Staphylococcus aureus. However, little is known about the molecular recognition events that mediate phage adsorption to the surface of S. aureus. The lysogenic siphophage ϕ11 infects S. aureus SA113. It was shown previously that ϕ11 requires α- or β-N-acetylglucosamine (GlcNAc) moieties on cell wall teichoic acid (WTA) for adsorption. Gp45 was identified as the receptor binding protein (RBP) involved in this process and GlcNAc residues on WTA were found to be the key component of the ϕ11 receptor. Here we report the crystal structure of the RBP of ϕ11, which assembles into a large, multidomain homotrimer. Each monomer contains a five-bladed propeller domain with a cavity that could accommodate a GlcNAc moiety. An electron microscopy reconstruction of the ϕ11 host adhesion component, the baseplate, reveals that six RBP trimers are assembled around the baseplate core. The Gp45 and baseplate structures provide insights into the overall organization and molecular recognition process of the phage ϕ11 tail. This assembly is conserved among most glycan-recognizing Siphoviridae, and the RBP orientation would allow host adhesion and infection without an activation step.Phages play key roles in the pathogenicity and adaptation of the human pathogen  Staphylococcus aureus is a Gram-positive bacterium that causes a wide range of infections. It is a leading cause of bacteremia, infective endocarditis, as well as osteoarticular, skin and soft tissue, pleuropulmonary and device related infectionsS. aureus (MRSA) remains a severe global problem threatening the health care system as resistance restricts treatment options to a few drugs of last resort.S. aureus genomes sequenced to date contain one or several prophages2S. aureus phages can be integrated into the bacterial chromosome or exist as extra-chromosomal elements. It is known that many of these phages encode a large variety of S. aureus virulence or fitness factors that allow the bacterium to escape the host immune system. Among all mobile genetic elements in S. aureus, phages are probably most efficient in mediating horizontal gene transfer of virulence or resistance genes between strains, and across species or even genus. Therefore, phages play important roles in staphylococcal pathogenicity and adaptation of S. aureus to different hostile environments2All The large number of staphylococcal phages sequenced to date display an extensive mosaicism in their gene structure, which is a consequence of gene shuffling among different phages that can infect staphylococcal species. The resulting mosaic gene organization is consistent with a modular evolution involving exchanges of genome modules by horizontal transfer and genetic recombination. The genetic exchanges of modules can involve single genes, protein domains, groups of genes, or even functional modulesAlthough phages are the most abundant and diversified biological entity on earth, each phage can only infect a limited number of bacterial strains. This specific phage-host interaction is determined, in part, by the protein recognition device located at the tip of the phage tail, which engages a receptor at the bacterial cell surface. Since bacterial cell wall polysaccharides or glycopolymers project from the cell surface and are thus easily accessible, they are the most common molecules targeted by bacteriophages45S. aureus typically contains poly-ribitol phosphate type wall teichoic acid (WTA), which is modified with D-alanine and N-acetyl-glucosamine (GlcNAc). S. aureus ϕ11 is often used as model to study horizontal gene transfer of virulence genesThe cell wall of S. aureus, we embarked on structural analyses of the Gp45 and baseplate of ϕ11 using X-ray crystallography and electron microscopy. The RBP structure reveals a trimer with a complex fold that can be divided, from N- to C-terminus, into a “stem”, a “platform” and a “tower”. The stem is formed by a long, severely bent triple α-helical coiled coil that features three interruptions: the first and third interruptions are both β-hairpin structures and the second is a short disordered region. A putative “hinge”-like feature is located between the second and the third interruption. The stem is followed by a “platform” of three β-propellers, and the protein terminates with a “tower” formed by a repetitive all-β domain. Platform and tower are interconnected by a fifth short triple helix buried inside of the protein on the molecule’s longest three-fold axis. An unusual iron is located at the C-terminal end of the first coiled coil and may play a role in mediating flexibility or conformational rearrangements within the helical domain. Six copies of the trimer assemble around the baseplate core. This hexameric organization is commonly observed in lactococcal Siphoviridae17Phages adopt a two-fold strategy for host adhesion. They first deploy adhesion modules on fibers or on capsid or tail that recognize the host’s cell wall glycan structures in a reversible way: this allows cell wall scanning in search for the final, specific receptor, to which they bind irreversibly1011124514161718gp45 gene was cloned into the pET28 vector (Novagen) for overexpression in E. coli as described elsewhere6Br12 derivative using single isomorphous replacement with anomalous scattering (SIRAS) and exploiting the non-crystallographic symmetry (NCS) present in the crystals. Initial refinement with PHENIXfree and Rwork values of 21.1% and 17.5%, respectively, for the final model  are buried in the interaction as calculated by PISA2 for the trimer. Most of the buried surface area is concentrated in the stem and C-terminal regions, while the propeller domains engage in few intermolecular contacts  , forcing His42X-Nε2 to point towards the Fe-center. A similar tautomerization effect might occur for the diametrically opposed His50Z, as it is in close distance to Gln54Z (3.24–3.27 Å), forcing His50Z-Nε2 to coordinate to Fe3+, resulting in an intertwined chelate-complex comprised of all three protein chains (see 2-Fe3+ distances are comparable (2.295 ± 0.025 Å) but significantly longer than the His50-Nε2-Fe3+ distances (2.21 ± 0.02 Å). Such a Fe3+ binding geometry has previously been observed in the membrane-piercing spike proteins of phages P2 (PDB code 3QR7) and ϕ92 (PDB code 3PQH)2-Fe distances were 2.20 ± 0.01, 2.23 ± 0.01 and 2.31 ± 0.06, respectively, compared to an average of 2.25 ± 0.07 Å for the distances observed in ϕ11 RBP. The distance values in ϕ11 RBP are also close to the averages of those reported for N-Fe3+ bonds in average-resolution and high-resolution protein structures deposited in the PDB, 2.25 ± 0.15 Å and 2.16 ± 0.13 Å, respectively3+ and the Nε2 of histidines coordinating it axially in myoglobin (2.00–2.11 Å)3+ binding regions in the phage P2, ϕ92 and T4 spike structures involve histidines within a His-X-His motif at the apex of an intertwined triple β-helix. It has been proposed that this ion binding structure might strengthen the puncturing device of phages that pierce the cell wall7-His50 motif lies at a junction between two helical bundles. We therefore suggest that it serves a different role, perhaps by helping to stabilize the bundles that undergo a sharp turn at the hinge. If the Fe lock would not be in place, the structure of the α1/α2 segment would likely not be maintained as a rigid unit.A strong electron density feature suggesting the presence of a metal ion was observed at the junction between the first two bundles. Using an RBP crystal and extended X-ray absorption fine structure (EXAFS) spectroscopy, the identity of this ion was determined to be iron, which probably exists in its oxidized form FePX1) See . The Fe3monomers . This giains see . It is nGln54Z 3.–3.27 Å, Gln54Z 3.–3.27 Å, Gln54Z 3.–3.27 Å, While the first and second helical bundles are collinear, the hinge introduces a sharp angle of ~30° between the second and the third bundle. This angle is the smallest possible since the second and third bundles are in contact at positions 61–64 and 116–121, while the first bundle contacts the propeller domain at position 219  and S1. 2 from solvent, and much of this surface is buried due to a helix-helix contact at the center of the trimer axis Tectiviridae member infecting Gram-negative bacteria (PDB code 1N7U)Pseudomonas myophage PhiKZ, forming a seven-bladed β-propeller domain (PDB code 4GBF)As in other propeller structures, sets of four anti-parallel β-strands form each blade, and the N-terminal β-strand closes the fold by forming the final blade (blade 5) with the three C-terminal β-strands of the domain . A DALI Zymomonas mobilis glutamine cyclotransferase structureWhen the The two C-terminal tower domains form a structure of dimensions 60 × 50 × 50 Å. These two domains are very similar in structure, which is confirmed by a superposition that yields a low r.m.s.d. value of 1.7 Å for their Cα atoms . A DALI To define the topology of the ϕ11 baseplate and allow location of the RBP, we conducted electron microscopy analysis using negative staining of the virion. This approach has been successful in other cases17183210The remainder of the RBP structure was left unaltered, and the modified trimers fit well in a peripheral region of the map that could accommodate the triangular shape of the platform domain’s platform. In order to explain the remaining density of the baseplate, we performed HHPREDWe have solved the crystal structure of ϕ11 RBP and located this protein in the tail spike of the assembled phage using electron microscopy. Our analysis defines the domain organization of RBP, which can be divided into a stem region, a platform domain and a tower-like C-terminal structure composed of two nearly identical domains. Interestingly, the stem displays a severely bent, hook-like conformation that may undergo a conformational change as the protein can only be fitted into the electron density of the tail spike in a less bent arrangement. Unexpectedly, the stem also contains a bound iron. The function of this iron is unknown as its location differs from irons found in other spike proteins. The platform region is formed by three propeller domains and likely harbours the binding site for the substrate GlcNAc. Although soaking and cocrystallization experiments with GlcNAc were not successful, modelling suggests a reasonable location for the GlcNAc binding site in the platform region. Of note, the propeller fold was identified in the endosialidases of several phages. These enzymes cleave polysialic acid at the surface of their host in order to obtain access to the capsular cell wall. For example, coliphages K1F and phi92 possess such endosialidases, which also exhibit trimeric propeller domains attached to a stem35Myoviridae infecting Gram-negative bacteria . In Gp45, only the first 30 amino-acids of the stem resemble those of phages TP901-1 or Tuc2009.Comparison with the phage TP901-1 tail spike assembly allows us to also assign a putative location of the Gp43 and Gp54 proteins of ϕ11. Gp43 likely forms the hexameric Dit, while the N-terminus of Gp54 resembles the first 160 residues of TP901-1 BppU T4 or Mu39, Siphoative T5  or Gram-ative T5 42 bacterT5 173343. It is wAlthough the phage TP901-1 BppU protein does not seem to share such an extensive evolution coverage, the presence of a large part of it either in the RBP (Gp45) or in the subsequent protein (Gp54) was quite surprising. This finding suggests that phages might not capture only widespread elements in the protein domains repertoire, but also less diffused components, even between remote phages with different hosts. In the present case, the role of Gp54 is not documented and difficult to predict. We think it likely that in phage ϕ11 Gp54, a large C-terminal domain might also accommodate the RBP N-terminus, but with different structural features compared to TP901-1 BppU, because of its much increased size. The electron microscopy low-resolution structure gives hints of the putative receptor binding sites, located below the five-bladed propeller domain. This arrangement allows for a correct orientation to capture the GlcNAc of the cell wall teichoic acids. Although, a well-defined cavity in the size of a monosaccharide exists, a much larger crevice surrounds this cavity, suggesting that other WTA components might complement the interaction. However, further structural data are necessary to develop this hypothesis.Gp45 was produced and purified as described elsewhere6Br12 derivative  was prepared by soaking native crystals in crystallization solution + 2mM Ta6Br12 for up to 2 weeks before backsoaking in crystallization condition and vitrification in liquid nitrogen. Anomalous data were processed according to MAD, SAD, MIRAS and SIRAS protocols using SHARP/autoSHARPData for native and derivative crystals were collected at the Swiss Light Source (SLS) on beamline X06DA (PXIII) using a PILATUS 2M hybrid pixel detector. For the determination of peak, inflection, high-remote and low-remote wavelengths from fluorescence spectra, the program CHOOCH was adjusted to the absorption edges of Ta-L-II (1.11325 Å peak) and Ta-L-III (1.25476 Å peak). Data were processed with the XDS packageA threefold NCS was elicited from the self-rotation function via polarrfn ccp4 , giving Due to the size of the multi-domain protein, it was split in various parts for further processing: two for the stem (before and after the ‘hinge’), the platform domain and the C-terminal tower domain. Molecular masks626365599 pfu) was applied to glow-discharged carbon-coated grids and left to adsorb for one min. Sample excess was blotted off and the grids were stained with 10 μL of 1% uranyl acetate for 30 sec. Micrographs (512) were recorded on a 2Kx 2K FEI Eagle CCD camera using a Tecnai Spirit electron microscope operated at 120 kV and a magnification of 48,500 (resulting in a pixel size of 4.83 Å/pixel) (Phages were purified as previously describedÅ/pixel) . The thrÅ/pixel) .Molecular graphics and analyses were performed with the UCSF Chimera package www.rcsb.org) under accession code 5EFV. The EM map of the baseplate reconstruction has been deposited at the Electron Microscopy Data Bank .X-ray structures and structure factors have been deposited with the Protein Data Bank .


Arabidopsis thaliana NLR protein RECOGNITION OF PERONOSPORA PARASITICA1 (RPP1), which recognizes the Hyaloperonospora arabidopsidis effector ARABIDOPSIS THALIANA RECOGNIZED1 (ATR1). Expression of the N-terminus of RPP1, including the Toll/interleukin-1 receptor (TIR) domain (“N-TIR”), elicited an effector-independent cell death response, and we used allelic variation in TIR domain sequences to define the key residues that contribute to this phenotype. Further biochemical characterization indicated that cell death induction was correlated with N-TIR domain self-association. In addition, we demonstrated that the nucleotide-binding (NB)-ARC1 region of RPP1 self-associates and plays a critical role in cell death activation, likely by facilitating TIR:TIR interactions. Structural homology modeling of the NB subdomain allowed us to identify a putative oligomerization interface that was shown to influence NB-ARC1 self-association. Significantly, full-length RPP1 exhibited effector-dependent oligomerization and, although mutations at the NB-ARC1 oligomerization interface eliminated cell death induction, RPP1 self-association was unaffected, suggesting that additional regions contribute to oligomerization. Indeed, the leucine-rich repeat domain of RPP1 also self-associates, indicating that multiple interaction interfaces exist within activated RPP1 oligomers. Finally, we observed numerous intramolecular interactions that likely function to negatively regulate RPP1, and present a model describing the transition to an active NLR protein.Upon recognition of pathogen virulence effectors, plant nucleotide-binding leucine-rich repeat (NLR) proteins induce defense responses including localized host cell death. In an effort to understand the molecular mechanisms leading to this response, we examined the  Many plant pathogens inject proteins known as effectors into the cells of their hosts in order to suppress host immune responses and promote pathogen growth. Over time, plants have evolved receptors, described as nucleotide-binding leucine-rich repeat (NLR) proteins, which recognize the activity of pathogen effectors and stimulate defense responses. Plant NLRs contain several domains that exhibit striking functional conservation with NLRs from other eukaryotes. Despite their important contribution to plant immunity, the molecular mechanisms that underlie effector recognition and subsequent immune activation by NLRs remain to be fully elucidated. Here, we focus on RPP1, an NLR from Arabidopsis that recognizes the oomycete effector ATR1. Using transient co-expression of proteins in plants, we demonstrate that recognition of ATR1 stimulates RPP1 oligomerization. This interaction involves multiple domains of RPP1 and is critical for immune activation. In the absence of ATR1, we documented interactions between domains within an individual RPP1 protein, likely occurring to prevent inappropriate immune activation. Finally, we examined differences between RPP1 alleles as well as structural data from animal NLRs to help identify specific amino acids that mediate interactions within and between RPP1 molecules. Collectively, these data allow us to propose a model for the activation of RPP1 following ATR1 recognition. Through interactions with a plethora of phytopathogenic organisms, plants have evolved a sophisticated molecular surveillance system that involves nucleotide-binding leucine-rich repeat (NLR) immune receptors that recognize pathogen-derived virulence effector proteins. Effector recognition elicits a strong immune response that is associated with programmed cell death, known as the hypersensitive response (HR), which ultimately restricts pathogen proliferation ,2. PlantThere is increasing evidence from the study of animal NLR immune receptors that intermolecular associations play a key role in the stimulation of immune responses. Similar to plant NLR proteins, these receptors include a nucleotide-binding domain flanked by a C-terminal ligand-sensing domain and an N-terminal signaling domain that recruits additional immunomodulatory proteins . FollowiArabidopsis thaliana protein RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1) [Hyaloperonospora arabidopsidis (formerly Peronospora parasitica) effector ATR1 (ARABIDOPSIS THALIANA RECOGNIZED1) [in planta biochemical evidence suggesting a direct mode of recognition [In light of this uncertainty, we examined the SITICA1) , a TIR dGNIZED1) . This reognition ,22. MultIn this study, we sought to functionally characterize the domains of RPP1 with regards to cell death induction following effector recognition. Using transient expression of epitope-tagged proteins, we demonstrate that RPP1 oligomerizes in the presence of a recognized allele of ATR1. Co-immunoprecipitation and mutagenesis experiments were used to investigate the specific domains involved in pre-activation intramolecular interactions and post-activation intermolecular interactions. Phenotypic variation amongst RPP1 alleles and structural data from various animal immune receptors were used to identify specific amino acids that facilitate these protein-protein interactions. Based on these observations, we suggest a model of the molecular events underlying only the second documented example of effector-dependent NLR protein oligomerization in plants.Nicotiana tabacum, an effector-independent HR developed within 24 hours  alleles of RPP1 . Est-1 w90-254 and WsB84-248 were 33.5% and 11.6% higher, respectively, than would be expected for a monomeric protein (1-254 and WsB1-248) further increased the measured molecular mass relative to the expected molecular mass of a monomer. Again, this difference was much more striking for NdA compared to WsB  could self-associate, while very weak interactions were observed for the NB region (aa 255–424) and no self-association was detected with NB-ARC1-ARC2 constructs (aa 255–597) . FurtherCaenorhabditis elegans CED-4 protein, whose assembly into an apoptosome is mediated by intermolecular contacts on the surface of the α/β fold [in planta expression was also lower. This pattern was consistent aside from the N-TIR-NB-ARC1-inactivating substitution V362A, which retained self-association, accompanied by relatively high levels of protein expression. Overall, these data suggested that the NB subdomain, in cooperation with ARC1, is a key mediator of cell death induction.The NB-ARC1 interactions that we observed are reminiscent of the α/β fold . In RPP1N. benthamiana revealed RPP1 self-association in the presence of a recognized allele of ATR1 (ATR1_Emoy2) but not an unrecognized allele   . The imm1_Cala2) . Based o1_Cala2) . Interes1_Cala2) . NeitherConsidering the differential impact of NB mutations on self-association between NB-ARC1 constructs versus full-length RPP1_NdA, it is possible that the LRR domain participates in the oligomerization event as well. Indeed, we detected LRR (aa 606–1154) and NB-ARC-LRR (aa 255–1154) self-association by co-immunoprecipitation, even in the absence of ATR1 . The RPPThe demonstration of effector-induced oligomerization provided a snapshot of RPP1 in a post-activation state, but we also sought to clarify the domain organization of this protein prior to effector recognition. Through co-immunoprecipitation experiments we found that non-autoactive N-TIR domains, including the R104A F106A and G229A Y230A mutants of the NdA allele, interacted with the NB-ARC1 subdomain . Less NBInteractions were also detected between the NB-ARC1 subdomain and the LRR domain. The NB subdomain was again sufficient for this association, while little or no interaction was observed between the LRR and NB-ARC1-ARC2 , S17 FigPlant NLR proteins are characterized by a number of conserved domains whose collective activity yields tightly regulated immune responses. When expressed on its own, the N-TIR domain from RPP1_NdA elicited cell death even in the absence of ATR1. Similar autoactivity was noted for the TIR domains of RPS4 from Arabidopsis  and L6 fIn planta, we noted that this N-terminal sequence is required for TIR domain autoactivity and for effector-dependent cell death induction by full-length RPP1_NdA. It is also worth noting that this region represents the most polymorphic sequence of the N-TIR domain between the NdA and WsB alleles, with 14 aa of insertions/deletions and 67% aa identity (versus 90% identity within the TIR domain).Interestingly, N-TIR domains from different RPP1 alleles varied substantially in their autoactivity, allowing us to ascribe specific sequences to this phenotypic polymorphism. Using chimeric constructs, we noted that a region of 50 amino acids directly N-terminal to the predicted TIR domain from RPP1_NdA conferred weak autoactivity to an otherwise inactive background of RPP1_WsB. The functional role of this region is not known and its structure is predicted to be disordered, although our SEC-MALS analysis suggested that it contributes to TIR domain self-association . In planet al. [The analysis of polymorphisms within the predicted TIR domain yielded a small number of residues with significant contributions to autoactivity. In a WsB background, the substitutions K98R I100F, K228S, or R230C enabled the elicitation of cell death at levels near that of the N-TIR domain from RPP1_NdA. Using the crystal structure of the L6 TIR domain [et al. . Based oet al. [et al. [The NB-ARC domain is generally considered to act as a molecular switch for NLR protein activation , but we et al.  previouset al. . A majoret al. . We obseet al. . In eith [et al. .Following the demonstration of self-association between NB-ARC1 proteins, we investigated the specific residues that influence this interaction. Substitution of a highly conserved lysine within the P-loop motif (K299L) eliminated NB-ARC1 self-association and N-TIR-NB-ARC1 autoactivity . This reC. elegans apoptosome comprises an octamer of CED-4 proteins forming a funnel-shaped structure, within which the α/β domains of each CED-4 member are closely aligned [The identification of residues at the physical protein interaction interface(s) of the NB-ARC1 region was guided by structural similarity with animal immune receptors. The activated  aligned . Structu aligned –37.An unusual observation was that the substitution E365A strengthened the intensity of cell death induced by an N-TIR-NB-ARC1 construct or by full-length RPP1_NdA co-inoculated with ATR1_Emoy2. While a glutamate-to-alanine substitution represents an increase in local surface hydrophobicity, similar increases in HR strength resulted from substitutions to the relatively hydrophilic amino acids glutamine and lysine . As suchin planta. We did not face such an obstacle with RPP1, and were able to demonstrate that residues within the RNBS-A motif and in the adjoining α-helix are involved in self-association of the NB-ARC1 subdomain. It is noteworthy that, while most of the substitutions retained some level of NB-ARC1 self-association, cell death induction was largely eliminated in both N-TIR-NB-ARC1 and full-length contexts. This may indicate that a certain threshold of association is required to activate cell death, and that substitutions of surface-exposed residues result in localized structural perturbations such that this threshold is not exceeded.While the assembly of higher-order immunoregulatory structures is well documented in animal systems ,15, dataAt the same time, the retention of effector-dependent oligomerization in mutants of full-length RPP1_NdA suggested that additional domains are involved in this interaction. Indeed, co-immunoprecipitation experiments revealed LRR self-association, even in the absence of ATR1. Similar homotypic interactions were shown for LRRs from RPS5 and Rp1-D21 ,41, as mcis. Additional examples of this interaction from other TIR domain-containing NLR proteins would help to clarify these issues.Resistance proteins control the induction of cell death programs and thus must be tightly regulated. For RPP1, we observed a series of conditional intramolecular interactions with potential negative regulatory functions. Firstly, interactions between the NB-ARC1 and N-TIR domains were most strongly detected with non-autoactive N-TIR domain mutants. Reduced protein accumulation of the wild-type NdA N-TIR domain likely influenced this result, although an appreciable quantity of the wild-type protein was immunoprecipitated. It is relevant to note that, while both CC:CC and CC:NB-ARC interactions were observed for RPS5 and Rp1-D21, the CC domain alone was not autoactive ,41. BaseIn addition to binding the N-TIR domain, we noted that the NB-ARC1 domain also interacts with the LRR domain of RPP1. Mutagenesis experiments revealed residues that differentially affected N-TIR:NB-ARC1 and LRR:NB-ARC1 interactions. Mutations in the NB P-loop motif significantly impaired N-TIR:NB-ARC1 binding but had little impact on the interaction of the LRR with NB-ARC1. A similar discrepancy was observed with the Rx protein from potato, where a P-loop mutation disrupted the CC:NB-ARC-LRR interaction but not the CC-NB-ARC:LRR interaction . The P-ltrans rather than in cis, although it is difficult to bypass this limitation for in planta analyses. For the N-TIR domain, the use of a non-autoactive mutant may have allowed a more stable interaction with the LRR, as the wild-type N-TIR domain did not appear to interact with the LRR. It is also possible that the activation of RPP1 may involve a series of subtle conformational changes that preserve these intramolecular interactions, possibly through shifts in the specific residues bound by each domain.For intramolecular interactions involving the LRR domain, we anticipated that effector binding might disrupt these associations, as seen in the NLR proteins Rx1 and Gpa2 ,45. We oOverall, our data are compatible with a model  in whichEscherichia coli, RPP1 TIR domain sequences were introduced into pLIC171  by ligation-independent cloning [Constructs for transient expression were prepared using the primers listed in  cloning . All poi cloning  combined cloning .Nicotiana tabacum var Turks and Nicotiana benthamiana were grown on potting soil in a controlled environment room with a 10-h photoperiod at 24°C. Agrobacterium tumefaciens GV3101 (pMP90) was grown at 28°C on Luria-Bertani agar media supplemented with 50 μg/mL gentamycin and 25 μg/mL kanamycin. For transient expression experiments, including co-immunoprecipitations, bacteria from plates were resuspended in 1 mL of induction media , diluted to OD600 = 0.45 with induction media, and incubated at room temperature for three hours. For all inoculations, leaves were pierced with a 22 gauge needle prior to infiltration with a 1 mL needleless syringe. Plants were subsequently maintained at room temperature under continuous light until tissue collection at 24 to 48 hours post-infiltration.To detect transiently expressed proteins, two leaf discs were obtained from inoculated tissues using a #7 cork borer (1.2 cm diameter). The leaf discs were placed in a 1.7 mL SnapLock microfuge tube (Thermo Fisher Scientific) along with a 3 mm glass bead and frozen in liquid nitrogen. Samples were homogenized for 1.5 min in a chilled aluminum block with a Mini-Beadbeater-96  and suspended in 130 μL of modified Laemmli buffer . Samples were vortexed for 30 s, boiled for 5 min, centrifuged at maximum speed for 1 min in a microcentrifuge at room temperature, and the supernatants were transferred to 1.5 mL microfuge tubes. Generally, 12 μL of each sample was separated on 7% or 12.5% discontinuous SDS-PAGE gels and transferred to a nitrocellulose membrane . Antibodies for protein detection included monoclonal ANTI-FLAG M2-Peroxidase , anti-HA-Peroxidase , mouse monoclonal anti-GFP , and monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich). The latter two antibodies were used in conjunction with a goat-anti-mouse HRP secondary antibody. Antibody binding was visualized with either SuperSignal West Pico or SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher Scientific).et al. [Co-immunoprecipitation experiments were conducted essentially as described by Krasileva et al.  with somAgrobacterium suspensions were prepared as described above and infiltrated into N. tabacum leaves such that each technical replicate comprised all constructs infiltrated into sections of the same leaf. When macroscopic HR symptoms became visible , four leaf discs were collected using a #7 cork borer  and floated on 5 mL of distilled water for 30 min. Leaf discs were then transferred to polypropylene culture tubes containing 6 mL of distilled water and placed under continuous light at room temperature. Twenty-four hours later, electrolyte leakage was measured using a Thermo Orion Model 105A Plus Conductivity Meter (Thermo Fisher Scientific).To quantify the extent of cell death elicited by various constructs, E. coli BL21 (DE3) cells was induced overnight at 20°C via autoinduction [The expression of RPP1 (N-)TIR domain proteins in nduction . ProteinFor protein molecular mass determination, 175 μg of purified protein was separated on a Superdex Increase 200 5/150 GL SEC column  in a buffer containing 10 mM HEPES pH 8.0, 150 mM NaCl, 1 mM DTT, with laser light scattering measured by a Dawn Heleos II 18-angle light-scattering detector coupled with an Optilab TrEX refractive index detector . Astra6.1 software (Wyatt Technology) was used to perform molecular mass calculations. Molecular mass is reported at the protein elution peak (corresponding to the refractive index peak) from the SEC column, where the signal from light-scattering is strong. Estimations of molecular mass were performed across the protein elution peak, with refractive increment  fixed at 0.186 mL/g, with the assumption that dn/dc is invariable for unmodified proteins .Sequence data for the genes examined in this study can be retrieved from the National Center for Biotechnology Information using the following accession numbers: RPP1_NdA (HM209027), RPP1_WsB (AAC72978), ATR1_Emoy2 (AAX51198), ATR1_Cala2 (AAX51204), and RPS2 (AAA21874).S1 Fig2 of tissue were collected at approximately 28 hours post-infiltration and electrolyte concentration (conductivity) was measured 24 hours after collection. Error bars indicate standard deviation, and letters above data points indicate statistical significance groups as determined by pairwise Student’s t-tests (α = 0.05). Experiments were performed at least three times with similar results.Data in (A) and (B) correspond to the constructs tested in (D) and (E) of (TIF)Click here for additional data file.S2 FigNicotiana tabacum via Agrobacterium-mediated transient expression and images of hypersensitive response (HR) phenotypes were captured at 48 hours post-infiltration. The presence or absence of HR is indicated by a “+” or “-“, respectively. The specific amino acids comprising each construct are indicated in subscript. (D) An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. The experiment was performed three times with similar results.(A) Amino acid alignment of N-TIR domain sequences from the Niederzenz (NdA), Wassilewskija (WsB), and Estland-1 (Est-1) alleles of RPP1. The TIR domain borders predicted by Pfam are delimited by square brackets, and polymorphic amino acids with potential relevance to protein function are highlighted in red. Residues shown to influence autoactivity are indicated with arrowheads. (B) Functionally relevant polymorphic residues are also indicated on a putative structure of the RPP1_WsB TIR domain, derived by homology modeling using structural data from the L6 TIR domain (PDB: 3OZI). Relevant α-helices are also annotated. (C) Autoactivity of N-TIR domains from different RPP1 alleles. Constructs were tested in (TIF)Click here for additional data file.S3 FigNicotiana tabacum via Agrobacterium-mediated transient expression and images of hypersensitive response (HR) phenotypes were captured at 48 hours post-infiltration. HR phenotypes are scored as negative (-), weak (w), or strong (+). The specific amino acids comprising each construct are indicated in subscript. An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. The experiment was performed three times with similar results.Constructs were tested in (TIF)Click here for additional data file.S4 FigNicotiana tabacum via Agrobacterium-mediated transient expression and images of hypersensitive response (HR) phenotypes were captured at 48 hours post-infiltration. The presence or absence of HR is indicated by a “+” or “-“, respectively. The specific amino acids comprising each construct are indicated in subscript. An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. The experiment was performed three times with similar results.Constructs were tested in (TIF)Click here for additional data file.S5 Fig2 of tissue were collected at approximately 28 hours post-infiltration and electrolyte concentration (conductivity) was measured 24 hours after collection. Error bars indicate standard deviation, and letters above data points indicate statistical significance groups as determined by pairwise Student’s t-tests (α = 0.05). The experiment was performed three times with similar results.Leaf discs representing approximately 4.5 cm(TIF)Click here for additional data file.S6 FigNicotiana tabacum via Agrobacterium-mediated transient expression and images were captured at 48 hours post-infiltration. HR phenotypes are scored as negative (-), very weak (vw), weak (w), or strong (+). (B) An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. Experiments were performed at least three times with similar results. (C) Predicted structure of the RPP1 NB-ARC domain based on homology modeling using the Drosophila Dark protein (PDB:4v4l) as a template. The three subdomains of this region are highlighted and the locations of each truncation tested in (A) are indicated by arrows.(A) Hypersensitive response (HR) phenotypes associated with C-terminal truncations of an N-TIR-NB-ARC1 construct from the NdA allele. The amino acids at the C-terminus of each truncation are indicated in subscript. Constructs were tested in (TIF)Click here for additional data file.S7 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Staining of RuBisCO with Ponceau S provides a loading control. The experiment was performed three times with similar results.Differentially epitope-tagged N-TIR-NB-ARC1 proteins from the Niederzenz (N) and Wassilewskija (W) alleles of RPP1 were transiently expressed in (TIF)Click here for additional data file.S8 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Staining of RuBisCO with Ponceau S provides a loading control. The experiment was performed three times with similar results.Constructs were transiently expressed in (TIF)Click here for additional data file.S9 FigNicotiana benthamiana and samples were collected at 24 hours post-infiltration (hpi) for (A) and (B) or 48 hpi for (C). Staining of RuBisCO with Ponceau S provides a loading control. The experiments were performed three times with similar results.(A) N-TIR domain addition constructs tested in (TIF)Click here for additional data file.S10 FigCaenorhabditis elegans CED-4 protein (PDB: 2A5Y) are shown on the left, with residues that contribute to oligomerization highlighted in red. The corresponding region of RPP1 is shown at center; residues required for induction of the hypersensitive response are highlighted in red, while those that were dispensable for this response are depicted in green. The predicted structure of the RPP1 NB-ARC domain is shown on the right and is derived from homology modeling using the Drosophila Dark protein (PDB:4v4l) as a template. The putative oligomerization interface region is highlighted in red. Note that the enlarged image of this region is rotated approximately 90° counterclockwise on the y-axis for improved visibility of the residues of interest.The α-helices that comprise the oligomerization interface of the (TIF)Click here for additional data file.S11 FigNicotiana tabacum via Agrobacterium-mediated transient expression, using a range of inoculum concentrations (as measured by OD600) to allow a comparison of the strength of cell death induction by wild-type (WT) and E365 substitution mutants. Images were captured at 48 hours post-infiltration (hpi). (B) An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. (C) Quantification of electrolyte leakage induced by various N-TIR-NB-ARC1 constructs. Leaf discs representing approximately 4.5 cm2 of tissue were collected at approximately 28 hpi and electrolyte concentration (conductivity) was measured 24 hours after collection. The non-autoactive F350A mutant and autoactive N-TIR domain were included as negative and positive controls, respectively. Error bars indicate standard deviation, and letters above data points indicate statistical significance groups as determined by pairwise Student’s t-tests (α = 0.05). Experiments were performed three times with similar results.(A) Hypersensitive response phenotypes elicited by N-TIR-NB-ARC1 constructs or by co-expression of full-length RPP1_NdA and ATR1_Emoy2. Constructs were tested in (TIF)Click here for additional data file.S12 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Asterisks indicate non-specific bands. Staining of RuBisCO with Ponceau S provides a loading control. The experiment was performed three times with similar results.Constructs were transiently expressed in (TIF)Click here for additional data file.S13 FigNicotiana benthamiana and samples were collected at 36 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Due to a non-specific signal at the expected molecular weight of ATR1:citrine when the elution fraction was probed with an α-GFP antibody, HA:ATR1:citrine constructs were used and ATR1 expression detected with an α-HA antibody. Staining of RuBisCO with Ponceau S provides a loading control. The experiment was performed three times with similar results.Differentially epitope-tagged RPP1_NdA proteins were transiently co-expressed with either ATR1_Emoy2 (E) or ATR1_Cala2 (C) in (TIF)Click here for additional data file.S14 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Staining of RuBisCO with Ponceau S provides a loading control. Experiments were performed three times with similar results.While self-association of the RPP1_NdA LRR is detected, this protein does not interact with the LRR domain of RPS2. Constructs were transiently expressed in (TIF)Click here for additional data file.S15 FigNicotiana tabacum via Agrobacterium-mediated transient co-expression with ATR1_Emoy2, and images of hypersensitive response phenotypes were captured at 48 and 72 hours post-infiltration (hpi). The constructs included RPP1_NdA1-1154 (1), RPP1_NdA30-1154 (2), and RPP1_NdA93-1154 (3), where the specific amino acids comprising each construct are indicated in subscript. An α-HA antibody was used to evaluate protein expression, and the expression of ATR1_Emoy2:citrine was detected with an α-GFP antibody. Staining of RuBisCO with Ponceau S provided a loading control. The experiment was performed three times with similar results.Constructs were tested in (TIF)Click here for additional data file.S16 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Staining of RuBisCO with Ponceau S provides a loading control. The experiment was performed three times with similar results.Constructs were transiently expressed in (TIF)Click here for additional data file.S17 FigNicotiana benthamiana and samples were collected at 48 hours post-infiltration for co-immunoprecipitation using α-Flag agarose beads. Staining of RuBisCO with Ponceau S provides a loading control. Asterisks indicate non-specific bands. The experiments were performed two times with similar results.An NB-ARC1-ARC2 construct only weakly binds the LRR domain, regardless of whether the epitope tag is located C-terminally (A) or N-terminally (B). Constructs were transiently expressed in (TIF)Click here for additional data file.S18 FigN. benthamiana and samples were collected at 48 hours post-infiltration . Co-immunoprecipitations were performed using α-Flag agarose beads. The expression of ATR1:citrine was detected with an α-GFP antibody. Asterisks indicate non-specific bands. Staining of RuBisCO with Ponceau S provides a loading control. Experiments were performed three times with similar results.A non-autoactive N-TIR domain mutant (R104A F106A) associates with the LRR, and the interaction is not disrupted by the presence of ATR1. Constructs were transiently expressed in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This paper focuses on the particle size distribution (PSD) changes during nixtamalized corn kernels (NCK) as a function of the steeping time (ST). The process to obtain powder or corn flour from NCK was as follows: (i) the NCK with different STs were wet-milled in a stone mill, (ii) dehydrated by a Flash type dryer, and (iii) pulverized with a hammer mill and sieved with a 20 mesh. The powder was characterized by measuring the PSD percentage, calcium percentage (CP), peak viscosity at 90°C (PV), and crystallinity percentage (CP). The PSD of the powder as a function of ST was determined by sieving in Ro-TAP equipment. By sieving, five fractions of powder were obtained employing meshes 30, 40, 60, 80, and 100. The final weight of the PSD obtained from the sieving process follows a Gaussian profile with the maximum corresponding to the average particle obtained with mesh 60. The calcium percentage as a function of ST follows a behavior similar to the weight of the PSD. The study of crystallinity versus the mesh number shows that it decreases for smaller mesh number. A similar behavior is observed as steeping time increases, except around ST = 8 h where the gelatinization of starch is observed. The trend of increasing viscosity values of the powder samples occurs when increasing ST and decreasing particle size. The ST significantly changes the crystallinity and viscosity values of the powder and, in both cases, a minimum value is observed in the region 7–9 h. The experimental results show that the viscosity increases (decreases) if the particle size decreases (increases). Zea mays L.) is the third most important cereal grain for human consumption in the world after wheat and rice and is a staple food in many regions. It is estimated that, in 2012, the global production of maize was around 900 million tons, with the United States of America, China, Brazil, and Argentina being the top maize-producing countries [Maize . This mixture was cooked for 40 min at 92°C. After the cooking process, the maize was steeping in the cooking water for 0, 3, 5, 7, 8, 9, 11, 13, 15, and 24 h, taking samples at the predetermined ST. Then the cooking water (nejayote) of each sample was drained off, and the nixtamal was washed twice with water for 2 minutes. Finally, the washed nixtamal was grounded in a stone mill  and then dehydrated in a Flash type dryer. To turn it into corn flour the material from the Flash type dryer was pulverized with in a hammer mill employing a 20 mesh.The following procedure was used to prepare the corn flour samples: 6 L of water, previously heated to a temperature of 92°C, was added to 3 kg of commercial white dent maize (Toluca region) (1 : 2 maize-water ratio). Immediately 60 g of Ca(OH)μm), 40 (400 μm), 60 (250 μm), 80 (177 μm), and 100 (149 μm). The sieving of the corn flour determined the PSD groups. The sieving procedure was done during 15 minutes employing 150 g of corn flour as reported in a previous work [The PSD of the corn flour was measured using Ro-TAP equipment with a set of meshes: 30 595 95 μm, 40The CP was obtained by atomic absorption spectroscopy . The relative viscosities of the PSD water suspensions of corn flour dough were determined using a pasting viscometer. Suspensions of corn flour produced at different ST were prepared to determine RVA profile. The total time to obtain the RVA graphs was 15 min. They were obtained by raising the temperature of the corn flour suspension from 50 to 90°C. The samples were maintained at the higher temperature during 5 min, and then the temperature was decreased at the same rate at which it had increased. For each sample, 4 g of corn flour, with a humidity of 12%, and 24 milliliters of distilled water were used.α radiation (wavelength λ = 1.5406 Å) was employed, on a Siemens D5000 diffractometer operating at 35 KV and 15 mA. The relative percentage values for the crystallinity of the PSD from corn flour were calculated using software integrated into the measurement system.The corn flour samples were compressed and placed in a sample holder. The X-ray diffraction study was made from 4° to 30° with a step of 0.05°. A monochromatic CuKThe weight of the set of PSDs (W-PSD) as a function of the mesh number and ST was determined (fixes the maximum particle size in the PSDs) for each of the ten STs. As shown in ≅ 8-9 h. The inset at the bottom of  peak viscosity and breakdown point depended on calcium percentage, ST, and particle size. The ST produces nonlinear changes in percentages of calcium, PDS, and crystallinity in the corn flour. For the corn flour obtained employing meshes 30, 40, and 60, the peak viscosity and breakdown point were observed as an effect of ST on the coarse particles.The several flour corn fractions showed significantly different RVA profiles, PDS, and calcium and crystallinite percentages as function of ST. Therefore the


Intensive investigations have been drawn on nanoscale ferroelectrics for their prospective applications such as developing memory devices. In contrast with the commonly used electrical means to process  the information carried by ferroelectric domains, at present, mechanisms of non-electrical processing ferroelectric domains are relatively lacking. Here we make a systematical investigation on the stability of 180° cylindrical domains in ferroelectric nanofilms subjected to macroscopic mechanical loads, and explore the possibility of mechanical erasing. Effects of domain size, film thickness, temperature and different mechanical loads, including uniform strain, cylindrical bending and wavy bending, have been revealed. It is found that the stability of a cylindrical domain depends on its radius, temperature and film thickness. More importantly, mechanical loads have great controllability on the stability of cylindrical domains, with the critical radius nonlinearly sensitive to both strain and strain gradient. This indicates that erasing cylindrical domain can be achieved by changing the strain state of nanofilm. Based on the calculated phase diagrams, we successfully simulate several mechanical erasing processes on 4 × 4 bits memory devices. Our study sheds light on prospective device applications of ferroelectrics involving mechanical loads, such as flexible memory devices and other micro-electromechanical systems. Ferroelectrics are natural candidates for data storage, as they possess switchable spontaneous polarization to carry bit information “0” and “1”. Their usefulness also originates from a wide spectrum of other important properties, such as piezoelectricity, pyroelectricity, photovoltaic effect, and nonlinear optic behaviors1456784567891315Nevertheless, at present there are still many challenges towards such a wonderful picture. The crucial issues include nanodomain stability and proper processing  mechanisms. In general, stability of a ferroelectric domain intrinsically depends on its size due to the collective nature. It is also sensitive to the environments surrounding it. Particularly for a ferroelectric nanofilm, its strain state and properties in the vicinity of surfaces/interfaces, e.g., defects, bondings and screening condition, can significantly affect the domain structure and thus the stability of specific domains1718192045678Due to the natural coupling between polarization and electric field, almost all reported reading/writing/erasing mechanisms of ferroelectric domains are electrical. In literature, it has been understood that film-substrate mismatch strains can be utilized to obtain novel ferroelectric phases and domain structures in ferroelectric thin films1723In this letter, based on systematical phase field simulations, we demonstrate that stability of polar domains in ferroelectric ultrathin films can be effectively controlled by macroscopic mechanical loads. As a consequence, ultrahigh density of stable polar domains can be obtained. Moreover, erasing of domains can be realized by adjusting the strain state of the nanofilm. With carefully taken into account the effects of inhomogeneous electromechanical fields, ambient temperature and surfaces, stability of 180° cylindrical domains has been comprehensively revealed as a function of domain size, temperature, film thickness and particularly various mechanical loads, such as uniform strain, cylindrical bending and wavy bending. The revealed mechanical controllability of domain stability should provide insight into the fundamental limits of ferroelectric memories and indicate prospective applications of ferroelectrics involving mechanical loads.3 (PTO) ferroelectric ultrathin films divided into rectangular memory units. The conclusions of this investigation are believed to be applicable to other systems such as Pb(ZrxTix1-)O3 and BaTiO3 nanofilms, which have also been predicted to exhibit 180° domains. The PTO films are initially poled into single domain state, with all memory units initially carrying bit information “0”. Some memory units are then written with 180° inverted cylindrical domains to carry bit information “1”. The domain size is characterized by a lateral radius r. To see the effects of both strain and strain gradient on the domain stability, and to explore effective mechanical erasing mechanisms, we apply various mechanical loads to the nanofilm written with cylindrical domains, including (i) uniform strain, (ii) cylindrical bending and (iii) wavy bending, respectively. Note that cylindrical domain and control of its size can be achieved in SPM experiments by controlling strength and period of the SPM electric field45678The basic idea of our investigation is illustrated in r = 16 nm , below which it is instable, shrinks and disappears in the simulation cell. Above the critical size, cylindrical domains with various sizes can maintain stable, indicating that they are actually metastable states. Obviously, this result reflects the multi-valleys morphology of the system's free energy. It is the barriers between the energy valleys that lead to a pinning force against domain wall shrinkage. We believe that this domain wall pinning effect is not an artificial effect but an intrinsic effect of real systems with periodic lattice potential. While it is difficult to be taken into account in analytic models, this intrinsic domain wall pinning effect is important for domain stability in defect-free systems.We continue to investigate the stability of cylindrical domains with considering the effects of film thickness and temperature. To achieve this, we write cylindrical domains with different radii into 128 nm × 128 nm × T = 300 K, the critical size of cylindrical domain decreases from 22 nm to 6 nm when the film thickness increases from 6 nm to 16 nm  structure, whereas in the last loading condition it is in a/b-domain  structure. This is due to that c-domain structure and a/b-domain structure can respectively accommodate the compressive and tensile biaxial strains, leading to a decrease of elastic energy, as has been experimentally verified from the domain abundance in epitaxial PTO thin filmsc-domain size is sensitive to the external strain. For example, as indicated by the green arrows, small domains are stabilized by compressive strain (−0.01). However, they disappear when the film is in a less compressive strain state (−0.005). More interestingly, the formed domain patterns are also different when the nanofilm is subjected to different strain gradient . This indicates that mechanical erasing can be achieved even though a/b-domains were induced. Nevertheless, this seems only possible when the abundance of induced a/b-domains is relatively small. For those patterns with a large abundance of induced a/b-domains at biaxial strain condition . This nonlinear feature is even more significant in the mixed bending-strain condition  at room temperature. The simulation cells are initially written into cylindrical domains, with their radii ranging from 1 nm to 64 nm. Ab in the x-z plane of the simulation cell under a wavy bending with λ = 128 nm. Here we also investigate the effect of wavy bending in the presence of a flexoelectric field, f12 = 10 V. The distribution of Ab in the x-z plane of the simulation cell is depicted in z-direction, i.e., <P3>, over the initial cylindrical region of the written domain, are also calculated and depicted in To see how wavy bending affects the stability of 180° cylindrical domain, simulations are conducted on 128 nm × 128 nm × 8 nm simulation cells under different wavy bending strains  to the memory system (Ab = 1.0 nm and λ = 128 nm) to the system (Ab = 0.2 nm and λ = 128 nm) can maintain 16 nm cylindrical domains at the valleys, due to enhance of domain stability by the flexoelectric effect. After removing the wavy bending, as 16 nm cylindrical domain is no longer stable, the information is successfully erased . Moreover, information can be stored at different regions according to their operating condition . This would be of practical importance for applications. All of these, although there is still a long path to go through, indeed present us wonderful possibility in memory applications involving mechanical loads.x- and y- directions. We mainly restrict ourselves to simulation cells containing one inverted cylindrical domain at the center region  technique based on Khachaturyan's microscopic elasticity theory176 time steps). The material parameters in calculations are listed f12 (~10 V) is a roughly estimated to lie between the theoretical estimations and experiment measurements, due to the uncertainty of this parameter of the materialThe domain structure is solved by a generally accepted and powerful theory for simulating domain structures of ferroelectrics, i.e., the phase field model based on the Landau-type formalism, in which we also carefully takes into account the effects of inhomogeneous mechanical field, electric filed and surfaces. Such approach has been widely used to simulate ferroelectric domain structure, and represents well the experimental results of ferroelectric domain structures in a wide range of materials and conditions333435Y.Z. initiated and performed this work and manuscript. W.J.C. and Y.Z. conceived and designed the basic idea, structures and simulations. X.F. and B.W. suggested the principle idea. W.J.C. performed the simulations. Y.Z., W.J.C., X.F., D.C.M. and Y.W. analyzed the results of simulations. Y.Z., W.J.C. and B.W. co-wrote the manuscript. All authors contributed to discussion and reviewed the manuscript.Supplementary Information


Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence.The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization.Complementary V. cholerae.These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of The online version of this article (doi:10.1186/s12866-014-0272-9) contains supplementary material, which is available to authorized users. Gluconacetobacter xylinus GTP  and 5 μL of 100 mM GTP buffer containing 75 mM Tris, pH 8, 250 mM NaCl, 25 mM KCl, and 10 mM MgCl2. Subsequent steps were carried out as described c-di-GMP for 0 min to 30 min at room temperature as previously described ?>of V. cholerae nor hydrolyzing c-di-GMP in vitro. VCA0931 possesses an N-terminal domain of unknown function, which may post-translationally regulate the HD-GYP domain in response to specific cues lacking in our assays, or mask activity of the HD-GYP domain [Using P domain ,65. We ein [PmGH . In addiin [PmGH ,66. We cP domain .in vivo and in vitro. The activities of a subset of the HD-GYP domain proteins could not be easily predicted due to lack of conservation of one or more residues expected to be required for activity: VC2497, VC2340 and VCA0681 [PmGH . The substitution of an aspartic acid with an asparagine in VCA0681 may preserve enzymatic activity, or the requirements of PmGH may not be universal to all HD-GYP domain PDEs. Further characterization of structure-function relationships of HD-GYP domain phosphodiesterases from a variety of organisms will be necessary to define the features required for activity.As we anticipated, VC1087 and VCA0895, which lack the HD dyad characteristic of HD metal-dependent phosphohydrolases, showed no evidence of c-di-GMP hydrolytic activity  VCA0681 ,61. Whil VCA0681 ,60. The V. cholerae showed that VC1348, VCA0210, VCA0681 and VCA0895 transcripts are significantly higher in biofilm-derived V. cholerae than in planktonic cells. Interestingly, three of these genes, VCA0210, VCA0681 and VCA0895 were among the four HD-GYP domain genes regulated by quorum sensing [V. cholerae biofilm may be the signal controlling expression of these genes.Analysis of the expression of the HD-GYP domain genes in  sensing . Thus, hV. cholerae under the conditions tested. Even removal of all active (and many inactive) HD-GYP domain proteins in a single strain had no effect on motility or biofilm production. There are several possible reasons for the lack of phenotypic differences from the parent strain. First, it remains possible that HD-GYP domain phosphodiesterases, individually or as a class, impact c-di-GMP regulated processes other than those investigated here. Second, not all HD-GYP domain proteins are likely to be produced or enzymatically active under the conditions tested, so eliminating them may not have an observable effect. Our qRT-PCR studies of HD-GYP gene expression in planktonic and biofilm cultures support this possibility—expression of each HD-GYP domain gene is variable within those conditions. The observation that a VC1295 mutant produces somewhat more biofilm in the presence of bile acids further indicate that specific extracellular cues may be necessary to stimulate HD-GYP production and/or activity [We found that mutation of individual HD-GYP domain genes did not affect c-di-GMP regulated phenotypes of activity .V. cholerae strain. For example, mutation of the EAL domain phosphodiesterase VieA in the classical biotype of V. cholerae (strain O395) causes marked effects on motility, biofilm formation and virulence gene expression [There may also be strain-dependent differences in the panel of c-di-GMP metabolic enzymes used, and different results might be obtained using a different pression ,40; the pression ,47.Xanthomonas spp., for example, the HD-GYP phosphodiesterase RpfG interacts with two diguanylate cyclases to regulate motility [Pectobacterium atrosepticum lacks apparent c-di-GMP phosphodiesterase activity (and lacks the conserved HD motif), yet still impacts c-di-GMP regulated processes, possibly via direct interactions with other proteins [V. cholerae may also rely on protein-protein interactions to mediate their effects, and the necessary interactions may not have been achieved through over-expression or mutagenesis.HD-GYP domain proteins can also perform their regulatory function via physical interactions with other proteins, independently of c-di-GMP phosphodiesterase activity. In motility ,66,67. Tproteins . The HD-V. cholerae, and only some mutants showed altered motility and/or biofilm development [V. cholerae to colonize the infant mouse small intestine, but a combination of mutations (ΔHDGYP7) significantly impaired colonization. It is possible that a combination of two or more HD-GYP domain proteins, or that the additive effects of multiple c-di-GMP PDEs, influence virulence of V. cholerae. These findings suggest that HD-GYP domain proteins as a family influence c-di-GMP levels during infection, which has previously been shown to impact V. cholerae virulence by regulating the virulence gene regulator ToxT [Finally, there is potential for phenotypic redundancy among the HD-GYP domain phosphodiesterases, as well as with EAL domain phosphodiesterases. Upon mutation of each of the GGDEF and EAL domain genes in elopment ,45. Thertor ToxT ,15.V. cholerae to induce virulence gene expression in the host despite the absence or mitigation of one specific extracellular signal. A different set of c-di-GMP metabolic enzymes could modulate biofilm or motility in response to a distinct set of signals.In the context of an infection, discrete extracellular signals may trigger the production or activity of a specific c-di-GMP phosphodiesterase (or diguanylate cyclase), with cumulative but targeted effects on virulence gene expression. Strictly speaking, the c-di-GMP phosphodiesterases involved would not be mechanistically redundant , but work together toward the same phenotypic output. Such a regulatory system may allow V. cholerae, only a subset of the HD-GYP domain genes encoded are functional c-di-GMP PDEs. The individual HD-GYP domain genes are dispensable for motility and biofilm formation under the conditions tested, but collectively two or more of the genes are important for virulence of V. cholerae. Additional studies of HD-GYP domain proteins from other bacteria are needed to gain a full understanding their roles in c-di-GMP signaling.Compared to GGDEF domain diguanylate cyclases and EAL domain phosphodiesterases, the HD-GYP domain phosphodiesterases have been understudied. This may be partly due to the fact that genes encoding HD-GYP domains are less widespread in bacterial genomes than those encoding GGDEF and EAL domains ,65. In V


Caulobacter crescentus system, which carries dozens of these pairs, the streamlined bacterial endosymbiont Wolbachia pipientis encodes only two pairs: CckA/CtrA and PleC/PleD. Here, we used bioinformatic tools to compare characterized two-component system relays from C. crescentus, the related Anaplasmataceae species Anaplasma phagocytophilum and Ehrlichia chaffeensis, and 12 sequenced Wolbachia strains. We found the core protein pairs and a subset of interacting partners to be highly conserved within Wolbachia and these other Anaplasmataceae. Genes involved in two-component signaling were positioned differently within the various Wolbachia genomes, whereas the local context of each gene was conserved. Unlike Anaplasma and Ehrlichia, Wolbachia two-component genes were more consistently found clustered with metabolic genes. The domain architecture and key functional residues standard for two-component system proteins were well-conserved in Wolbachia, although residues that specify cognate pairing diverged substantially from other Anaplasmataceae. These findings indicate that Wolbachia two-component signaling pairs share considerable functional overlap with other α-proteobacterial systems, whereas their divergence suggests the potential for regulatory differences and cross-talk.Two-component regulatory systems are commonly used by bacteria to coordinate intracellular responses with environmental cues. These systems are composed of functional protein pairs consisting of a sensor histidine kinase and cognate response regulator. In contrast to the well-studied  Rhizobium and Agrobacterium, as well as virulence properties of pathogens like Vibrio sp., Brucella sp., and Pseudomonas sp. , consisting of a sensor histidine kinase (HK) and paired response regulator (RR) . A largeonas sp. . In addiCaulobacter crescentus, which has a complex, dimorphic life cycle, encodes 62 HKs and 44 RRs (Anaplasma phagocytophilum and Ehrlichia chaffeensis have retained only 3 HKs and 3 RRs (c-di-GMP), and NtrY/NtrX, which coordinate nitrogen sensing with changes in gene expression . On activation, the CA domain of CckA transfers a phosphate from hydrolyzed ATP to a conserved histidine (His) in the DHp domain . This enables CtrA to function in both transcriptional regulation and inhibition of chromosome replication  is referred to as a “hybrid” histidine kinase . It has lication .Figure S1B). The CA phosphorylates the conserved His within the DHp, which transfers the phosphate to the cognate RR, PleD or NtrX, respectively . Genomes documented as fully complete or near-complete were selected for further analysis and classified according to supergroup identity, as indicated by prior phylogenetic analyses   . All sucWolbachia strains wOo, wBm, wMel, wPip Pel, wHa, wNo, and wRi, as well as for A. phagocytophilum and E. chaffeensis. First, the position and orientation of the origin of replication (ori) relative to hemE were identified  was included in Genomic positions for TCS genes and the associated ORFs of interest were determined for the completely sequenced and assembled http://archaea.u-psud.fr/absynte/) (www.microbesonline.org) and the program DOOR: Database of prOkaryotic OpeRons (http://csbl.bmb.uga.edu/DOOR/)  and 9-mer (TTAA-N7-TTAAC) sequences were identified on + or − strands, using the “find” function in CLC Sequence Viewer (version 7.5) . To confirm the wAlbB cckA sequence, genomic DNA samples of wAlbB were collected from Sau5B mosquito tissue culture cells and wAlbB-infected A. albopictus mosquitoes, kindly provided by Jason Rasgon, Pennsylvania State University. The DNeasy Blood and Tissue extraction kit was used to extract purified DNA . Wolbachia DNA was also harvested from several Wolbachia-infected Drosophila stocks using the same method. D. melanogaster stocks of the genotype w; Sp/Cyo; Sb/TM6B were used, which had been infected previously with the wMelPop or wMel Wolbachia strains . For wAlbB, cckA fragments were PCR-amplified from both tissue culture and whole mosquito DNA samples using forward 5′-AAGGAAGCGATTGAACATGG and reverse 5′-AGCAAAGGCTGTYGAYAAAT primers. Thirty rounds of PCR were performed at an annealing temperature of 56° for 30 sec and product extension was performed at 72° for 2 min. Resulting PCR fragments were analyzed on a 1% agarose gel and prepared for sequencing using ExoSAPIT according to the manufacturer’s protocol . ABI BigDye (R) Terminator v3.1 cycle sequencing reactions using the terminal forward and reverse primers, as well as specific internal primers, were analyzed on an ABI 3100 Genetic Analyzer with sequencing analysis and Genescan software . Coverage of greater than 6× was obtained for each sequence, and nucleotide identities were manually checked against alignments. Sequence information for the entire pleD region from each of the Wolbachia fly–host combinations was also obtained, confirming deposited sequences.Full-length C. crescentus, A. phagocytophilum, E. chaffeensis, and all Wolbachia strains were complied and cross-referenced to CLC Sequence Viewer-deduced sequences . Corresponding protein accessions, annotated lengths, and percent identity/similarity to E. chaffeensis homologs were compiled. Domain structure and conserved motifs/residues were then identified using Pfam database annotations and the Simple Modular Architecture Research Tool    to determine the probability of membrane spanning helixes (to a cut-off of P = 0.8) as well as the Phyre 2.0 server (http://www.sbg.bio.ic.ac.uk/phyre2) to determine the likelihood of secondary structure formation consistent with other predictions  was used to further investigate PAS-domain secondary structure predictions in Wolbachia CckA. This program confirmed alpha-helix and beta-strand predictions consistent with the classic 5-beta strand PAS-fold for CckA from all Wolbachia strains. Additional ligand-binding potential was indicated by the Phyre 2.0 3DLigandSite server (http://www.sbg.bio.ic.ac.uk/3dligandsite/) for which confidence values had an average LnE ≥10 for all PAS domains  . In accordance with this, we used predicted amino acid sequences from the closer phylogenetic relative, E. chaffeensis, to identify TCS homologs in Wolbachia . This is seen for pleD of wOo as well as for cckA of wAlbB and wMel.The widespread use of TCS by eubacteria raises the question of how widely these genes have been retained in endosymbiotic olbachia . We searde hosts . This redetected . One of Wolbachia TCS gene could dramatically affect signaling processes in a system lacking functionally redundant genes. To confirm the basis for the split ORF predictions, we re-examined the deposited sequences of wOo pleD, wAlbB cckA, and wMel cckA genes. For wOo pleD, nucleotide sequence alignments and visual inspection revealed multiple nucleotide substitutions leading to four stop codons between wOo_06950 and wOo_06960. A frameshift was also detected that positions these ORFs in different reading frames. Because these data cannot be substantiated by any single sequencing error in relation to other Wolbachia pleD genes, these findings are consistent with a split ORF prediction in the wOo pleD locus.A split ORF in any wAlbB cckA locus revealed five in-frame stop codons, partitioning the gene into two annotated ORFs, WALBB_620009 and WALBB_620010. Because all of these changes could be attributed to a single nucleotide deletion, it was unclear whether this change was genuine or reflected an artifact in the deposited sequence. Our re-sequencing of this cckA region, using wAlbB DNA isolated from both A. albopictus tissue culture cells and intact mosquitoes, revealed an exact match with the deposited sequence. Thus, data obtained from our two independent samples confirm the split ORF prediction for wAlbB cckA.Investigating the basis for the prediction in wMel cckA also indicated that the split ORF prediction was potentially attributable to a single nucleotide addition in the deposited sequence, creating a stop codon that partitioned wMel CckA into the ORFs WD1215 and WD1216. To verify whether this split ORF prediction is accurate, we sequenced cckA of wMel carried by D. melanogaster . This indicates that wMel cckA is more likely encoded by a single ORF, analogous to cckA in other Wolbachia strains. Further analysis of wMel CckA, presented below, is done in accordance with this finding.Analysis of the genomic region for nogaster  and in aWolbachia raises other questions about how well the overall TCS regulatory network is conserved. In the Caulobacter system, a complex network of kinases and phosphotransfer proteins affects the signaling ability of CckA and PleC . This sequence, encoding an approximately 400-amino-acid-long N-terminal fragment of DivL, will be referred to as dvlL (for DivL-like) in this analysis. A. phagocytophilum, E. chaffeensis, and 11 of 12 Wolbachia strains analyzed all contained dvlL. The status of dvlL was inconclusive in the wUni Wolbachia strain due to lack of sequence coverage in that region of the genome . Because these strains also carried 2 CcrM methylation sites within 400 base pairs of the ctrA start site (unpublished observation), the presence of ccrM has possible implications for Wolbachia TCS and cell cycle regulation.α-Proteobacteria are known to carry other factors that modulate CtrA activity as well . These iori) for DnaA to bind and initiate DNA replication; RcdA and PopA, which facilitate CtrA interaction with ClpX/P; and EAL-domain phosphodiesterase proteins, which hydrolyze the c-di-GMP second messenger produced by PleD . These results taken together indicate that Wolbachia have retained a subset of factors that regulate TCS activity.Many α-proteobacteria have been shown to use additional regulatory proteins to drive shutdown of CtrA and PleD outputs through degradation . These iWolbachia share core TCS-related genes with Anaplasma and Ehrlichia, we asked whether the overall positioning of these genes is also conserved in Wolbachia. To address this, we created syntenic alignments using the genomes of completely assembled Wolbachia strains. These were aligned with respect to the ori locus and oriented according to the proximal hemE gene .The positioning of genes throughout the bacterial genome has a strong impact on relative expression throughout the cell cycle . This differential positioning raises the possibility that Wolbachia TCS gene dosage may differ appreciably from A. phagocytophilum and E. chaffeensis during the cell cycle   DNA binding protein and downstream by xnse, which encodes a 3′–5′ exonuclease family protein . This genomic arrangement was similar in wBm and wOo, although the neighboring operon may be fragmented or incomplete. The 3′ end of Wolbachia ctrA was flanked by a variety of genetic regions that differed according to supergroup . Interestingly, a gdhB-hp-pleD–containing operon was also consistently predicted in Wolbachia, with the addition of a chaperonin gene, clpB, included at the 5′ end of the operon  . Two of these “PAS-like” domains were found in A. phagocytophilum and one was found in E. chaffeensis. Neighboring this N-terminal “sensor” portion, a dimerization/histidine phosphotransfer (DHp) domain was predicted. The DHp contained the conserved His residue, as well as two closely flanking residues that impart both kinase and phosphatase capabilities to the DHp (Table S5) (If  the DHp  (Willett the DHp . Followiable S5) .Wolbachia CckAs were predicted to carry internal DHp and CA domains, a C-terminal REC domain, and all the key functional residues associated with those domains (Table S5) . The conservation of these structural features suggests a functional role for CckA has been conserved in Wolbachia. Furthermore, examination of DvlL domain structure indicated the three previously identified PAS domains, as well as complete conservation of DvlL between all Wolbachia strains  . The C-terminal helix-turn-helix (HTH) domain was also confirmed, and all Wolbachia strains carried the conserved α3-helical residues required for DNA binding .The response regulator CtrA was strikingly conserved in its domain structure between  binding  . The Wolbachia PleCs were similarly organized in nearly all strains, carrying a pair of transmembrane domains, an internal DHp domain, a C-terminal CA domain, and all key residues. PleC of wPip JHB was distinctive in the loss of a transmembrane domain, and wOo was, as noted, predicted to lack PleC altogether . One exception was wAlbB PleD, which carried an Asp-to-Tyrosine substitution in the internal REC domain. The other exception was wOo PleD, in which the REC domains were separated by a split ORF, and the dissociated REC carried an Asp to Asn substitution. The GGDEF domain at the PleD C-terminus was also shared between Wolbachia and other Anaplasmataceae .This study has revealed that the core TCS factors CckA, CtrA, PleC, and PleD and several of their interacting proteins were conserved between C. crescentus  (dnaA in all Wolbachia strains analyzed also supports an important role for CtrA in regulating genome replication. A recent study analyzing eight strains of Wolbachia identified three DnaA binding sites and up to five CtrA consensus binding sites per ori (Wolbachia genus.Of all Caulobacter compared with the Anaplasmataceae. Although PleC is generally conserved between these species, no sensory PAS domains were detected in A. phagocytophilum, E. chaffeensis, or Wolbachia PleC (Wolbachia PleC functions in an unregulated manner. Because PleC contains residues essential for both kinase and phosphatase activity, its function may also be heavily influenced by ATP availability. It is also possible that Anaplasmataceae PleC senses periplasmic cues through non-PAS structural features or is regulated by factors associated with the plasma membrane, as has been shown in Caulobacter (TCS domain comparisons highlight distinctions between the PleC sensing capacity in Wolbachia PleD function are also suggested by variation in the PleD REC domains of two Wolbachia strains. Previous work suggests that the PleD N-terminal REC is mainly responsible for regulating PleD GGDEF activity (Wolbachia, loss of an Asp residue from the internal REC domain of wAlbB PleD may have little functional impact. In wOo PleD, however, the original N-terminal REC lacks this key Asp residue and is further predicted to be physically separate from the fully conserved GGDEF domain. In this case, the remaining REC domain may regulate the GGDEF, analogous to the WspR protein in P. aeruginosa (c-di-GMP binding may have a primary regulatory role (Wolbachia PleDs, including wOo, is consistent with this possibility. Because the complexity of second messenger signaling by c-di-GMP has been unaddressed in Wolbachia and many other symbiotic bacteria, this remains a poorly understood area of host–microbe interaction studies.Insights into ruginosa . AlternaWolbachia and other systems including A. phagocytophilum and E. chaffeensis (Wolbachia CckA largely matched those of Ehrlichia, Wolbachia CtrA, PleC, and PleD cognate specificity residues varied extensively, consistent with the potential for loss of HK/RR interaction specificity of other systems (Wolbachia strains. Perhaps CckA/CtrA and PleC/PleD have co-evolved in a manner that preserved spatially constrained, specific interactions between these TCS pairs. An alternative explanation is that cross-talk is common and necessary in the streamlined Wolbachia system (Wolbachia in the context of the host environment. Together, work on this important endosymbiont and its divisional regulation will help to inform the mechanisms underlying Wolbachia titer regulation and interactions between Wolbachia and host.Analysis of the key residues that specify pairing between TCS proteins has also shown differences between a system . Future


Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity.C-di-GMP  is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis  from  C-di-GMP is a ubiquitous second messenger in bacteria. It was first reported as an activator regulating cellulose synthesis134789101112Xanthomonas campestris, the HAMP and TM domains are essential for the dimerization of DGC domainsThe GGDEF domain is responsible for the catalytic activity of DGCs. Previous studies on GGDEF domain-containing proteins  have proposed that the activity of DGCs requires the cooperative action of two GGDEF domains that individually bind one molecule of GTP1416In addition to activation by oligomerization, the activity of GGDEF domains can also be regulated by product inhibition. More than 60% of GGDEF domains contain a conserved RxxD motif that is bound to the product c-di-GMP and that facilitates product feedback inhibition20PA2771 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is a major drug-resistant secondary infection source in hospitals. The observation that PA2771 shows the same distribution as the type III-delivered cytotoxin-encoding exoSAlthough the structures of the previously studied GGDEF domain-containing proteins have provided a general model for the mechanism of oligomerized activation and product inhibition, exceptions do exist in some proteins, especially in those with various additional signaling domains. Our study was focused on the protein product of the P. aeruginosa strainsGMP-specific phosphodiesterase, adenylyl cyclases and FhlA) domain, which is a common signaling domain, and a C-terminal GGDEF domain. This domain organization indicates that PA2771 synthesizes c-di-GMP. However, little is known regarding its enzymatic activity and the regulation of its activity. In addition, there is little known about its effect on bacterial behaviors, such as biofilm formation and motility.The PA2771 gene is located on the genomic island of strain PAO1, which has not been reported in strain PA14 and six other In vitro activity assays show that PA2771 is a highly active diguanyl cyclase, a result consistent with its possession of a GGDEF domain. To study the mechanism of PA2771 catalysis and regulation, we also crystallized full-length PA2771 and the GGDEF domain of PA2771 in complex with c-di-GMP. The crystal structure of full-length PA2771 revealed the active site and product inhibition site of the enzyme. In the full-length structure, PA2771 forms dimers via the GAF domain. Intriguingly, the two monomers in the dimer block the I-site of each other with a loop (protecting loop) extending from the GAF domain. However, in the GGDEF domain/c-di-GMP complex structure, the GGDEF domain dimerizes via the two c-di-GMPs from the inhibition sites. Because I-site product inhibition self blockage is a special feature of PA2771, we named the protein Dcsbis (diguanylate cyclase with a self-blocked I-site). From the structural and biochemical data, we propose a potential regulatory model for the regulation of the DGC activity of Dcsbis.Here, we show that PA2771 controls cell motility without substantially affecting biofilm formation. ΔPA2771) of PAO1 and compared its behavior with that of the wild type strain (WT). According to previous studies, GGEDF-domain containing proteins, such as SadCTo study the biological function of PA2771 in PAO1, we constructed a PA2771 deletion mutant  of the RxxD motif also exists in Dcsbis, and it is located upstream of the GGEEF active site. The sequence is RPED (Arg-Pro-Glu-Asp). To investigate whether the I-site facilitates product inhibition by binding to c-di-GMP in Dcsbis, we explored the DGC activity of full-length Dcsbis in the presence of 5 μM c-di-GMP. The release of PPi was reduced in the presence of additional c-di-GMP, a result indicating that the production of new c-di-GMP was hindered . These rP21 and contained four molecules in the asymmetric unit.To investigate the molecular mechanism underlying the high activity of Dcsbis and the regulation mechanism of DGC activity by the N-terminal GAF domain, we determined the crystal structure of full-length Dcsbis. We first crystallized selenomethionine-incorporated Dcsbis. The structure was solved at a resolution of 2.9 Å by using the single-wavelength anomalous dispersion (SAD) technique. The higher resolution native structure (2.5 Å) was solved by molecular replacement, using one molecule from the selenomethionine structure as the search model. The details of the refinement data are shown in The overall structure showed that the protein contains an N-terminal GAF domain and a C-terminal GGDEF domain, as predicted. The crystal packing analysis revealed the presence of a tightly associated homodimer, which was verified by gel filtration analysis (data not shown). The dimerization is mediated by helices α2 and α5 from the GAF domain of the two monomers .Acinetobacter sp. phosphoenolpyruvate-protein phosphotransferase . However, the sequence identity was only 15%, which may imply differences in the function of the GAF domain in these two proteins, despite the similarity in their three dimensional folding.A DALI server searchFollowing the GAF domain, the GGDEF domain has a typical DGC structure. It superposes well with the GGDEF domain of the known DGC, PleD (with an RMSD of 2.3 Å for 162 Cα atoms) and WspR (with an RMSD of 2.3 Å for 162 Cα atoms). Similarly, Dcsbis contains a conserved active site in the GGEEF motif (A-site) and a conserved product inhibition site in the RxxD motif (I-site) located antipodal to the A-site. Interestingly, in the closely packed Dcsbis dimer, the A-sites of the two monomers face away from each other, and the I-site is buried in the structure . This reC2 space group. The structure was determined using molecular replacement with the GGDEF domain from the full-length protein structure as the search model. There were two molecules in the asymmetric unit. The two monomers face each other, with the I-sites from both molecules in proximity. After molecular replacement, differences in electron density appeared between the I-sites of the two monomers. This extra density may be explained by building in two mutually intercalated c-di-GMP molecules  and protein dimerization29The Dcsbis molecules in the crystal are tightly dimerized by a four-helix bundle . In this structure, the α1 helix does not contribute to the dimerization , unlike There is a large pocket located between the six-stranded β sheet floor and the roof made of α-helices α3 and α4 in the GAF domain . To inve31in vitro, it was speculated that the GAF domain promotes the DGC activity of the GGDEF domain. This increased activity may result from the dimerization via the GAF domain. The unknown ligand might play a role in the regulation in vivo.Given the higher activity of full-length Dcsbis compared with that of the GGDEF domain alone  in vitroAs shown in 2021One hypothesis to explain this observation is that the product, c-di-GMP, does not bind as efficiently to full-length Dcsbis as it does to other proteins, reducing the product inhibition effects. To test this hypothesis, we measured the binding of c-di-GMP to Dcsbis with ITC, and no apparent interaction was detected , thus inFurther analysis of the crystal structures provided the molecular basis of the special property of the Dcsbis protein. Although the overall folding between the apo GGDEF domain in the full-length protein is very similar to that in the c-di-GMP bound state, the environment around the I-sites is very different. The superposition of the GGDEF domain/c-di-GMP complex onto the monomers of the Dcsbis full-length dimer revealed intermolecular interactions in the I-site. A protecting loop  from the GAF of one monomer (MonoA) inserts into the I-site of MonoB and clashes with the docked c-di-GMP molecule . Given tThus, we propose that the protecting loop in the GAF domain prevents c-di-GMP from binding to the I-site (self-blocking I-site) of Dcsbis until c-di-GMP reaches a very high level in the reaction solution. This hypothesis is supported by the observation that the catalytic reaction turned off earlier when c-di-GMP was present, whereas the initial rate of the reaction was almost not affected . This hyVmax and an unchanged Km in the presence of c-di-GMP when the reaction begins. If this is true, then we would be able to conclude that c-di-GMP truly is a noncompetitive inhibitor of Dcsbis. Km of 88 μM and a Vmax of 2.1 μM/m without c-di-GMP. The enzyme had a much higher Km of 300 μM and a slightly lower Vmax of 1.6 μM/m when 50 μM of c-di-GMP was added prior to GTP. This result fundamentally deviates from the widely accepted theory of noncompetitive inhibition for c-di-GMP. The kinetics can be explained by a self-blocked I-site, which may be partially occupied at high c-di-GMP concentrations. The greatly increased Km suggests that c-di-GMP mainly binds to the active site as a competitive inhibitor, as in the case of XCC4471. Although our ITC data showed that c-di-GMP did not tightly bind to Dcsbis, the kinetic data suggest that c-di-GMP outcompetes GTP for the active site at comparable concentrations.Previous studies on DGCs have proposed a widely accepted mechanism that c-di-GMP functions as an noncompetitive inhibitor that binds to the I-site of the canonical DGC domain, thus shutting down catalysis through an allosteric effect. Our structures and preliminary biochemical data, however, suggest a new mechanism for Dcsbis. Dicsbis has an intact I-site but is not inhibited by c-di-GMP as intensely as other canonical DGCs with an intact I-site. To clarify the inhibition mechanism of Dcsbic, we performed a kinetic study of catalysis with or without c-di-GMP in the reaction solutions. We hypothesized that if c-di-GMP is still a noncompetitive inhibitor for Dcsbis, the enzyme would show a much lower P. aeruginosa PAO1 and PA1436 C-di-GMP is a ubiquitous signaling molecule in bacteria. The DGC and PDE proteins involved in the metabolism of c-di-GMP are redundant. A total of 39 proteins related to c-di-GMP homeostasis have been identified in Rhodobacter sphaeroidesMycobacterium smegmatisOur studies showed that full-length Dcsbis possesses a high level of DGC activity, whereas its GGDEF domain has residual diguanylate cyclase activity. This result is similar to that of DgcA from From the crystal structure of full-length Dcsbis, we found that the GAF domain acts as a bridge in the dimerization of Dcsbis. This dimerization brings the GAF domain of one monomer close to the GGDEF domain of the other monomer. Furthermore, the I-site of the GGDEF domain from one monomer is blocked by the protecting loop between β5 and β6 of the neighboring GAF domain. The observation that c-di-GMP is not a noncompetitive inhibitor of full-length Dcsbis is supported by the crystal structure. The blockage of the I-site might be the primary cause of the high catalytic activity of Dcsbis and the loss of product inhibition. With this novel characteristic, Dcsbis is a highly efficient diguanylate cyclase that may be used in industrial applications to produce large amounts of c-di-GMP at a low cost.in vitro, GTP binds to the active site and induces a conformation change in the active dimer, thus leading to the proximity of the two active sites in both monomers. Two GTPs are then brought close to each other and form c-di-GMP. When the environment changes, bacteria favor a highly motive life style, and some unknown small molecules may bind to the GAF domain as a chemical signal. The binding of the signal molecule to the GAF domain would, in turn, trigger a significant conformation change and cause the protecting loop to leave the I-site. C-di-GMP in the cell would then bind to the exposed I-site, which would shut down the synthesis of c-di-GMP through an allosteric mechanism. Over time, the environment may change to favor lower motility, and the concentration of the signal molecule may drop, thus resulting in the release of a signal molecule from the GAF domain. The protecting loop would then block the I-site again and prevent the binding of c-di-GMP, which would turns on the high enzymatic activity once again. Because the peptide segment of the protecting loop also occurs in many Dcsbis homologues of Pseudomonas species, this mechanism may occur in many other bacteria .From our structural and functional analyses, we propose a model for the activation and inhibition of Dcsbis . The cryWe demonstrated that Dcsbis is a functional diguanylate cyclase that tightly coordinates cell motility. However, the entire signaling pathway is largely unknown. Our results suggest that there should be an upstream signal molecule capable of binding to the GAF domain. The relevant downstream c-di-GMP receptor of the pathway is also unclear. We showed that the PA2771 gene is related to cell motility. Whether this gene is relevant to other cellular functions, such as drug resistance, virulence and cell division, remains an open question. All these issues are expected to be addressed through further research.P. aeruginosa and E. coli strains were cultured in LB broth, except that the selective medium for P. aeruginosa was VBMM with citrate as a carbon sourceE. coli and with 300 μg/ml carbenicillin and 100 μg/ml gentamicin for P. aeruginosa.All relevant strains, plasmids and primers are listed in E. coli DH5α cells containing the plasmid pRK2013 and E. coli DH5α cells containing PKO2771, thus generating a double-crossover recombinant. The ΔPA2771 mutant was isolated for gentamicin resistance and sucrose sensitivity.An unmarked nonpolar deletion strategy was used to construct the ΔPA2771 mutant41The PA2771 gene was cloned into pUCP18 to generate the plasmid pPA2771, which was electroporated into WT PAO1 and the ΔPA2771 mutant, respectively.LB medium with 0.3% (wt/vol) agar was used for the swimming motility assay. The bacteria from an overnight culture in LB agar  were placed into swim plates with a sterile toothpick and incubated at 37 °C for 12 h. Swarming motility assays were performed in LB broth with 0.5% (wt/vol) agar.Static biofilm assays were performed with 96-well microtiter platesP. aeruginosa PAO1 and cloned into the pGL01 vector. E. coli BL21 (DE3) was used to express proteins. The transformed cells were grown at 37 °C until the absorbance (OD600) reached approximately 0.8, and this was followed by overnight induction with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation ; resuspended in lysis buffer , and lysed by sonication on ice. The soluble fraction containing Dcsbis was isolated from cell debris by ultra-centrifugation  and subsequently purified to homogeneity by combining nickel affinity , ion exchange , and size-exclusion chromatography  in 10 mM Tris-HCl pH 8.0, 100 mM NaCl, and 3 mM dithiothreitol.The full-length and the GGDEF domain (residues 173-341) of the PA2771 gene were amplified from the genome of The selenomethionine substituted protein was expressed in M9 medium with L-selenomethionine, and the purification procedure was the same as that of the native proteins. The pH of the medium was adjusted to 7.0 with 1 M Tris-HCl pH 8.0 (approximately 10 ml/L medium) before induction with IPTG.Native Dcsbis crystals were grown in sitting drops at 20 °C by mixture of equal volumes of protein (7 mg/ml) with reservoir solution . The selenomethionine-substituted protein was crystallized using a reservoir solution containing 10% PEG 10,000, 0.1 M HEPES pH 7.5 and 5% MPD. The crystals of Dcsbis (173-341) were obtained in a reservoir solution containing 0.5 M sodium citrate tribasic dehydrate and 0.1 M Tris-HCl pH 8.5. All diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 and processed with the software package HKL200046A coupled spectrophotometric assay was performed to monitor the production of inorganic pyrophosphate (PPi) released from the cyclization reaction by using the EnzCheck Pyrophosphate Assay Kit Accession codes: 4ZMM, 4ZMU.How to cite this article: Chen, Y. et al. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation. Sci. Rep. 6, 29499; doi: 10.1038/srep29499 (2016).