Isolation of RNA polymerase from Clostridium difficile and characterization of glutamate dehydrogenase and rRNA gene promoters in vitro and in vivo

Clostridium difficile is the primary causative agent of antibiotic-associated diarrheal disease. To facilitate molecular genetic analysis of gene expression in this organism, methods were developed to study transcriptional regulation in vitro and in vivo. That is, C. difficile RNA polymerase was partially purified and shown to bind to and initiate transcription in vitro from bona fide C. difficile promoters for rRNA and glutamate dehydrogenase genes. In addition, primer extension analyses and a beta-glucuronidase reporter system were used to quantitate transcription from these promoters in vivo. With these tools in hand, it is now possible to characterize the behavior of any C. difficile gene in vivo and to study the regulation of its expression in detail.