Chlamydia trachomatis, the causative agent of blinding trachoma and many sexually transmitted diseases, exists in two developmental forms: an infectious elementary body (EB) and a replicative reticulate body (RB). The transition between EB and RB forms is an essential step in the life cycle of this obligate bacterial pathogen. However, remarkably little is known about the environmental cues and molecular mechanism underlying these developmental changes. We recently determined that C. trachomatis EBs accumulate cyclic di-adenosyl monophosphate (c-di-AMP), a newly identified nucleic acid metabolite that contributes to cell wall homeostasis in gram-positive bacteria. In gram-negative bacteria, the structurally related cyclic dinucleotide c-di-GMP is an allosteric regulator of enzymes, gene expression and riboswitches important for biofilm formation and microbial virulence. We hypothesize that c-di-AMP regulates protein and/or RNA functions to promote the RB to EB transition and possibly virulence. In this exploratory research program, we outline experiments to identify proteins that regulate the Chlamydia diadenylate cyclase, and protein(s) and RNA(s) that may be targets of c-di-AMP-mediated regulation. We will employ new biochemical and genetic methods developed in our laboratory to assess the role of c-di-AMP and its identified target(s) in chlamydial development and pathogenesis.