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.     

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.     

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.     

H‐NOX regulation of c‐di‐GMP metabolism and biofilm formation in Legionella pneumophila

Haem Nitric oxide/OXygen (H‐NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H‐NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H‐NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper‐biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF‐EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well‐studied diguanylate cyclase, vca0956, results in a hyper‐biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)‐NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper‐biofilm phenotype back to wild‐type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c‐di‐GMP production by lpg1057 and biofilm formation in response to NO.     

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.     

The c‐di‐GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c‐di‐GMP) phosphodiesterase‐encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free‐living and virulence‐related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour‐inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real‐time monitoring of olive plants infected with green fluorescent protein (GFP)‐tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild‐type strain. All free‐living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c‐di‐GMP signalling in the virulence of this group of plant pathogens.     

Cyclic‐di‐GMP and cyclic‐di‐AMP activate the NLRP3 inflammasome

The cyclic dinucleotides 3'‐5'diadenylate (c‐diAMP) and 3'‐5' diguanylate (c‐diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN‐Is) through the c‐diGMP‐binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL‐1β through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c‐diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen‐associated molecular patterns associated with intracellular infections.     

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.     

Molecular dynamics simulation on the allosteric analysis of the c‐di‐GMP class I riboswitch induced by ligand binding

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand‐bound aptamer complex structures have been solved, it is important to know ligand‐free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand‐free and ligand‐bound aptamer domain of the c‐di‐GMP class I (GEMM‐I) riboswitch. The results revealed that the ligand‐free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c‐di‐GMP binding. The P1 helix forms when c‐di‐GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM‐I riboswitch contributing to the formation of P1 helix have been found. The binding of the c‐di‐GMP ligand to the GEMM‐I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM‐I riboswitch from the c‐di‐GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.     

Surface sensing and lateral subcellular localization of WspA,the receptor in a chemosensory‐like system leading to c‐di‐GMP production

Pseudomonas aeruginosa responds to growth on agar surfaces to produce cyclic‐di‐GMP, which stimulates biofilm formation. This is mediated by an alternative cellular function chemotaxis‐like system called Wsp. The receptor protein WspA, is bioinformatically indistinguishable from methyl‐accepting chemotaxis proteins. However, unlike standard chemoreceptors, WspA does not form stable clusters at cell poles. Rather, it forms dynamic clusters at both polar and lateral subcellular locations. To begin to study the mechanism of Wsp signal transduction in response to surfaces, we carried out a structure–function study of WspA and found that its C‐terminus is important for its lateral subcellular localization and function. When this region was replaced with that of a chemoreceptor for amino acids, WspA became polarly localized. In addition, introduction of mutations in the C‐terminal region of WspA that rendered this protein able to form more stable receptor–receptor interactions, also resulted in a WspA protein that was less capable of activating signal transduction. Receptor chimeras with a WspA C‐terminus and N‐terminal periplasmic domains from chemoreceptors that sense amino acids or malate responded to surfaces to produce c‐di‐GMP. Thus, the amino acid sequence of the WspA periplasmic region did not need to be conserved for the Wsp system to respond to surfaces.     

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.     

The phytopathogen Dickeya dadantii 3937 cpxR locus gene participates in the regulation of virulence and the global c‐di‐GMP network

Bacteria use signal transduction systems to sense and respond to their external environment. The two‐component system CpxA/CpxR senses misfolded envelope protein stress and responds by up‐regulating envelope protein factors and down‐regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS‐inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c‐di‐GMP regulators were also up‐regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c‐di‐GMP phenotypes in biofilm formation and swimming. Increased production of cellular c‐di‐GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c‐di‐GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.     

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.     

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c‐di‐GMP production by the DgcQ protein of Escherichia coli

Production of cellulose, a stress response‐mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c‐di‐GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N‐carbamoyl‐aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N‐carbamoyl‐aspartate appears to be favoured by protein‐protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N‐carbamoyl‐aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.