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, 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 drechiB 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 similar to 7.5-fold increase in the expression level of chiB. 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 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.

苏云金芽孢杆菌产生几丁质酶，其参与抗真菌活性并促进杀虫活性。在我们近期的工作中，我们发现几丁质酶B基因（chiB）最小启动子区域下游的一个16bp序列dre(chiB)（AGACTTCGTGATGTCT）是苏云金芽孢杆菌Bti75中chiB诱导表达的关键位点。在这项工作中，我们通过电泳迁移率变动分析（EMSAs）和等温滴定量热法（ITC）分析表明，一个与枯草芽孢杆菌的YvoA同源的GntR家族转录调节因子（命名为YvoA(Bt)）能够在体外特异性结合drechiB寡核苷酸序列。实时定量逆转录 - PCR（qRT - PCR）和蛋白质印迹的结果表明，yvoA的缺失导致chiB表达水平增加约7.5倍。此外，氨基葡萄糖 - 6 - 磷酸（GlcN - 6 - P）可消除纯化的YvoA(Bt)与其靶DNA的结合。我们还证实，在磷酸化蛋白Hpr - Ser45 - P存在的情况下，纯化的CcpA(Bt)特异性结合到chiB的启动子上，该启动子包含“cre(chiB)”序列（ATAAAGCGTTTACA）。根据qRT - PCR和蛋白质印迹的结果，ccpA的缺失导致chiB表达水平增加39倍，并且葡萄糖不再影响chiB的表达。我们证实，在Bti75中，chiB受到CcpA(Bt)和YvoA(Bt)的负调控。