## 3 Challenges for the Future  
### The Complexity of Second Messenger Regulatory Networks  
Second messenger homeostasis genes are widespread in the genomes of cyanobacteria and a single second messenger molecule can regulate several phenotypes. For instance, Ca\({}^{2+}\) can control gliding motility, heterocyst differentiation, and degradation of phycobilisomes (Figure 2). (p)ppGpp regulates ribosomal RNA accumulation, heat shock proteins, and heterocyst differentiation (Figure 3). Cyclic AMP is involved in photoproduction, heterocyst differentiation, photoheterotrophic growth, and nitrogen and phosphorous uptake (Figure 4). In addition to a single second messenger controlling multiple phenotypes, multiple genes may encode proteins that regulate the synthesis or degradation of a single second messenger molecule. One extreme case is apparent with the c-di-GMP signaling system in bacteria. Among cyanobacteria possessing c-di-GMP proteins, there is an average of 20 enzymes that synthesize or degrade c-di-GMP for each species in which they are found [16]. There are several mechanisms by which c-di-GMP signaling specificity is achieved in systems exhibiting such complexity. One mechanism involves regulating the timing of accumulation of c-di-GMP relative to the presence of c-di-GMP receptors or effectors in cells. For instance, expression of c-di-GMP homeostasis enzymes could occur under environmental conditions that support accumulation of c-di-GMP specific targets. This has been demonstrated in _E. coli_ for c-di-GMP in the control of biofilm formation during stationary phase [102]. Alternatively, in light of evidence that c-di-GMP enzymes are constitutively expressed in many species, a different mechanism could explain signaling specificity. In this instance, temporal or spatial sequestration of individual c-di-GMP components could be used to control signaling, in which case c-di-GMP molecules would target co-localized receptors. The use of c-di-GMP sensors has demonstrated distinct patterns of spatial localization of c-di-GMP in some bacterial cells [103]. Individual second messenger homeostasis enzymes also could distinctly effect second messenger pools and thereby control distinct phenotypes in the organism. Such signaling is referred to as high-specificity signaling and has been reported for distinct c-di-GMP synthesis enzymes in _Vibrio cholera_[104]. Also, by controlling the presence of receptors of a second messenger that have differences in binding affinities, the activation of distinct receptors could be achieved at different intracellular second messenger concentrations, thereby allowing specificity of control of distinct phenotypes [105]. Although compartmentalization of several second messengers has been well demonstrated in eukaryotic cells [106],signaling specificity in cyanobacterial systems has not been well investigated. Technologies already in use in pathogenic bacteria, such as fluorescence resonance energy transfer (FRET) [103] or single fluorescent protein-based indicators [107] could be used to monitor c-di-GMP or cAMP concentrations to permit visualization of asymmetrical distributions of these second messengers in cyanobacteria.