Cyclic di-GMP (c-di-GMP) is a bacterial second messenger that is widely utilized by bacteria, with more than 80% of sequenced bacteria predicted to use this signal [1, 2]. C-di-GMP controls a variety of phenotypes, including biofilm formation, motility, and virulence in multiple bacteria [3–8]. In Vibrio cholerae, high levels of c-di-GMP induce biofilm formation, reduce swimming motility, and inhibit colonization in a murine infection model [9, 10]. These c-di-GMP associated behaviors are enacted through c-di-GMP binding to a variety of receptor proteins [5, 11–13] and, potentially to one of two classes of riboswitches [14, 15]. Intracellular levels of c-di-GMP are controlled through synthesis from two molecules of GTP by diguanylate cyclases (DGC)  and degradation to pGpG or GMP by phosphodiesterases (PDE) [6, 17].
DGCs are characterized by the presence of a conserved GGDEF domain composed of approximately 200 amino acids . These domains are believed to require the specific amino acid sequence GG(D/E)EF in their active site (referred to as A-site here) in order to retain their enzymatic activity. In addition to their active site motif, approximately 53% of GGDEFs contain an RXXD motif . The RXXD motif is a feedback inhibition site (referred to as the I-site here) located near the active site, which specifically binds to dimeric c-di-GMP to non-competitively inhibit enzyme activity . EAL domains are one of the two enzymatic domains that contain c-di-GMP specific phosphodiesterase (PDE) activity, the other being the HD-GYP domain [6, 17]. Approximately 67% of DGCs are multi-domain proteins, containing at least one partner domain, with the most common partner domain being the EAL domain . Interestingly, in more than 40% of these EAL-GGDEF proteins (and in 24% of DGCs in general) the GGDEF active site is degenerate at one or more amino acids, suggesting that many of these proteins are incapable of c-di-GMP synthesis .
One explanation for the high frequency of degenerate DGCs is that these proteins in some cases act as c-di-GMP receptors by binding to c-di-GMP at either their degenerate active site or their RXXD inhibition site. These degenerate DGCs respond to c-di-GMP in several ways. C-di-GMP may mediate participation of the DGC in a regulatory cascade, as does PopA from C. crescentus, PelD from Pseudomonas aeruginosa, and CdgA from Bdellovibrio bacteriovorus which bind c-di-GMP to regulate cell cycle progression, biofilm formation, and predation, respectively [20–22]. Binding of c-di-GMP may be required for proper localization of the protein akin to both FimX from P. aeruginosa and SgmT from Myxococcus xanthus[23, 24]. Additionally, degenerate DGCs may also retain other roles independent of c-di-GMP binding. CdpA in V. cholerae requires its degenerate GGDEF domain, but not c-di-GMP, to retain its PDE activity, and the highly degenerate GGDEF domain of YybT from Bacillus subtilis exhibited ATPase activity [10, 25]. In one case, a degenerate DGC has been shown to be active as the Pectobacterium atrosepticum DGC ECA3270 retains DGC activity despite having a degenerate SGDEF active site motif . Interestingly, evidence is accumulating that DGCs and PDEs themselves form protein complexes, and it is intriguing to speculate degenerate DGCs impact these processes [27, 28].
Here we investigate the degenerate V. cholerae DGC VCA0965. We examined the ability of all 40 V. cholerae DGCs to inhibit motility in semisolid agar, and we determined that VCA0965 was active in this assay. This result was surprising as VCA0965 is a DGC that encodes a degenerate AGDEF active site. Rather than functioning as a receptor for c-di-GMP, our results suggest that VCA0965, despite its degenerate active site motif, is capable of c-di-GMP synthesis.