YvoA and CcpA Repress the Expression of chiB in Bacillus thuringiensis

Bacillus thuringiensis produces chitinases, which are involved in its antifungal activity and facilitate its insecticidal activity. In our recent work, we found that a 16-bp sequence,drechiB (AGACTTCGTGATGTCT), downstream of the minimal promoter region of the chitinase B gene (chiB ) was a critical site for the inducible expression ofchiB inB. thuringiensis Bti75. In this work, we show that a GntR family transcriptional regulator (named YvoABt ), which is homologous to YvoA ofBacillus subtilis , can specifically bind to thedrechiB oligonucleotide sequencesin vitro by using electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC) assays. The results of quantitative real-time reverse transcription-PCR (qRT-PCR) and Western blotting indicated that deletion ofyvoA caused an ∼7.5-fold increase in the expression level ofchiB . Furthermore, binding of purified YvoABt to its target DNA could be abolished by glucosamine-6-phosphate (GlcN-6-P). We also confirmed, in the presence of the phosphoprotein Hpr-Ser45 -P, that purified CcpABt bound specifically to the promoter ofchiB , which contains the “crechiB ” sequence (ATAAAGCGTTTACA). According to the results of qRT-PCR and Western blotting, deletion ofccpA resulted in a 39-fold increase in thechiB expression level, and glucose no longer influenced the expression ofchiB . We confirm thatchiB is negatively controlled by both CcpABt and YvoABt in Bti75.