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.
Pérez-García
Germán
G
Departamento de Biotecnología y Bioquímica, CINVESTAV-Irapuato, 36500 Irapuato, Mexico. gepega62@gmail.com
Basurto-Ríos
Regina
R
Ibarra
Jorge E
JE
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis*; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D006460:Hemolysin Proteins / Q000235:genetics* / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D015854:Up-Regulation
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; D012808:Sigma Factor; C030956:insecticidal crystal protein, Bacillus Thuringiensis