SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

On the stage of bacterial signal transduction and regulation, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has long played the part of Sleeping Beauty. c-di-GMP was first described in 1987, but only recently was it recognized that the enzymes that 'make and break' it are not only ubiquitous in the bacterial world, but are found in many species in huge numbers. As a key player in the decision between the motile planktonic and sedentary biofilm-associated bacterial 'lifestyles', c-di-GMP binds to an unprecedented range of effector components and controls diverse targets, including transcription, the activities of enzymes and larger cellular structures. This Review focuses on emerging principles of c-di-GMP signalling using selected systems in different bacteria as examples.

Principles of c-di-GMP signalling in bacteria.

Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.

Signals, regulatory networks, and materials that build and break bacterial biofilms

Bacteria use diverse small molecules for extra- and intracellular signaling. They scan small-molecule mixtures to access information about both their extracellular environment and their intracellular physiological status, and based on this information, they continuously interpret their circumstances and react rapidly to changes. Bacteria must integrate extra- and intracellular signaling information to mount appropriate responses to changes in their environment. We review recent research into two fundamental bacterial small-molecule signaling pathways: extracellular quorum-sensing signaling and intracellular cyclic dinucleotide signaling. We suggest how these two pathways may converge to control complex processes including multicellularity, biofilm formation, and virulence. We also outline new questions that have arisen from recent studies in these fields.

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Bacterial small-molecule signaling pathways.

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.

https://www.pnas.org/content/pnas/102/40/14422.full.pdf

A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels.

Recent discoveries suggest that a novel second messenger, bis-(3′→5′)-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and/or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product c-diGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer–monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotide-binding mode is substantially different. The guanine base is H-bonded to Asn-335 and Asp-344, whereas the ribosyl and α-phosphate moieties extend over the β2-β3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem–DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.

https://www.pnas.org/content/pnas/101/49/17084.full.pdf

Dietrich Samoray

Papers

Structural basis of activity and allosteric control of diguanylate cyclase

Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP.