Potential effect of a putative sigma(H)-driven promoter on the over expression of the Cry1Ac toxin of Bacillus thuringiensis.

Sequence analysis of the upstream region of the cry1Ac gene in the HD-73 strain of B. thuringiensis showed a putative sigma(H)-like promoter. The potential regulating role of this sequence was tested by transforming an acrystaliferous mutant of the HD-73 strain with three different constructs: (1) a construct consisting of the sigma(H)-, sigma(E)- and sigma(K)-like promoters, the 0A box, and the cry1Ac coding sequence (EK0AH); (2) a derivative construct that lacked the sigma(H)-promoter (EK0A); and (3) a second derivative construct that lacked the sigma(H)-promoter and the 0A box (EK). Crystals from the recombinant and the wild-type (Bt HD-73) strains were measured by transmission electron microscopy. Statistically significant differences in crystal size were detected between all the transformed and the wild-type strains, with averages of 1.54, 1.31, 1.05, and 0.95microm for the EK0AH, EK0A, HD-73, and EK constructs, respectively. SDS-PAGE analyses of the EK0AH construct corroborated a higher expression level of the cry1Ac gene than that of the EK0A construct, as well as the lower expression of the EK construct. Interestingly, RT-PCR analyses indicated that the recombinant strain carrying the construct EK0AH started the transcription of the cry gene earlier than the Bt HD-73 strain, as observed when a kinetics study was carried out, which may explain the larger crystals and the higher expression of the construct with the putative sigma(H)-like promoter, along with the vector's high copy number.