Abstract
During sporulation, Bacillus thuringiensis produces inclusions comprised of different amounts of several related protoxins, each with a unique specificity profile for insect larvae. A major class of these genes designated cry1 have virtually identical dual overlapping promoters, but the upstream sequences differ. A gel retardation assay was used to purify a potential regulatory protein which bound with different affinities to these sequences in three cry1 genes. It was identified as the E2 subunit of pyruvate dehydrogenase. There was specific competition for binding by homologous gene sequences but not by pUC nor Bacillus subtilis DNA; calf thymus DNA competed at higher concentrations. The B. thuringiensis gene encoding E2 was cloned, and the purified glutathione S-transferase-E2 fusion protein footprinted to a consensus binding sequence within an inverted repeat and to a potential bend region, both sites 200-300 base pairs upstream of the promoters. Mutations of these sites in the cry1A gene resulted in decreased binding of the E2 protein and altered kinetics of expression of a fusion of this regulatory region with the lacZ gene. Recruitment of the E2 subunit as a transcription factor could couple the change in post exponential catabolism to the initiation of protoxin synthesis.
Highlights
Many but not all of these genes contain very similar overlapping promoters [2, 3] recognized in the mother cell during sporulation by E and K forms of RNA polymerase [2, 4]
Three of these cry1 genes, the sequences upstream for ϳ1 kbp2 were examined and found to differ substantially [10].3,4. These regions appear to be important for regulation because expression of cry1-lacZ fusion plasmids in B. thuringiensis was enhanced by their presence
BamHI site and 5Ј-ATAGGGAAATCTCGAGCTACCATAACATTA-3Ј containing a XhoI site were based on the sequences of the B. subtilis pdhC gene [23] and used to clone a 1350-bp region of DNA from B. thuringiensis corresponding to its pdhC gene
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF039908. Three of these cry genes, the sequences upstream for ϳ1 kbp were examined and found to differ substantially [10].3,4. These regions appear to be important for regulation because expression of cry1-lacZ fusion plasmids in B. thuringiensis was enhanced by their presence.. Employing a gel retardation assay, a novel DNA binding protein was identified and purified, and its gene was cloned. The binding sites in the upstream regions of two of these cry genes were determined by footprinting. The effects of mutations in these sites indicated that this protein is likely to have a role in regulating the expression of this class of protoxin genes
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