PROJECT SUMMARY/ABSTRACT The research goals of my laboratory are focused on advancing our understanding of physiologic processes that provide the necessary flexibility to the metabolic network of prokaryotic cells. Such flexibility is needed for cells to survive challenges encountered under the diverse conditions they find themselves in, be it free-living, in association with a host as a commensal, or during the process of infection. We are particularly interested in the molecular basis of metabolism and on the physiological consequences brought about by genetic and epigenetic events that change the function of proteins either permanently or temporarily. Here we propose to define the function of two enzymes that chemically modify other proteins altering their activities. The first protein-modifying enzyme appears to target a global regulatory protein, whilst the second one appears to target an enzyme whose activity can balance the biosynthesis of building blocks with energy generation via oxidative phosphorylation. We are also continuing our work on the biosynthesis of coenzyme B12, the largest coenzyme known that is not a polymer. Specifically, we are interested in the late steps of the pathway, which occur as a multi-enzyme complex anchored in the cell membrane. Our work has advanced to the point that allows us to assemble the complex using liposome methodologies, and in so doing we can now investigate the molecular details of the assembly process. Ultimately, we will engage a microscopy expert to help us visualize what is likely to be a magnificent molecular structure. Finally, we will continue to work on a recently discovered transcription factor that we have shown modulates the expression of a large set of genes involved in iron metabolism during a Salmonella infection. We will engage a colleague working on Salmonella pathogenesis to provide insights into how genes affected by the function of this regulator impact the ability of Salmonella to establish and maintain an infection. To perform the proposed we will use innovative combinations of genetic, molecular biology, biochemical, bioinformatics and global approaches and will complement those through collaborations with spectroscopists, crystallographers, molecular biophysicists, and membrane biologists to provide comprehensive, rigorous testing of hypotheses and working models. We seek answers to questions in metabolism of microbes relevant to human health, and in so doing contribute to the mission of the NIH.