Cyclic dinucleotides (CDNs) are ubiquitous signaling molecules in bacteria. As second messengers, CDNs direct a bacterial response from an extracellular input. These signaling cascades dictate physiology, in the form of changes in movement, or control survival, in the form of biofilm formation and antibiotic resistance. Cyclic di-GMP (cdiG) was the first CDN discovered in 1987. In the 30 plus years since this achievement, our understanding of the writers, readers, and erasers that govern cdiG processes has expanded. This research has also led to new discoveries, including new CDNs like cyclic GMP-AMP (cGAMP). Unlike cdiG, comparatively little is known about the determinants of cGAMP processes in bacteria. Furthermore, the scope of cGAMP signaling is limited to only two species, Vibrio cholerae and Geobacter sulfurreducens.

The discoveries presented in this thesis begin to unmask cGAMP signaling. GMP-AMP cyclases (GACs), in collaboration with Hallberg and Wang, were elucidated as the second class of cGAMP synthases, adding many deltaproteobacteria as genera that produce cGAMP. Biochemical characterization of a GAC from Myxococcus xanthus yielded cyclic AMP (cAMP) as an enzyme activator, while also revealing the first evidence of CDN cross-regulation by cdiG through inhibition of cGAMP synthase activity. A GMP-AMP phosphodiesterase (GAP), evolved to degrade cGAMP, was identified in the same species. This enzyme class was determined as selective for cGAMP and, due to a conserved mutation, is predicted to exist beyond Proteobacteria. Firmicutes, including Clostridia and Bacilli, may use cGAMP signaling. Finally, one would hypothesize cGAMP effectors are also encoded by M. xanthus. Differential RNA-seq of cGAMP signaling mutant strains generated a subset of protein and RNA targets for analysis, screening, and characterization. Together, these discoveries and investigations of bacterial cGAMP pathways suggest it approaches cdiG in its complexity and breadth.