Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain

Proteins containing GGDEF domains are encoded in the majority of sequenced bacterial genomes. In several species, these proteins have been implicated in biosynthesis of exopolysaccharides, formation of biofilms, establishment of a sessile lifestyle, surface motility, and regulation of gene expression. However, biochemical activities of only a few GGDEF domain proteins have been tested. These proteins were shown to be involved in either synthesis or hydrolysis of cyclic-bis(3'-->5') dimeric GMP (c-di-GMP) or in hydrolysis of cyclic AMP. To investigate specificity of the GGDEF domains in Bacteria, six GGDEF domain-encoding genes from randomly chosen representatives of diverse branches of the bacterial phylogenetic tree, i.e., Thermotoga, Deinococcus-Thermus, Cyanobacteria, spirochetes, and alpha and gamma divisions of the Proteobacteria, were cloned and overexpressed. All recombinant proteins were purified and found to possess diguanylate cyclase (DGC) activity involved in c-di-GMP synthesis. The individual GGDEF domains from two proteins were overexpressed, purified, and shown to possess a low level of DGC activity. The oligomeric states of full-length proteins and individual GGDEF domains were similar. This suggests that GGDEF domains are sufficient to encode DGC activity; however, enzymatic activity is highly regulated by the adjacent sensory protein domains. It is shown that DGC activity of the GGDEF domain protein Rrp1 from Borrelia burgdorferi is strictly dependent on phosphorylation status of its input receiver domain. This study establishes that majority of GGDEF domain proteins are c-di-GMP specific, that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria, and that it is highly regulated.     

GGDEF domain is homologous to adenylyl cyclase

The GGDEF domain is detected in many prokaryotic proteins, most of which are of unknown function. Several bacteria carry 12-22 different GGDEF homologues in their genomes. Conducting extensive profile-based searches, we detect statistically supported sequence similarity between GGDEF domain and adenylyl cyclase catalytic domain. From this homology, we deduce that the prokaryotic GGDEF domain is a regulatory enzyme involved in nucleotide cyclization, with the fold similar to that of the eukaryotic cyclase catalytic domain. This prediction correlates with the functional information available on two GGDEF-containing proteins, namely diguanylate cyclase and phosphodiesterase A of Acetobacter xylinum, both of which regulate the turnover of cyclic diguanosine monophosphate. Domain architecture analysis shows that GGDEF is typically present in multidomain proteins containing regulatory domains of signaling pathways or protein-protein interaction modules. Evolutionary tree analysis indicates that GGDEF/cyclase superfamily forms a large diversified cluster of orthologous proteins present in bacteria, archaea, and eukaryotes. Proteins 2001;42:210-216.     

Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP

Cyclic diguanylic acid (c-di-GMP) is a global second messenger controlling motility and adhesion in bacterial cells. Synthesis and degradation of c-di-GMP is catalyzed by diguanylate cyclases (DGC) and c-di-GMP-specific phosphodiesterases (PDE), respectively. Whereas the DGC activity has recently been assigned to the widespread GGDEF domain, the enzymatic activity responsible for c-di-GMP cleavage has been associated with proteins containing an EAL domain. Here we show biochemically that CC3396, a GGDEF-EAL composite protein from Caulobacter crescentus is a soluble PDE. The PDE activity, which rapidly converts c-di-GMP into the linear dinucleotide pGpG, is confined to the C-terminal EAL domain of CC3396, depends on the presence of Mg2+ ions, and is strongly inhibited by Ca2+ ions. Remarkably, the associated GGDEF domain, which contains an altered active site motif (GEDEF), lacks detectable DGC activity. Instead, this domain is able to bind GTP and in response activates the PDE activity in the neighboring EAL domain. PDE activation is specific for GTP (K(D) 4 microM) and operates by lowering the K(m) for c-di-GMP of the EAL domain to a physiologically significant level (420 nM). Mutational analysis suggested that the substrate-binding site (A-site) of the GGDEF domain is involved in the GTP-dependent regulatory function, arguing that a catalytically inactive GGDEF domain has retained the ability to bind GTP and in response can activate the neighboring EAL domain. Based on this we propose that the c-di-GMP-specific PDE activity is confined to the EAL domain, that GGDEF domains can either catalyze the formation of c-di-GMP or can serve as regulatory domains, and that c-di-GMP-specific phosphodiesterase activity is coupled to the cellular GTP level in bacteria.     

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.     

MHYT, a new integral membrane sensor domain

MHYT, a new conserved protein domain with a likely signaling function, is described. This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098. In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins. In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein. A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.     

Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity

Cyclic-di-GMP is a bacterial second messenger that controls the switch between motile and sessile states. It is synthesized by proteins containing the enzymatic GGDEF domain and degraded by the EAL domain. Many bacterial genomes encode several copies of proteins containing these domains, raising questions on how the activities of parallel c-di-GMP signalling systems are segregated to avoid potentially deleterious cross-talk. Moreover, many ‘hybrid’ proteins contain both GGDEF and EAL domains; the relationship between the two apparently opposing enzymatic activities has been termed a ‘biochemical conundrum’. Here, we present a computational analysis of 11 248 GGDEF- and EAL-containing proteins in 867 prokaryotic genomes to address these two outstanding questions. Over half of these proteins contain a signal for cell-surface localization, and a majority accommodate a signal-sensing partner domain; these indicate widespread prevalence of post-translational regulation that may segregate the activities of proteins that are co-expressed. By examining the conservation of amino acid residues in the GGDEF and EAL catalytic sites, we show that there are predominantly two types of hybrid proteins. In the first, both sites are intact; an additional regulatory partner domain, present in most of these proteins, might determine the balance between the two enzymatic activities. In the second type, only the EAL catalytic site is intact; these—unlike EAL-only proteins—generally contain a signal-sensing partner domain, suggesting distinct modes of regulation for EAL activity under different sequence contexts. Finally, we discuss the role of proteins that have lost GGDEF and EAL catalytic sites as potential c-di-GMP-binding effectors. Our findings will serve as a genomic framework for interpreting ongoing molecular investigations of these proteins.     

GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility

Cyclic nucleotides represent second messenger molecules in all kingdoms of life. In bacteria, mass sequencing of genomes detected the highly abundant protein domains GGDEF and EAL. We show here that the GGDEF and EAL domains are involved in the turnover of cyclic-di-GMP (c-di-GMP) in vivo whereby the GGDEF domain stimulates c-di-GMP production and the EAL domain c-di-GMP degradation. Thus, most probably, GGDEF domains function as c-di-GMP cyclase and EAL domains as phosphdiesterase. We further show that, in the pathogenic organism Salmonella enterica serovar Typhimurium, the nosocomial pathogen Pseudomonas aeruginosa and the commensal species Escherichia coli, GGDEF and EAL domains mediate similar phenotypic changes related to the transition between sessility and motility. Thus, the data suggest that c-di-GMP is a novel global second messenger in bacteria the metabolism of which is controlled by GGDEF and EAL domain proteins.     

The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.     

Allosteric control of cyclic di-GMP signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.     

The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg(2+) or Mn(2+), strongly inhibited by Ca(2+), and independent of protein oligomerization. Linear dimeric GMP was shown to be 5'pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5'pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.     

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.     

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis-(3',5') cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient-deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB-G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di-GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.     

Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-Di-GMP phosphodiesterase with a putative hypoxia-sensing domain

Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.     

YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity

The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K_m) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.     

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.     

Identification of the YfgF MASE1 domain as a modulator of bacterial responses to aspartate

Complex 3′-5′-cyclic diguanylic acid (c-di-GMP) responsive regulatory networks that are modulated by the action of multiple diguanylate cyclases (DGC; GGDEF domain proteins) and phosphodiesterases (PDE; EAL domain proteins) have evolved in many bacteria. YfgF proteins possess a membrane-anchoring domain (MASE1), a catalytically inactive GGDEF domain and a catalytically active EAL domain. Here, sustained expression of the Salmonella enterica spp. Enterica ser. Enteritidis YfgF protein is shown to mediate inhibition of the formation of the aspartate chemotactic ring on motility agar under aerobic conditions. This phenomenon was c-di-GMP-independent because it occurred in a Salmonella strain that lacked the ability to synthesize c-di-GMP and also when PDE activity was abolished by site-directed mutagenesis of the EAL domain. YfgF-mediated inhibition of aspartate chemotactic ring formation was impaired in the altered redox environment generated by exogenous p-benzoquinone. This ability of YfgF to inhibit the response to aspartate required a motif, ²¹³Lys-Lys-Glu²¹⁵, in the predicted cytoplasmic loop between trans-membrane regions 5 and 6 of the MASE1 domain. Thus, for the first time the function of a MASE1 domain as a redox-responsive regulator of bacterial responses to aspartate has been shown.     

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.     

Diversity and distribution of hemerythrin-like proteins in prokaryotes

Hemerythrins are oxygen-binding proteins found in the body fluids and tissues of certain invertebrates. Oxygen is bound at a nonheme iron centre consisting of two oxo-bridged iron atoms bound to a characteristic set of conserved histidine: aspartate and glutamate residues with the motifs H-HxxxE-HxxxH-HxxxxD. It has recently been demonstrated biochemically that two bacterial proteins bearing the same motifs do in fact possess similar iron centres and bind oxygen in the same way. The recent profusion of prokaryotic genomic sequence data has shown that proteins bearing hemerythrin motifs are present in a wide variety of bacteria, and a few archaea. Some of these are short proteins as in eukaryotes; others appear to consist of a hemerythrin domain fused to another domain, generally a putative signal transduction domain such as a methyl-accepting chemotaxis protein, a histidine kinase, or a GGDEF protein (cyclic di-GMP synthase). If, as initial evidence suggests, these are in fact hemerythrin-like oxygen-binding proteins, then their diversity in prokaryotes far exceeds that seen in eukaryotes. Here, a survey is presented of prokaryotic protein sequences bearing hemerythrin-like motifs, for which the designation 'bacteriohemerythrins' is proposed, and their functions are speculated.