## 2 Synthesis of c-di-AMP  
In contrast to the variety of GGDEF domain-containing proteins that synthesize c-di-GMP [33], only a few c-di-AMP synthesizing enzymes have thus far been discovered in bacteria and archaea. These c-di-AMP synthesizing enzymes are found primarily in Gram-positive Firmicutes and Actinobacteria but are also present in some Gram-negative bacteria including Bacteroidetes, Deltaproteobacteria, and Cyanobacteria [3]. Moreover, the signaling pathways for c-di-GMP and c-di-AMP rarely co-exist in the same organisms. For example, _Staphylococcus_, _Streptococcus_, and _Corynebacterium_ species do not contain functional c-di-GMP synthesizing enzymes [17; 34; 35]. Notable exceptions to this rule include _Bacillus_, _Clostridium_, _Listeria_, _Mycobacterium_, and _Streptomyces_, which produce both signaling molecules [3; 32].  
c-di-AMP is synthesized from ATP or ADP by cyclase domain-containing proteins known as diadenylate cyclases (DACs) (Figure 1). DAC enzymes catalyze the synthesis of a single molecule of c-di-AMP from two molecules of ATP or ADP through a condensation reaction [3; 14; 32; 34; 35]. Structural and functional analysis of individual DAC domains and operons have enhanced the current understanding of the functions of the various classes of these proteins [32; 35]. The DAC domain was first identified by X-ray crystallography in DisA (DNA integrity scanning protein A), which functions as a DNA check point protein scanning the chromosome for lesions [18; 29]. Later, DAC domain-containing proteins were found in many bacterial and archaeal species [34]. Four classes of DACs have been identified: DisA, CdaA, CdaS, and CdaM. While most organisms contain only one type of DAC, some contain multiple enzymes including _Clostridium_ spp., which contain two types of DACs (CdaA and DisA) and _B.subtilis_, which has DisA, CdaA, and CdaS [36]. Several Gram-positive human pathogenic bacteria including _S. pyogenes_, _S. pneumoniae_, and _L. monocytogenes_ encode CdaA [14; 16; 25] while _M. tuberculosis_ expresses MtDisA, a DisA homolog [11; 37]. Previous studies have demonstrated that DAC mutant strains display altered physiologies such as loss of resistance to heat, salt, and DNA-damaging molecules due to the synthesis of a weak cell wall, making the bacteria vulnerable to its environment [15; 32]. The impact of DAC on numerous aspects of cell physiology highlights the essential nature of DACs in bacteria [15; 19; 29; 32].  
All DAC domain proteins possess conserved motifs, most commonly DGA and RHR motifs, which perform the cyclase activity in the c-di-AMP synthesis reactions [38; 39]. However, these domains do not share any structural or amino acid similarities with the GGDEF domain found in c-di-GMP-catalyzing enzymes suggesting these signaling pathways evolved independently [40]. Structural analysis of the three types of DACs have demonstrated that the DAC domains in CdaA and CdaS share 40% amino acid identity, while the DAC domain in the DisA protein is more distantly related, sharing only 19% identity with CdaA and CdaS [32; 34]. The association of neighboring genes with each class of DAC points to the physiological role of the c-di-AMP. For example, the association of _cdaA_ with a gene that encodes an enzyme synthesizing a cell wall building block (_glmM_) suggests a role for CdaA and c-di-AMP in the maintenance of cell wall homeostasis [32; 34].