Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis

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Abstract
Summary: The promoter region of the cryIIIA toxin gene of Bacilius thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions ‐635 to ‐553 (with reference to the translational start codon of cryIIIA), an internal region extending from nucleotide positions ‐553 to ‐367, and a downstream region extending from nucleotide position 367 to +18. Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region. Primer extension experiments reveal a major cryIIIA transcript (designated T‐129) starting at nucleotide position ‐129 and another transcript (designated T‐558) starting at nucleotide position ‐558. Mutation in the ‐35 region of the promoter responsible for the initiation of T‐558 indicates that the upstream promoter is essential for full expression of cryIIIA, although not sufficient. Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5 end of T‐129. Taken together, these results indicate that the 5 end of T‐129 is not a transcriptional start site. Therefore, we propose that T‐129 results from the processing of the mRNA initiated at the upstream promoter (T‐558), generating a stable mRNA with a 5’extremity at nucleotide position ‐129. From primer extension analysis and transcriptional fusions to lacZ, it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporula‐tion and remains active at least until t5. However, unlike the promoters of other cry genes, this promoter is similar to σa‐dependent promoters rather than sporulation‐specific promoters. This promoter may therefore be transcribed by the EσA form of RNA polymerase. Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary‐phase‐specific activator.