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.     

The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.     

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.     

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.     

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.     

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.     

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.     

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.     

Cellulose production,activated by cyclic di‐GMP through BcsA and BcsZ,is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.     

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.     

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.     

Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.     

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.     

Nitric oxide‐mediated dispersal in single‐ and multi‐species biofilms of clinically and industrially relevant microorganisms

Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilm formation and biofilm‐related infections. Nitric oxide (NO), an important messenger molecule in biological systems, was previously identified as a signal for dispersal in biofilms of the model organism Pseudomonas aeruginosa. In the present study, the use of NO as an anti‐biofilm agent more broadly was assessed. Various NO donors, at concentrations estimated to generate NO levels in the picomolar and low nanomolar range, were tested on single‐species biofilms of relevant microorganisms and on multi‐species biofilms from water distribution and treatment systems. Nitric oxide‐induced dispersal was observed in all biofilms assessed, and the average reduction of total biofilm surface was 63%. Moreover, biofilms exposed to low doses of NO were more susceptible to antimicrobial treatments than untreated biofilms. For example, the efficacy of conventional chlorine treatments at removing multi‐species biofilms from water systems was increased by 20‐fold in biofilms treated with NO compared with untreated biofilms. These data suggest that combined treatments with NO may allow for novel and improved strategies to control biofilms and have widespread applications in many environmental, industrial and clinical settings.