Elucidation of cellulase gene expression mechanisim in cellulolytic bacteria

利用PCR和集落杂交技术，从纤维素分解厌氧菌梭菌中克隆出一个具有螺旋-螺旋-螺旋DNA结合基序的分解代谢控制蛋白A （CcpA）基因。利用枯草芽孢杆菌ccpA缺陷突变体，检测了ccpA基因与枯草芽孢杆菌ccpA基因启动子区融合的补体试验。葡萄糖酸激酶活性测定来补充突变体。结果，来自C， cellulovorans的ccpA基因不能补充缺陷突变体。利用Ni-NTA和Resource Q柱纯化大肠杆菌中表达的CcpA蛋白。纯化后的蛋白与C. cellulovorans脚手架蛋白基因启动子区和枯草芽孢杆菌amyE基因启动子区进行凝胶移位实验，该启动子区被分解代谢物调节。DNA条带没有移位；这表明需要其他因素将蛋白质与DNA结合。该蛋白序列与枯草芽孢杆菌CcpA相似，但功能不同。此外，还进行了磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）编码基因的克隆实验。成功克隆了纤维素葡萄球菌PTS酶I和酶II Glc基因。PTS基因的存在提示纤维素酵母裂解物调控机制介导了PTS的发生。克隆并测序了几丁质溶解副果壳梭菌的ccpA基因。它们有相似的蛋白质序列。

A gene encoding the catabolite control protein A (CcpA) having helix-turn-helix DNA binding motif was cloned from the cellulolytic anaerobe, Clostridium cellulovorans, using PCR and colony hybridization. A fusion ccpA gene with the promoter region of Bacillus subtilis ccpA gene was examined to complement test using ccpA defect mutant of B. subtilis. A gluconate kinase activity was measured to complement the mutant. As a result, the ccpA gene from C, cellulovorans was not able to complement the defect mutant. The CcpA protein expressed in Escherichia coli was purified using Ni-NTA and Resource Q column. The purified protein was examined for gel shift assay with the promoter region of C. cellulovorans scaffolding protein gene and B. subtilis amyE gene, being reglutated by catabolite. The DNA bands showed no shifting ; suggesting other factors need to bind the protein to DNA. And the protein sequence has similarity to B. subtilis CcpA, but the function is not same. In addition, cloning experiments of genes encoding phosphoenolpyruvate dependent phosphotransferase system (PTS) were done. PTS enzyme I and enzyme II Glc gene were cloned from C. cellulovorans, successfully. The presence of PTS genes suggest the catabolite control mechanism mediated PTS in C. cellulovorans. A ccpA gene from chitinolytic Clostridium paraputrificum was also cloned and sequenced. These have similar protein sequence.