Elucidation of cellulase gene expression mechanisim in cellulolytic bacteria

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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.