Xenorhabdus” (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner – the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The “peptide-antimicrobial- The activated amino acid reacts with the terminal thiol group of the Ppant moiety to form a thioester. The T domain is followed by a condensation domain (C) which catalyses the formation of peptide bonds between the amino acid bound to the T domain of its own module and an amino acid or a peptide chain bound to the T domain of the downstream module. Additional domains with enzymatic activities such as methylation or epimerization can be included in individual modules and may further modify the peptide product. Finally, the nascent peptide is released by a thioesterase (TE) domain localized at the C-terminus of a specialized termination module. TE domains can release either linear, cyclic or branched cyclic peptides  use amino acids as building blocks for the synthesis of complex natural products. Each amino acid is incorporated into the final product by an individual specialized functional module of the NRPS. In turn, each individual module contains a number of enzymatically distinct catalytic domains. A typical NRPS elongation module contains at least an adenylation (A), a thiolation (T) and a condensation (C) domain. The adenylation (A) domain uses ATP to activate a specific amino acid as aminoacyl adenylate and then transfers the activated amino acid to the neighbouring thiolation (T) domain. The T domain in its The individual functional modules necessary for the stepwise incorporation of each amino acid into the final product can be located either on a single long protein chain or on multiple proteins. When all modules are arranged on a single protein chain their linear order directly predicts  in PaxA was predicted to be very short in comparison to other structurally characterized types of docking domains. Thus, in this case the T domain directly preceding the CDD of PaxA might play a supporting role in establishing a specific non-covalent interaction with the N-terminal docking domain (NDD) of PaxB. As a prerequisite for investigating a putative role of the T domain in this DD interaction and for establishing a structural basis for the specific interaction between PaxA and PaxB in the PaxS NRPS from Xenorhabdus cabanillasii JM26 we present here the NMR resonance assignments for a PaxA T1-CDD di-domain construct and for the PaxB NDD in their free states and in a 1:1 non-covalent complex. The T1-CDD (amino acids 981–1084 of PaxA) comprises 104 amino acids (12 kDa), whereas the NDD (amino acids 1–30 of PaxB) contains only 30 amino acids (3.6 kDa).The PaxS NRPS found in Gram-negative entomopathogenic bacteria from the genus 1-CDD and PaxB NDD were generated by PCR amplification using the genomic DNA from Xenorhabdus cabanillasii JM26 as the template. All protein sequences referred to in this work are based on the UniProt Archive (UniParc) entries for PaxA (UPI0003E57C57) and PaxB (UPI000C04EDD1) based on a genome assembly for Xenorhabdus cabanillasii JM26 produced in our group -tag followed by a SUMO (SMT3)-tag which also functions as a cleavage site for the ULP1 protease ΔentD  Gold (Agilent Technologies/Stratagene) cells.The DNA coding sequences for PaxA T600 ~ 0.7 with 1 mM IPTG at 20 °C for ~ 18 h in media supplemented with ampicillin (100 µg/ml). The expression of uniformly 15 N- and 15 N,13C-labelled proteins was achieved by using M9 minimal medium supplemented with 1 g/l 15NH4Cl or 1 g/l 15NH4Cl and 2.5 g/l 13C6-D-glucose (Cambridge Isotope Laboratories). For the stereospecific NMR assignment of γ1/2 and δ1/2 methyl groups of valine and leucine, the PaxA T1-CDD as well as the PaxB NDD were expressed as fractionally 13C-labelled proteins in M9 minimal medium containing a mixture of 0.25 g L−1 13C6-D-glucose and 2.25 g L−1 unlabeled glucose as the sole carbon source  and cOmplete protease inhibitor (Roche). The cell debris was cleared from the lysate by centrifugation  and the supernatant was run through a HisTrap HP column . With the help of a recombinantly produced His6-tagged ULP1 protease the His6-tagged SUMO (SMT3)-tag was cleaved off from the recombinant fusion proteins, leading to native target protein sequences. Both His6-tagged ULP1 and the SUMO-tag were separated from the protein of interest by a second immobilized metal ion affinity chromatography using a HisTrap HP column, followed by gel filtration chromatography with a HiPrep 16/60 Sephacryl S-100 high resolution column . For NMR measurements, the samples of the individual proteins (300 µM protein concentration) and the PaxA T1-CDD:PaxB NDD/PaxB NDD:PaxA T1-CDD complexes (300 µM protein A concentration: 360 µM protein B concentration) were prepared in a buffer containing 50 mM sodium phosphate, pH 6.5, 100 mM NaCl, 2 mM β-mercaptoethanol and 5% (v/v) D2O.Cell lysis of 1H chemical shifts were internally referenced to DSS, whereas the heteronuclear 13C and 15 N chemical shifts were indirectly referenced with the appropriate conversion factors NH, CBCA(CO)NH NH, H(CCO)NH (mixing time 12 ms) and (H)C(CO)NH (mixing time 12 ms) experiments NH experiments  Sattler  experime1-CDD contains amino acids 981 to 1084 of the PaxA protein from Xenorhabdus cabanillasii JM26. It has a molecular weight of 12 kDa and consists of 104 amino acid residues. Most of the backbone amid signals are well dispersed in the 1H,15 N-HSQC spectrum of the unbound PaxA T1-CDD as is typical for a well-folded protein. The 1H,15 N-HSQC is expected to contain 101 backbone amide signals since there is no observable amide signal for the N-terminal residue D981 and there are two proline residues  present in the sequence. However, only 99 backbone amide signals were observed and assigned  α-helix in the linker region between the canonical helices α1 and α2. Such an additional helix is also present at this position in some other carrier protein structures  and the non-native Y31. The TALOS-N analysis of the secondary structure suggests the presence of a single continuous α-helix including residues L11 to K22 in the free PaxB NDD (Fig. 13Cγ and 13Cβ chemical shifts of proline P10 are in agreement with a trans conformation for this residue (Schubert et al. 1/2 and leucine δ1/2 methyl groups of the unbound PaxB NDD were stereospecifically assigned.The PaxB 1-CDD and PaxB NDD form a stable 1:1 complex according to analytical gel filtration and NMR titration experiments which is in slow exchange on the NMR time scale. Assignments for both proteins in their bound states were made using samples containing one binding partner in 13C,15 N-labelled form and a 1.2-fold excess of the unlabelled binding partner. The backbone resonances of PaxA T1-CDD bound to the PaxB NDD could be assigned with the same degree of completeness as for the free protein. All expected backbone amide signals except those for H982 and S1027 (99/101, 98.0%; Fig. 1/2 and leucine δ1/2 methyl groups were stereospecifically assigned. In addition, 56.3% of all aromatic side chain proton-bound carbon and carbon-bound proton signals were assigned.PaxA T1-CDD with TALOS-N in its bound state suggests that its secondary structure is very similar to that in its free state but that there is now an additional α-helix present at the very C-terminus suggesting that the predicted docking domain becomes structured upon binding (Fig. The analysis of the backbone chemical shifts of the PaxA Ting Fig. a, bottomNDD (Fig. 1/2 and leucine δ1/2 methyl groups were stereospecifically assigned. In addition, 50% of all aromatic side chain proton-bound carbon and carbon-bound proton signals were assigned. The TALOS-N derived secondary structure for the bound PaxB NDD indicates the presence of two α-helices (Fig. trans conformation in the bound state according to its 13Cγ and 13Cβ chemical shifts (Schubert et al. The backbone assignment of the bound PaxB NDD Fig. b, top in1-CDD construct, the free PaxB NDD and the complex, respectively.The assigned chemical shifts have been deposited in the BMRB under the accession numbers 50,594, 34,576 and 34,575 for the free PaxA T


Molecular simulations reveal that multiple lipid interactions are needed for high-affinity binding of protein domains to membranes. 3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP Cell signaling and trafficking are regulated by peripheral membrane proteins that associate with cell membrane surfaces in a lipid-dependent fashion  bound at a canonical binding site (CA) formed by positively charged residues of the β1/β2 and β3/β4 loops see  2+ ions  simulations enable investigation of both protein-lipid interactions  for the interaction of the GRP1 PH domain with PIP-containing lipid bilayers, we performed REUS-MD simulations  state, in agreement with previous estimates  and a more tightly bound state (B) of the PH domain on the membrane surface. As the PIP3 concentration is increased, the bound state B is increasingly stabilized relative to the encounter state E such that the free energy difference between these two states is 4 kJ/mol for the 1 PIP3 system, increasing to 20 kJ/mol for the 5 PIP3 system.PMFs as a function of the GRP1-PIP3 molecules in each leaflet of the membrane. Thus, free energy surfaces  is characterized by a minimum distance of 0.73 nm and a COM distance of 4.3 nm. The bound state, characterized by a minimum distance of 0.48 nm, can be seen to be split into two orientational states ABound (AB) and BBound (BB), which differ in their COM distance such that BB is closer to the center of the bilayer. From the projection of the free energy landscape in AB and BB states are of comparable stability.From these free energy surfaces, it is evident that there are actually three states of the PH domain interacting with the membrane. The 3 within the membrane  = 2.1 × 10−4, 3 × 10−6, 1.6 × 10−7, 6.3 × 10−9, and 3.9 × 10−10 M, respectively, corresponding to free energies ΔG of −23, −34, −42, −51, and − 58 kJ/mol, respectively. Plotting the free energy minimum in PMFs  suggested that, on average, ~4 PIP3 molecules were bound to the PH domain. Comparing the free energies of binding as a function of the number of PIP3 molecules present with the range of experimental estimates suggests that the most likely state of the PH domain is bound to between three and five PIP3 molecules.Experimental estimates of the dissociation constants for PIP3 binding to the GRP1 PH domain  or to the corresponding di-C8-phosphoinositide in aqueous solution. The second bound state, BB, is not heavily populated for the K273A mutant in the presence of three PIP3 molecules (see fig. S3) in contrast to the wild-type (WT) PH domain under similar conditions (see above). This suggests the mutation perturbs both the strength and mode of interaction of the domain with a PIP3-containing membrane.A single mutation K273A within the canonical PIP binding site results in loss of experimentally detectable PIP3 gives good agreement with the range of measured dissociation constants for this interaction.Our simulations and PMF calculations indicate that a PH domain can bind simultaneously to multiple PIP molecules in a bilayer, via both the canonical and noncanonical sites alongside a third site and further less well spatially defined interactions. Interactions at these three binding sites increase the overall avidity of the PH domain for a PIP-containing membrane. This is seen in the dependence of the binding free energy (above), which indicates that assuming the GRP1 PH domain binds to at least three molecules of PIPEncounter state to two distinct bound states AB and BB that differ in the orientation of the PH domain and the depth of penetration of the bilayer  concentration of PIP3. Thus, the nature (strength and orientation) of the interaction of the PH domain with the bilayer surface will be influenced by nanoscale clustering of PIP3 and possibly of other molecules within the membrane. This correlates with previous studies of clustering of PIP molecules around bound PH domains  in a bilayer can influence the affinity of GRP1 PH for PIPOverall, our studies suggest that recognition of specific cell membranes by PH domain may be achieved by coincidence detection, either of locally clustered PIP molecules or of PIP molecules alongside other anionic lipids P4 molecule (Protein Data Bank ID: 1FGY). A single mutation on the GRP1 PH domain (K273A) was modeled using MODELLER . The systems were solvated with 6000 CG water molecules, and NaCl ions at a concentration of 150 mM were added to neutralize the system. The Martini 2.1 force field . An elastic network model was applied to all backbone particles within a cutoff distance of 0.7 nm to model secondary and tertiary structure . Only for the system of WT (10 PIP3), 20-μs simulation was performed for each replica. Multiple binding and unbinding transitions were observed in the continuous trajectories obtained by following the replicas. The unbiased PMFs after subtracting the effect of the umbrella potentials were calculated by the weighted histogram analysis method  by reweighting the trajectories obtained by the REUS-MD simulation biasing along a single CV, ξ1   is the PMF for association on the membrane. F(r) should be the constant zero above the bound distance b, and then we getzL → ∞, we getb = 1 nm.For binding of a protein to a membrane in a periodic box with the membrane perpendicular to the


The extracellular matrix (ECM) is a macromolecules network, in which the most abundant molecule is collagen. This protein in triple helical conformation is highly resistant to proteinases degradation, the only enzymes capable of degrading the collagen are matrix metalloproteinases (MMPs). This resistance and maintenance of collagen, and consequently of ECM, is involved in several biological processes and it must be strictly regulated by endogenous inhibitors (TIMPs). The deregulation of MMPs activity leads to development of numerous diseases. This review shows MMPs complexity. The extracellular matrix (ECM) is a macromolecules network, composed of collagen, enzymes and proteins , that prThe ECM has many components : Fibers 1 and α2), with approximate dimensions of 300 × 1.5 nm [d-segments with D1–D4 having a length of 67 nm and D5 equal to 0.46 nm [The collagen is the most abundant protein in ECM which gives structural support for cells ,3. DepenN-terminal signal sequence is removed; (2) hydroxylation of lysine and proline by prolyl hydroxylase and lysyl hydroxylases takes place, together with (3) glycosylation of lysine, forming the pro-collagen. This structure is a triple helix chain, but with the unwound terminals. The removal of these terminals occurs in extracellular medium, by collagen peptidases, forming tropocollagen. The microfibril collagen is formed by lysyl oxidase that packs together five tropocollagen chain [d-banding pattern [The collagen synthesis involves several steps  Figure . The mRNher with  glycosylher with  glycosylN-terminal and this is crucial for collagen degradation [Its triple helix conformation makes collagen resistant to many proteases. The enzymes able to cleave this structure are capthesin K and enzymes with collagenolytic activity  [radation ,7  , the desThe MMPs were first described in 1949 , as depoThe MMPs are a family of proteolytic enzyme that have different substrates, but which share similar structural characteristics ,11,13,23MMPs have been recently recognized as biomarkers in several fields  , since tThe principal biologic function of MMPs is degradation of ECM proteins and glycoproteins ,19,24,31The deregulation of MMP activity leads to the progression of various pathologies, that can be grouped into ,19,24,25The MMPs are involved in the development and progression of atherosclerosis, which is correlated with cardiovascular diseases . Their aSome studies show intracellular localization of MMP-2 in cardiac myocytes and colocalization of MMP-2 with troponin I in cardiac myofilaments . The MMPNon-furin regulated MMPs ;MMPs bearing three fibronectin-like inserts in the catalytic domain (MMP-2 and -9);C-terminal glycosylphosphatidylinositol (GPI) moiety ;MMPs anchored to the cellular membrane by a MMPs bearing a transmembrane domain  andAll the other MMPs .In vertebrates, there are 28 different types of MMPs ,13,16,17But, MMPs can be also subdivided according to substrate specificity, sequential similarity and domain organization  into: CoC-terminal and 3/4 N -terminal [Collagenases  cleave sterminal ,14,17,21Gelatinases  play an Gelatinases are involved in physiological and pathological states, such as, embryonic growth and development, angiogenesis, vascular diseases, inflammatory, infective diseases, degenerative diseases of the brain and tumor progression . Tumor mStromelysines  have the2+- binding site [The main characteristic of the matrilysins  is the ling site .C-terminal [Membrane-type metalloproteinases MT-MMP;  contain terminal ,8,17,18,terminal ,17,18 anterminal ,17,18. TLovejoy et al. reported the first structure of MMP-inhibitor complex . This st1-N-terminal peptide with variable length, that targets the peptide for secretion;A signal 2-A pro-domain (with about 80 aa), which keeps MMP inactive and is removed when the enzyme is proteolytically activated;3-A catalytic domain (with about 160 aa), with a zinc ion, that consists of five β-sheets, three α-helixes and three calcium ions;4-A linker of variable length (14–69 aa), which links the catalytic domain to hemopexin-like domain—“hinge region”;5-A hemopexin-like domain (with about 210 aa) that is characterized by four β-propeller and6-C-terminal domain, only present in MMPs-14, -15, -16 and -24.An additional transmembrane domain with the small cytoplasmatic The most common structural features are  1,2,8,9,18,19,23N-terminal signal sequence, which is removed in the endoplasmic reticulum, producing pro-enzymes [All MMPs are synthesized with an -enzymes .The pro-domain  contains2+, that has a structural function [2+ [Active site  consistsfunction ,17,19,21function ,17,19,23function ,19,21 thtion [2+ . The cattion [2+ . The section [2+ ,16,21.Catalytic and hemopexin-like domains are linked by a proline-rich linker ,13,17 ofC-terminal of the catalytic domain [The hemopexin-like domain has four β-propeller structural elements ,12,13,171, S2, S3, S1′, S2′, and S3′) [1, P2, P3, P1′, P2′, and P3′) [1, S2, and S3 pockets are unprimed pockets, which are localized on the right side of zinc ion [1′, S2′, and S3′ pockets are the primed pockets, which are localized on the left side of zinc ion [1′ is the most variable pocket in MMPs [2, S3′, S1, and S3 pockets with an equivalent degree of variance, while the S2′ has the lowest variability. The S2′ and S3′ pockets are shallower than S1′ and are more exposed to solvent [3 pocket may also contribute to substrate specificity [MMPs possess six pockets (Sand S3′)  and the and P3′) . The S1,zinc ion . The S1′zinc ion . It has  in MMPs ,8,15,16,1′ pocket is the most important since it is a determining factor for substrate specificity [1′ subsites, MMPs can be divided into three different subgroups [214 and Tyr214 residue, respectively [1′ pocket [Scificity ,8,16, Itcificity , formed cificity  is a higubgroups ,17,19,36ectively ,36, inst′ pocket .MMP expression is closely controlled for transcription, secretion, activation, and inhibition of the activated enzyme ,9,14,18.MMPs are produced by various tissues and cells . MMPs arC-terminal domain of TIMP-2 [N-terminal of TIMP-2 [Pro-MMP-2 forms a complex with TIMP-2 through interactions of the hemopexin-like domain with the non-inhibitory f TIMP-2 ,17,18,25N-ethylmaleimide) [2+ coordination [The MMP activation can also occur by physiochemical agents, such as heat, low pH, chaotropic agents and thiol-modifying agents  , which cdination .Pro-enzymes and active forms of MMP are controlled by the specific stereochemical binding of metalloproteinase-specific inhibitors  ,18,21,25Over the past 30 years, the detailed structural characterization of different MMPs allowed disentangling of the individual catalytic steps that occur at the active site during proteolysis .2+ is penta-coordinated with a substrate’s carbonyl oxygen atom, one oxygen atom from the glutamate-bound water and the three conserved histidine residues [N-terminal residues and S1′ pocket [N-terminal, glutamate and water [1′ pocket [The catalytic activity requires catalytic zinc ion and a water molecule flanked by three conserved histidine and a conserved glutamate, with a conserved methionine acting as a hydrophobic base to support the structure surrounding the catalytic zinc ion ,2,14 Fi. In the residues ,14,17,18residues ,16,18,21residues ,11,23 wiresidues . This re′ pocket , and by nd water ,16,18,231′ pocket in MMPs and its interaction with the substrate.MMPs are the most important enzymes for ECM maintenance. They are also involved in several biological and pathological diseases. However, MMPs remain a challenge for science, due to their high complexity, both in terms of regulation and activity. To that end, understanding of their role in the development of diseases and their mode of action require further studies. The development synthetic inhibitors in particular, critically depend on the full understanding of the structural details about S


GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies. Bacterial biofilm depends on the modulation of cyclic diguanylate (c-di-GMP) levels in response to environmental cues. This second messenger is also involved in bacterial signal transduction, regulation, virulence and antibiotic resistance. It is synthesized by diguanylate cyclases (DGCs) harboring the GGDEF domain, starting from two GTP molecules. Conversely, it is degraded by dedicated phosphodiesterases (PDEs), belonging to two families namely EAL and HD-GYP, to 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG). Moreover, the HD-GYP family is also able to carry out a second hydrolytic cleavage to produce GMP through the so-called PDE-B activity, via the c-di-GMP → pGpG → GMP pathway  1]. GGD. GGD1]. The variety of domain architectures in c-di-GMP metabolizing enzymes and the heterogeneity of its receptors, allow bacteria to react promptly to many different environmental conditions by modulating their c-di-GMP based adaptation strategy. As a consequence, it has been very hard to draw a unique mechanism for the c-di-GMP-mediated regulation. The only general rule that has emerged to date, is that the c-di-GMP dependent modulation of protein function occurs mainly via restriction of the possible conformations accessible to these multi-domain or multi-subunit proteins ,4.The GGDEF domain of DGCs is emblematic of this control; since only one GTP molecule can bind in the active site, a dimerization step is mandatory for the cyclization reaction to occur and yield c-di-GMP. Consequently, multidomain DGCs usually regulate their activity by allowing the GGDEF domains to dimerize (active dimer) or forcing them apart (inactive conformations), functioning as conformational switches .On the other hand, although in principle the hydrolysis of c-di-GMP could be performed by a single EAL domain, EAL containing proteins have been also shown to function as dimers and, as the GGDEF domains, to function as conformational switches ,6,7,8. FHere we report a re-evaluation of the kinetic profile of selected hybrid proteins as truncated catalytic constructs, showing that the GGDEF domain can slowly convert GTP to GMP as a side reaction. We also show that different GGDEF-containing proteins display this unexpected reactivity, and that this side reaction occurs when the physiological cyclase activity does not take place due to conformational or dimerization issues. We found that when using GTP as a substrate the GGDEF domain may catalyze opposite reactions (condensation or hydrolysis) depending on the presence of upstream domain(s) or on the assay conditions. This variability may bias the kinetic characterization of these proteins and raises some concerns on the design of soluble constructs of GGDEF-containing proteins. This work aims at highlighting these peculiar properties of the GGDEF domain and to propose some useful guidelines to minimize the structural and kinetics bias due to a possible disruption of the GGDEF conformational constraints in engineered constructs.P. aeruginosa harboring only the GGDEF and the C-terminal EAL domains were purchased from Life Technologies and subcloned in Pet28a vectors with an N-terminal His-tag between NdeI and XhoI/HindIII. YfinN GGDEF and PleD were obtained as reported in [Escherichia coli BL21 (DE3) strain. Freshly transformed colonies were grown at 37 °C in Luria-Bertani (LB) liquid medium supplemented with 30 μg/mL Kanamycin (Merck KGaA-Germany). At OD600 ~0.8, protein expression was induced by adding 0.5 mM IPTG  and the temperature was reduced to 20 °C. Cells were harvested by centrifugation after 20 h and stored at −20 °C. All bacterial pellets were suspended in the lysis buffer and then they were lysed by sonication. The supernatant was separated on a Ni2+-IMAC HisTrap column  equilibrated in Buffer A (® concentrators (Merck KGaA-Germany). Monodisperse protein was obtained by FPLC, according to their molecular weights, and eluted with buffer A using an FPLC apparatus   . For Ple2 were incubated with 100 μM GTP and the reaction mixture was kept at 25 °C for different time intervals. No activity was observed when Zn2+ or Ca2+ were used as divalent cations (data not shown). The rates of hydrolase activity were also assayed in the presence of 30 or 60 μM c-di-GMP, if indicated. To test the putative GTPase activity of PleD, 5 μM of enzyme in 20 mM TrisHCl pH 8, 100 mM NaCl and 10 mM MgCl2 was incubated for 10 min at room temperature. To populate the active DGC form, if indicated, the enzyme was incubated at room temperature for 30 min with 10 mM NaF and 1 mM BeCl2. All the reactions, which were carried out with different enzymes, were stopped as described in [The (α-β)-GTPase activity (hereinafter GTPase) of different RmcA derived constructs as well as of PleD, YfiN, RbdA and MorA was analyzed using Reverse Phase HPLC (RP-HPLC) as reported in . Small vv/v, 0.3 mL/min) a mobile phase, setting the UV detector at 254 nm. The product eluted was lyophilized and Mass Spectrometry analysis was done. HPLC/DAD/ESI-TRAP studies were performed using an Agilent 1100 series and an Ion Trap LC/MSD Trap SL G2445D from Agilent Technologies  equipped with an ESI source operating in negative ionization mode.To assess the identity of the GTPase activity product, we optimized the RP-HPLC separation. We separated the unknown species using a 150 × 4.6 mm reverse phase column . Due to the incompatibility of 100 mM phosphate buffer with MS analysis, catalytic products were eluted with 0.1% Formic Acid/Methanol  by mixing equal volumes of 2.5 mg/mL protein solution  with precipitant solution . Before flash freezing in liquid nitrogen, crystals were soaked in a solution of mother liquor containing 20% glycerol as cryoprotectant. A 360° data-set (oscillation of 0.1°) was collected at the BESSY synchrotron on ID-14.1 beamline, at 0.918 wavelength. The best crystal diffracted to 2.8 Å resolution. Data were integrated using XDS [YfiNsing XDS  within tsing XDS  softwaresing XDS  Cycles osing XDS  and RefmP. aeruginosa strain PAO1, a transmembrane GGDEF-EAL containing phosphodiesterase able to recognize L-Arginine in the periplasmic space [We recently characterized the catalytic moiety of RmcA from the ic space  and to dic space . We demoic space . Given tInterestingly, when GTP and c-di-GMP are both present in the reaction mixture, we observed that GMP accumulates after a lag-phase of ~35 min B, contraP. aeruginosa, whose kinetic characterization has been previously carried out [To investigate whether GTP hydrolysis is a general feature of the GGDEF-EAL DUAL moiety, we qualitatively analyze the reactivity of the motility regulator protein MorA from ried out . BrieflyP. aeruginosa [We then analyzed the reactivity of another well-characterized hybrid protein: the positive regulator of biofilm dispersal RbdA from ruginosa . Under oruginosa . NeverthGiven that GMP production is a common trait of the GGDEF-EAL tandem, a series of experiments were done to exclude a side  PDE-B activity. As mentioned above, pGpG may be further hydrolyzed into 2 molecules of GMP , as observed in the HD-GYP subtype of PDEs . To probMoreover, we tested the reactivity of the double mutant of DUAL in the EAL domain D1136N/D1137N (hereinafter DUAL-DD), where the replacement of the two aspartic acid residues hampers the holo active site construction and finally PDE turnover . As showC. crescentus, since it represents the reference system for studying DGCs: this protein populates a stable inhibited state, which is released by phosphorylation to allow the GGDEF moieties to interface and enter catalysis upon GTP binding [If this background reactivity is a general feature of the GGDEF domains, it could bias kinetic studies describing GGDEF-containing proteins; therefore, we probed the reactivity of GGDEF domains with GTP in protein with domain architectures lacking the DUAL motif. We analyzed the reactivity of PleD from  binding . As show binding , is confP. aeruginosa, an inactive truncated version, previously shown to be a monomer able to bind GTP with high affinity [Since the dimer formation is a pre-requisite to enter DGC catalysis, we investigated the possible GMP production of the isolated GGDEF of YfiN from affinity . AccordiGGDEF protein in complex with Mg2+ by X-ray crystallography at 2.8 Å resolution. The crystal contained 3 monomers in the asymmetric unit (https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver) showed that all protein-protein interfaces of the crystal lattice would not be stable in solution, confirming that YfiNGGDEF is monomeric : this may explain the alternative reactivity of the same protein towards GTP, depending on the allosteric status of the protein. This hypothesis has been confirmed by solving the 3D structure of the YfiNric unit A and prosolution ). A magnsolution , shows tM value measured for the EAL domain, [Proteins involved in controlling c-di-GMP levels (and c-di-GMP itself) are characterized by an extraordinary plasticity and their function is deeply governed by allosteric control of other signals or domains. The structural analysis, where possible, reports on multiple conformation and oligomeric state governing protein function ,29. This domain, ), the EA domain,  suggest It is worth mentioning that the GGDEF reactivity deeply changes depending on the experimental setup: in the case of MorA, the experimental conditions reported in Phippen and coworkers includes a very high enzyme concentration (40 µM) and little GTP excess (100 µM), which promotes a DGC turnover. It is known that DGC reaction is dependent on protein concentration for both the active dimer formation step ,28 and tcat comparison). The role of additional domains flanking the catalytic one is not new in the field of GTP-dependent proteins. In circularly permutated GTPases (cpGTPases) the presence on an additional domain located at the C-terminus with respect to the catalytic one is required [Taken together, this analysis confirms that GGDEF-containing multi-domain proteins show not only conformational plasticity but also versatility in their activities at least in vitro: they may act as DGCs, GTP-dependent conformational switches or, to a lower extent, as GTPases  [Up to now, we have no evidence that the GTPase activity has physiological relevance; most likely it is a spurious readout due to a certain experimental set up. According to in vitro parameters, GTPase occurs when c-di-GMP levels are low (close to the K-di-GMP) . Looking-di-GMP) ,36,37, tDespite that the GGDEF domain is evolutionarily conserved and structurally stable, its function is strictly dependent on the neighboring domain(s) or sequences that, independently of their nature, are required to ensure the proper dimerization to enter DGC catalysis. It is possible to obtain recombinant stable and properly folded GGDEF domain from different proteins , both in isolation or in combination with other domains, but their intrinsic reactivity is difficult to be clearly assessed. Their putative DGC and/or allosteric switch roles could be significantly affected or even abolished by the boundaries chosen in the engineering design step. Last but not least, the dependence on protein concentration should be carefully evaluated to characterize these proteins biochemically and to assign to a certain GGDEF domain its role in a multidomain protein.


Q as a function of strain rate and temperature for nickel-based superalloy was presented. The processing maps were generated upon the basis of Prasad stability criterion for true strains ranging from 0.2 to 1 at the deformation temperatures range of 900–1150 °C, and strain rates range of 0.01–100 s−1. Based on the flow stress curves analysis, deformation activation energy map, and processing maps for different true strains, the undesirable and potentially favorable hot deformation parameters were determined. The microstructural observations confirmed the above optimization results for the hot workability of the investigated superalloy. Besides, the numerical simulation and industrial forging tests were performed in order to verify the obtained results.The stress-strain curves for nickel-based superalloy were obtained from isothermal hot compression tests at a wide range of deformation temperatures and strain rates. The material constants and deformation activation energy of the investigated superalloy were calculated. The accuracy of the constitutive equation describing the hot deformation behavior of this material was confirmed by the correlation coefficient for the linear regression. The distribution of deformation activation energy  The hot deformation of nickel-based superalloys is often associated with the difficulty of selecting optimal parameters of processing. The main difficulties with processing such alloys at low deformation temperatures are connected with excessively high flow stresses while high deformation temperatures are accompanied by rapid grain growth . One sucThe microstructure of Waspaloy consists of γ′ precipitations in the γ matrix. Solution heat treatment and subsequent air cooling promote the formation of primary γ′, while multistage aging promotes precipitation of secondary γ′ particles. Chang et al.  noted a −1 dynamic recrystallization (DRX) in this alloy is observed at the temperatures above 1000 °C and low strain rates below 0.01 s−1, while dynamic recovery (DRV) occurred at the temperature of 950 °C. Semiatin et al.  ] − (1/T) relation  and supersolvus (1060–1140°C) deformation for Waspaloy at strain rates ranging from 0.001 s−1 to 1 s−1 as 1400 ± 372 kJ/mol and 462 ± 95 kJ/mol, respectively. The calculated value of Q for Waspaloy (630.17 kJ/mol) is much larger than the values presented by the authors of works [Q are presented in Based on linear regression of the relationships between ln inh(ασ)] a–c at direlation d at diff0 kJ/mol ,36,37,380 kJ/mol , McQueen0 kJ/mol , and Mat0 kJ/mol  calculat0 kJ/mol  calculatof works ,33,39 buof works . It shouof works ,42. The Z parameter. The high correlation coefficient for the linear regression (R2 = 0.997) confirms the accuracy of the constitutive equation (Equation (8)) describing the hot-deformation behavior of Waspaloy.The variation of the Zener–Hollomon parameter depending on steady flow stress e is apprThe constitutive equation of hot deformation for Waspaloy can be expressed as:Q at various temperatures and strain rates does not take into account the microstructural changes during hot working. On the other hand, it is necessary to know the distribution of Q depending on the hot deformation conditions to identify processes associated with DRV, DRX, nucleation, and the growth of precipitation [Q as a function of strain rate and temperature for Waspaloy at true strains of 0.2, 0.6, and 1 is presented in Q varies in the range from 150 kJ/mol to 1200 kJ/mol during the deformation conditions, which corresponds to the results noted earlier for this superalloy [Q parameter with a decrease in the deformation temperature for all true strains, which is due to the increased volume of γ′ precipitations. High deformation temperatures are characterized by the smallest value of activation energy due to the dissolution of γ′ precipitations. Moreover, the increase in the kinetics of dislocation movement, which facilitates deformation, is associated with an increase in temperature  [Generally, a constant value of pitation . The disperalloy , and parperalloy . In genechanism) . This faQ presented on the activation energy map, and as noted earlier, it can be argued that the DRX is a dominant softening mechanism. It should also be noted that a more constant domain of Q value on activation energy map should be considered as optimal for hot working. However, despite the above, for optimization of hot workability of Waspaloy, a comprehensive analysis, including also the processing maps and microstructural observation is needed.It is considered that DRV is characterized by the deformation activation energy equal or close to the activation energy for self-diffusion. From the distribution of G), and the other part, represented by microstructural changes (J), is related to the microstructural evolution related to DRV, DRX and phase transformations [−1); σ—flow stress (MPa).The processing maps in accordance with Prasad’s criterion consist of a power dissipation efficiency and flow instability maps. In DMM hot working is strongly connected with power dissipation. One part of this power is dissipated as heat caused by plastic deformation, represented by heat generation  determines the ratio between heat generation (G) and microstructural changes (J), and can be determined as [T—temperature (°C); and ε—constant true strain value.The strain rate sensitivity parameter (mined as :(10)m=(∂η) as a function of the strain rate sensitivity can be calculated from:The efficiency of power dissipation  corresponds to DRX, while the lower value (20–30%) is associated with DRV. However, it should be noted that high values of η do not always correspond to the safe domains for material workability because the criterion for the occurrence of flow instabilities (ξ) should also be taken into account. This instability criterion is given by the following Equation [Based on the results of the distribution of the efficiency of power dissipation Equation ,29,30:. These processing maps can be designed by the superimposition of instability maps on power dissipation maps. The black isocline lines describe the distribution of the efficiency of power dissipation (η%), while the shaded areas indicate a negative value of instability criterion (ξ). Generally, the value of η tends to increase with increasing deformation temperature and, to a greater extent, with decreasing strain rate. It can also be noted, that the variation of true strains does not have a significant effect on the distribution of η. At all processing maps the domain in which the peak efficiency of power dissipation increases from 40% to 54% with increasing true strain at the temperature of 1050 °C and strain rate 0.01 s−1 is observed. This phenomenon can be the result of an increase in the amount of recrystallized grains with increasing true strain, which increases the amount of energy dissipated by ongoing changes in the microstructure. Taking into account that the higher value of this parameter reflects better workability, the hot-working parameters of this domain for the investigated superalloy can be considered as optimal. η ≥ 30%) is located mainly at the high temperatures ranging from 1000 to 1150. Moreover, each of the maps shown in As can be seen from ξ (shaded areas) presented in The instability domains with negative vales of −1, and decreases with increasing true strain (−1), is decreasing with increasing values of true strain to 0.4  combined with a processing temperature range of 900–1050 °C and strain rates ranging from 0.04 s−1 to 100 s−1. They are characterized by the lowest efficiency of power dissipation  deformation activation energy values  leads to an increase in the exposure of the material to a given temperature, resulting in a large part of γ′ precipitations and carbides as well as in recrystallized equiaxed grain growth (−1. Higher strain rates (0.04–100 s−1) will reduce the exposure time of the material to deformation temperature and, therefore, will increase the temperature of dissolution of large volume fraction of γ′ precipitations.n growth a. An inc−1. These microstructures are characterized by almost full DRX and good stability of the microstructures. As can be seen from The microstructures presented in Based on the results obtained, relating to hot deformation conditions for the investigated superalloy, the numerical simulation as well as the drop forging test of the selected part (gear wheel) in industrial conditions was performed. Generally, it was found that the stable process of hot working of the investigated alloy took place in the ranges of high deformation temperatures (≥1050 °C). Moreover, high deformation temperatures are characterized by lower values of deformation activation energy . Due to The numerical distributions of temperature, mean stress and effective strain in the forged gear wheel in a final stage are shown in The verification based on a complex analysis of the previously obtained results was carried out by industrial drop forging tests of a gear wheel  performeAfter drop-forging tests, the quality of the obtained gear wheel was evaluated. As can be seen in −1 to 100 s−1, and at temperatures in the range of 900–1150 °C. Based on these tests, calculations, and microstructural observations, the following conclusions can be summarized:Q) value, calculated for the investigated Waspaloy, was 630.17 kJ/mol. The high correlation coefficient for the linear regression R2 = 0.997 confirmed the accuracy of the constitutive equation describing the hot-deformation behavior of the investigated superalloy. The distribution of activation energy as a function of strain rate and temperature for Waspaloy at different strain rates varied in the range from 150 kJ/mol to 1200 kJ/mol.The average activation energy (−1) combined with the processing temperature range of 900–1050 °C, and strain rates ranging from 0.04 s−1 to 100 s−1.The undesirable processing conditions include low temperatures (from 900 °C to 1000 °C) and low strain rates range (0.01–0.04 s−1.The most favorable hot-deformation conditions for Waspaloy can be described by the processing map area for the temperatures from 1100 °C to 1150 °C, and at all investigated strain rates as well as the area for the temperatures range of 1025–1100 °C, and strain rates ranging from 0.01 to 0.04 sThe microstructures of the specimens obtained in the instability conditions confirmed the occurrence of the flow instability during deformation. The microstructures of the specimens compressed at optimal hot deformation conditions were characterized by almost full DRX and good deformation stability.The numerical analyses as well as drop forging tests of a gear wheel confirmed the results related to optimization of hot-forging parameters for Waspaloy.The stress-strain curves for Waspaloy were obtained from isothermal hot compression tests performed to a total true strain of 1 at strain rates ranging from 0.01 s


With the demand for high resolution remote sensing, load array technology has gradually become an effective measure to improve imaging resolution. However, the external flow and internal engine vibration disturbance may lead to the flexible deformation of wings. The traditional rigid baseline error compensation method cannot solve the problem of serious coupling movement error caused by flexible deformation. To address the problem, a transfer alignment model based on fiber Bragg grating for distributed position and orientation system is proposed in this paper. Firstly, based on the multidimensional requirements of flexible deformation information, the layout scheme of fiber Bragg grating was designed, then the continuous strain in the wing surface was obtained after the quadratic fitting of strain measured by fiber Bragg gratings, and the deformation displacement and angle are calculated. Thirdly, flexible deformation compensation for distributed position and orientation system based on fiber Bragg grating was studied. The state equation including position error, velocity error, misalignment angle, and inertial device error was established. The position and attitude information compensated by the flexible lever arm was used as the quantitative measurement. The filtering estimation improved the measurement accuracy of the slave inertial navigation systems. At last, the experiment was carried out and showed that the accuracy of the transfer alignment has been improved significantly. With the development of the flight platform technology, it is possible to realize simultaneous observation of multi-remote sensing devices on the same platform using multiple observation windows, such as SAR, visible light camera, imaging spectrometer, and laser radar, which can operate at the same time ,2,3. BecThe subarray antenna is often installed at different wing nodes, which is limited by volume weight and even cost and conducts distributed motion measurement often using MEMS-IMU, and its precision is far lower than that of the main system. When the array antenna SAR is three-dimensional imaging, not only is the motion compensation required at each node, but also the required precision of the baseline length between nodes is very high . At presThe measurement of deflection deformation based on the inertial navigation system could realize the full dimensional information measurement of the position, velocity, attitude, acceleration, and angular velocity of the sub-nodes, which especially realizes the high-precision measurement of the attitude. However, the error of the inertial navigation system is accumulated with time and it is difficult to achieve the sub-millimeter accuracy measurements of baseline vectors that solely relied on the inertial measurements. At the same time, the fiber Bragg grating (FBG) had small volume and light quality, which has become an important means of monitoring the flexible structural parts in the fields of aerospace large buildings and medical devices, while it is widely used in the internal stress, strain, temperature measurement, and detection of structural fracture and deformation . VariousIn summary, the deformation measurement based on FBG and the inertial measurement had their own advantages, which are complementary in flexible baseline measurements for array antenna SAR and InSAR. Therefore, this paper used FBG to obtain the three-dimensional deformation between the main node and the sub nodes, which includes the flexural deformation displacement vector and the deflection angles between the main system and the sub-nodes, thus the high-precision measurement information is obtained, and at the same time, the transfer alignment model based on FBG is established. In addition, the according experiment is designed and implemented for wing deformation measurement.The measurement system consists of a high precision main POS, several sub IMUs, a distributed POS computer system (DPCS), a post-processing software (PPS), and multiple fiber Bragg grating sensors. The main POS is generally assembled on the middle of the plane or in the cabin, while the sub IMUs are assembled on or close to every imaging sensor symmetrically distributed along the wings. DPCS is a multifunctional computer system and mainly completes the IMU data sampling and storage, data synchronization, and real-time fusion computation, which includes SINS/GNSS integration for main POS, transfer alignment algorithm for sub IMUs, and communication with other systems. Multiple fiber Bragg grating sensors can provide measurement information about mechanical distortion, which can improve the accuracy of transfer alignment. The motion parameters of DPOS can be calculated in real-time by DPCS or in post-processing by PPS. The PPS is available to calculate a complex model of massive computation without a time limit and can further improve the precision of DPOS. As a result, DPOS can successively provide high-precision position, velocity, and attitude information to further improve imaging accuracy and resolution for multi-task remote sensing sensors. The operation principle of DPOS is shown in Y-axis, the Z-axis and the torsion variables around the X-axis.The FBG measured the flexure deformation displacement vector that was generated by the flexible baseline and the deflection angle around the three axes between the main system and the sub-nodes, that is, the bending variable around the ith pair of FBG sensor is measured as n long segments on the wing surface, while each segment could calculate the curvature of the center point by strain. The curvature values at each point are obtained by linear interpolation from the curvature values of the n discrete points. Finally, the deformation angle and the line displacement vector are solved by the geometric relationship.Several fiber Bragg grating sensors are pasted on the wing surface, while assuming that the ith pair FBG sensor is symmetric and stickupThe sensitive strain of the X-axis tensile force, X, Y, and Z-axis bending, respectively. Then the line strain produced by the Y-axis bending could be calculated as:In the formula, the linear strain caused by the X-Y plane displacement vector and the deformation angle along the Z-axis; the decoupling of the line strain caused by the pure Z-axis bending is as follows in In the same way, the strain modeling calculated by the FBG is used to calculate the ith pair of lower surface is:The susceptibility strain of the FBG sensor symmetrically pasted by the Z-axis bending could be calculated asThen the line strain produced by the The small arc segments after the wing bending deformation are shown in the Available from geometric relationships:So thatX-Z plane and the deformation angle Y axis in the node coordinate system, the displacement vector X-Y plane and the direction of deformation angle Z axis in the nodal coordinate system could be expressed as:According to the stress and strain analysis of the wing, the strain of the wing root is the largest and the tip of the wing is the smallest. It can be known from Formula (7) that the strain is inversely proportional to the radius of curvature obtained by the modeling calculation, that is, when the root strain of the wing tends to zero, the radius of curvature tends to infinity. When there is a small error in the strain measured by the fiber Bragg grating sensor, the radius of curvature would produce a large error, and the displacement vector calculated by the radius of curvature would also have a large error. Since the strain at the tip of the wing is small, the deformation angle is small, and it is assumed that the length before and after the baseline deformation is unchanged, that is B at 0 degrees, and the measured value of the FBG E, F with symmetric paste measured at 45° is FBG sensor is only sensitive to the line strain in one direction, so the shear strain generated by the wing under the measurement of the torsional moment could only be obtained by modeling. X-axis tensile force, X, Y, and Z-axis bending, respectively. The line strain angle 45° generated at the measuring point under X-axis torsion could be calculated as:In the formula, the oblique 45° linear strain caused by the According to the geometric relationship of the FBG installation, it could be concluded that there is a relationship between the sensitive line strain of FBG installed in the direction of oblique 45°and the FBG installed at 0°.Substitution of Formula (17) into (16), which could obtain:Modeling and analysis of FBG sensors before and after deformation are shown in In the diagram, the deformation angle The geometric relationship could be obtained:It could be solved by Formulas (13) and (14):From When the Substituting Equation (15) into Equations (17) and (18), the deformation angle The navigation accuracy of the strap-down inertial navigation system is influenced by various error sources, which include the self-error of the inertial sensor, the quantization error of the inertial sensor, the initial condition error, the data truncation error, and various interference errors. In this section, from the mechanism of error propagation, the velocity, position and attitude error equations are derived, and the general error model of strap-down inertial navigation system is also given.The velocity errors equation is described as follows:The position error equation of the location differential equation is derived from the longitude, the position error equation is:The attitude error angle is defined as follows:The inertial sensors error is approximated as a random constant and white noise, and the random constant could be described by the following differential equations:Based on the analysis of the transfer alignment error model, the fifteen dimensional state variables of the transfer alignment system model are selected:Among them: Among them, The wing deformation displacement Since the position and attitude measurement information of the main POS could be obtained by using the fiber grating correction, the position and attitude matching method could be used to establish the measurement equation of the sub-IMU transmission alignment:In the equation, (1)Position measurement information correctionThe two level flexible lever arms between the main child nodes included a fixed lever arm and a flexible deformation. Then the position correction formula for the transfer alignment is:(2)Attitude measurement information correctionThe attitude error angle between the main POS and the sub-IMU The attitude relationship between the main POS and the sub-IMU is as follows:The above formula is developed to eliminate the second-order small amount, and the approximate value is taken:Remember ith row and jth column of the main POS pose matrix. The two sides are expanded according to the Taylor series while the second order and above are ignored.In the formula, The corrected attitude measurement information could be obtained and its expression is as follows:The wing deformation test and verification system based on FBG is composed of a simulated wing structure, the FBG deformation measurement system, the DPOS, and six degree of freedom motion simulator. The simulation wing junction contains 10 sub-nodes on the left and right sides, and the whole wing structure is fixed by the mounting base. It is fixed on the six degree of freedom motion simulator, and the six degree of freedom motion simulator could simulate the typical motion environment of the aircraft, which includes the typical line motion and angular motion. The FBG deformation measurement system is composed of FBG sensors and a demodulator, in which the FBG sensor is installed on the upper surface and lower surface of the simulated wing structure. The surface corresponding position information is used to measure the wing deformation. The main IMU is mounted on the base of the analog wing structure, and the 10 sub-IMU are installed on the left and right side of the simulated wing structure, respectively. A schematic diagram of the system composition and installation is shown in During the test, the dynamic deformation of the wing structure is simulated and the wing deformation is measured. The system provides simulated wing deformation environment under the simulated airborne environment; the FBG deformation measurement system transfers the high-precision wing deformation measurement to DPOS. Then the transfer alignment based on the FBG is completed using the high-precision main POS and the wing deformation measurement information, and the motion parameters of the sub-nodes is measured.The simulated wing structure is based on the real wing shape and structure, which is consistent with the real wing modal characteristics. The wing structure on both sides is connected in a stitching mode, which is fixed on the motion simulator by the upper pressure plate. Both sides of the wing used the classic flat convex airfoil CLARK-Y on the upper surface streamline and lower surface plane. The wing is 3 m long and the string length is 0.32 m; the other string length is 0.24 m. In addition, the main IMU is set on the surface of the upper pressure plate. The base is set up with five sub-IMUs on the lower surface of the wings of both sides, respectively. At the same time, the reference axis of the sub-IMUs and FBG are set on the upper and lower surfaces of the simulated wing. The specific parameters of the wing structure are shown in the FBG sensors are used to directly sense the strain changed at the mounting points. Its layout would directly affect the accuracy of deformation measurement of the simulated wing structure. FBG sensors should be installed as far as possible to enhance the signal-to-noise ratio of the signal. At the same time, the installation of the sensor could not affect the load and IMU installation.In order to reduce the influence of common measurement errors such as temperature and vibration on FBG sensors, the FBG sensors are installed at the corresponding position of the upper and lower surfaces of the wing structure, and the effect of the common error on the deformation measurement results is reduced through the reduction of the sensor signals on the upper and lower surfaces. In addition, the distance between the corresponding sensors should be increased as far as possible, so as to improve the division degree of the deformation signal. For the designed wing structure, six FBG sensors are installed on each wing, while there are three on the upper surface and three on the lower surface. Each fiber Bragg grating sensor contains 15 measuring points and also five sensors near the wing of the wing and the spacer 20 cm installation of 10 sensors. Through the arrangement of the FBG sensors, the network distribution measurement of the FBG sensors on the simulated wing structure is realized, as shows in Y-axis direction.The test equipment is shown in The wavelength data measured by FBG sensors on the upper and lower surface as well as the strain values of the measured points of the FBG sensors are calculated after the difference. X-axis and the Y-axis are the end displacement values of the wing and the Z-axis is the strain value at each load weight.According to the force condition of the flexible wing, the strain value is fitted two times. The strain curve after the fitting is plotted using second-order integration again, and the wing end displacement is measured by micrometer through loading 600, 1100 and 2100 g weight. In addition, the micrometer measurement is used as the reference data to calibrate the wing deflection curve obtained by the calculation, which is shown in The calculated displacement of the wing tip is different from the displacement measured by the micrometer. The experimental results of measuring the deformation displacement for the simulated wing with FBG showed that the measuring accuracy of FBG sensor for one-dimensional linear displacement could reach 0.151 mm, as shown in The high-precision IMU of distributed POS is placed at the position of the main node, and two sub nodes are selected to place high-precision IMU and low-precision IMU on one side, that is slave system The vertical displacement changes of two subsystems First of all, the main system-based laser gyros and subsystem based on MEMS gyros conduct transfer alignment according to position and attitude matching method above, and obtain the attitude of the subsystem and the flexible baseline length of three dimension; second, the main system and the high-precision subsystem based on fiber gyros also conduct transfer alignment to obtain the attitude of the subsystem and the flexible baseline length of three dimensions. Finally, the two transfer alignment results are compared to verify the accuracy of transfer alignment. In the process, MEMS IMU and fiber IMU are rigidly connected at the end of the simulated wing. The comparison of the processed attitude results is shown in A DPOS transfer alignment method based on FBG for flexible deformation compensation is studied. Firstly, the measurement error of matching parameter caused by flexible baseline is compensated by the deformation displacement and deformation angle measured using FBG sensors, then the transfer alignment model based on FBG assisted inertial measurement is established, and the position and attitude accuracy of the subsystem are improved by filtering estimation. Finally, the effectiveness of the transfer alignment method is verified by the DPOS ground demonstration system. In the future, some real field test will be conducted in the vehicle and during flight.


UBE3A gene codes for a protein with two known functions, a ubiquitin E3-ligase which catalyzes ubiquitin binding to substrate proteins and a steroid hormone receptor coactivator. UBE3A is most famous for its critical role in neuronal functioning. Lack of UBE3A protein expression leads to Angelman syndrome (AS), while its overexpression is associated with autism. In spite of extensive research, our understanding of UBE3A roles is still limited. We investigated the cellular and molecular effects of Ube3a deletion in mouse embryonic fibroblasts (MEFs) and Angelman syndrome (AS) mouse model hippocampi. Cell cultures of MEFs exhibited enhanced proliferation together with reduced apoptosis when Ube3a was deleted. These findings were supported by transcriptome and proteome analyses. Furthermore, transcriptome analyses revealed alterations in mitochondria-related genes. Moreover, an analysis of adult AS model mice hippocampi also found alterations in the expression of apoptosis- and proliferation-associated genes. Our findings emphasize the role UBE3A plays in regulating proliferation and apoptosis and sheds light into the possible effects UBE3A has on mitochondrial involvement in governing this balance.The  UBE3A gene that encodes for the ubiquitin E3-ligase protein UBE3A is located in the q11–q13 region of chromosome 15 in humans and at 28.65 cm of chromosome 7 in mice. UBE3A possesses five well-characterized functional domains: an HECT domain, E6 binding domain, p53 binding domain, three nuclear receptor interaction domains, and an activation domain [UBE3A gene. Around birth, the paternal copy of UBE3A is imprinted in most brain areas, including the hippocampus, and only the maternal copy is expressed [UBE3A deletion in Angelman syndrome, a mouse model that carries the maternal deletion of exon 2 of the Ube3a gene [The n domain . In genen domain ,10,11. In domain . Alteratn domain ,15,16. Yn domain , while in domain . In mostxpressed ,20. Thuse3a gene  was genee3a gene ,22,23.To date, previous studies by us and others have suggested that UBE3A may play a role in regulating apoptosis  and mitoOne of the major intersections in regulating the apoptotic response is the mitochondria. Apoptosis usually entails alterations of mitochondrial production of reactive oxygen species (ROS) and the release of cytochrome c, which initiate the post-mitochondrial apoptotic cascade ,37. MitoSeveral types of neurodevelopmental disorders and diseases, such as autism , schizopUbe3a deletion in mouse embryonic fibroblasts (MEFs) and in the hippocampi of AS model mice.In spite of vast efforts invested in studying UBE3A activity and function, the basic molecular mechanisms governing AS pathology are still unclear. This emphasizes the need to discover the basic molecular pathways governed by this multifunctional protein. For this reason, we chose to investigate the cellular and molecular effects of Ube3a−/−) and wild-type (Ube3a+/+) 13.5-day-old embryos were generated by breeding Ube3a-/+ mice [g for 5 min at 4 °C) and resuspended in fresh DMEM medium with 15% fetal bovine serum . Cells (1 × 106) were cultured on 100-mm dishes at 37 °C with 5% CO2. In this study, we used MEFs within three to five passages to avoid replicative senescence. Housing, handling, and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the University of Haifa animal ethics committee.Mice used were all on a C57BL/6 background. The MEFs from null . After 1 h, the cells were washed with PBS, trypsinized, and washed with PBS again. Immediately after the final wash, the cells were fixed, permeabilized, stained, and analyzed by Fluorescence-activated cell sorting (FACS) according to the manufacturer’s instructions .Cells were cultured in 96-well plates . Cell proliferation assay (XTT-based)  was added to the wells for 2 h to measure cell proliferation according to the manufacturer’s instructions. The absorbance was recorded at 475 nm .Apoptosis of MEFs was determined by staining cells with Fluorescein isothiocyanate (FITC)-conjugated annexin-V/PI using a MEBCYTO Apoptosis Kit  and analyzed by FACS according to the manufacturer’s recommendations.® 3/7 Assay kit  following the manufacturer’s protocols. Briefly, 1 × 103 and 5 × 103 MEFs were seeded in coated 96-well white plates with clear bottoms. After 48 h of incubation in 100-μL culture medium, 100-μL caspase 3/7 substrates were added. Immediately before this assay, the wells were examined under the microscope to confirm no over-confluence and no detached cells. After incubation at 37 °C for 30 min, the luminescence was measured to determine the caspase 3/7 activity. The luminescence signal was normalized to dimethyl sulfoxide (DMSO)-treated cells.Caspase 3/7 activity was measured using the Caspase-GloUbe3a−/− and Ube3a+/+ MEFs. The cells were washed with PBS, collected, and homogenized by sonication with an ice-cold lysis buffer containing the following (in mM): 10 HEPES pH 7.5, 150 NaCl, 50 NaF, 1 EDTA, 1 EGTA, 10 Na4P2O7 , PMSF , and protease inhibitor cocktail . The samples (15 µg) were loaded on an SDS PAGE 4–20% gradient, followed by a transfer to polyvinylidene difluoride  membranes and probed with primary antibodies using standard techniques. The primary antibodies and the dilutions for the Western blots were as follows: BAX 1:2000 , BCL-2 1:2000 , and UBE3A 1:1000 . β-ACTIN 1:40,000  was used as a loading control. Secondary antibodies were used, respectively. The blots were developed and imaged using the Image Quant LAS 4000 system. All signals were normalized by the total protein and quantified using Image Studio Lite Ver 5.2 software.Western blot analysis was carried out for Ube3a−/− and Ube3a+/+ MEFs were cultured in 12-well plates (35 × 104 cells/well). Imaging was started 24 h after seeding. Multilocation imaging was performed inside the incubator scope for the duration of 12 h. Bright-field images were acquired with a frequency of 16 frames every 5 min, for proliferation capacity of the cells was analyzed using software tools (tTt) for single-cell tracking and quantification of the cellular and molecular properties [operties .Staining was performed as recommended by the supplier . In brief, cells were incubated with CytoPainter cell proliferation red fluorescence reagent, and the median fluorescence intensity was measured by flow cytometry on BD-FACSCanto II at T0 and 48 h post-labeling. At least 10,000 events were collected in each analysis. Data analysis was performed using FlowJo software .Ube3a−/− and Ube3a+/+ MEFs were cultured for 5 passages in SILAC medium  according to the manufacturer’s protocols. After collecting the cells, proteins were extracted from the cell pellets in 9-M urea, 400-mM ammonium bicarbonate, and 10-mM DTT and 2 cycles of sonication. Protein (1 mg) from each sample were mixed heavy (H) with light (L), reduced (60 °C for 30 min), modified with 35-mM iodoacetamide in 400-mM ammonium bicarbonate , and digested in 2-M urea and 90-mM ammonium bicarbonate with modified trypsin  at a 1:50 enzyme-to-substrate ratio overnight at 37 °C. Additional second trypsinization was done for 4 h in 1-M urea.m/z 350–1800, resolution 70,000) using repetitively full MS scan followed by high collision-induced dissociation (HCD) at 35 normalized collision energy of the 10 most dominant ions (>1 charge) selected from the first MS scan. A dynamic exclusion list was enabled with an exclusion duration of 20 s. The mass spectrometry data were analyzed using MaxQuant software 1.5.2.8. (www.maxquant.org) for peak picking identification and quantification using the Andromeda search engine, searching against the Mus musculus proteome from the UniProt database with a mass tolerance of 20 ppm for the precursor masses and 20 ppm for the fragment ions. Oxidation of methionine and protein N-terminus acetylation were accepted as variable modifications, and carbamidomethyl on cysteine was accepted as a static modification. Minimal peptide length was set to six amino acids, and a maximum of two miscleavages was allowed. Peptide- and protein-level false discovery rates (FDRs) were filtered to 1% using the target-decoy strategy. Protein tables were filtered to eliminate the identifications from the reverse database, common contaminants, and single peptide identifications. Heavy (H)/light (L) ratios for all peptides belonging to a particular protein species were pooled, providing a ratio for each protein. The resulting tryptic peptides were desalted using C18 tips . The proteins were analyzed by LC-MS/MS using a Q Exactive Plus mass spectrometer  fitted with a capillary HPLC . The peptides were loaded onto a homemade capillary column  packed with Reprosil C18-Aqua  in solvent A (0.1% formic acid in water). The peptide mixture was resolved with a (5–28%) linear gradient of solvent B (95% acetonitrile with 0.1% formic acid) for 105 min followed by 15 min gradient of 28–95% and 15 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μL/min. Mass spectrometry was performed in a positive mode  according to the manufacturer’s instructions. The isolated RNA concentration and quality were determined by Qubit® quantitation assay using a Qubit® 2.0 fluorometer  and Agilent 4200 TapeStation System . Samples were prepared for Illumina sequencing using NEB’s Ultra RNA Library Prep Kit for Illumina (NEB#7530)  according to the manufacturer’s protocols. Libraries were sequenced with a 2 × 150 bp PE run on Illumina HiSeq 2500 using a V3 flow cell.https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/, using the Bowtie2 algorithm [p-value of less than 0.01. All bioinformatics analyses were performed on the T-BioInfo Platform (http://tauber-data2.haifa.ac.il:3000/). DAVID Bioinformatics [http://PANTHERdb.org/) were used to classify the differentially expressed genes into functional groups. To identify proteins that are localized to mitochondria, we used a curated database of mitochondrial localized proteins—the MitoCarta2.0 database [RNA-seq fastQ files were filtered and trimmed from adaptors using the Trimmomatic algorithm . The realgorithm . Gene exlgorithm . Differelgorithm . Log2 foormatics ,54 and tormatics  , Biocarta (genes annotated by GO term GO: 0008632), and KEGG (https://www.genome.jp/kegg-bin/show_pathway?mmu04210). Proliferation-associated genes (56) were identified based on the GO term GO: 008283.Apoptosis-related genes (121) were extracted based on Hallmark  using the Bowtie2 algorithm [RNA-seq fastQ files were filtered and trimmed from adaptors using the Trimmomatic algorithm . The realgorithm . First, lgorithm . Genes algorithm ,54 and tlgorithm  were uselgorithm .Ube3a−/− and Ube3a+/+ groups of MEFs were identified by utilizing regression-based analysis on the T-BioInfo Platform. In brief, for each position, the frequency of substitution was calculated. The 95% confidence interval for frequency of editing was calculated via the log-likelihood ratio of binomial distributions that generate the chi-square-distributed statistics (Wilks theorem) [Next, the differentially edited sites in the theorem) . The regtheorem) ,54 and Ptheorem)  softwaret-test was used for in vitro data analysis. Two-tailed p-values of 0.05 or less were considered to be statistically significant. Analysis of variance (ANOVA) or repeated-measures analysis of variance (RM-ANOVA) were performed whenever required. Bonferroni correction was used for post-hoc multiple comparisons. Student’s unpaired Ube3a−/− and Ube3a+/+ MEFs are available in GEO (PRJNA575629).Fastq files of RNA-seq data from the Ube3a−/− and Ube3a+/+ 13.5-day-old embryos, Ube3a−/− MEFs reached confluency faster than their Ube3a+/+ controls. In order to quantify this observation, we determined the growth rate by seeding 0.3 × 106 cells in tissue culture dishes in DMEM with 15% FBS at day zero (T0). Once the Ube3a−/− cells reached 80 percent confluence, all cultures were trypsinized, counted, and replated in a ratio of 0.3 × 106 cells per dish. Both at the end of the first and the second passages, the cell numbers of the Ube3a−/− MEFs were higher than those of the Ube3a+/+ MEFs  = 43.61, p < 0.0001 for the interaction of the genotype by time in a two-way RM-ANOVA) (2), the final averages of MEF counts were 1.37 × 106 and 0.56 × 106 for Ube3a−/− and Ube3a+/+, respectively (t(18) = 12.7, p < 0.0001 post-hoc Bonferroni corrected comparison)  = 11.05, p < 0.01 post-hoc Bonferroni corrected comparison)  = 3.43, p < 0.05 post-hoc Bonferroni corrected comparison) and 1.35 after 48h (t(16) = 6.47, p < 0.0001 post-hoc Bonferroni corrected comparison and F = 28.20, p < 0.0001 for the interaction of the genotype by time in a two-way RM-ANOVA) (5Ube3a−/− and Ube3a+/+ MEFs in 12-well tissue culture plates and utilized live-cell imaging and tracking software to randomly track 75 cells in a single well for 12 h (Ube3a−/− MEFs had a higher percentage of dividing cells compared to the Ube3a+/+ MEFs (27.33% versus 9.3%)   = 29.11, p < 0.001 for the interaction of the genotype by division in a two-way ANOVA and t(4) = 4.04, p < 0.05 in a post-hoc Bonferroni corrected comparison).Upon preparation and culturing of the MEFs from M-ANOVA) A. At theparison) A. Furtheparison) B,C. To fM-ANOVA) D. PerforM-ANOVA) . Though Ube3a−/− MEFs, we examined the effects of Ube3a deletion on cell cycle progression. Measuring BrdU incorporation following 1-h incubation showed a difference in the cell-cycle phases between the two genotypes  = 29.7, p < 0.0001 for the interaction of genotype by the cell-cycle phase in a two-way ANOVA). Ube3a deletion increased the percentage of cells found in the S-phase by 1.53-fold and in G0/G1 by 1.24-fold (t(16) = 3.3, p < 0.05 and t(16) = 5.9, p < 0.0001, respectively, in a post-hoc Bonferroni corrected comparison). In the G2/M cell population, the significant differences between Ube3a−/− and Ube3a+/+ were also observed (t(16) = 4.3, p < 0.01 in a post-hoc Bonferroni corrected comparison). The percentage of cells found in the sub-G1 phase was, however, reduced by almost four-fold (t(16) = 5, p < 0.001 in a post-hoc Bonferroni corrected comparison) (Due to the observed enhancement in the growth capacity of parison) .Ube3a−/− MEFs led us to suspect that the differences observed in cell counts between the Ube3a+/+ and Ube3a−/− may also arise from the altered mortality rate of these MEFs and not only from enhanced proliferation. For this reason, we evaluated the cellular apoptosis by utilizing the annexin-V/PI assay, which suggested differences in apoptosis/viability  = 23.13, p < 0.0001 for the interaction of the genotype by apoptosis/viability in a two-way ANOVA). Ube3a deletion induced a significant decrease in the percentage of early apoptotic cells: 21.4% versus 12.3% for Ube3a+/+ and Ube3a−/−, respectively (t(12) = 3.2, p < 0.05 post-hoc Bonferroni corrected comparison)  = 8.6, p < 0.0001 in an unpaired t-test). In the 5000-cell experiments, caspase 3/7 activity levels were nearly 50% lower in the Ube3a−/− MEFs compared to the controls (t(8) = 11.4, p < 0.0001 in an unpaired t-test)  and BrdU. 7-AAD is usually used to determine the DNA ploidy status . The celparison) A,B. Nextpoptosis . To furt t-test) D. This fUbe3a deletion on molecular pathways, we performed polyA RNA sequencing of Ube3a+/+ and Ube3a−/− MEF samples. First, we confirmed that our MEFs were correctly labeled as Ube3a+/+ and Ube3a−/−. We analyzed the alignment of reads on the deleted second exon of the Ube3a gene [Ube3a+/+ and Ube3a−/− samples. While in Ube3a+/+ samples the second exon was enriched by reads, in Ube3a−/− samples, the reads were not aligned to this exon   B, known tor (Ar) C. All ofFurthermore, we also observed that the expression of several genes coding for proteins localized to the mitochondria were altered  . Ube3a−/− and Ube3a+/+ MEFs. At first, we examined each sample separately for the editing sites utilizing the GIREMI algorithm [Ube3a−/− MEFs was similar to the frequency of editing in the Ube3a+/+ MEFs  or mitochondria-related  in accordance with GO terms. From the 48 genes edited only in Ube3a−/− MEFs, eight were either apoptosis-related  or mitochondria-related  in accordance with their GO terms , we found 338 differentially edited nucleotides in 113 genes . UBE3A has been shown to indirectly regulate p16 expression in non-small cell lung cancer [It is known that mRNA expression does not always directly translate to protein abundance . Therefog cancer .p < 0.05 for the mRNA analysis in the t-test). Amongst these 24 genes, 13 were upregulated and 11 were downregulated  [Having examined the effect of Methods) . MachineMethods) . Using iUbiquitin protein ligase E3A (UBE3A), also known as human papillomavirus E6-associated protein (E6-AP), is one of the E3 ligases in the ubiquitin-proteasome system. Alterations in UBE3A levels, either deletion or overexpression, culminate in severe neurodevelopmental disorders such as Angelman syndrome or autism, respectively. This suggests that cells, especially neurons, are UBE3A dosage-sensitive. For the last few decades, since the discovery of UBE3A as an E3 ligase  and the Ube3a starts to be imprinted, and mice with maternal deletion do not express the UBE3A protein in neurons. Given the lack of UBE3A protein expression in AS patients [Ube3a−/− mouse embryonic fibroblasts (MEFs) to investigate the basic molecular and cellular mechanisms affected by UBE3A. MEFs, in general, have been shown in the past to be a powerful discovery tool for the identification of novel molecular pathways relevant to neurodegenerative disorders [Around birth, when apoptosis is still an ongoing process in neurons , Ube3a spatients  and in Apatients   was differentially edited in the mplex IV . Mutatiomplex IV . This fimplex IV ) exhibitmplex IV . In addimplex IV . A latermplex IV . All of Ube3a in AS mice, immediately after birth, rescued some but not all of AS phenotypes and deficits [Taken together, the fact that MEFs have reduced apoptosis in the absence of UBE3A expression suggests that similar deviations might be present in the brains of AS model mice during early development. It was previously shown that reinstating deficits . Neverthdeficits ,101. Inddeficits ,25,102. deficits  showed adeficits . Our findeficits ,104. TheBased on our MEFs and AS mice studies, we suggest that, in the absence of UBE3A expression, the crosstalk between proliferation, apoptosis, and the mitochondrial functioning is disrupted. These findings imply that the fine-tuning of the interaction between the mitochondrial and the proliferation/apoptosis pathways may be of great value when addressing the novel therapeutic approaches for Angelman syndrome patients.The herein study indicates that dysregulated levels of UBE3A affects apoptosis and proliferation. Accurate neuronal proliferation during embryonic stages and precise neuronal apoptosis during late embryonic and perinatal stages are required for healthy brain development. This suggests that brains of AS and UBE3A duplication individuals are disrupted at critical early developmental milestones. Hence, later interventions of balancing UBE3A levels might be limited in rescuing some of the phenotypes. The effects of UBE3A dose on apoptosis and proliferation during early brain developmental stages must be further elucidated, so therapeutic strategies beyond correcting UBE3A levels could be considered and investigated.


Vibrio cholerae, the causative agent of the acute diarrheal disease cholera, is able to thrive in diverse habitats such as natural water bodies and inside human hosts. To ensure their survival, these bacteria rely on chemosensory pathways to sense and respond to changing environmental conditions. These pathways constitute a highly sophisticated cellular control system in Bacteria and Archaea. Reflecting the complex life cycle of V. cholerae, this organism has three different chemosensory pathways that together contain over 50 proteins expressed under different environmental conditions. Only one of them is known to control motility, while the function of the other two remains to be discovered. Here, we provide an overview of the chemosensory systems in V. cholerae and the advances toward understanding their structure and function. V. cholerae, has three chemosensory pathways. The multiple proteins of these systems are involved in the control of cellular functions at different stages of its complex life cycle. Currently, we know that one of these systems controls motility, while the role of the other two remains unknown. Here, we provide an overview of the latest research advances aimed toward understanding the chemosensory systems in V. cholerae.The bacterium causing cholera disease,  Here, they regulate the autophosphorylation of the histidine kinase CheA. This kinase is a five‐domain enzyme that forms homodimers. Each domain plays a distinct role in the function of CheA: P1 carries the substrate histidine that receives the phosphoryl group during autophosphorylation, P2 is the binding site for the response regulator CheY. Both, the P1 and P2 domains are attached to the rest of the protein via flexible linkers. P3 is the dimerization domain, P4 is the kinase domain with the ATP binding pocket, and P5 is the receptor‐binding domain  rotation pulls the cells backward. The switch from cw to ccw rotation of the motor is followed by a so‐called “flick” of the flagellum, which allows the cell to randomly reorient itself    and a concatenated triple CheW (VC1402) Table , sandwicet al., Cryo‐electron tomography data (cryoET) revealed that this receptor arranges as an extended rod with one signaling domain at each end Figure . This al6et al., et al., The Cluster II/F6 system is the only one that has been shown to affect chemotactic behavior under any tested conditions so far. This was first determined by the analysis of deletion mutants of the three CheA kinases on motility swarm assays in LB soft agar  domain, and a ParC‐interaction domain. The AIF domain binds to the receptors as well as the LID domain of CheA. This Location and Inheritance Domain is an extra domain located between the response‐regulator docking domain P2 and the dimerization domain P3. It only occurs in species that also encode the proteins ParC and ParP in their genomes , Table et al., ParP is not the only extra component that integrates into the baseplate of the Cluster II/F6 arrays. The genome of et al., An earlier study using swarm assays and fluorescence microscopy discovered that this CheV plays a role in chemotaxis , CheV1(VC2006), CheV2(VC2202), and CheV3(VCA0954). So the labeling “CheV” used in the study   had no effect on the presence or abundance of the III/F7 system arrays. VCA1088 is a McpA‐like receptor: McpA‐like chemoreceptors are only found in genomes that encode an F7 system, such as in Pseudomonas‐like and Vibrio‐like systems, and always neighboring the F7 chemosensory gene cluster  class  senses amino acids such as l‐arginine, L‐proline, and L‐serine (Nishiyama et al., et al., While the ligands for most of the et al., Another 40H receptor, VCA0658, has been shown to be involved in aerotaxis (Boin and Hase, et al., et al., et al., Other less well‐specified functions of the receptors have been reported in the past. For example, the receptor VCA0220/Mlp30/HlyB has been implicated in pathogenicity and Hemolysin secretion control (Alm and Manning, V. cholerae chemotaxis system are not fully understood, we predict it senses a large variety of input signals. Several of the chemoreceptors in V. cholerae contain cytoplasmic PAS domains that are known to sense different small molecules, most notably oxygen (Henry and Crosson, V. cholerae.Although the signaling capabilities of the 9V. cholerae remains a formidable target to study the complex arrangements of chemosensory systems. The similarities with other emergent model organisms, such as P. aeruginosa, uncovered by recent bioinformatics work, serve to bridge the gap between these systems and allow for extrapolation of hypothesis and a better understanding of the signaling system as a whole. While the functions of two out of three chemosensory systems in V. cholerae remain unknown, recent work has significantly advanced the field. Taken together, these recent studies allow for the generation of a complex signaling hypothesis in this organism. It supports a functional cross talk of I/F9 and III/F7 clusters with II/F6 cluster via specialized chemoreceptors.The rich chemosensory system in V. cholerae's chemosensory systems studies:What is the function of I/F9 and III/F7 and how are they related to the II/F6 system?What is the mechanism that attaches the III/F7 system to the membrane?How does the II/F6 system balance signals of so many chemoreceptors?In our opinion three intriguing questions remain at the center of Finally, further experimental research will be essential to confirm our proposed prediction of matching chemoreceptors to signaling pathways. This will further improve predicting models of chemosensory signaling systems in bacteria and archaea.Table S1Click here for additional data file.