PROJECT SUMMARY The small nucleotide c-di-AMP is a ubiquitous second messenger produced by thousands of bacterial and archeal species. Many bacteria, especially those in the Firmicutes phylum, require c-di-AMP for growth in standard laboratory media, and the mechanisms for c-di-AMP essentiality have been extensively studied. However, in a wide range of c-di-AMP-producing bacteria, including those for which c-di-AMP is completely dispensable, unregulated c-di-AMP accumulation is toxic, diminishing bacterial growth and stress response. Thus, c-di-AMP homeostasis is critical for bacteria that produce it. Nevertheless, the mechanisms that regulate c-di-AMP levels in the bacterial cell, and the mechanisms by which c-di-AMP regulates different cellular pathways to achieve optimal physiology, are both poorly understood. My lab found that c-di-AMP levels are dynamic in the bacterial cell, and c-di-AMP hydrolysis is a key mechanism to modulate c-di-AMP levels. Furthermore, we have generated bacterial mutants lacking c-di-AMP phosphodiesterases, called pde mutants, and extensively studied their phenotypic defects. In the next five years, we will investigate two major aspects of c-di-AMP signaling: i) the signal detection and regulatory mechanisms of c-di-AMP phosphodiesterase, and ii) the mechanisms by which c-di-AMP coordinates its molecular targets to achieve optimal physiology and stress response. For the second theme, we will take two complementary approaches. One, we will elucidate the biological functions of c- di-AMP molecular targets based on targeted phenotypic, genetic, and biochemical analyses. Two, we will identify the molecular targets underlying the toxicity phenotypes of pde mutants, through genetic fitness assays. Our findings will reveal fundamental mechanisms of c-di-AMP signaling, and these mechanisms will provide a framework to understand the global regulatory roles of c-di-AMP in the physiology of different bacterial species.