cry1Ac toxin of Bacillus thuringiensis. J Invertebr Pathol 104:140–146
Poncet S, Dervyn E, Klier A, Rapoport G (1997) SpoOA represses transcription of the cry toxin genes in Bacillus thuringiensis. Microbiology 143:2743–2751
Porcar M, Juárez-Pérez VM (2003) PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiol Rev 26:419–432
Ruiz de Escudero I, Estela A, Porcar M, Martínez C, Oguiza JA, Escriche B, Ferré J, Caballero P (2006) Molecular and insecticidal characterization of a Cry1I protein toxic to insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. Appl Environ Microbiol 72:4796–4804
Sanchis V (2011) From microbial sprays to insect-resistant transgenic plants: history of the biospesticide Bacillus thuringiensis. A review. Agron Sustain Dev 31:217–231
Schnepf HE (2012) Bacillus thuringiensis Recombinant Insecticidal Protein Production. In: Sansinenea E (ed) Bacillus thuringiensis Biotechnology. Springer, Heidelberg, pp 259–281
Walter T, Aronson A (1999) Specific binding of the E2 subunit of pyruvate dehydrogenase to the upstream region of Bacillus thuringiensis protoxin genes. J Biol Chem 274:7901–7906
Wang J, Mei H, Qian H, Tang Q, Liu X, Yu Z, He J (2012) Expression profile and regulation of spore and parasporal crystal formation-associated genes in Bacillus thuringiensis. J Proteom Res 12:5487–5501
Wu D, Federici BA (1993) A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J Bacteriol 175:5276–5280
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
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
10482_2014_160_MOESM1_ESM.jpg
Growth curves from strains LBIT-7, LBIT-404 and LBIT-418. Lines are defined by the best quadratic fit (logistic curve) of data
10482_2014_160_MOESM2_ESM.jpg
Growth curves from strains LBIT-127, LBIT-287 and LBIT-290. Lines are defined by the best quadratic fit (logistic curve) of data
10482_2014_160_MOESM3_ESM.jpg
Detection of transcripts from genes cry1A, cry1E, cry1I, and cry2 by RT-PCR analysis from strain LBIT-7. All transcripts were detected at the three sampling times (including the cry1I transcript at t5). For details, see Table 3
Rights and permissions
About this article
Cite this article
Porcar, M., Déleclusse, A., Ibarra, J.E. et al. Early transcription of Bacillus thuringiensis cry genes in strains active on Lepidopteran species and the role of gene content on their expression. Antonie van Leeuwenhoek 105, 1007–1015 (2014). https://doi.org/10.1007/s10482-014-0160-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-014-0160-1