Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATC C 824 was developed by using the lacZ gene from Thermoanaerobacterium therm osulfurogenes EM1 as the reporter gene, In order to test the reporter syste m, promoters of three key metabolic pathway genes, ptb (coding for phosphot ransbutyrylase), thl (coding for thiolase), and ade (coding for acetoacetat e decarboxylase), were cloned upstream of the reporter gene in pHT3 in orde r to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of bet a-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the report er gene produced a functional beta-galactosidase in C. acetobutylicum, In a ddition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stabil ity of the beta-galactosidase produced by the reporter gene was also examin ed,vith strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chIoramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-ga lactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was st able in the exponential phase of growth, In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb p romoter and phosphotransbutyrylase formation from its own autologous promot er were found to be similar.