Regulation effect of CcpA on chitinase gene chiA and chiB in Bacillus thuringiensis

Abstract:

[Objective] Purpose of this work was to research the regulation effect of catabolite control protein A (CcpA) on chitinase gene chiA and chiB in Bacillus thuringiensis subsp. israelensis 75 (Bti75). [Methods] We used the PREDetector software program to analyze the upstream regulatory region of chiA and chiB in Bti75, in addition, specific binding of CcpA protein to the promoter region of chiA and chiB was determined by electrophoretic mobility shift assay (EMSA). In order to acquire ccpA deletion mutant ΔccpA, we constructed a ccpA knockout vector. Then, the influence of CcpA to chiA and chiB in Bti75 strains with and without glucose was detected by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot. [Results] PREDetector software program analysis showed that there is a potential CcpA binding site crechiB in the promoter region of the chiB, while the similar site is not found in the chiA promoter region. In vitro, CcpA could bind specifically to the chiB promoter with the assist of Hpr-Ser45-P, but the binding of CcpA to chiA promoter is non-specific. The results of qRT-PCR and western blot revealed that ccpA deletion led to a significantly increased expression of chiB, however, the change of chiA expression was not obvious. [Conclusion] The expression of chiB not chiA in Bti75 was negative controlled by CcpA in the presence of glucose.