Bacterial microcompartments are large subcellular structures composed of metabolic enzymes encapsulated within a protein shell built from multiple subunits. They are widespread among bacteria, functionally diverse, play vital metabolic roles, are linked to pathogenesis, and appear to incorporate unique mechanistic and structural principles. Their function is to sequester and regulate the production of toxic or volatile intermediates found in certain metabolic pathways. However, little is known about how this is occurs at the mechanistic level. The long-term goal of the proposed research is to elucidate the molecular principles and to build up a 3-dimensional structure of the microcompartments involved in 1,2-propanediol degradation by Salmonella. The Salmonella system is unmatched with regard to the knowledge and tools available for mechanistic studies of microcompartments. The proposed studies combine genetic, biophysical, and structural methods to elucidate the cellular function of the Salmonella Pdu microcompartment at a mechanistic level. Three specific aims are proposed: 1. determine the structures of the microcompartment shell proteins and enzymes;2. elucidate biochemical interactions and higher-order architecture in the Pdu microcompartment;and 3. conduct mutational analysis of microcompartment function. Structures will be investigated and analyzed by x-ray crystallography, biophysical, and computational methods. Interactions studies will include two-hybrid analyses, labeling studies, and crystallography. Functional and mechanistic insights will be derived from structure-guided mutagenesis in conjunction with growth studies, enzyme and transport assays, and reverse two-hybrid analyses. Completion of the proposed investigations will elucidate the mechanistic and structural principles of the Salmonella pdu microcompartment. This will provide general insights into bacterial microcompartments. Furthermore, since bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, the proposed studies may ultimately lead to new opportunities for interfering with pathogenic processes. PUBLIC HEALTH RELEVANCE: Bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, but they are very poorly understood at the present time. This proposal seeks to understand how the Pdu microcompartment functions in the human pathogen, Salmonella enterica serovar Typhimurium. The studies will lead to a deeper fundamental and mechanistic understanding of these important bacterial organelles, and ultimately to new opportunities for interfering with pathogenic processes.