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.     

Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic‐di‐guanosine monophosphate in motility and virulence

The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic‐dimeric guanosine monophosphate (cyclic‐di‐GMP) metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic‐di‐GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic‐di‐GMP with a K_m of 0.054 µM, confirming it is a functional cyclic‐di‐GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic‐di‐GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic‐di‐GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic‐di‐GMP, B. burgdorferi motility and Lyme disease pathogenesis. A mechanism by which cyclic‐di‐GMP influences motility and infection is proposed.     

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.     

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.     

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.     

The extremophile Acidithiobacillus ferrooxidans possesses a c-di-GMP signalling pathway that could play a significant role during bioleaching of minerals

Aims: The primary goal of this study was to characterize the existence of a functional c‐di‐GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans. Methods and Results: A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c‐di‐GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c‐di‐GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c‐di‐GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells. Conclusions: At. ferrooxidans possesses a functional c‐di‐GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes. Significance and Impact of the Study: This is the first global study about the c‐di‐GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro‐organisms during the bioleaching process.     

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.     

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.     

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.     

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.     

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.     

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.