## 5 Conclusions  
Second messenger molecules are critical elements in signal transduction pathways utilized by many organisms. They are involved in the regulation of various key cellular processes in both prokaryotes and eukaryotes. In recent years, c-di-AMP signaling has been identified as a central factor in many Gram-positive bacteria regulating cell wall synthesis, potassium ion channels, DNA repair, and biofilm formation [32]. c-di-AMP is also essential for cell growth, survival, and virulence of several well-known human pathogenic bacteria including _S. aureus_, _L. monocytogenes_, _S. pyogenes_, and _Mycobacterium_ spp. [17; 20; 22; 26; 35]. Although many cellular functions of c-di-AMP are known, the detailed mechanisms of the cellular processes have yet to be revealed. The c-di-AMP synthesizing enzymes and degradation enzymes regulate the homeostasis of intracellular c-di-AMP levels. Four classes of DAC (DisA, CdaA, CdaS, and CdaM) and three types of PDE (Gdpp, Pde2 and PgpH) are found in c-di-AMP-producing bacteria, and the types of enzymes and their functions vary by bacterial species [1; 32]. Due to their essential role in bacteria, the construction of DAC mutant strains is often difficult. For example, the construction of DAC deletion strains in _L. monocytogenes_ was not possible in rich media unless accompanied by a secondary mutation in guanosine pentaphosphate (ppGpp) synthases. Similarly, simultaneous depletion of all three DACs in _B. subtilis_ was synthetic-lethal [36]. However, deletion of the DAC enzyme in _S. mutans_ was not lethal, so c-di-AMP might be not essential for the growth of some bacteria even under standard lab growth conditions. Unlike the c-di-GMP network, where more than twenty enzymes are involved in the production and degradation of the cyclic di-nucleotide, the c-di-AMP synthesis and hydrolysis network appears much less complex. DACs and PDEs regulate intracellular c-di-AMP homeostasis, while the receptor proteins and RNA are the key factors that carry out the cellular processes upon binding to c-di-AMP [32; 35]. As DAC is often an essential enzyme in many bacteria, many roles of c-di-AMP in cellular processes have been revealed by examining PDE mutant strains. The alteration of c-di-AMP levels following PDE depletion often results in decreased survival of the bacteria. Furthermore,altered cell wall synthesis and dysregulation of potassium uptake have been observed in PDE mutant strains [17]. To understand the regulatory network of c-di-AMP at the cellular level, further intensive molecular studies are necessary. Research has been directed towards the characterization of the receptor molecules to decipher the regulation mechanisms by c-di-AMP. Several proteins and one type of RNA molecule have so far been identified that bind to c-di-AMP (Table 2) [32; 35]. It appears that there is not much overlap between c-di-AMP binding proteins among bacteria except the proteins involved in potassium ion homeostasis. Thus, identifying c-di-AMP-binding proteins using the bacteria previously not studied might discover new types of c-di-AMP-binding proteins, resulting in discovering new cellular pathways controlled by c-di-AMP. The only c-di-AMP-binding RNA identified so far is the _ydaO_ riboswitch, and many bacteria producing c-di-AMP do not contain this riboswitch. New types of c-di-AMP-binding RNAs might also be discovered by the screening of c-di-AMP-binding RNA in those bacteria. Thus, investigations with diverse organisms might lead to a better understanding of the mechanisms of regulation in c-di-AMP signaling pathways. The specific environmental stimuli in c-di-AMP signaling pathways are still a mystery, so identifying the signals is also necessary for better understanding of how bacteria sense environmental stimuli and adapt to environmental changes. Agents that disrupt c-di-AMP signaling, such as inhibitors of DACs and PDEs, may also prove useful for antibacterial therapy in the future. We would like to thank [PERSON] and [PERSON] for proofreading of the manuscript.