Diguanylate Cyclase Activity Research Articles

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae.

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo 6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

Diguanylate Cyclases in Vibrio cholerae: Essential Regulators of Lifestyle Switching

Biofilm formation in Vibrio cholerae empowers the bacteria to lead a dual lifestyle and enhances its infectivity. While the formation and dispersal of the biofilm involves multiple components—both proteinaceous and non-proteinaceous, the key to the regulatory control lies with the ubiquitous secondary signaling molecule, cyclic-di-GMP (c-di-GMP). A number of different cellular components may interact with c-di-GMP, but the onus of synthesis of this molecule lies with a class of enzymes known as diguanylate cyclases (DGCs). DGC activity is generally associated with proteins possessing a GGDEF domain, ubiquitously present across all bacterial systems. V. cholerae is also endowed with multiple DGCs and information about some of them have been pouring in over the past decade. This review summarizes the DGCs confirmed till date in V. cholerae, and emphasizes the importance of DGCs and their product, c-di-GMP in the virulence and lifecycle of the bacteria.

N-terminal truncation of VC0395_0300 protein from Vibrio cholerae does not lead to loss of diguanylate cyclase activity

The bacterial secondary messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

A non-flagellated, predatory soil bacterium reprograms a chemosensory system to control antifungal antibiotic production via cyclic di-GMP signalling.

Lysobacter enzymogenes is a non-flagellated, soil proteobacterium that secretes a diffusible antibiotic known as heat-stable antifungal factor (HSAF) to kill nearby fungi for food. The genome of the model strain OH11 encodes a homologous Wsp system, which is generally deployed by flagellated bacteria to achieve flagella-dependent outputs via a c-di-GMP-FleQ complex, in which c-di-GMP is a ubiquitous dinucleotide second messenger and FleQ is a transcription factor (TF). Here, we show that the Wsp system in the non-flagellated OH11 participates in a unique c-di-GMP-dependent signalling pathway and forms a WspR-CdgL binary complex to alter HSAF production, in which WspR and CdgL act as a c-di-GMP diguanylate cyclase (DGC) and a non-TF binding protein respectively. We found that the phosphorylation of WspR activates its DGC activity and enhances c-di-GMP production while inhibiting HSAF biosynthesis. The phosphorylation of WspR also plays a key role in weakening WspR-CdgL binding and HSAF generation. Interestingly, c-di-GMP binding to CdgL did not seem to induce the disassociation of the WspR-CdgL complex. These observations, along with our earlier findings, lead us to propose a model in which L. enzymogenes re-programs the Wsp system via c-di-GMP signalling to regulate HSAF biosynthesis for the benefit of ecological adaptation.

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR RpfR uniquely integrates two major signaling systems-quorum sensing and the motile-sessile switch mediated by cyclic-di-GMP-by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions.

The Expression of NOX From Synthetic Promoters Reveals an Important Role of the Redox Status in Regulating Secondary Metabolism of Saccharopolyspora erythraea.

Redox cofactors play a pivotal role in primary cellular metabolism, whereas the clear link between redox status and secondary metabolism is still vague. In this study we investigated effects of redox perturbation on the production of erythromycin in Saccharopolyspora erythraea by expressing the water-forming NADH oxidase (NOX) from Streptococcus pneumonia at different levels with synthetic promoters. The expression of NOX reduced the intracellular [NADH]/[NAD+] ratio significantly in S. erythraea which resulted in an increased production of erythromycin by 19∼29% and this increment rose to 60% as more oxygen was supplied. In contrast, the lower redox ratio resulted in a decreased production of another secondary metabolite, the reddish pigment 7-O-rahmnosyl flaviolin. The metabolic shifts of secondary metabolism results in a higher NADH availability which compensates for its oxidization via NOX. The expression of the erythromycin biosynthesis gene cluster (BGC) in the NOX-expression strains was upregulated as the activity of diguanylate cyclase was inhibited moderately by NADH. This study also suggested that lower intracellular [NADH]/[NAD+] ratio benefits the biosynthesis of erythromycin by potentially affecting the biosynthesis of the secondary messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), which may stimulate the positive regulation of erythromycin BGC via BldD. The present work provides a basis for future cofactor manipulation in S. erythraea to improve the industrial production of erythromycin.

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of “local” c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

Signal Sensing and Signal Transduction with Heme and Hemeproteins

The heme-based sensor proteins become a new family of hemeproteins that show a new biological function as the sensor for gas molecules such as O2, CO, and NO.These gas molecules can act as signaling molecules for the regulation of biological signal transduction systems. These regulatory systems consist of a sensor and regulatory domains/proteins, in which the sensor domain/protein senses the physiological gas molecule and then regulates the biological function of the regulatory domain/protein via intra- or inter-molecular signal transduction between the sensor and regulatory domains/proteins. The heme-based sensor domain/protein senses a gas molecule as its physiological effector by binding it to the heme in the sensor domain/protein. Though the heme-based sensor proteins adopt several different domains including H-NOX, globin, PAS, and GAF domains as the sensor domain in which the heme is accommodated, it is common among the heme-based sensors/proteins that gas-binding to the heme triggers the signal transduction process to regulate the regulatory domain/protein, which is initiated by a conformational change around the heme upon gas-binding. The interactions between the heme-bound ligand and surrounding amino acid residues will play an important role for distinguishing the physiological gas molecule among other heme-binding ligands and for the conformational change responsible for signal transduction. Thus, structural information of the sensor domain/protein is essential to elucidate the molecular mechanisms of gas-sensing and functional regulation by the heme-based sensor proteins.In this work, we have elucidated the structure-function relationships of several heme-based sensor proteins including HemAT, Aer2, HemDGC. While HemAT and Aer2 are signal transducer proteins in the aerotaxis regulatory systems, HemDGC is a heme-containing diguanylate cyclase that consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain.We have determined the X-ray crystal structures of Aer2-N384 (residues 1-384 that consists of three-HAMP, PAS, and di-HAMP) and Aer2-PH (residues 173-384 that consists of PAS and di-HAMP) to elucidate the mechanism by which intramolecular signal transduction proceeds between the HAMP and PAS domains in Aer2. Aer2-N384 is a homodimer having a non-crystallographic 2-fold symmetry. The three-unit poly-HAMP, PAS, and di-HAMP domains are ordered in linear configuration. A heme exists in a hydrophobic pocket in the PAS domain. His234 serves as the proximal ligand of the heme. The structural analyses of Aer2 suggests that the heme-bound O2 forms a hydrogen bond with Trp283. Trp283 is located on the C-terminal of the strand b5. This strand connects to the helix a5, which is the starting region of the C-terminal di-HAMP domain, suggesting that the hydrogen bond between Trp283 and O2 are responsible for intramolecular signal transduction.The enzymatic activity of HemDGC is regulated by the ligand binding to the heme. The enzymatic activity for the formation of c-di-GMP from GTP was measured for ferric, cyanomet, deoxy, oxy, CO-bound, and NO-bound HemDGC. Only oxy HemDGC showed the activity of diguanylate cyclase for the formation of c-di-GMP from GTP, but ferric, cyanomet, deoxy, CO-bound, and NO-bound HemDGC did not at all. These results indicate that O2 will be a physiological effector of HemDGC and will regulate the enzymatic activity of HemDGC via the formation of the oxy-complex of the heme. Thus, the globin domain of HemDGC acts as a sensor that can strictly discriminate O2 among external ligands capable to bind to the heme. HemDGC shows different hydrogen bonding patterns between the O2- and CO-bound forms. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O2, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O2 sensing responsible for the regulation of the enzymatic activity.

Screening for Diguanylate Cyclase (DGC) Inhibitors Mitigating Bacterial Biofilm Formation.

The majority of bacteria in the natural environment organize themselves into communal biofilms. Biofilm formation benefits bacteria conferring resistance to harmful molecules (e.g., antibiotics, disinfectants, and host immune factors) and coordinating their gene expression through quorum sensing (QS). A primary signaling molecule promoting bacterial biofilm formation is the universal second messenger cyclic di-GMP. This dinucleotide predominantly controls the gene expression of motility, adhesins, and capsule production to coordinate biofilm formation. Cyclic di-GMP is synthesized by diguanylate cyclases (DGCs) that have a GGDEF domain and is degraded by phosphodiesterases (PDEs) containing either an EAL or an HD-GYP domain. Since high cellular c-di-GMP concentrations are correlated with promoting the ability of bacteria to form biofilms, numerous research endeavors to identify chemicals capable of inhibiting the c-di-GMP synthesis activity of DGCs have been performed in order to inhibit bacterial biofilm formation. This review describes currently identified chemical inhibitors that disturb the activity of DGCs and the methods of screening and assay for their discovery.

The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa

Bacterial cyclic-di-GMP (c-di-GMP) production is associated with biofilm development and the switch from acute to chronic infections. In Pseudomonas aeruginosa, the diguanylate cyclase (DGC) SiaD and phosphatase SiaA, which are co-transcribed as part of a siaABCD operon, are essential for cellular aggregation. However, the detailed functions of this operon and the relationships among its constituent genes are unknown. Here, we demonstrate that the siaABCD operon encodes for a signaling network that regulates SiaD enzymatic activity to control biofilm and aggregates formation. Through protein-protein interaction, SiaC promotes SiaD diguanylate cyclase activity. Biochemical and structural data revealed that SiaB is an unusual protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD and regulate c-di-GMP levels and subsequent virulence phenotypes. Collectively, our data provide insights into the molecular mechanisms underlying the modulation of DGC activity associated with chronic infections, which may facilitate the development of antimicrobial drugs.

π-Helix controls activity of oxygen-sensing diguanylate cyclases.

The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

Azorhizobium caulinodans c-di-GMP phosphodiesterase Chp1 involved in motility, EPS production, and nodulation of the host plant.

Establishment of the rhizobia-legume symbiosis is usually accompanied by hydrogen peroxide (H2O2) production by the legume host at the site of infection, a process detrimental to rhizobia. In Azorhizobium caulinodans ORS571, deletion of chp1, a gene encoding c-di-GMP phosphodiesterase, led to increased resistance against H2O2 and to elevated nodulation efficiency on its legume host Sesbania rostrata. Three domains were identified in the Chp1: a PAS domain, a degenerate GGDEF domain, and an EAL domain. An in vitro enzymatic activity assay showed that the degenerate GGDEF domain of Chp1 did not have diguanylate cyclase activity. The phosphodiesterase activity of Chp1 was attributed to its EAL domain which could hydrolyse c-di-GMP into pGpG. The PAS domain functioned as a regulatory domain by sensing oxygen. Deletion of Chp1 resulted in increased intracellular c-di-GMP level, decreased motility, increased aggregation, and increased EPS (extracellular polysaccharide) production. H2O2-sensitivity assay showed that increased EPS production could provide ORS571 with resistance against H2O2. Thus, the elevated nodulation efficiency of the ∆chp1 mutant could be correlated with a protective role of EPS in the nodulation process. These data suggest that c-di-GMP may modulate the A. caulinodans-S. rostrata nodulation process by regulating the production of EPS which could protect rhizobia against H2O2.

Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating.

Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.

A tandem GGDEF-EAL domain protein-regulated c-di-GMP signal contributes to spoilage-related activities of Shewanella baltica OS155.

Cyclic diguanylate (c-di-GMP) is a second messenger involved in the regulation of various physiological processes in bacteria. However, its function in spoilage bacteria has not yet been addressed. Here, we studied the function of a tandem GGDEF-EAL domain protein, Sbal_3235, in the spoilage bacterium Shewanella baltica OS155. The deletion of sbal_3235 significantly reduced the c-di-GMP level, biofilm formation, and exopolysaccharide, trimethylamine (TMA), and putrescine production; sbal_3235 deletion also downregulated the expression of the torS and speF genes and affected membrane fatty acid composition. Site-directed mutagenesis in conserved GGDEF and EAL motifs abolished diguanylate cyclase (DGC) and phosphodiesterase (PDE) activity, respectively. These data indicate that Sbal_3235 is an essential contributor to the c-di-GMP pool with bifunctional DGC and PDE activity, which is involved in the biofilm formation and spoilage activity of S. baltica OS155. Our findings expand the biochemical role of c-di-GMP and uncover its link to spoilage activities, providing novel targets for food quality and safety controlling.

A fluorescence-based high-throughput screening method for determining the activity of diguanylate cyclases and c-di-GMP phosphodiesterases.

The dinucleotide 3′,5′-cyclic diguanylic acid (c-di-GMP) is a critical second messenger found in bacteria. High cellular levels of c-di-GMP are associated with a sessile, biofilm lifestyle in many bacteria, which is associated with more than 70% of clinically resistant infections. Cellular c-di-GMP concentrations are regulated by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which are responsible for the production and degradation, respectively, of c-di-GMP. Therefore, DGCs and PDEs might be attractive drug targets for controlling biofilm formation. In this study, a simple and universal high-throughput method based on a c-di-GMP-specific fluorescent probe for the determination of DGC and PDE activity was described. By using the proposed method, the c-di-GMP content in samples was rapidly quantified by measuring the fluorescence intensity in a 96-well plate by using a microplate reader. In addition, the probe molecule A18 directly interacted with the substrate c-di-GMP, and the method was not limited by the structure of enzymes.

Structure of the active GGEEF domain of a diguanylate cyclase from Vibrio cholerae

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.

A crosstalk between c-di-GMP and cAMP in regulating transcription of GcsA, a diguanylate cyclase involved in swimming motility in Pseudomonas putida.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis

Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5–9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were KmGTP 84 ± 21 μM and kcat 1.2 min−1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min−1, 0.95 min−1, and 0.3 min−1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min−1, 0.26 min−1 and 0.54 min−1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.

Bordetella bronchiseptica Diguanylate Cyclase BdcA Regulates Motility and Is Important for the Establishment of Respiratory Infection in Mice.

Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.

The Response of Cupriavidus metallidurans CH34 to Cadmium Involves Inhibition of the Initiation of Biofilm Formation, Decrease in Intracellular c-di-GMP Levels, and a Novel Metal Regulated Phosphodiesterase.

Cadmium is a highly toxic heavy metal for biological systems. Cupriavidus metallidurans CH34 is a model strain to study heavy metal resistance and bioremediation as it is able to deal with high heavy metal concentrations. Biofilm formation by bacteria is mediated by the second messenger bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). The aim of this study was to characterize the response of C. metallidurans CH34 planktonic and biofilm cells to cadmium including their c-di-GMP regulatory pathway. Inhibition of the initiation of biofilm formation and EPS production by C. metallidurans CH34 correlates with increased concentration of cadmium. Planktonic and biofilm cells showed similar tolerance to cadmium. During exposure to cadmium an acute decrease of c-di-GMP levels in planktonic and biofilm cells was observed. Transcription analysis by RT-qPCR showed that cadmium exposure to planktonic and biofilm cells induced the expression of the urf2 gene and the mercuric reductase encoding merA gene, which belong to the Tn501/Tn21 mer operon. After exposure to cadmium, the cadA gene involved in cadmium resistance was equally upregulated in both lifestyles. Bioinformatic analysis and complementation assays indicated that the protein encoded by the urf2 gene is a functional phosphodiesterase (PDE) involved in the c-di-GMP metabolism. We propose to rename the urf2 gene as mrp gene for metal regulated PDE. An increase of the second messenger c-di-GMP content by the heterologous expression of the constitutively active diguanylate cyclase PleD correlated with an increase in biofilm formation and cadmium susceptibility. These results indicate that the response to cadmium in C. metallidurans CH34 inhibits the initiation of biofilm lifestyle and involves a decrease in c-di-GMP levels and a novel metal regulated PDE.