Our major interest is to understand the mechanisms of transcriptional regulation that govern gene expression at the initiation of spore formation in Bacillus subtilis (a non-pathogen) and toxin production in Clostridium difficile (the major causative agent of antibiotic-associated colitis). In both cases, nutrient limitation is the primary environmental condition that induces specialized gene expression.

To study the regulation of early sporulation genes in B. subtilis, we used a genetic selection to identify a novel regulatory protein, CodY, that is necessary for repression of these genes during rapid exponential growth. CodY has proved to be active as a repressor only when the intracellular concentrations of two metabolites, GTP and isoleucine, are high. When cells experience nutrient limitation, the pools of these metabolites decrease and CodY loses its repressor activity, leading to derepression of many genes whose products allow the cell to respond to nutritional stress and initiate sporulation. Current work is designed to understand the detailed mechanism of CodY-GTP and CodY-isoleucine interaction, using genetic analysis of mutants altered in metabolite binding, biochemical analysis of CodY-metabolite complexes and X-ray crystallography.

We have also used genetic and biochemical analysis to identify the regulatory proteins that control the synthesis of the first three enzymes of the Krebs citric acid cycle and the three most critical nitrogen metabolism enzymes (glutamate synthase, glutamate dehydrogenase and glutamine synthetase) in B. subtilis and Listeria monocytogenes. These enzymes couple carbon and nitrogen metabolism; their activities must be tightly controlled for efficient growth and response to environmental conditions. We have now identified nine transcription factors that contribute to regulation of these genes. We are studying many of these transcription factors in detail in order to understand at the molecular level how they sense the carbon and nitrogen status of the cell and adjust gene expression accordingly.

To unravel the mechanism of pathogenesis by C. difficile, we have focused on two questions: What mechanisms control the synthesis of the two major virulence factors, Toxin A and Toxin B? What is the role of spore formation in pathogenesis? Our studies of toxin gene expression have revealed that both genes are transcribed from promoters recognized by a special form of RNA polymerase containing a novel sigma factor encoded by tcdR, the gene immediately upstream of the toxin genes. In addition, we have found that homologs of TcdR in C. botulinum, C. tetani, and C. perfringens regulate expression of major toxin and bacteriocin genes. Thus, a unified mechanism of toxin gene expression exists in several major clostridial pathogens.

Environmental regulation of C. difficile toxin synthesis is mediated at the level of tcdR gene expression. That is, the tcdR gene is only expressed when cells enter stationary phase. We are currently seeking to identify the regulatory proteins that control tcdR expression, one of which appears to be the C. difficile homolog of CodY. We are also exploring the role of CodY homologs in several other pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, and Bacillus anthracis.