Crystal structure of an HD‐GYP domain cyclic‐di‐GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Bis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.     

GIL,a new c‐di‐GMP‐binding protein domain involved in regulation of cellulose synthesis in enterobacteria

In contrast to numerous enzymes involved in c‐di‐GMP synthesis and degradation in enterobacteria, only a handful of c‐di‐GMP receptors/effectors have been identified. In search of new c‐di‐GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope‐labelled c‐di‐GMP. We uncovered three new candidate c‐di‐GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c‐di‐GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, G GDEF I ‐site l ike domain. The RxGD motif of the GIL domain is required for c‐di‐GMP binding, similar to the c‐di‐GMP‐binding I‐site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c‐di‐GMP‐binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c‐di‐GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two‐tiered c‐di‐GMP‐dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.     

A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis

Cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide‐binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di‐GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo‐complex, demonstrated the site of cyclic GMP binding that modulates cyclic di‐GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di‐GMP signalling.     

The response threshold of Salmonella PilZ domain proteins is determined by their binding affinities for c‐di‐GMP

c‐di‐GMP is a bacterial second messenger that is enzymatically synthesized and degraded in response to environmental signals. Cellular processes are affected when c‐di‐GMP binds to receptors which include proteins that contain the PilZ domain. Although each c‐di‐GMP synthesis or degradation enzyme metabolizes the same molecule, many of these enzymes can be linked to specific downstream processes. Here we present evidence that c‐di‐GMP signalling specificity is achieved through differences in affinities of receptor macromolecules. We show that the PilZ domain proteins of Salmonella Typhimurium, YcgR and BcsA, demonstrate a 43‐fold difference in their affinity for c‐di‐GMP. Modulation of the affinities of these proteins altered their activities in a predictable manner in vivo. Inactivation of yhjH, which encodes a predicted c‐di‐GMP degrading enzyme, increased the fraction of the cellular population that demonstrated c‐di‐GMP levels high enough to bind to the higher‐affinity YcgR protein and inhibit motility, but not high enough to bind to the lower‐affinity BcsA protein and stimulate cellulose production. Finally, PilZ domain proteins of Pseudomonas aeruginosa demonstrated a 145‐fold difference in binding affinities, suggesting that regulation by binding affinity may be a conserved mechanism that allows organisms with many c‐di‐GMP binding macromolecules to rapidly integrate multiple environmental signals into one output.     

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c‐di‐GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme‐substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c‐di‐GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c‐di‐GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c‐di‐GMP to promote the O3'? P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670–1680. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the Legionella pneumophila lem10 effector reveals a new member of the HD protein superfamily

Legionella pneumophila, the intracellular pathogen that can cause severe pneumonia known as Legionnaire's disease, translocates close to 300 effectors inside the host cell using Dot/Icm type IVB secretion system. The structure and function for the majority of these effector proteins remains unknown. Here, we present the crystal structure of the L. pneumophila effector Lem10. The structure reveals a multidomain organization with the largest C‐terminal domain showing strong structural similarity to the HD protein superfamily representatives. However, Lem10 lacks the catalytic His‐Asp residue pair and does not show any in vitro phosphohydrolase enzymatic activity, typical for HD proteins. While the biological function of Lem10 remains elusive, our analysis shows that similar distinct features are shared by a significant number of HD domains found in Legionella proteins, including the SidE family of effectors known to play an important role during infection. Taken together our data point to the presence of a specific group of non‐catalytic Legionella HD domains, dubbed LHDs, which are involved in pathogenesis. Proteins 2015; 83:2319–2325. © 2015 Wiley Periodicals, Inc.     

XC1028 from Xanthomonas campestris adopts a PilZ domain‐like structure without a c‐di‐GMP switch

The crystal structure of XC1028 from Xanthomonas campestris has been determined to a resolution of 2.15 Å using the multiple anomalous dispersion approach. It bears significant sequence identity and similarity values of 64.10% and 70.09%, respectively, with PA2960, a protein indispensable for type IV pilus‐mediated twitching motility, after which the PilZ motif was first named. However, both XC1028 and PA2960 lack detectable c‐di‐GMP binding capability. Although XC1028 adopts a structure comprising a five‐stranded β‐barrel core similar to other canonical PilZ domains with robust c‐di‐GMP binding ability, considerable differences are observed in the N‐terminal motif; XC1028 assumes a compact five‐stranded β‐barrel without an extra long N‐terminal motif, whereas other canonical PilZ domains contain a long N‐terminal sequence embedded with an essential “c‐di‐GMP switch” motif. In addition, a β‐strand (β1) in the N‐terminal motif, running in exactly opposite polarity to that of XC1028, is found inserted into the parallel β3/β1′ strands, forming a completely antiparallel β4↓β3↑β1↓β1′↑ sheet in the canonical PilZ domains. Such dramatic structural differences at the N‐terminus may account for the diminished c‐di‐GMP binding capability of XC1028, and suggest that interactions with additional proteins are necessary to bind c‐di‐GMP for type IV fimbriae assembly. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A post‐translational,c‐di‐GMP‐dependent mechanism regulating flagellar motility

Elevated levels of the second messenger cyclic dimeric GMP, c‐di‐GMP, promote transition of bacteria from single motile cells to surface‐attached multicellular communities. Here we describe a post‐translational mechanism by which c‐di‐GMP initiates this transition in enteric bacteria. High levels of c‐di‐GMP induce the counterclockwise bias in Escherichia coli flagellar rotation, which results in smooth swimming. Based on co‐immunoprecipitation, two‐hybrid and mutational analyses, the E. coli c‐di‐GMP receptor YcgR binds to the FliG subunit of the flagellum switch complex, and the YcgR–FliG interaction is strengthened by c‐di‐GMP. The central fragment of FliG binds to YcgR as well as to FliM, suggesting that YcgR–c‐di‐GMP biases flagellum rotation by altering FliG‐FliM interactions. The c‐di‐GMP‐induced smooth swimming promotes trapping of motile bacteria in semi‐solid media and attachment of liquid‐grown bacteria to solid surfaces, whereas c‐di‐GMP‐dependent mechanisms not involving YcgR further facilitate surface attachment. The YcgR–FliG interaction is conserved in the enteric bacteria, and the N‐terminal YcgR/PilZN domain of YcgR is required for this interaction. YcgR joins a growing list of proteins that regulate motility via the FliG subunit of the flagellum switch complex, which suggests that FliG is a common regulatory entryway that operates in parallel with the chemotaxis that utilizes the FliM‐entryway.     

Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii

Cyclic diguanylate (c‐di‐GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c‐di‐GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c‐di‐GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c‐di‐GMP, RpoN and T3SS and the potential role of c‐di‐GMP in T3SS regulation among a wide range of bacterial pathogens.     

Activation and polar sequestration of PopA,a c‐di‐GMP effector protein involved in Caulobacter crescentus cell cycle control

When Caulobacter crescentus enters S‐phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co‐option as c‐di‐GMP effector protein. While the C‐terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N‐terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N‐terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c‐di‐GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S‐phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.     

Beyond antibiotic resistance: integrating conjugative elements of the SXT/R391 family that encode novel diguanylate cyclases participate to c‐di‐GMP signalling in Vibrio cholerae

In Vibrio cholerae, the second messenger bis‐(3′?5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) increases exopolysaccharides production and biofilm formation and decreases virulence and motility. As such, c‐di‐GMP is considered an important player in the transition from the host to persistence in the environment. c‐di‐GMP level is regulated through a complex network of more than 60 chromosomal genes encoding predicted diguanylate cyclases (DGCs) and phosphodiesterases. Herein we report the characterization of two additional DGCs, DgcK and DgcL, encoded by integrating conjugative elements (ICEs) belonging to the SXT/R391 family. SXT/R391 ICEs are self‐transmissible mobile elements that are widespread among vibrios and several species of enterobacteria. We found that deletion of dgcL increases the motility of V. cholerae, that overexpression of DgcK or DgcL modulates gene expression, biofilm formation and bacterial motility, and that a single amino acid change in the active site of either enzyme abolishes these phenotypes. We also show that DgcK and DgcL are able to synthesize c‐di‐GMP in vitro from GTP. DgcK was found to co‐purify with non‐covalently bound flavin mononucleotide (FMN). DgcL's enzymatic activity was augmented upon phosphorylation of its phosphorylatable response‐regulator domain suggesting that DgcL is part of a two‐component signal transduction system. Interestingly, we found orthologues of dgcK and dgcL in several SXT/R391 ICEs from two species of Vibrio originating from Asia, Africa and Central America. We propose that besides conferring usual antibiotic resistances, dgcKL‐bearing SXT/R391 ICEs could enhance the survival of vibrios in aquatic environments by increasing c‐di‐GMP level.     

Transmembrane redox control and proteolysis of PdeC,a novel type of c‐di‐GMP phosphodiesterase

The nucleotide second messenger c‐di‐GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c‐di‐GMP being controlled by diverse N‐terminal sensor domains. Here, we describe a novel class of widely occurring c‐di‐GMP phosphodiesterases (PDE) that feature a periplasmic “CSS domain” with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K‐12, we show that DsbA/DsbB‐promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.     

Elevated levels of the second messenger c‐di‐GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa

Biofilms are highly structured, surface‐associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that ΔsagS biofilm cells harbour the secondary messenger c‐di‐GMP at reduced levels similar to those observed in wild‐type cells grown planktonically rather than as biofilms. Restoring c‐di‐GMP levels to wild‐type biofilm‐like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of ΔsagS biofilm cells. We likewise found that increasing c‐di‐GMP levels present in planktonic cells to biofilm‐like levels (≥ 55 pmol mg^?1) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c‐di‐GMP levels of biofilm cells to ≤ 40 pmol mg^?1 correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signalling pathway involving a specific c‐di‐GMP pool regulated by SagS contributes to the resistance of P. aeruginosa biofilms.     

Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile‐to‐sessile switch

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a u nip olar p olysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c‐di‐GMP) lead to surface‐contact‐independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP‐negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c‐di‐GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c‐di‐GMP phosphodiesterase also elevate UPP production and attachment, consistent with c‐di‐GMP activation of surface‐dependent adhesin deployment.     

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram‐negatives, increased levels of the second messenger cyclic diguanylate (c‐di‐GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c‐di‐GMP include cell division, differentiation and virulence. Among Gram‐positive bacteria, where the function of c‐di‐GMP signalling is less well characterized, c‐di‐GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c‐di‐GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c‐di‐GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c‐di‐GMP signalling between B. subtilis and B. cereus group bacteria.     

Crystal structure of master biofilm regulator CsgD regulatory domain reveals an atypical receiver domain

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C‐terminal DNA binding domain and an N‐terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical β5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non‐canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c‐di‐GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.