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High-level transcription of the cryIIIA toxin gene of Bacillus thuringiensis depends on a second promoter located 600 bp upstream of the translational start site

Marlene Teixeira De-Souza^I; Herve Agaisse^II; Didier Lereclus^II,III

^ILaboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília DF, Brasil. Fax: +55 061 349-8411. E-mail: marlts@guarany.cpd.unb.br. Send correspondence to M.T. De-S.
^IIUnite de Biochimie Microbienne, Departement des Biotechnologies, URA 1300 CNRS, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
^IIIStation de Recherches de Lutte Biologique, Institut National de la Recherche Agronomique, La Minière, 78280 Guyancourt, France

ABSTRACT

The cryIIIA gene expression was analyzed in a low-copy number plasmid background by using deletion derivatives of a larger DNA fragment carrying the toxin gene and additional adjacent regions. The results indicated that a 1-kb DNA segment (the H₂-H₃ fragment) located 400 bp upstream from the presumed promoter strongly increases CryIIIA toxin production in Bacillus thuringiensis sporulating cells at the transcriptional level (De-Souza et al., J. Bacteriol. 175: 2952-2960, 1993). The delineation and functional analysis of this region showed that a functional promoter is located within the H₂-H₃ fragment, situated 400 bp from the promoter previously described and about 600 bp upstream of the crylllA gene coding region. The association of the region containing this promoter with the DNA sequence directly upstream from the cryIIIA gene is required to obtain full expression of the transcriptional fusions. The putative -35 e -10 regions of this upstream promoter do not match the sporulation-specific cry genes promoter sequences, but are similar to those recognized by the RNA polymerase bound to s^A.

Keywords: transcription; cryIIIA; toxin gene; Bacillus thuringiensis.

REFERENCES

Adams, L.F., Brown, K.L. and Whiteley, H.R. (1991). Molecular cloning and characterization of two genes encoding sigma factors that direct transcription from a Bacillus thuringiensis crystal protein gene promoter. J. Bacteriol. 173: 3846-3854.

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

Brown, K.L. and Whiteley, H.R. (1988). Isolation of a Bacillus thuringiensis RNA polymerase capable of transcribing crystal protein genes. Proc. Natl. Acad. Sci. USA 85: 4166-4170.

Brown, K.L. and Whiteley, H.R. (1990). Isolation of the second Bacillus thuringiensis RNA polymerase that transcribes from a crystal protein gene promoter. J. Bacteriol. 172: 6682-6688.

Chaufaux, J., Marchal, M., Müller-Cohn, J., Gilois, N. and Lereclus, D. (1993). Cloning and expression of a Bacillus thuringiensis gene encoding a S-endotoxin active against coleopteran larvae. C.R. Acad. Agric. 79: 103-110.

De-Souza, M.T., Lecadet, M.-M. and Lereclus, D. (1993). Full expression of the crylllA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription. J. Bacteriol. 175: 2952-2960.

DiMari, J.F. and Bechhofer, H.D. (1993). Initiation of mRNA decay in Bacillus subtilis. Mol. Microbiol. 7: 705-717.

Donovan, W.P., Gonzalez Jr., J.M., Gilbert, M.P. and Dankocsik, C. (1988). 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. 214: 365-372.

Gibson, T.J. (1984). Studies on the Epstein-Barr virus genome. Doctoral thesis, University of Cambridge, Cambridge, UK.

Herrnstadt, C., Gilroy, T.E., Sobieski, D.A., Bennett, B.D. and Gaertner, F.H. (1987). Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego. Gene 57: 37-46.

Höfte, H. and Whiteley, H.R. (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242-255.

Höfte, H., Seurinck, J., Houtven, A.V. and Vaeck, M. (1987). Nucleotide sequence of a gene encoding an insecticidal protein of Bacillus thuringiensis var. tenebrionis toxic against Coleoptera. Nucleic Acids Res. 15: 7189.

Lereclus, D., Bourgouin, C., Lecadet, M.-M., Klier, A. and Rapoport, G. (1989). Role, structure, and molecular organization of the genes coding for the parasporal d-endotoxins of Bacillus thuringiensis. In: Regulation or Procaryotic Development (Smith, I., Slepecky, R.A. and Setlow, P., eds.). American Society of Microbiology, Washington, DC, USA, chapter 13, pp. 255-276.

Meissner, P.S., Sisk, W.P. and Berman, M.L. (1987). Bacteriophage X cloning system for the construction of directional cDNA libraries. Proc. Natl. Acad. Sci. USA 84: 4171-4175.

Melin, L., Rutberg, L. and von Gabain, A. (1989). Transcriptional and posttranscriptional control of the Bacillus subtilis succinate dehydrogenase operon. J. Bacteriol. 171: 2110-2115.

Melin I Friden H., Dehlin E., Rutberg L. and von Gabain, (1990). The importance of the 5' - region in regulating the stability of sdh mRNA in Bacillus subtilis. Mol. Microbiol. 4: 1881-1889.

Ribier, J. and Lecadet, M.-M. (1973). Etude ultrastructurale et cinétique de la sporulation de Bacillus thuringiensis var. berliner 1715. Remarques sur la formation de l'inclusion parasporale. Ann. Microbiol. (Inst. Pasteur) 124A: 311-344.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, pp. 1.23-1.24; 4.12; 4.25; 4.37-4.38.

Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.

Sekar, V., Thompson, D.V., Maroney, M.J., Bookland, R.G. and Adang, M.J. (1987). Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. Proc. Natl. Acad. Sci. USA 84: 7036-7040.