Bacteriophage-mediated interference of the c-di-GMP signalling pathway in Pseudomonas aeruginosa

C-di-GMP is a key signalling molecule which impacts bacterial motility and biofilm formation and is formed by the condensation of two GTP molecules by a diguanylate cyclase. We here describe the identification and characterization of a family of bacteriophage-encoded peptides that directly impact c-di-GMP signalling in Pseudomonas aeruginosa. These phage proteins target Pseudomonas diguanylate cyclase YfiN by direct protein interaction (termed YIPs, YfiN Interacting Peptides). YIPs induce an increase of c-di-GMP production in the host cell, resulting in a decrease in motility and an increase in biofilm mass in P. aeruginosa. A dynamic analysis of the biofilm morphology indicates a denser biofilm structure after induction of the phage protein. This intracellular signalling interference strategy by a lytic phage constitutes an unexplored phage-based mechanism of metabolic regulation and could potentially serve as inspiration for the development of molecules that interfere with biofilm formation in P. aeruginosa and other pathogens.     

A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.     

Determinants for the Activation and Autoinhibition of the Diguanylate Cyclase Response Regulator WspR

The bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) controls secretion, cell adhesion, and motility, leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity.     

Systematic Analysis of Diguanylate Cyclases That Promote Biofilm Formation by Pseudomonas fluorescens Pf0-1

Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.     

Tyrosine Phosphatase TpbA of Pseudomonas aeruginosa Controls Extracellular DNA via Cyclic Diguanylic Acid Concentrations

Inactivating the tyrosine phosphatase TpbA of Pseudomonas aeruginosa PA14 induces biofilm formation by 150-fold via increased production of the second messenger cyclic diguanylic acid (c-di-GMP). Here, we show the tpbA mutation reduces extracellular DNA (eDNA) and that increased expression of tpbA increases eDNA; hence, eDNA is inversely proportional to c-di-GMP concentrations. Mutations in diguanylate cyclases PA0169, PA4959, and PA5487 and phosphodiesterase PA4781 corroborate this trend. The tpbA mutation also decreases cell lysis while overexpression of tpbA increases cell lysis. To further link c-di-GMP concentrations and eDNA, the gene encoding phosphodiesterase PA2133 was overexpressed which increased eDNA and decreased biofilm formation by decreasing c-di-GMP. Furthermore, the effect of the tpbB mutation along with the tpbA mutation was examined because loss of TpbB restored the phenotypes controlled by enhanced c-di-GMP in the tpbA mutant. The tpbA tpbB double mutations restored eDNA to that of the PA14 wild-type level. These findings suggest that c-di-GMP, rather than TpbA, controls eDNA. Hence, TpbA acts as a positive regulator of eDNA and cell lysis by reducing c-di-GMP concentrations.     

BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.     

Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors

In Gram-negative bacteria, production of bis-(3′,5′)-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.     

'Life-style' control networks in Escherichia coli: signaling by the second messenger c-di-GMP

Most bacteria can exist in either a planktonic-motile single-cell state or an adhesive multicellular state known as a biofilm. Biofilms cause medical problems and technical damage since they are resistant against antibiotics, disinfectants or the attacks of the immune system. In recent years it has become clear that most bacteria use cyclic diguanylate (c-di-GMP) as a biofilm-promoting second messenger molecule. C-di-GMP is produced by GGDEF-domain-containing diguanylate cyclases and is degraded by phosphodiesterases featuring EAL or HD-GYP domains. Many bacterial species possess multiple proteins with GGDEF and EAL domains, which actually belong to the most abundant protein families in genomic data bases. Via an unprecedented variety of effector components, which include c-di-GMP-binding proteins as well as RNAs, c-di-GMP controls a wide range of targets that down-regulate motility, stimulate adhesin and biofilm matrix formation or even control virulence gene expression. Moreover, local c-di-GMP signaling in macromolecular complexes seems to allow the independent and parallel control of different output reactions. In this review, we use Escherichia coli as a paradigm for c-di-GMP signaling. Despite the huge diversity of components and molecular processes involved in biofilm formation throughout the bacterial kingdom, c-di-GMP signaling represents a unifying principle, which suggests that the enzymes that make and break c-di-GMP may be promising targets for anti-biofilm drugs.     

Investigating the Allosteric Regulation of YfiN from Pseudomonas aeruginosa: Clues from the Structure of the Catalytic Domain

Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.     

Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.     

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.     

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae

The bacterial second messenger bis-(3′-5′)-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain–containing diguanylate cyclases and degraded by HD-GYP domain–containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain–containing PDE-As to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.     

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains,and Its Role in the Development of Pierce’s Disease

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.     

Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.     

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB,the c-di-GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air–liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.     

The Pseudomonas aeruginosa Diguanylate Cyclase GcbA,a Homolog of P. fluorescens GcbA,Promotes Initial Attachment to Surfaces,but Not Biofilm Formation,via Regulation of Motility

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.     

c-di-GMP turn-over in Clostridium difficile is controlled by a plethora of diguanylate cyclases and phosphodiesterases

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen.