High levels of cyclic‐di‐GMP in plant‐associated Pseudomonas correlate with evasion of plant immunity

The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.     

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.     

Listeria monocytogenes exopolysaccharide: origin,structure, biosynthetic machinery and c‐di‐GMP‐dependent regulation

Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production.     

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.     

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.     

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.     

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.     

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.     

Diguanylate cyclase NicD‐based signalling mechanism of nutrient‐induced dispersion by Pseudomonas aeruginosa

Dispersion enables the transition from the biofilm to the planktonic growth state in response to various cues. While several Pseudomonas aeruginosa proteins, including BdlA and the c‐di‐GMP phosphodiesterases DipA, RbdA, and NbdA, have been shown to be required for dispersion to occur, little is known about dispersion cue sensing and the signalling translating these cues into the modulation c‐di‐GMP levels to enable dispersion. Using glutamate‐induced dispersion as a model, we report that dispersion‐inducing nutrient cues are sensed via an outside‐in signalling mechanism by the diguanylate cyclase NicD belonging to a family of seven transmembrane (7TM) receptors. NicD directly interacts with BdlA and the phosphodiesterase DipA, with NicD, BdlA, and DipA being part of the same pathway required for dispersion. Glutamate sensing by NicD results in NicD dephosphorylation and increased cyclase activity. Active NicD contributes to the non‐processive proteolysis and activation of BdlA via phosphorylation and temporarily elevated c‐di‐GMP levels. BdlA, in turn, activates DipA, resulting in the overall reduction of c‐di‐GMP levels. Our results provide a basis for understanding the signalling mechanism based on NicD to induce biofilm dispersion that may be applicable to various biofilm‐forming species and may have implications for the control of biofilm‐related infections.     

Pseudomonas aeruginosa uses a cyclic‐di‐GMP‐regulated adhesin to reinforce the biofilm extracellular matrix

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic‐resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic‐di‐GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c‐di‐GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two‐partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod‐shaped protein harbouring a β‐helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto‐aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co‐immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl‐specific lectin staining suggests that CdrA either cross‐links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.     

Cyclic di‐GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization.     

De‐ubiquitinases on the move: an emerging field in plant biology

A balance between the synthesis and degradation of active proteins governs diverse cellular processes in plants, spanning from cell‐cycle progression and circadian rhythm to the outcome of several hormone signalling pathways. Ubiquitin‐mediated post‐translational modification determines the degradative fate of the target proteins, thereby altering the output of cellular processes. An equally important, and perhaps under‐appreciated, aspect of this pathway is the antagonistic process of de‐ubiquitination. De‐ubiquitinases (DUBs), a group of processing enzymes, play an important role in maintaining cellular ubiquitin homeostasis by hydrolyzing ubiquitin poly‐proteins and free poly‐ubiquitin chains into mono‐ubiquitin. Further, DUBs rescue the cellular proteins from 26S proteasome‐mediated degradation to their active form by cleaving the poly‐ubiquitin chain from the target protein. Any perturbation in DUB activity is likely to affect proteostasis and downstream cellular processes. This review illustrates recent findings on the biological significance and mechanisms of action of the DUBs in Arabidopsis thaliana, with an emphasis on ubiquitin‐specific proteases (UBPs), the largest family among the DUBs. We focus on the putative roles of various protein–protein interaction interfaces in DUBs and their generalized function in ubiquitin recycling, along with their pre‐eminent role in plant development.     

Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility

The c‐di‐GMP‐binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c‐di‐GMP‐bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re‐annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ‐mediated downregulation of motility is fine‐tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c‐di‐GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c‐di‐GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c‐di‐GMP‐dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella‐driven swimming and swarming motility.