Messenger Cyclic di-GMP Research Articles

The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP.

The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels. Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.

Structural basis of functional diversification of the HD-GYP domain revealed by the Pseudomonas aeruginosa PA4781 protein, which displays an unselective bimetallic binding site.

The intracellular level of the bacterial secondary messenger cyclic di-3',5'-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site. In bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 from Pseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs.

Intestinal GPS: bile and bicarbonate control cyclic di-GMP to provide Vibrio cholerae spatial cues within the small intestine

The second messenger cyclic di-GMP (c-di-GMP) regulates numerous phenotypes in response to environmental stimuli to enable bacteria to transition between different lifestyles. Here we discuss our recent findings that the human pathogen Vibrio cholerae recognizes 2 host-specific signals, bile and bicarbonate, to regulate intracellular c-di-GMP. We have demonstrated that bile acids increase intracellular c-di-GMP to promote biofilm formation. We have also shown that this bile-mediated increase of intracellular c-di-GMP is negated by bicarbonate, and that this interaction is dependent on pH, suggesting that V. cholerae uses these 2 environmental cues to sense and adapt to its relative location in the small intestine. Increased intracellular c-di-GMP by bile is attributed to increased c-di-GMP synthesis by 3 diguanylate cyclases (DGCs) and decreased expression of one phosphodiesterase (PDE) in the presence of bile. The molecular mechanisms by which bile controls the activity of the 3 DGCs and the regulators of bile-mediated transcriptional repression of the PDE are not yet known. Moreover, the impact of varying concentrations of bile and bicarbonate at different locations within the small intestine and the response of V. cholerae to these cues remains unclear. The native microbiome and pharmaceuticals, such as omeprazole, can impact bile and pH within the small intestine, suggesting these are potential unappreciated factors that may alter V. cholerae pathogenesis.

Stimulation of innate immunity by in vivo cyclic di-GMP synthesis using adenovirus.

The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP

BackgroundDiguanylate cyclases (DGCs) regulate biofilm formation and motility in bacteria by synthesizing the second messenger cyclic di-GMP (c-di-GMP) in response to environmental stimuli. DGC enzymatic activity is believed to be dependent on the presence of a GG(D/E)EF active site motif, however approximately 25% of known DGCs contain a degenerate active site. The Vibrio cholerae protein VCA0965 contains an AGDEF active site and is presumed to be an inactive DGC.ResultsEctopic expression of VCA0965 in V. cholerae causes a 3-fold reduction in flagellar-based motility. Additionally, an RXXD allosteric inhibition mutant of VCA0965 strongly inhibited motility and stimulated biofilm formation. This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP. In support of this, ectopic expression of VCA0965 and VCA0965 containing a mutation in its RXXD motif significantly increased the intracellular c-di-GMP levels in V. cholerae and Escherichia coli. Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro. Systematic mutation of the first amino acid in the AGDEF motif of VCA0965 revealed that glycine, methionine, and histidine also produced an active DGC capable of inhibiting motility and increasing the intracellular concentration of c-di-GMP in V. cholerae.ConclusionsBased on these results, we conclude that VCA0965 is capable of c-di-GMP synthesis and that the first amino acid of the GG(D/E)EF motif is more tolerant of substitutions than currently appreciated.

What is the role of cyclic di-GMP signaling within the human gut microbiome?

AbstractThere is currently little understanding of the role of the bacterial second messenger cyclic di-GMP (c-di-GMP) in the human gut microbiome. C-di-GMP is synthesized by highly conserved diguanylate cyclase (DGC) enzymes and degraded by highly conserved phosphodiesterase (PDE) enzymes. To begin to assess the prevalence of c-di-GMP signaling in the gut microbiome, we found on average 1.0 DGC and 0.8 PDE enzymes per million base pairs of metagenomic DNA derived from stool samples. Specific species encoding substantial numbers of GGDEF and EAL domains were the commensal species

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.

The LuxR-Type Regulator VpsT Negatively Controls the Transcription of rpoS, Encoding the General Stress Response Regulator, in Vibrio cholerae Biofilms

Cholera is a waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. Expression of the general stress response regulator RpoS and formation of biofilm communities enhance the capacity of V. cholerae to persist in aquatic environments. The transition of V. cholerae between free-swimming (planktonic) and biofilm life-styles is regulated by the second messenger cyclic di-GMP (c-di-GMP). We previously reported that increasing the c-di-GMP pool by overexpression of a diguanylate cyclase diminished RpoS expression. Here we show that c-di-GMP repression of RpoS expression is eliminated by deletion of the genes vpsR and vpsT, encoding positive regulators of biofilm development. To determine the mechanism of this regulation, we constructed a strain expressing a vpsT-FLAG allele from native transcription and translation signals. Increasing the c-di-GMP pool induced vpsT-FLAG expression. The interaction between VpsT-FLAG and the rpoS promoter was demonstrated by chromatin immunoprecipitation. Furthermore, purified VpsT interacted with the rpoS promoter in a c-di-GMP-dependent manner. Primer extension analysis identified two rpoS transcription initiation sites located 43 bp (P1) and 63 bp (P2) upstream of the rpoS start codon. DNase I footprinting showed that the VpsT binding site at the rpoS promoter overlaps the primary P1 transcriptional start site. Deletion of vpsT significantly enhanced rpoS expression in V. cholerae biofilms that do not make HapR. This result suggests that VpsT and c-di-GMP contribute to the transcriptional silencing of rpoS in biofilms prior to cells entering the quorum-sensing mode.

Finally! The structural secrets of a HD-GYP phosphodiesterase revealed

The major sessility-motility lifestyle change and additional fundamental aspects of bacterial physiology, behaviour and morphology are regulated by the secondary messenger cyclic di-GMP (c-di-GMP). Although the c-di-GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD-GYP domain c-di-GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al. provide an important step towards functional and structural characterization of the previously neglected HD-GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri-nuclear catalytic iron centre involved in c-di-GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c-di-GMP to GMP by HD-GYP domains.

Cyclic Di-GMP Receptor PlzA Controls Virulence Gene Expression through RpoS in Borrelia burgdorferi

As an obligate pathogen, the Lyme disease spirochete Borrelia burgdorferi has a streamlined genome that encodes only two two-component signal transduction systems, Hk1-Rrp1 and Hk2-Rrp2 (in addition to CheA-CheY systems). The output of Hk1-Rrp1 is the production of the second messenger cyclic di-GMP (c-di-GMP), which is indispensable for B. burgdorferi to survive in the tick vector. The output of Hk2-Rrp2 is the transcriptional activation of the global regulator RpoS, which is essential for the pathogen to accomplish its tick-mouse transmission and to establish mammalian infection. Although evidence indicates that these two systems communicate with each other, how they are connected is not fully understood. In this study, we showed that the c-di-GMP-binding protein PlzA, a downstream effector of Rrp1, positively modulates the production of RpoS, a global regulator and downstream target of Rrp2. Thus, PlzA functions as a connector that links Hk1-Rrp1 with Hk2-Rrp2. We further showed that PlzA regulates rpoS expression through modulation of another regulator, BosR, at both the transcriptional and the posttranscriptional levels. In addition, PlzA was also capable of regulating rpoS expression independently of Rrp1, suggesting that besides being a c-di-GMP-binding protein, PlzA has other functions. Along with the previous finding of PlzA controlling motility, these studies demonstrate that PlzA is a multifunctional protein. These findings further reinforce the notion that B. burgdorferi utilizes its limited signaling systems and regulators to govern multiple cellular processes during its complex enzootic cycle between ticks and mammals.

Proteomics Analysis of the Regulatory Role of Rpf/DSF Cell-to-Cell Signaling System in the Virulence of Xanthomonas campestris

The black rot pathogen Xanthomonas campestris utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis, whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in X. campestris that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC, or rpfG mutants compared with the wild type. These proteins belonged to several functional categories, including biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance, and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors among these proteins, including elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Disintegration of aerobic granules: Role of second messenger cyclic di-GMP

Loss of structural stability of aerobic granular process is the challenge for its field applications to treat wastewaters. The second messenger, cyclic diguanylate (c-di-GMP), is widely used by bacteria to regulate the synthesis of exopolysaccharide. This study for the first time confirmed the correlation between concentration of intracellular c-di-GMP and the granular stability under sequencing batch reactor (MBR) mode. In the presence of manganese ions (Mn(2+)), the concentrations of intracellular c-di-GMP and of extracellular polysaccharides and proteins in granules were declined. Clone library study revealed that the polysaccharide producers. Acinetobacter sp., Thauera sp., Bdellovibrio sp. and Paracoccus sp. were lost after Mn(2+) addition. The findings reported herein confirmed that the c-di-GMP is a key chemical factor epistatic to quorum sensing to determine granular stability. Stimulation of synthesis of intracellular c-di-GMP presents a potential way to enhance long-term stability of aerobic granules.

Cis-2-dodecenoic acid quorum sensing system modulates N-acyl homoserine lactone production through RpfR and cyclic di-GMP turnover in Burkholderia cenocepacia

BackgroundBurkholderia cenocepacia employs both N-Acyl homoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) quorum sensing (QS) systems in regulation of bacterial virulence. It was shown recently that disruption of BDSF synthase RpfFBc caused a reduction of AHL signal production in B. cenocepacia. However, how BDSF system influences AHL system is still not clear.ResultsWe show here that BDSF system controls AHL system through a novel signaling mechanism. Null mutation of either the BDSF synthase, RpfFBc, or the BDSF receptor, RpfR, caused a substantial down-regulation of AHL signal production in B. cenocepacia strain H111. Genetic and biochemical analyses showed that BDSF system controls AHL signal production through the transcriptional regulation of the AHL synthase gene cepI by modulating the intracellular level of second messenger cyclic di-GMP (c-di-GMP). Furthermore, we show that BDSF and AHL systems have a cumulative role in the regulation of various biological functions, including swarming motility, biofilm formation and virulence factor production, and exogenous addition of either BDSF or AHL signal molecules could only partially rescue the changed phenotypes of the double deletion mutant defective in BDSF and AHL signal production.ConclusionsThese results, together with our previous findings, thus depict a molecular mechanism with which BDSF regulates AHL signal production and bacterial virulence through modulating the phosphodiesterase activity of its receptor RpfR to influence the intracellular level of c-di-GMP.

Exploring Environmental Control of Cyclic di-GMP Signaling in Vibrio cholerae by Using theEx VivoLysate Cyclic di-GMP Assay (TELCA)

Vibrio cholerae senses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. V. cholerae encodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system in V. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named the ex vivo lysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show that V. cholerae grown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP, and we present a new method that is widely applicable to the systematic analysis of complex c-di-GMP signaling networks.

Cyclic di-GMP signalling and the regulation of bacterial virulence

Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3′-5′)-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small ‘adaptor’ protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein–protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence.

Structures of the catalytic EAL domain of theEscherichia colidirect oxygen sensor

The direct oxygen sensor DosP is a multidomain protein that contains a gas-sensing haem domain and an EAL effector domain. EAL domains are omnipresent signal transduction domains in bacteria. Many EAL domains are active phosphodiesterases and are involved in breakdown of the ubiquitous bacterial second messenger cyclic di-GMP. Despite a great deal of information on the functional and structural aspects of active and inactive EAL domains, little is known about the structural basis of their regulation by their associated sensory domains. Here, two crystal structures of the Escherichia coli DosP EAL domain derived from cubic and monoclinic crystal forms that were obtained under tartrate and PEG conditions, respectively, are described. Both of the structures display the typical TIM (triosephosphate isomerase) barrel fold with one antiparallel β-strand. However, unlike other EAL structures, access to the active site in DosP EAL is sterically restricted by the presence of a short helical stretch (Ser637-Ala-Leu-His640) in loop L3 between strand β3 and helix α3. This element, together with an unordered fragment, replaces the short α-helix (named α5 in Tbd1265 EAL) that is found in other EAL-domain structures. Since DosP EAL is an active c-di-GMP phosphodiesterase, the observed inactive conformation is suggested to be of functional relevance for the regulation mechanism of DosP.

Cyclic Di-GMP modulates the disease progression of Erwinia amylovora.

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD