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 P-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.