# The World of Cyclic Dinucleotides in Bacterial Behavior
## 3 Cyclic Dinucleotide Receptors
Moreover, another c-di-GMP receptor is also involved in exopolysaccharideaderiate production, the PelD of _Pseudomonas aeruginosa_ that regulates the synthesis of the Pel exopolysaccharideaderiate (Table 1). PilZ proteins interact with c-di-GMP by two conserved sequence motifs: RxxxR and DxSxxG motifs (Figure 7D). In the RxxxR motif located in a loop at the N-terminal part of the PiIZ domain, each arginine is interacting with the phosphate group and the base of the ligand. In the case of DxSxxG motif, the aspartic acid, serine and glycine residues bind the base and the pentose ring of the c-di-GMP molecule (Figure 7G). Other residues not conserved within members of the PiIZ family are also involved in ligand binding and some of them are located at the \(\beta\)-strand 7 of the PiIZ protein (Figure 7D,F,G). Some PiIZ proteins lost their canonical residues to bind c-di-GMP and are not c-di-GMP receptors anymore but may work as protein-protein adaptors, as happens with the complex regulates the twitching motility in _Xanthomonas citri_[8]. This ternary complex is an example of a full set of \"degenerate\" GGDEF, EAL, and PiZ domains, in which GGDEF does not synthesize c-di-GMP, PiIZ does not bind c-di-GMP, and the EAL domain does not cleave c-di-GMP but kept the ability to bind it [8]. Degenerate EALs proteins lost their ability to cleave c-di-GMP to pGpG, and some of them still bind c-di-GMP molecules but do not cleave them changing its function from enzyme to a CDN receptor. The residues involved in c-di-GMP interaction are described in Table 1 and Figure 7H,I. The loss of the EAL domain catalytic function seems to be related with a change in the residues important for the coordination of a magnesium cation (Figure 7H,I). In _Xanthomonas citri_, _Xanthomonas camepstris_, and _Pseudomonas aeruginosa_, FimX proteins regulate twitching motility by sensing c-di-GMP levels via interaction with degenerate EAL domain and regulates type IV pilus machinery [8]. LapD from _Pseudomonas fluorescens_ is a transmembrane protein that binds c-di-GMP through its C-terminal degenerate EAL domain to prevent cleavage of the surface adhesin LapA and therefore activates biofilm formation [126]. Different classes of RNA riboswitches sense different kinds of CDNs (Table 1). Riboswitches are structured RNAs located in the 5'-untranslated regions of mRNAs and some can sense CDNs molecules to change its structure to regulate expression of downstream genes that could be involved with virulence, motility, biofilm formation, cell wall metabolism, synthesis and transport of osmoprotcants, sporulation, and other important biological processes [127; 128]. There are three distinct classes of riboswitches that bind specific CDNs and have had their structures solved in complex with their ligand and deposited in the Protein Data Bank: c-di-GMP I riboswitch (RF01051), c-di-GMP II riboswitch (RF01786), and c-di-AMP riboswitch (ydaO-yuaA riboswitch, RF00379). C-di-GMP I riboswitch and c-di-GMP II riboswitch bind c-di-GMP molecules while c-di-AMP riboswitch binds c-di-AMP molecules [28; 129; 130]. The c-di-GMP I riboswitch was originally annotated as a conserved RNA-like structure of Genes Related to the Environment, Membranes and Motility (GEMM motif) and later another c-di-GMP riboswitch class was identified, the c-di-GMP II riboswitch. They have the same function but do not share any sequence motif or structural similarities. The c-di-AMP riboswitch is one of the most common riboswitches in various bacterial species and is found in the vicinity of genes related to cell wall metabolism, sporulation in Gram-positive bacteria, and other important biological processes [127; 128]. These structures reveal that the RNAs use different ways to bind CDNs. The TetR-like transcriptional factor, DarR, from _Mycobacterium smegmatis_ was the first c-di-AMP receptor discovered [27], where c-di-AMP stimulate the DNA binding activity of this protein. DarR is a repressor that negatively regulates the expression of its target genes [27].