Molecular cloning and the nucleotide sequence of the Mr28 000 crystal protein gene ofBacillus thuringiensissubsp.israelensis

The Mr 28.000 crystal protein gene of Bacillus thuringiensis subspecies israelensis has been cloned into pBR322 as part of a 9.7 kb HindIII fragment. From hybridization experiments of recombinant p425 DNA with B.t. subspecies israelensis RNA from different stages of growth it was concluded that transcription of the gene is restricted to early sporulation stages. Nucleotide sequence analysis revealed the presence of a large open reading frame with a coding capacity of 249 amino acids (Mr 27.340). Nuclease S1 mapping demonstrated that transcription starts 44 nucleotides upstream of the initiation codon. A Shine-Dalgarno sequence (AAGGAG) was found 10 nucleotides upstream of the translation startpoint. At the 3'-end of the gene a complex secondary structure was found immediately after the stop-codon. Despite the presence of these regulation signals only limited expression in E. coli was detected. This can be explained by assuming that B.t. subsp. israelensis promotor sequences are poorly recognized by E. coli RNA polymerase.