Identification of the Promoter in the Intergenic Region between orf1 and cry8Ea1 Controlled by Sigma H Factor

摘要

Bacillus thuringiensis Cry8Ea toxin is specifically toxic to larvae of the Asian cockchafer, Holotrichia parallela . Here we investigated the mechanism of transcriptional regulation of the cry8Ea1 gene. Reverse transcription-PCR (RT-PCR) results indicated that cry8Ea1 and an upstream gene ( orf1 ) were cotranscribed. Transcriptional fusions with the lacZ gene demonstrated that transcription of the cry8Ea1 gene started from two promoters: P_( orf1 ), which is located upstream of the orf1 gene, and P_( cry8E ), located in the intergenic region mapping between orf1 and cry8Ea1 . Of the known, similar orf1-cry operons, this is the first report of the existence of a promoter in the intergenic region between the orf1 and cry genes. The transcriptional activity of P_( orf1 ) was found during sporulation in B. thuringiensis subsp. kurstaki HD-73 and was almost abolished in the sigE mutant, while the transcriptional activity of P_( cry8E ) was detected after the end of the exponential phase in HD-73 and was considerably lower in the sigH mutant. The transcription start sites generated by the two cry8Ea1 promoters were determined by the 5′ -SMARTer rapid amplification of cDNA ends (RACE) method. The ?35 and ?10 regions of P_( orf1 ) and P_( cry8E ) showed high sequence similarity with the σ~(E) and σ~(H) promoters, respectively. These results indicated that P_( orf1 ) is controlled by the σ~(E) factor and P_( cry8E ) by the σ~(H) factor.