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

Journal of Bacteriology - Tập 175 Số 10 - Trang 2952-2960 - 1993
M.T. de Souza, M.‐M. Lecadet, Didier Lereclus1
1ULB - Unité expérimentale de Lutte Biologique (Site principal : 0605 INRA Valbonne - France)

Tóm tắt

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.

Từ khóa


Tài liệu tham khảo

Adams , L. F. , K. L. Brown , and H. R. Whiteley . 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 D. Lereclus . 1991 . Construction of cloning vectors for Bacillus thuringiensis . Gene 108 : 115 - 119 .

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

Chaufaux J. M. Marchal J. Muller-Cohn N. Gilois and D. Lereclus. Cloning and expression of a Bacillus thuringiensis gene encoding a b-endotoxin active against coleopteran larvae. C. R. Acad. Agric. Fr. in press.

Crickmore , N. , and D. J. Ellar . 1992 . Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA b-endotoxin gene in Bacillus thuringiensis . Mol. Microbiol. 6 : 1533 - 1537 .

Debarbouillie , M. , and 0. Raibaud . 1983 . Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter . J. Bacteriol. 153 : 1221 - 1227 .

Donovan , W. P. , J. M. Gonzalez , Jr. , M. P. Gilbert , and C. Dankocsik . 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 .

Fourney , R. M. , J. Miyakoshi , R. S. Day III , and M. C. Pederson . 1988 . Northern blotting: efficient RNA staining and transfer . Focus 10 : 5 - 7 .

Gibson T. J. 1984. Ph.D. thesis. University of Cambridge Cambridge.

Glatron , M. F. , and G. Rapoport . 1972 . Biosynthesis of the parasporal inclusion of Bacillus thuringiensis: half-life of its corresponding messenger RNA . Biochimie 54 : 1291 - 1301 .

Herrnstadt , C. , T. E. Gilroy , D. A. Sobieski , B. D. Bennett , and F. H. Gaertner . 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 .

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

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

Kaneko , T. , R. Nozaki , and K. Aizawai . 1978 . Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis . Microbiol. Immunol. 22 : 639 - 641 .

Lecadet , M. M. , M. 0. Blondel , and J. Ribier . 1980 . Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54 Ber . J. Gen. Microbiol. 121 : 203 - 212 .

Lecadet , M. - M. , J. Chaufaux , J. Ribier , and D. Lereclus . 1992 . Construction of novel Bacillus thuringiensis strains with different insecticidal specificities by transduction and by transformation . Appl. Environ. Microbiol. 58 : 840 - 849 .

Lecadet , M. - M. , V. Sanchis , G. Menou , P. Rabot , D. Lereclus , and D. Martouret . 1988 . Identification of a b-entoxin 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. 54 : 2689 - 2698 .

Lederberg , E. M. , and S. N. Cohen . 1974 . Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid . J. Bacteriol. 119 : 1072 - 1074 .

Leonhardt , H. , and J. C. Alonso . 1988 . Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis . J. Gen. Microbiol. 134 : 605 - 609 .

Lereclus D. Unpublished data.

Lereclus , D. , and 0. Arantes . 1992 . spbA locus ensures the segregational stability of pHT1030, a novel type of Grampositive replicon . Mol. Microbiol. 7 : 35 - 46 .

Lereclus , D. , 0. Arantes , J. Chaufaux , and M. -M. Lecadet . 1989 . Transformation and expression of a cloned b-endotoxin gene in Bacillus thuringiensis . FEMS Microbiol. Lett. 60 : 211 - 218 .

Lereclus D. C. Bourgouin M.-M. Lecadet A. Klier and G. Rapoport. 1989. Role structure and molecular organization of the genes coding for the parasporal 8-endotoxins of Bacillus thuringiensis p. 255-276. In I. Smith R. A. Slepecky and P. Setlow (ed.) Regulation of procaryotic development. American Society for Microbiology Washington D.C.

Martin-Verstraete I. Unpublished data.

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

Msadek , T. , F. Kunst , D. Henner , A. Klier , G. Rapoport , and R. Dedonder . 1990 . 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. 172 : 824 - 834 .

Perkins , J. B. , and P. J. Youngman . 1986 . Construction and properties of Tn 917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis . Proc. Natl. Acad. Sci. USA 83 : 140 - 144 .

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

Sambrook J. E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual 2nd ed. Cold Spring Harbor Laboratory Cold Spring Harbor N.Y.

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

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

Tinoco , I. J. , P. N. Borer , B. Dengler , M. D. Levine , 0. C. Uhlenbeck , D. M. Crothers , and J. Gralla . 1973 . Improved estimation of secondary structure in ribonucleic acids . Nature (London) New Biol. 246 : 40 - 41 .

Visick , J. E. , and H. R. Whiteley . 1991 . Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on pro- duction of the CytA protein by Escherichia coli . J. Bacteriol. 173 : 1748 - 1756 .

Wong , H. C. , and S. Chang . 1986 . Identification of a positive retroregulator that stabilizes mRNAs in bacteria . Proc. Natl. Acad. Sci. USA 83 : 3233 - 3237 .

Thông tin
Thông tin xuất bản

Journal of Bacteriology

Tập 175 Số 10

2952-2960

Thông tin tác giả