YvoA and CcpA Repress the Expression of chiB in Bacillus thuringiensis

Abstract

ABSTRACT 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, dre chiB (AGACTTCGTGATGTCT), downstream of the minimal promoter region of the chitinase B gene ( chiB ) was a critical site for the inducible expression of chiB in B. thuringiensis Bti75. In this work, we show that a GntR family transcriptional regulator (named YvoA Bt ), which is homologous to YvoA of Bacillus subtilis , can specifically bind to the dre chiB oligonucleotide sequences in 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 of yvoA caused an ∼7.5-fold increase in the expression level of chiB . Furthermore, binding of purified YvoA Bt to its target DNA could be abolished by glucosamine-6-phosphate (GlcN-6-P). We also confirmed, in the presence of the phosphoprotein Hpr-Ser 45 -P, that purified CcpA Bt bound specifically to the promoter of chiB , which contains the “ cre chiB ” sequence (ATAAAGCGTTTACA). According to the results of qRT-PCR and Western blotting, deletion of ccpA resulted in a 39-fold increase in the chiB expression level, and glucose no longer influenced the expression of chiB . We confirm that chiB is negatively controlled by both CcpA Bt and YvoA Bt in Bti75.