Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription.
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.
de Souza
M T
MT
Département des Biotechnologies, 1300 Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France.
Lecadet
M M
MM
Lereclus
D
D
D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D004731:Endotoxins; D005798:Genes, Bacterial / Q000235:genetics; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D001616:beta-Galactosidase