Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks

In Caulobacter crescentus, phosphorylation of key regulators is coordinated with the second messenger cyclic di-GMP to drive cell-cycle progression and differentiation. The diguanylate cyclase PleD directs pole morphogenesis, while the c-di-GMP effector PopA initiates degradation of the replication inhibitor CtrA by the AAA+ protease ClpXP to license S phase entry. Here, we establish a direct link between PleD and PopA reliant on the phosphodiesterase PdeA and the diguanylate cyclase DgcB. PdeA antagonizes DgcB activity until the G1-S transition, when PdeA is degraded by the ClpXP protease. The unopposed DgcB activity, together with PleD activation, upshifts c-di-GMP to drive PopA-dependent CtrA degradation and S phase entry. PdeA degradation requires CpdR, a response regulator that delivers PdeA to the ClpXP protease in a phosphorylation-dependent manner. Thus, CpdR serves as a crucial link between phosphorylation pathways and c-di-GMP metabolism to mediate protein degradation events that irreversibly and coordinately drive bacterial cell-cycle progression and development.     

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.     

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.     

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

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.     

Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor‐binding specificities in different bacterial species

ClpXP, an AAA+ protease, plays key roles in protein‐quality control and many regulatory processes in bacteria. The N‐terminal domain of the ClpX component of ClpXP is involved in recognition of many protein substrates, either directly or by binding the SspB adaptor protein, which delivers specific classes of substrates for degradation. Despite very limited sequence homology between the E. coli and C. crescentus SspB orthologs, each of these adaptors can deliver substrates to the ClpXP enzyme from the other bacterial species. We show that the ClpX N domain recognizes different sequence determinants in the ClpX‐binding (XB) peptides of C. crescentus SspBα and E. coli SspB. The C. crescentus XB determinants span 10 residues and involve interactions with multiple side chains, whereas the E. coli XB determinants span half as many residues with only a few important side chain contacts. These results demonstrate that the N domain of ClpX functions as a highly versatile platform for peptide recognition, allowing the emergence during evolution of alternative adaptor‐binding specificities. Our results also reveal highly conserved residues in the XB peptides of both E. coli SspB and C. crescentus SspBα that play no detectable role in ClpX‐binding or substrate delivery.     

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.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

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.     

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.     

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.     

Finally! The structural secrets of a HD‐GYP phosphodiesterase revealed

The major sessility‐motility lifestyle change and additional fundamental aspects of bacterial physiology, behaviour and morphology are regulated by the secondary messenger cyclic di‐GMP (c‐di‐GMP). Although the c‐di‐GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD‐GYP domain c‐di‐GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al. provide an important step towards functional and structural characterization of the previously neglected HD‐GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri‐nuclear catalytic iron centre involved in c‐di‐GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c‐di‐GMP to GMP by HD‐GYP domains.     

Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg²⁺, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker‐A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N‐terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped‐down ‘active site’. Homology modelling of the N‐terminal atypical receiver domain of CpaE indicates that it has a conserved protein–protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.     

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.