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.     

The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase

The newly recognized bacterial second messenger 3',5'-cyclic diguanylic acid (cyclic diguanylate (c-di-GMP)) has been shown to regulate a wide variety of bacterial behaviors and traits. Biosynthesis and degradation of c-di-GMP have been attributed to the GGDEF and EAL protein domains, respectively, based primarily on genetic evidence. Whereas the GGDEF domain was demonstrated to possess diguanylate cyclase activity in vitro, the EAL domain has not been tested directly for c-di-GMP phosphodiesterase activity. This study describes the analysis of c-di-GMP hydrolysis by an EAL domain protein in a purified system. The Vibrio cholerae EAL domain protein VieA has been shown to inversely regulate biofilm-specific genes (vps) and virulence genes (ctxA), presumably by decreasing the cellular pool of c-di-GMP. VieA was maximally active at neutral pH, physiological ionic strength, and ambient temperatures and demonstrated c-di-GMP hydrolytic activity with a Km of 0.06 microM. VieA was unable to hydrolyze cGMP. The putative metal coordination site of the EAL domain, Glu170, was demonstrated to be necessary for VieA activity. Furthermore, the divalent cations Mg2+ and Mn2+ were necessary for VieA activity; conversely, Ca2+ and Zn2+ were potent inhibitors of the VieA phosphodiesterase. Calcium inhibition of the VieA EAL domain provides a potential mechanism for regulation of c-di-GMP degradation.     

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.     

The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3'-5')-cyclic dimeric GMP synthesis

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.     

Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.     

Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production

Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.     

环二鸟苷酸——新型的细菌第二信使

环二鸟苷酸(cyclic diguanylate,c-di-GMP)是新近发现的在细菌中普遍存在的第二信使分子,参与调节多种生理功能,包括细胞分化、从运动状态到生物被膜状态的转变、致病因子产生等.基于其对细菌抗生素耐药的物理屏障—生物被膜形成的影响,c-di-GMP的研究越来越受到人们的关注.细胞内c-di-GMP的产生受二鸟苷酸环化酶(diguanylate cyclase,DGC)合成和磷酸二酯酶(phosphodiesterase,PDE)降解两条途径调控.在结构上,通常DGC含有GGDEF结构域,PDE含有EAL结构域.c-di-GMP的作用靶点包括PilZ结构域和GEMM 核开关两种类型.本文综述了c-di-GMP的代谢途径、调控机理、生物学功能等方面的最新研究进展,并对c-di-GMP在今后研究中的应用和发展趋势进行展望.     

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.     

Indirect modulation of the intracellular c-Di-GMP level in Shewanella oneidensis MR-1 by MxdA

The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.     

'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.     

An unorthodox bacteriophytochrome from Rhodobacter sphaeroides involved in turnover of the second messenger c-di-GMP

Bacteriophytochromes are bacterial photoreceptors that sense red/far red light using the biliverdin chromophore. Most bacteriophytochromes work as photoactivated protein kinases. The Rhodobacter sphaeroides bacteriophytochrome BphG1 is unconventional in that it has GGDEF and EAL output domains, which are involved, respectively, in synthesis (diguanylate cyclase) and degradation (phosphodiesterase) of the bacterial second messenger c-di-GMP. The GGDEF-EAL proteins studied to date displayed either diguanylate cyclase or phosphodiesterase activity but not both. To elucidate the function of BphG1, the holoprotein was purified from an Escherichia coli overexpression system designed to produce biliverdin. The holoprotein contained covalently bound biliverdin and interconverted between the red (dark) and far red (light-activated) forms. BphG1 had c-di-GMP-specific phosphodiesterase activity. Unexpectedly for a photochromic protein, this activity was essentially light-independent. BphG1 expressed in E. coli was found to undergo partial cleavage into two species. The smaller species was identified as the EAL domain of BphG1. It possessed c-di-GMP phosphodiesterase activity. Surprisingly, the larger species lacking EAL possessed diguanylate cyclase activity, which was dependent on biliverdin and strongly activated by light. BphG1 therefore is the first phytochrome with a non-kinase photoactivated enzymatic activity. This shows that the photosensory modules of phytochromes can transmit light signals to various outputs. BphG1 is potentially the first "bifunctional" enzyme capable of both c-di-GMP synthesis and hydrolysis. A model for the regulation of the "opposite" activities of BphG1 is presented.     

Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.     

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.     

Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa

Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a DeltafimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP-FimXDeltaREC, unlike RFP-FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although DeltafimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia.     

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.