Bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a soluble molecule that functions as a secondary messenger in bacterial cells. One effector of C-di-GMP signaling is RNA, which acts as a riboswitch (an RNA regulatory element that alters the expression profile of the cell).

supergalactic | Shutterstock

C-di-GMP is small and diffusible, a characteristic typical of second messengers. It is involved in two distinct bacterial ‘lifestyle’ choices; to remain sedentary and adherent or to adopt planktonic motility, that is, swim.

The general effect of c-di-GMP is to reduce the expression of proteins that constitute the flagella and increase the expression of molecular mediators of adherence. These adherent molecules are called adhesins and biofilm-associated exopolysaccharides (EPSs). C-di-GMP also plays a subsidiary role in virulence of animal and plant pathogens as well as cell cycle control in certain bacteria.

Synthesis of c-di-GMP

Signal transduction pathways must be tightly controlled. As such, messengers must be synthesized and degraded rapidly to initiate and terminate a signaling event, respectively. Diguanylate cyclases (DGCs) synthesize c-di-GMP and are degraded by c-di-GMP phosphodiesterases (PDEs).

The enzymatic activity of both DGCs and PDEs are provided by domains which are named after the amino acid motifs that directly contribute to enzymatic activity: GGDEF domains in DGCs and EAL or HD-GYP domains in PDEs.

The c-di-GMP signaling module

DGCs, PDEs, and effector proteins are all targets of c-di-GMP; this multiplicity of targeting allows several signals to be integrated into one cascade to produce a specific response. Additionally, parallel pathways may be activated, allowing multiple functional outcomes based on spatial sequestration of c-di-GMP in these pathways. These separate pathways are comprised of partnered or 'cognate' DGCs, PDEs, effectors, and targets which together comprise a specific c-di-GMP control module.

Partnering is specific and is accomplished by the amino-terminal sensory domains of DGCS and PDEs. Binding of c-di-GMP occurs on a secondary site (I site) in most DGCs. This binding also has the effect of eliciting feedback inhibition, limiting the amount of c-di-GMP that accumulates in the cell. The operation of each module requires direct macromolecular interactions, therefore these effects are localized to a specific region of the cell.

The cognate nature of c-di-GMP modules has facilitated the evolution of proteins containing ‘degenerate’ GGDEF and EAL domains. These proteins can, therefore, operate without c-di-GMP control; however, these proteins still operate in systems that control the formation of biofilms or affect motility – they are 'evolutionarily trapped' in this regard.

How is c-di-GMP made and destroyed?

The active DGC is a dimer of two subunits that carry a GGDEF domain, each of which binds a single GTP molecule. At their interface lies the A site (active site) which corresponds to the GGDEF domain. The mechanism of c-di-GMP production is analogous to that seen in structurally related adenylate cyclases and polymerases.

PDEs possess the EAL domain which enables conversion of c-di-GMP to 5′-pGpG, which is then further degraded by non-specific PDEs. The second type of c-di-GMP-specific PDE is those that contain HD-GYP domains; these also produce 5′-pGpG but can additionally degrade this further to produce GMP.

Usually, these three domains with their antagonistic functions are separated. In some bacteria, however, they occur as composite (fusion) proteins. In this case, GGDEF domain is covalently linked to either EAL or HD-GYP domains. Alternatively, bifunctional enzymes that can switch, in a binary fashion, between DGC and PDE activity also existhe t.

c-di-GMP effectors

c-di-GMP is a secondary messenger that exerts its function through binding to the target effector component. It acts allosterically, binding to the effector component at a site away from the effector’s active site and inducing a conformational change in the effector to modify its function.

c-di-GMP effectors can be categorized into four families:

1. PilZ family of proteins (named after a type IV pilus control protein in P. aeruginosa): these may be attached to the carboxyl terminus of the GGDEF, EAL and/or HD-GYP domains or a domain that produces a molecular product such as alginate or cellulose.
2. C-di-GMP-binding transcription factor FleQ of P. aeruginosa: this is a repressor. Inactivation by c-di-GMP alleviates its repressive function, subsequently allowing transcription to take place.
3. PelD of P. aeruginosa: this is activated by binding c-di-GMP at a site that resembles the I site in proteins bearing the GGDEF domain.
4. Effectors regulated through the I site: The first example of effectors that operate in this way is the C. crescentus PopA protein; when  c-di-GMP binds to its I site, PopA sequesters CtrA, a replication inhibitor.

Cellular processes are altered as a result of c-di-GMP targeting of effector proteins. The process affected is dependent on the identity of the targeted effector. For example

• Transcription is altered if the effector is a transcription factor.
• Elongation or translation if the effector is a riboswitch
• Proteolysis if the effector is a protein-degrading enzyme
• Cell motility if the effector is the flagella or cell adhesion of the effector is an adhesion

In addition to these processes, c-di-GMP signaling plays an essential role in virulence; small concentrations of c-di-GMP molecules are required to induce virulence gene expression in the host. Further, c-di-GMP signaling has been implicated in cell cycle progression, antibiotic production and other cellular functions. The mechanisms that underpin these effects are, however, still under study.

Adhesion and biofilm formation

The c-di-GMP effector system in P. fluorescens illustrates how the switch between motile and sessile (biofilm) states occurs via c-di-GMP signaling. LapD is an inner membrane effector protein that binds c-di-GMP through its EAL domain. Inside-out signaling mechanisms, in which intracellular signals induce extracellular effects, allow LapD signal inputs to impact cell-surface levels of the biofilm adhesin LapA.

In the absence of c-di-GMP, LapD exists in an autoinhibited state, whereby the GGDEF domain prevents C-di-GMP from gaining access to the EAL domain. As such, LapD’s periplasmic domain (the area that exists between the inner cytoplasmic membrane and the bacterial outer membrane) cannot bind the LapG protease. LapG is thus free to cleave LapA from the cell surface, thereby preventing biofilm formation.

When cytoplasmic C-di-GMP levels increase, its allosteric effect on LapD results in a conformational change within the GGDEF that allows EAL domains to dimerize. This conformational change is relayed to the periplasmic face of LapD, which is permissive to  LapA binding. LapG is no longer able to cleave LapA , allowing biofilm formation to occur.

Further Reading

• All Microbiology Content
• Gram Negative Bacteria
• Differentiating Between Microbial Species
• Quorum Sensing and Pseudomonas aeruginosa
• Single Cell Microbiology

Written by

Hidaya Aliouche

Hidaya is a science communications enthusiast who has recently graduated and is embarking on a career in the science and medical copywriting. She has a B.Sc. in Biochemistry from The University of Manchester. She is passionate about writing and is particularly interested in microbiology, immunology, and biochemistry.