Cyclic di‐GMP signaling—Where did you come from and where will you go?

Microbes including bacteria are required to respond to their often continuously changing ecological niches in order to survive. While many signaling molecules are produced as seemingly circumstantial byproducts of common biochemical reactions, there are a few second messenger signaling systems such as the ubiquitous cyclic di‐GMP second messenger system that arise through the synthesis of dedicated multidomain enzymes triggered by multiple diverse external and internal signals. Being one of the most numerous and widespread signaling system in bacteria, cyclic di‐GMP signaling contributes to adjust physiological and metabolic responses in all available ecological niches. Those niches range from deep‐sea and hydrothermal springs to the intracellular environment in human immune cells such as macrophages. This outmost adaptability is possible by the modularity of the cyclic di‐GMP turnover proteins which enables coupling of enzymatic activity to the diversity of sensory domains and the flexibility in cyclic di‐GMP binding sites. Nevertheless, commonly regulated fundamental microbial behavior include biofilm formation, motility, and acute and chronic virulence. The dedicated domains carrying out the enzymatic activity indicate an early evolutionary origin and diversification of “bona fide” second messengers such as cyclic di‐GMP which is estimated to have been present in the last universal common ancestor of archaea and bacteria and maintained in the bacterial kingdom until today. This perspective article addresses aspects of our current view on the cyclic di‐GMP signaling system and points to knowledge gaps that still await answers.

Initially discovered as an allosteric regulator of the cellulose synthase in 1987, cyclic di-GMP was subsequently realized as being nearly ubiquitous within the bacterial kingdom (Galperin et al., 2001;Ross et al., 1987).The domains which predominantly encode diguanylate cyclase and phosphodiesterase activities, the GGDEF, and F I G U R E 1 Timeline of nucleotide-based second messenger discovery, occurrence of cyclic di-GMP and cyclic di-AMP signaling in major investigated phyla, and focus of investigations.(a) Discovery of nucleotide second messengers.Indicated is the date of discovery of the molecule either in vivo or by a biochemical assay.Cyclic AMP, the first second messenger and cyclic nucleotide identified, has been discovered in eukaryotes in 1956 (red) and subsequently in prokaryotes (Escherichia coli) in 1963 (blue).Cyclic di-GMP has been identified in 1987 (red; as other molecules upon first recognition of existence beyond chemical synthesis) and was "rediscovered" in 2004 (green; as other molecules upon "rediscovery").Note that date of first discovery in pro-and eukaryotes is only noted for cyclic AMP and cyclic AMP-GMP/cyclic GMP-AMP (cyclic [AG/GA]MP), the latter molecules being chemically distinct.(b) Phylogenetic tree of the bacterial domain of life with major investigated phyla.The presence of at least one GGDEF domain protein indicating the presence of a cyclic di-GMP signaling network in the respective phylum is indicated by a red dot (no data available for candidate phyla OBP41, LD1, and GNG1).The presence of at least one DAC domain protein indicative for cyclic di-AMP signaling is marked by a green spot.A light green spot indicates the presence of few DAC gene products.Dark green and dark red spots indicate unconventional cyclic di-GMP and cyclic di-AMP turnover proteins.Presence of cyclic di-GMP and cyclic di-AMP signaling in the respective phyla has been verified by Blast search using AdrA (P0AAP1) of E. coli MG1655, CdaA (A0A8U0AEL2) of Synechococcus sp.A10-1-5-1 and DisA (Q9WY43) of Thermus thermophilus DSM 3109 as queries.Phylogenetic tree taken from Schulz et al. (2017).(c) Publications of original research work that address investigation of cyclic di-GMP signaling in the respective bacterial species or genus as assessed by PubMed using the keywords cyclic di-GMP/c-di-GMP/diguanylate cyclase/GGDEF in combination with species/genus name.Number of publications is indicated for the five most often investigated genera/ species.(d) Publications of original research work that address investigation of the regulatory role of cyclic di-GMP signaling for microbial phenotypes, the contribution of distinct cyclic di-GMP turnover proteins to regulation and biochemical and structural characterization of network proteins and aptamers.Publications were assessed by PubMed using the keywords cyclic di-GMP/c-di-GMP/diguanylate cyclase/ GGDEF in combination with respective selected phenotypes.
EAL and HD-GYP domains, the latter a subcategory of the HD domain, constitute the most abundant bacterial superfamily domains.
Occasionally, though surprisingly few, eukaryotes such as the social amoeba Dictyostelium, captured diguanylate cyclases and integrated cyclic di-GMP signaling into their morphological development (Chen & Schaap, 2012;Forbes et al., 2022).The promiscuous dinucleotide cyclase DncV, only sporadically occurring in investigated bacterial species, had been captured among all structural and functional paralogs which synthesize cyclic (di-and oligo-)nucleotides and evolved in metazoans as a central innate immune sensor for viral defense.
Why just this enzyme that synthesizes cyclic dinucleotides by a fundamentally different enzymatic mechanism than GGDEF domains had been selected, but cyclic di-GMP-based signaling has been (nearly) dismissed (though the cyclic AMP synthase is a structural homology of the GGDEF domain; Pei & Grishin, 2001) remains an open question.Equally, why cyclic di-GMP, as judged from the currently available information of the sequenced bacterial kingdom, had been selected as the nearly ubiquitous signaling molecule with broad physiological impact remains a mystery.

| ORI G IN OF C YCLI C di-G MP S IG NALING
Based on bioinformatics, cyclic di-GMP is found in the deepest branching bacteria and, as predicted from the presence of genes encoding turnover proteins, also synthesized by archaea (Romling, 2023;Ryjenkov et al., 2005).Thus, cyclic di-GMP, as its equally deeply branching analog cyclic di-AMP (Braun et al., 2021;Römling, 2008;Witte et al., 2008), are ancient second messengers predicted to have been present in the proposed last universal common ancestor, LUCA, of bacteria and archaea.Indeed, cyclic di-GMP and cyclic di-AMP regulate physiological and metabolic processes in microbial cells that are at the origin of the emergence of cellular life.Those processes include the production of protective extracellular matrix components against primordial hazards including ultraviolet light, radiation, and antimicrobials; and the regulation of osmolytes including K + and glycine betaine homeostasis, biosynthesis and remodeling of peptidoglycan, and membrane permeability (Corrigan et al., 2013;Poudyal & Sauer, 2018;Ross et al., 1987).Furthermore, the chemical properties of cyclic dinucleotides such as thermal stability and conformational relaxation due to the two phosphodiester bonds compared to cyclic AMP and cyclic GMP indicate an early evolutionary origin in the timeline of the emergence of the first RNA molecules beyond ribonucleotides that are even today biochemically assembled from basic components such as amino acids, CO 2 (via carbonate), and formate.The nonenzymatic synthesis of cyclic di-GMP in the presence of catalytically active minerals is hypothesized to be possible as it has been demonstrated for other biological molecules and their oligomers such as lipids, short peptides and linear RNA molecules (Lambert, 2008;Wachtershauser, 1988).Such a scenario would place cyclic dinucleotides at the emergence of cellular life.

| C YCLIC di-G MP A S A UB I QU ITOUS LIFE S T YLE REG UL ATOR IN BAC TERIA
Indeed, cyclic di-GMP is found in all major known bacterial phyla, and also cyclic di-AMP turnover proteins can be identified in most of the phyla either consistently or sporadically (Figure 1b).Cyclic di-AMP signaling seems to have been diminished almost entirely in many phyla though, predominantly in alpha-, beta-, and gamma-proteobacteria. Equally, but by far less frequently, cyclic di-GMP signaling has been reduced or entirely disappeared rapidly in evolutionary terms within Pseudomonadota (previously proteobacteria) and other phyla (Liu et al., 2020;Ojha et al., 2021;Römling et al., 2023).This evolutionary flexibility is remarkable with the number of cyclic di-GMP turnover proteins to span from 0 to 86 for a genome of even minor size within a phylum (http://www-ncbi-nlm-nih-gov.proxy.kib.ki.se/Compl ete_Genom es/c-di-GMP.html).Currently, cyclic di-GMP signaling has been investigated in more than 186 species belonging to >105 genera and 16 phyla.The majority of investigated species belong to the Pseudomonadota, in particular gamma proteobacteria (Figure 1c).Thus, cyclic di-GMP signaling has been investigated in only a narrow spectrum of representatives of the estimated 1300 existing phyla and our view on the impact of second messenger molecules might be biased.Relevant human pathogens and probiotics where the role of cyclic di-GMP signaling is not or underexplored include Salmonella enterica serovar Typhi, Shigella spp., Klebsiella pneumoniae, and the lactic acid bacteria.The investigations conducted, however, confirmed initially observed universal physiological and metabolic traits mediated by this multifunctional molecule namely to regulate in environmental and host-associated bacteria the fundamental single-cell lifestyle transition between motility and sessility (biofilm formation) and associated, distinctly regulated, physiological traits such as antimicrobial tolerance (Lichtenberg et al., 2022;Poudyal & Sauer, 2018;Simm et al., 2004).This common physiological trait of cyclic di-GMP signaling, unique among ubiquitous second messenger signaling molecules, is surprising as the cyclic di-GMP binding sites of receptors can be highly diverse with cyclic di-GMP binding in multiple conformations (Chou & Galperin, 2016).Equally can the components of the extracellular matrix, which include conserved moonlighting ribosomal and glycolytic proteins such as the ribosomal protein elongation factor EF-Tu and glyceraldehyde-3-phosphate dehydrogenase, respectively, be chemically, structurally, and evolutionary unrelated (Lorite et al., 2022).Furthermore, the environmental conditions under which distinct biofilms are formed can vary substantially even within a single isolate (Cole et al., 2014).Interference with other associated fundamental traits such as the cell cycle, recombination, and DNA repair and alteration in cell morphology seems to require specific cyclic di-GMP turnover proteins (Fernandez et al., 2018;Gupta et al., 2016;Hwang & Harshey, 2023;Kaczmarczyk et al., 2020;Manikandan et al., 2018).This diversity of biofilm traits and their flexibility to respond to different environmental conditions partly answers the conundrum of the | 567 RÖMLING abundant presence of numerous cyclic di-GMP turnover proteins encoded by bacterial genomes (Amikam & Galperin, 2006;López-Ochoa et al., 2017;Lorite et al., 2022;Roelofs et al., 2015;Römling et al., 2005).Cell lysis and release of DNA moonlighting as extracellular eDNA biofilm matrix component is stimulated at low cyclic di-GMP and cyclic di-AMP levels though (DeFrancesco et al., 2017;Ueda & Wood, 2010).In addition, also seemingly catalytically incompetent GGDEF domain proteins still contribute to biofilm formation.Thus stimulates GdpS in S. aureus eDNA release and protects against cold shock and osmotic stress (Fischer et al., 2014;Zhu et al., 2022).If those observations can be generalized, then it remains to be shown whether and, if so, how these seemingly contradictory regulatory schemes are integrated in biofilm formation at the single-cell level (Allesen-Holm et al., 2006).
Indeed, occurrence and mechanisms of local versus global signaling have been addressed early on and subsequently experimentally demonstrated (Romling et al., 2005).In the same line, it has also been demonstrated that dispersal of biofilms initially requires cyclic di-GMP signaling (Petrova & Sauer, 2012).Recently investigated extracellular matrix composites include twisted stalks, Fe 3+oxyhydroxide-exopolysaccharide composites derived through ferrous ion oxidation by members of beta and zeta proteobacteria (Koeksoy et al., 2021).However, the ubiquitousness to stimulate biofilm formation might be based on cyclic di-GMP's fundamental impact with emergence of motility as a second subsequent trait.
Exemplarily dissected, cyclic di-GMP signaling pathways were shown to be integrated into cascades of temporally and spatially intertwined second messenger signaling events on the single-cell level (Lee et al., 2022;Webster et al., 2022).Indeed, deviations from this "rule," in agreement with bioinformatic and subsequently biochemical investigations, have led to the identification and characterization of noncanonical functionalities of cyclic di-GMP turnover proteins (Ahmad et al., 2013;Simm et al., 2007;Yilmaz et al., 2020).

| ADAP TATION OF CONS ERVED C YCLIC di-G MP REG UL ATED PRO CE SS E S THROUG HOUT THE PHYLOG ENE TI C TREE
However, to what extent and how the temporal and spatial expression of different biofilm extracellular matrix components is coordinated by cyclic di-GMP signaling still needs to be resolved in greater detail (Aragon et al., 2015;Monteiro et al., 2012).To which extent cyclic di-GMP-regulated signaling pathways are conserved or have been refunctionalized throughout the phylogenetic tree has been started to be addressed for extracellular matrix components such as the exopolysaccharide cellulose (Bundalovic-Torma et al., 2020;Romling & Galperin, 2015).The acid-and alkaline-resistant macromolecule cellulose is an extracellular matrix component produced by bacteria such as deeply branching thermophilic microbes and in hot spring sulfur-turf microbial mats, mediating environmental survival due to protection from ultraviolet light and other environmental stresses.In the course of evolution, cellulose production has been refunctionalized for microbial-plant and microbial-fungal interactions and in higher organisms such as humans cellulose biosynthesis serves even as an extra-and intracellular antivirulence factor in microbial host interactions (Balbontin et al., 2014;Lamprokostopoulou et al., 2010;Napoli et al., 1975;Ogawa & Maki, 2003;Pontes et al., 2015;Williams & Cannon, 1989).In the course of the evolution, however, regulation of the catalytic activity of the cellulose synthase by cyclic di-GMP signaling has been occasionally lost (Nobles et al., 2001).Equally has the catalytic activity of the poly-N-acetylglucoseamine synthase producing the alternative exopolysaccharide PNAG been occasionally uncoupled from regulation by cyclic di-GMP signaling.
In microbial-host interactions, cyclic di-GMP ubiquitously mediates the fundamental lifestyle transition between acute and chronic virulence.Alternatively, though not often addressed, a distinct low cyclic di-GMP network status might also be associated with commensalism and symbiosis (Li et al., 2021).This acute to chronic virulence transition is reflected by a substantial alteration in the physiology in animal and plant pathogens which lead to a low immunogenic state of the organism (Ahmad et al., 2011;Lamprokostopoulou et al., 2010;Lamprokostopoulou & Romling, 2022;Pfeilmeier et al., 2016) and biofilm production within immune cells (Pontes et al., 2015).On the other hand, can immune cells induce cyclic di-GMP-promoted biofilm formation through secreted antimicrobial agents such as the short-lived hypochlorous acid HOCl.In Escherichia coli, HOCl targets the diguanylate cyclase DgcZ which upon oxidation of the Zn 2+ binding cysteines in the chemoreceptor zinc binding, CZB, sensor domain becomes activated (Perkins et al., 2021).

| COMPLE XIT Y OF C YCLI C di-G MP S IG NALING
The complexity of the cyclic di-GMP signaling system is not only reflected by the number of cyclic di-GMP turnover proteins encoded by one bacterial genome.Cyclic di-GMP turnover proteins can form multiprotein complexes equally as they can physically interact with their receptors with the broad impact of the interactions still to be fully unraveled (Abidi et al., 2022;Andrade et al., 2006;Kader et al., 2006;Sarenko et al., 2017).In the majority of cases, cyclic di-GMP turnover proteins are multidomain proteins with N-terminal sensory domains, many of which still uncharacterized.Although sensory domains such as PAS, GAF, and receiver are most frequently associated with cyclic di-GMP turnover proteins, sensory domains sense signals as fundamental and diverse as light, temperature, phosphotransfer, the redox status, and quorum sensing molecules (Chou et al., 2020;Deng et al., 2012;Johnson et al., 2005;Jung et al., 2005;Randall et al., 2022;Ryan et al., 2010).Furthermore, as in all superfamily domains, the enzymatic functionality of cyclic di-GMP turnover domains is subject to evolution (Galperin & Chou, 2022;Gao et al., 2015;Holland et al., 2008;Rao et al., 2010).While proteins with not preserved signature motifs can be readily identified as catalytically incompetent, investigation of seemingly catalytically competent, but experimentally enzymatically inactive turnover proteins has led to the identification of novel regulatory entities such as the loop 6 of EAL domains (Rao et al., 2009).Some of the most intriguing recent examples of functional evolution include the identification of cyclic AMP-GMP activity of GGDEF domain proteins governed mainly by one amino acid substitution (Hallberg et al., 2019;Lowry et al., 2022), guanylate cyclase activity outside the GGDEF domain, and the seemingly reversal of catalytic activity in cyclic di-AMP turnover proteins as observed in Porphyromonas gingivalis a member of the Bacteroidetes phylum (Moradali et al., 2022;Ximinies et al., 2023).Some of the cyclic di-GMP turnover proteins such as those associated with the Wsp chemotaxis system and the YfiN diguanylate cyclase with its periplasmic YfiR/YfiB regulators are more often than other identified in currently designed screens and show up as regulatory units in investigated phenotypes (Figure 1d).

| HE TEROG ENEIT Y AND DIFFERENTIAL S ING LE-CELL P OL ARIT Y OF C YCLI C di-G MP S IG NALING
Although cell heterogeneity has long been considered to be a hallmark of biofilm populations (Grantcharova et al., 2010), it is equally observed in the planktonic cell population (Koirala et al., 2014;Sporing et al., 2018).Monitored by aptamer or protein receptorbased fluorescent reporter systems that respond with differential transcriptional activity or conformational changes upon cyclic di-GMP binding, the concentration of cyclic di-GMP in a bacterial population has been shown to be heterogeneous with high cyclic di-GMP levels in cells producing extracellular matrix components and undergoing sporulation (Chou et al., 2020;Christen et al., 2010;Weiss et al., 2019).Thereby, upon cell division, localized cyclic di-GMP turnover proteins become unequally distributed to the daughter cell causing cell heterogeneity (Christen et al., 2010;Kulasekara et al., 2013).Remarkably, upon asymmetric surface sensing by, for example, a polar flagellum, the cyclic di-GMP concentration can be modulated within seconds and cell polarity in cyclic di-GMP synthesis reverted upon cell division to produce surface exploring spreader cells (Manner et al., 2023).With the transition between motility and sessility to be gradually conducted by surface appendages such as the flagellum and subsequently surface-induced type IV pili with functionalities in both lifestyles (Conrad et al., 2011;Nakane et al., 2022;Zhao et al., 2013), observations on the single-cell level indicated transient, but lasting cellular states that conduct both biosynthesis of extracellular matrix equally as surface motility that are promoted by low-and high-cyclic di-GMP, respectively.Local cyclic di-GMP signaling might also play a role in the direction of the rotational bias of the periplasmic flagella produced on both poles of the spirochaete Borrelia burgdorferi (Sultan et al., 2011).These opposite (local) signaling events within a single bacterial cell indicate also again that a direct correlation between the overall cyclic di-GMP concentration with physiological, behavioral, and metabolic events can be misleading.

| INTER AC TI ON WITH ALTERNATIVE S ECOND ME SS ENG ER S IG NALING NE T WO RK S
As another level of complexity, the product of the EAL phosphodiesterase activity, the linear dinucleotide pGpG is hypothesized to be a signaling molecule on its own with its degradation distinctively regulated (Cohen et al., 2015;Orr et al., 2015).Furthermore, cyclic di-GMP signaling is highly integrated with other signaling systems such as phosphotransfer systems, cyclic AMP, cyclic di-AMP, cyclic AMP-GMP, and stringent response ppGpp signaling in a bidirectional way (Figure 2; Chou et al., 2020;Shyp et al., 2021).Indeed, on a first sight, however, the horizontally transferred second messenger cyclic AMP-GMP in an animal commensal E. coli strain does not seem to interfere with cyclic di-GMP regulation of rdar biofilm formation and motility (Li et al., 2019).In this context, cyclic AMP-GMP signaling, however, does inhibit both lifestyles, sessility and motility, addressing alternative targets on another level in the regulatory cascade than cyclic di-GMP.

| D IVER S IT Y AND PL A S TI CIT Y OF C YCLIC di-G MP RECEP TOR S
In all environments, binding of cyclic di-GMP over a wide affinity range from nano to μM to a multitude of protein and RNA aptamer receptors with distinct binding sites, often, but not commonly determined by an RxxxR or DxxR motif, enables the signaling system to conduct a coordinated multilayer response.Cyclic di-GMP can thereby bind to effectors such as transcriptional regulators and F I G U R E 2 Selected interactions between cyclic di-GMP and different alternative cyclic dinucleotide signaling systems and their overall effect on major phenotypes of Gram-negative bacteria.Green and blue arrows indicate positive interactions, while red and yellow arrows indicate negative interactions.ppGpp inhibits the enzymatic activity of the cyclic di-AMP specific DHH/DHHA1 phosphodiesterase and vice versa cyclic di-AMP can indirectly inhibit synthesis of ppGpp in Gram-positive bacteria (Covaleda-Cortes et al., 2023;Rao et al., 2010).Although cyclic di-AMP has been detected as a byproduct of the enzymatic activity of the dinucleotide cyclase DncV and of GGDEF domains (Davies et al., 2012;Li et al., 2019;Lowry et al., 2022), whether and, if so, which role it exerts in Gram-negative bacteria needs to be determined.Green/blue arrow, positive ineraction; red/yellow arrow, inhibiton.
| 569 RÖMLING enzymes equally as to non-canonical GGDEF and EAL domains of GGDEF/EAL domain proteins thereby affecting central physiological reactions such as protein production equally as proteolysis on a millisecond to an hour time scale (Chou et al., 2020;Cooley et al., 2016).
Depending on the determinative interactions and degree of enzymatic activities, cyclic di-GMP signaling can thus act entirely locally with (almost) no overall alteration in cyclic di-GMP concentration and globally through combination of enzymatic activities equally as globally by locally affecting component(s) with broad physiological impact (Römling et al., 2013).Intriguingly, the ribosome is a major target of cyclic di-GMP signaling.Binding of cyclic di-GMP to the elongation factor P facilitates translation of proline tracks in biofilm proteins and to the α-L-glutamate ligase RimK stimulates its enzymatic activity leading to C-terminal glutaminylation of the ribosomal protein RpsF to modulate translation (Guo et al., 2022;Little et al., 2016).On the overall evolutionary time scale equally as the cyclic di-GMP network encoded by genomes, cyclic di-GMP binding sites of individual proteins can rapidly appear and disappear in evolution (Römling, 2023).

| MOONLIG HTING FUN C TIONS OF C YCLIC di-G MP
Besides its role as second messenger, cyclic di-GMP possesses also moonlighting extracellular functions.Remarkably, in bacteria that have a highly reduced cyclic di-GMP signaling system, not known to produce cyclic di-GMP at all, but also in cyclic di-GMP-proficient bacteria, extracellularly provided cyclic di-GMP was shown to inhibit biofilm formation, disperse biofilms, and promote chemotaxis (Elgamoudi et al., 2022;Karaolis et al., 2005;Yang et al., 2023).The receptor(s) and signaling pathways that mediate this behavior still need to be identified.Although first observed for the second messenger cyclic di-AMP secreted by intracellular Listeria monocytogenes overexpressing multidrug efflux pumps (Woodward et al., 2010), at which occasion and to what extent cyclic di-GMP signaling is secreted by diverse bacterial species or the microbiome, either to inhibit the immune response or to stimulate inflammatory processes via the cyclic dinucleotide sensing host protein STING, needs to be more broadly defined (Cui et al., 2019;Shmuel-Galia et al., 2021).Phages, often considered ideal alternatives for antimicrobial therapy of chronic biofilm-associated infections, not only can exploit cyclic di-GMP-dependent exopolysaccharides as degradable receptors, but also induce or enhance biofilm formation with peptides that target the YfiN cyclic di-GMP signaling pathway (De Smet et al., 2021;Sellner et al., 2021).

| IMPAC T OF C YCLI C di-G MP S I G NALING IN THE ECOLOG I C AL , AG RI CULTUR AL , AND INDUS TRIAL CONTE X T
Also in a wider perspective with respect to regulation of biofilm formation and beyond, cyclic di-GMP signaling has been found to be of ecological, agricultural, and industrial importance.The complex intimate interactions between microbes and higher organisms are regulated by complex crosstalks that involve signal perception by cyclic di-GMP turnover proteins and cyclic di-GMP-regulated signals.This is exemplified frequently in the aquatic environment.For example, microbial biofilms in marine environments can induce or inhibit settlement and metamorphosis of invertebrate organisms.
In a strongly settlement, inducing Pseudoalteromonas marina strain alternating cyclic di-GMP levels are associated with the alteration of these developmental traits in the macrofouling edible mussel Mytilus coruscus (Peng et al., 2020).Stimulated intrinsic nitric oxide signaling in a Ruegeria (previously Silicibacter) isolate mediates cyclic di-GMP-dependent biofilm formation to interact with the abundant marine nitrogen-fixing cyanobacterium Trichodesmium erythraeum (Rao et al., 2015).Inhibition of a diguanylate cyclase activity by hostderived amino acids proline, valine, or isoleucine promotes motility to subsequently colonize the gnotobiotic zebrafish host (Robinson et al., 2021).Whether and how pollutants might interfere with signal perception and downstream cyclic di-GMP signaling to potentially disturb these or other delicate ecological interactions remains to be shown.On the other hand, cyclic di-GMP signaling might be exploited for enhanced performance of microbial strains and communities for the biodegradation of pollutants and in wastewater treatment processes partly also due to the enhanced tolerance, degradation potential, and stabilized biofilm formation mediated by cyclic di-GMP signaling (Benedetti et al., 2016;Dorado-Morales et al., 2021;Schmutzler et al., 2017;Wan et al., 2016;Wang & Shao, 2021;Yuan et al., 2022).
The microbiome of plants fulfills multiple functions and includes symbionts such as nitrogen-fixing Sinorhizobium to promote growth.Within nodules, cyclic di-GMP signaling is downregulated (Li et al., 2021), which might reflect a host-associated immune-protected status similar as in human commensals.In plant endophytes, cyclic di-GMP signaling regulates protection from plant pathogens by various mechanisms including biofilm formation and production of antifungal compounds (Chen et al., 2022;Mousa et al., 2016).
Production of antimicrobial compounds can, however, be differentially regulated by cyclic di-GMP signaling (Chou et al., 2020) and extends beyond plant endophytes.

| ROLE OF C YCLIC di-G MP S I G NALING IN B I OG EO CHEMIC AL C YCLING
Being fundamental and providing a wider perspective, but not often explored yet is the role of cyclic di-GMP signaling in biogeochemical cycling.Promotion of biofilm formation on sulfur and pyrite, iron disulfide, the regulation of cytochrome c-type proteins on the transcriptional level, involvement in the regulation of the abiotic sulfur cycle, and enhanced biofilm formation by anaerobic, oceanic ecosystem relevant electron acceptors couples cyclic di-GMP signaling to the global abiotic sulfur cycle and biogeochemical and ancient energy conservation processes (Cai et al., 2023;Martin-Rodriguez et al., 2021;Ng et al., 2020;Ruiz et al., 2012).
Whether cyclic di-GMP signaling is involved to a larger extent in the global abiotic or biotic sulfur cycle equally as in the global nitrogen and carbon cycles remains to be shown.In any case, sensing of nitric oxide can be tightly associated with cyclic di-GMP signaling to promote or to prevent biofilm formation (Williams & Boon, 2019).
Aquatic sediments and soils with their rich microbial community equally play a determinative ecological role in biogeochemical cycling and promotion of nutrient dynamics.An ecological function of Geobacter, a genus found in those sediments, namely cyclic AMP-GMP-mediated reduction of ferric and Mn 4+ ions, is exploited in microbial fuel cells for energy conservation (Hallberg et al., 2019;Nelson et al., 2015).
Beyond the marine environment, the abundant Roseobacter clade which harbors marine microorganisms is involved in multiple biogeochemical cycles and climate change as they produce to a large extent volatile dimethyl sulfide which is causative in cloud nuclei formation.
As cyclic di-GMP signaling is found in the R. clade (D'Alvise et al., 2014), one can put forward the challenging hypothesis that cyclic di-GMP signaling might, directly or indirectly, affect the global climate.

| CON CLUS ION
Although cyclic di-GMP signaling has been the subject of intensive investigations since its rediscovery in 2004 (Paul et al., 2004;Simm et al., 2004;Tischler & Camilli, 2004) and regulation of major phenotypes, molecular mechanisms and signal integration pathways have been unraveled, there are still major relevant open questions to be answered some of them put forward in this perspective article.Those open questions include the evolutionary origin of second messenger signaling by cyclic di-GMP and analogs, the evolutionary forces of expansion, diversification, and disappearance of the cyclic di-GMP signaling system and, equally, whether there is a broad metabolic impact on turnover of this molecule beyond GGDEF, EAL, and HD-GYP domain proteins.Equally, studies on the single-cell level with innovative experimental approaches have just started to address the diversity of regulation of physiological, metabolic, and morphological traits and the corresponding diversity of molecular mechanisms on an unprecedented time scale and in unprecedented details.Beyond cyclic di-GMP signaling, the recent discovery of additional (cyclic di-and oligo-)nucleotides (Braun et al., 2021;Davies et al., 2012;Tal et al., 2021) and the rediscovery of "old" nucleotides previously considered primarily to be just a byproduct of enzymatic activities (Chou et al., 2020) add to the complexity of nucleotidebased second messenger signaling.