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. 1993 May;175(10):2952–2960. doi: 10.1128/jb.175.10.2952-2960.1993

Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription.

M T de Souza 1, M M Lecadet 1, D Lereclus 1
PMCID: PMC204613  PMID: 8491716

Abstract

The cryIIIA gene encoding a coleopteran-specific toxin is poorly expressed in Bacillus thuringiensis when cloned in a low-copy-number plasmid. This weak expression is observed when the gene is cloned only with its promoter and its putative terminator. cryIIIA gene expression was analyzed by using deletion derivatives of a larger DNA fragment carrying the toxin gene and additional adjacent sequences. The results indicate that a 1-kb DNA fragment located 400 bp upstream of the promoter strongly enhances CryIIIA production in B. thuringiensis sporulating cells. Similar results were obtained when the low-copy-number plasmid pHT304 carrying transcriptional fusions between upstream regions of cryIIIA and the lacZ gene was used. Analysis of the start sites, the sizes, and the amounts of cryIIIA-specific mRNAs shows that the enhancement occurs at the transcriptional level by increasing the number of cryIIIA-specific transcripts from the onset of sporulation to about 6 h after the onset of sporulation. The nucleotide sequence of the 1-kb activating fragment and of the 700 bp containing the promoter region and the 5' end of cryIIIA were determined. No potential protein-coding sequences were found upstream of the promoter. The major characteristic of the 1-kb activating fragment is the presence of a 220-bp A + T-rich region.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams L. F., Brown K. L., Whiteley H. R. Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter. J Bacteriol. 1991 Jun;173(12):3846–3854. doi: 10.1128/jb.173.12.3846-3854.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arantes O., Lereclus D. Construction of cloning vectors for Bacillus thuringiensis. Gene. 1991 Dec 1;108(1):115–119. doi: 10.1016/0378-1119(91)90495-w. [DOI] [PubMed] [Google Scholar]
  3. Brown K. L., Whiteley H. R. Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4166–4170. doi: 10.1073/pnas.85.12.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown K. L., Whiteley H. R. Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J Bacteriol. 1990 Dec;172(12):6682–6688. doi: 10.1128/jb.172.12.6682-6688.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crickmore N., Ellar D. J. Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. Mol Microbiol. 1992 Jun;6(11):1533–1537. doi: 10.1111/j.1365-2958.1992.tb00874.x. [DOI] [PubMed] [Google Scholar]
  6. Donovan W. P., Gonzalez J. M., Jr, Gilbert M. P., Dankocsik C. Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene. Mol Gen Genet. 1988 Nov;214(3):365–372. doi: 10.1007/BF00330468. [DOI] [PubMed] [Google Scholar]
  7. Débarbouillé M., Raibaud O. Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter. J Bacteriol. 1983 Mar;153(3):1221–1227. doi: 10.1128/jb.153.3.1221-1227.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elleman T. C., von Ahlefeldt D. A. Nucleotide sequence of the pilin gene from Bacteroides nodosus strain 238 (serogroup G). Nucleic Acids Res. 1987 Sep 11;15(17):7189–7189. doi: 10.1093/nar/15.17.7189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glatron M. F., Rapoport G. Biosynthesis of the parasporal inclusion of Bacillus thuringiensis: half-life of its corresponding messenger RNA. Biochimie. 1972;54(10):1291–1301. doi: 10.1016/s0300-9084(72)80070-1. [DOI] [PubMed] [Google Scholar]
  10. Herrnstadt C., Gilroy T. E., Sobieski D. A., Bennett B. D., Gaertner F. H. Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego. Gene. 1987;57(1):37–46. doi: 10.1016/0378-1119(87)90174-0. [DOI] [PubMed] [Google Scholar]
  11. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaneko T., Nozaki R., Aizawa K. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol Immunol. 1978;22(10):639–641. doi: 10.1111/j.1348-0421.1978.tb00414.x. [DOI] [PubMed] [Google Scholar]
  13. Lecadet M. M., Blondel M. O., Ribier J. Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber. J Gen Microbiol. 1980 Nov;121(1):203–212. doi: 10.1099/00221287-121-1-203. [DOI] [PubMed] [Google Scholar]
  14. Lecadet M. M., Chaufaux J., Ribier J., Lereclus D. Construction of Novel Bacillus thuringiensis Strains with Different Insecticidal Activities by Transduction and Transformation. Appl Environ Microbiol. 1992 Mar;58(3):840–849. doi: 10.1128/aem.58.3.840-849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lecadet M. M., Sanchis V., Menou G., Rabot P., Lereclus D., Chaufaux J., Martouret D. Identification of a delta-Endotoxin Gene Product Specifically Active against Spodoptera littoralis Bdv. among Proteolysed Fractions of the Insecticidal Crystals of Bacillus thuringiensis subsp. aizawai 7.29. Appl Environ Microbiol. 1988 Nov;54(11):2689–2698. doi: 10.1128/aem.54.11.2689-2698.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leonhardt H., Alonso J. C. Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis. J Gen Microbiol. 1988 Mar;134(3):605–609. doi: 10.1099/00221287-134-3-605. [DOI] [PubMed] [Google Scholar]
  18. Lereclus D., Arantes O. spbA locus ensures the segregational stability of pTH1030, a novel type of gram-positive replicon. Mol Microbiol. 1992 Jan;6(1):35–46. doi: 10.1111/j.1365-2958.1992.tb00835.x. [DOI] [PubMed] [Google Scholar]
  19. Lereclus D., Arantès O., Chaufaux J., Lecadet M. Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett. 1989 Jul 15;51(1):211–217. doi: 10.1016/0378-1097(89)90511-9. [DOI] [PubMed] [Google Scholar]
  20. Meissner P. S., Sisk W. P., Berman M. L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4171–4175. doi: 10.1073/pnas.84.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Msadek T., Kunst F., Henner D., Klier A., Rapoport G., Dedonder R. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J Bacteriol. 1990 Feb;172(2):824–834. doi: 10.1128/jb.172.2.824-834.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perkins J. B., Youngman P. J. Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Jan;83(1):140–144. doi: 10.1073/pnas.83.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ribier J., Lecadet M. M. Etude ultrastructurale et cinétique de la sporulation de Bacillus thuringiensis var. Berliner 1715. Remarques sur la formation de l'inclusion parasporale. Ann Microbiol (Paris) 1973 Apr;124(3):311–344. [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sekar V., Thompson D. V., Maroney M. J., Bookland R. G., Adang M. J. Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7036–7040. doi: 10.1073/pnas.84.20.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  27. Visick J. E., Whiteley H. R. Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli. J Bacteriol. 1991 Mar;173(5):1748–1756. doi: 10.1128/jb.173.5.1748-1756.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wong H. C., Chang S. Identification of a positive retroregulator that stabilizes mRNAs in bacteria. Proc Natl Acad Sci U S A. 1986 May;83(10):3233–3237. doi: 10.1073/pnas.83.10.3233. [DOI] [PMC free article] [PubMed] [Google Scholar]

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