ECF) that targets the cytosolic response by modifying gene expression and a cytoplasmic membrane anti-sigma factor that keeps the σECF in an inactive state in the absence of the signal and transduces its presence from the outer membrane to the cytosol. Although CSS systems regulate bacterial processes as crucial as stress response, iron scavenging and virulence, the exact mechanisms that drive CSS are still not completely understood. Binding of the signal to the CSS receptor is known to trigger a signaling cascade that results in the regulated proteolysis of the anti-sigma factor and the activation of the σECF in the cytosol. This study was carried out to generate new insights in the proteolytic activation of CSS σECF. We performed a random mutagenesis screen of the unique IutY protein of Pseudomonas putida, a protein that combines a cytosolic σECF domain and a periplasmic anti-sigma factor domain in a single polypeptide. In response to the presence of an iron carrier, the siderophore aerobactin, in the extracellular medium, IutY is processed by two different proteases, Prc and RseP, which results in the release and activation of the σIutY domain. Our experiments show that all IutY mutant proteins that contain periplasmic residues depend on RseP for activation. In contrast, Prc is only required for mutant variants with a periplasmic domain longer than 50 amino acids, which indicates that the periplasmic region of IutY is trimmed down to ~50 amino acids creating the RseP substrate. Moreover, we have identified several conserved residues in the CSS anti-sigma factor family of which mutation leads to constitutive activation of their cognate σECF. These findings advance our knowledge on how CSS activity is regulated by the consecutive action of two proteases. Elucidation of the exact mechanism behind CSS activation will enable the development of strategies to block CSS in pathogenic bacteria.Cell-surface signaling (CSS) is a signal transfer system that allows Gram-negative bacteria to detect environmental signals and generate a cytosolic response. These systems are composed of an outer membrane receptor that senses the inducing signal, an extracytoplasmic function sigma factor (σ Pseudomonas aeruginosa and Pseudomonas putida typically synthesize one main siderophore (e.g. pyoverdine) and some strains also a second lower-affinity siderophore. Nevertheless, they can produce up to 30 TonB-dependent receptors, which reflects the potential of these bacteria to use siderophores synthesized by other organisms  sigma factor (σECF). CSS receptors form large 22-stranded β-barrels in the outer membrane and contain, in contrast to TonB-dependent receptors that are not involved in signal transduction, a periplasmic N-terminal extension referred to as the signaling domain  that binds the σECF, a single transmembrane segment, and a large periplasmic C-terminal domain that receives the signal from the CSS receptor , a transmembrane anti-sigma factor and a cytosolic Koebnik, . The sigKoebnik, . σECF arP. putida, the Iut system, which is employed by the bacterium to regulate the uptake of aerobactin, a siderophore produced by certain E. coli species  and a periplasmic anti-sigma factor domain that are separated by a single transmembrane segment  in order to liberate and activate the cytosolic σIutY domain  in 1:1 proportion . Construction of null mutants was performed by allelic exchange using the suicide vector pKNG101 as previously described : ampicillin (Ap), 100; piperacillin (Pip), 25; streptomycin (Sm), 50 for E. coli and 100 for Pseudomonas; tetracycline (Tc), 12.5 for E. coli and 20 for Pseudomonas.Strains used in this study are listed in Table P. putida or P. aeruginosa by electroporation  D473G*  substrate (Sigma-Aldrich) as previously described .β-Galactosidase measurements were performed in soluble cell extracts using the P. putida was grown to late log-phase in CAS medium containing 200 μM of 2,2′-bipyridyl and 1 mM IPTG, in the presence of either aerobactin deficient or aerobactin containing supernatant. Subsequently, cells were pelleted by centrifugation and heated for 10 min at 95°C following solubilization in SDS-PAGE sample buffer. Sample normalization was done according to the OD660 of the bacterial culture. Proteins were separated by SDS-PAGE containing 15% (w/v) acrylamide and electrotransferred to nitrocellulose membranes (Millipore). As a loading control staining with a Ponceau S dye (Serva) was performed. Subsequently, HA-tagged proteins were detected using a monoclonal antibody directed against the influenza hemagglutinin epitope  as previously described  acquired a premature stop codon in the iutY gene and therefore produced truncated IutY variants. This indicates that the most efficient method to create constitutive activity of IutY is to introduce a premature stop codon and these mutations will be discussed first. The shortest truncated variant (until amino acid 168) contained only the cytosolic σIutY domain, while the longest truncated variant obtained (until amino acid 365) only lacked the last nine amino acids of the protein . We also included in this analysis four additional IutY constructs, encoding HA-IutY-225, -236, -293, or -338  was not affected by the deletion of Prc, while the activity of the longer truncated variants was either completely abolished (IutY-293) or significantly reduced  were significantly lower than that of the fully active proteins . In order to avoid amino acid substitutions in the cytosolic region of the protein, we repeated the screen but with a different cloning strategy to exclude mutations in σIutY . Subsequently, the plasmids were transferred to P. putida KT2440 and constitutive σIutY activity was detected on LB plates supplemented with X-gal. Bright blue clones were first analyzed by Western-blot to select for strains expressing a complete IutY protein and then sequenced. This approach identified in total 20 full-length active IutY mutants , indicating that these single mutations did not confer constitutive activity to the protein. In contrast, the activity of the IutY-D210A, -R231Q, -A240E, -F251S, -V253D, -W301G, and -LtoP  was detected in high amounts only upon growth with aerobactin, it was highly present in all constitutively active mutants even in absence of the siderophore , both in the periplasmic anti-sigma domain and the cytosolic σs Figure . Two of s Figure  and intrs Figure . The funt Figure . It shout Figure , whereast Figure . This ise Figure  and corre Figure . TogetheiutA::lacZ activity in ΔiutY Δprc and ΔiutY ΔrseP double mutants upon bacterial growth in aerobactin. Similar to the wild-type protein, activity of all mutant proteins was completely abolished in the ΔiutY ΔrseP mutant  was considerably reduced but not completely abrogated, since the protein retained ~7% of the lacZ expression measured in the ΔiutY single mutant , only low amounts were detectable for those in which activity was compromised (IutY-R231Q and -LtoP), and they were hardly detectable for proteins with impaired activity  and FiuR . Activity of these proteins was assayed by β-galactosidase measurements in P. aeruginosa ΔfoxR and ΔfiuR mutant strains  using the Protein Homology/analogy Recognition Engine (Phyre2) server  and FoxR  . In a previous study, we identified the C-terminal processing protease Prc as the protease that mediates the site-1 cleavage of the unique sigma/anti-sigma hybrid protein IutY  is a conserved mechanism in which a transmembrane protein is subjected to several processing steps in order to liberate and activate a cytosolic effector . It is possible that point mutations in IutY disrupt the binding of this hypothetical protein thereby making the periplasmic anti-sigma domain susceptible to proteolytic cleavage even in non-inducing conditions. However, several attempts to identify this putative protecting protein by pull-down experiments did not yield significant results (data not shown). The Asp-210, Arg-231, Phe-251, and Val-253 residues are all located on the same side of the N-terminus region of the periplasmic domain of IutY  responsible for this are currently unclear, but this implies that proteolytic regulation of σECF activity is even more complex than previously anticipated.Importantly, despite differences in the proteolytic regulation of CSS systems, in this study we have identified several conserved residues of CSS anti-sigma factors of which single substitution leads to activation of their cognate σn Figure  indicateE. coli Fec system that showed that introduction of mutations that compromises the interaction between the FecA receptor and the FecR anti-sigma factor reduce the activity of σFecI in response to the inducing signal ferric citrate  did not abolish CSS activity, and, in contrast, the mutation resulted in constitutive activity of the σIutY domain. This indicates that the IutA-IutY interaction can be impaired without inhibiting the sigma factor activity, and suggests that the CSS receptor could function in a different way as initially thought. The signaling domain of CSS receptors display a common fold, in which two α-helices are flanked by two β-sheets . These data indicate that only the binding of the signaling domain to the anti-sigma factor is not sufficient to trigger activity of the CSS pathway and suggest that this interaction can protect the anti-sigma factor from downstream proteolytic degradation. This introduces an additional level of complexity in the CSS signal transduction cascade and shows that the signaling domain of the receptor deserves more attention in future research.None of the IutY full-length mutant proteins we have analyzed depended on the IutA receptor for activation. In the current CSS model, perception of the CSS signal by the receptor promotes the interaction between the signaling domain of the receptor and the anti-sigma factor in the periplasm Figure . This moKB, WB, and ML conceived and designed the study. KB and CC performed the experiments. KB, WB, and ML analyzed and interpreted the data, and wrote the manuscript.This work was supported by the EU Seventh Framework through a Marie Curie CIG grant (3038130), the Netherlands Organization for Scientific Research (NWO) through an ECHO grant (2951201), and the Spanish Ministry of Economy with grants inside the Ramon&Cajal (RYC2011-08874) and the Plan Nacional for I+D+i (SAF2012-31919 and SAF2015-68873-P) programs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella typhimurium is responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacterium possesses an inner and an outer membrane (OM), the latter allowing its survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Successful structure determination of YejM will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately,yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium. Salmonella typhimurium is a Gram-negative bacterium responsible for over 35% of all foodborne illness-related hospitalizations in the United States  with an asymmetric lipid composition, that serves as a barrier to the environment allowing its survival and replication within host tissues  (Uniprot ID P40709) in pBAD24 and the periplasmic domain of YejM 191-586 in pET28a plasmid are described in .YejM (15 mg/ml) and Monoolein (Hampton Research) were mixed at a 1:1.5 ratio at 23°C in a coupled Hamilton syringe using a LCP Mixer Station (ARI) until LCP mixture appeared clear. LCP crystallization screens were set up using a Gryphon robot (ARI) pipetting 50 nl LCP boli and 500–800 nl precipitant solution of the following screens: MemGold 1 and 2 (Molecular Dimensions), MembFac (Hampton Research), and JBScreen Membrane (Jena Bioscience). LCP crystallization was set up using LaminexTM plates (Molecular Dimensions).TM HT crystal imaging and detection tower (Rigaku). Optimal crystal growth was obtained at a protein concentration of 4mg/ml.Vapor diffusion crystallization of YejM 241-586 (5–50 mg/ml) was set up using 96-well crystallization plates (Hampton Research) with a Phoenix robot (ARI). Various sparse matrix screens were used to set up sitting drops with a drop size between 500 nl to 1 µl. Crystallization plates were incubated at 20°C and monitored for crystal growth in a MinstrelCrystals were harvested using Litho loops (Molecular Dimensions) and Nylon loops (Hampton Research), submerged into paraffin and blotted until no phase separation was visible between paraffin and the excess crystallization solvent. Diffraction data of YejM241-586 crystals were collected at the Advanced Light Source beamline 4.2.2 in Berkeley CA at 100K, using an oscillation of 0.1–0.2° per image. Diffraction data were processed using iMosflm  and scalhttp://cxidb.org/id-42.html) and are available under CXIDB ID 42, DOI10.11577/1252489  of YejM, an inner membrane protein potentially responsible for transport of glycerophospholipid cardiolipin. The authors also tested interaction between YejL and YejM using blue native gel electrophoresis and size exclusion chromatography. In general, it is an interesting study, yet the following issues need to be addressed before accepted for indexation:In this manuscript, GabeleIt is not proper to claim that YejL is a ligand of YejM. “Ligand” is dedicated for “receptors” only. Also, there is no strong genetic evidence to show that YejL is involved in cardiolipin transport.The authors claimed that YejM is a dimer based on its periplasmic domain, and probably, also because of YejL forming a dimer. This is not clear as the authors did not study the oligomerization state of the full-length YejM . Other than this, when running SEC on superpose 6, the authors did not include a standard marker for reference. It ishard to judge if the periplasmic domain of YejM forms a dimer in solution.The author believed that the negative charge surfaces of YejL might bind with the arginine-rich loop of YejM. Why the fragment of YejM (241-586) that seems to not include the arginine-rich loop still binds YejL ? Please explain.  I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1.) We apologize for the incorrect use of terminology and would rather term YejL as potential interaction partner of YejM.“Our initial X-ray data analysis of YejM241-586 crystals suggests a dimer assembly of the periplasmic domain, therefore the membrane domain is very likely needed to form the YejM tetramer”.2.) We carefully checked within the manuscript what section/sentence could have caused this reviewer’s understanding that we claimed YejM to be a dimer. We found the following sentence in the “Conclusions” section of our originally submitted manuscript:et al. 2015) that full-length YejM forms a tetramer when purified with the detergent DDM. Therefore we reasoned in the above stated sentence that further oligomerization into a tetramer must be facilitated by the trans-membrane domain. Whether is exists as a dimer or a monomer either in the native membrane, in the presence of specific lipids or purified using a different detergent is a matter of current investigation by other laboratories .In this sentence, we suggested the existence of a periplasmic domain YejM241-586 dimer based on our analysis of the crystallographic unit. In this analysis the content of the asymmetric unit that builds the crystal resulted in a dimer of YejM 241-586, and therefore we proposed it to be the likely physiologically relevant state of the periplasmic domain alone. From the SEC-MALS studies in  Indeed, the arginine-rich linker region of YejM would be the most obvious binding region for YejL. However, we cannot exclude the possibility that YejL binds to solvent-accessible positively charged residues on the YejM241-586 surface. Here, the authors report the expression, purification and isolation of diffractable crystals of the periplasmic domain of the essential salmonella protein YejM, putatively involved in transport of cardiolipin to the outer membrane. They also report interaction between the periplasmic domain of YejM and the small protein YejL produced from a gene immediately upstream and likely cotranscribed with YejM. YejM was identified by a screen for mutants defective in PhoPQ mediated outer membrane remodeling and was renamed PbgA (PhoPQ barrier gene A). The regions of the protein and their proposed functions are as follows: 1-190, transmembrane domain of the inner membrane; 191-240, arginine-rich linker that binds to cardiolipin and 240-586, the periplasmic domain seemingly required for interaction with OM. The authors report isolation of crystals of the YejM241-596 periplasmic domain and interaction of YejL with YejM241-586 and YejM191-586 truncated mutants. Sadly, the inability to crystalize YejM191-586 will forestall attempts to examine cardiolipin-YejM interactions. This work represents a preliminary attempt to understand at the molecular level an extremely interesting process required for Salmonella virulence namely OM lipid remodeling. YejM/PbgA represents a novel virulence factor and potential drug target. The importance of its identification cannot be understated.This reviewer questions the physiological relevance of the interaction between YejL and the YejM periplasmic domains. YejL is a 75 amino acid protein encoded by the gene immediately upstream of YejM. It is not predicted to contain a secretion signal and the PDB itself predicts it to be a soluble cytoplasmic protein. It is difficult to envision how it would interact with the periplasmic domain of YejM in a living cell. Cellular fractionation studies showing a periplasmic localization of YejL would need to accompany the data in Figure 2 in order for it to be meaningful. Genetic studies may also be useful- for example, does the YejL mutant show alterations in PhoPQ mediated survival or YejM tetramer formation?The BNE gels are not convincing. Why does YejL migrate at such a large molecular weight? YejM migrates as a smear in both gels when loaded by itself. Adding YejL reduces the smear but it cannot be said that YejL alters the MW of YejM in either gel. Major issues:Why not call YejM by its new name PbgA throughout? It would cause less confusion since the senior author was associated with the publication reporting the discovery last year.Figure 1A, the membrane domain in the figure should be labeled 0-190 not 191-240. Also the “j” in “YejL structure” is partially obscured.I take issue with describing YejL as a ‘ligand”. To me and most biochemists a ligand is a small molecule that interacts with a target protein (receptor) causing some kind of conformational change and usually generation of a signal of some sort. What they are describing here is a protein-protein interaction and should be referred to as such, i.e. “YejL interacts with the periplasmic domain of YejM”. Minor issues:I have read this submission. I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Major issues:1.) We performed cellular fractionation studies based on the reviewer's suggeestions and present the results in the supplemental section in the updated version of the manuscript. The suggested genetic studies are beyond our laboratory’s main focus and scope of revised manuscript; however these studies are being currently performed in the laboratory of Dr. Dalebroux .2.) We improved the quality of our BNE experiments and show the results in the supplemental section of the updated version of the manuscript.Minor issues:1.) PbgA is not yet officially listed as an alternative name for YejM. Uniprot lists entries for PbgA as proteins that are Phospho-beta-glucosidases. To prevent confusion, we chose to use the official Uniprot database nomenclature of YejM (http://www.uniprot.org/uniprot/P40709).2.) This figure is not included in the updated version of the manuscript.3.) We agree that this was not the correct term to use and would rather term YejL as potential interaction partner of YejM.We thank both reviewers for their helpful critique.


Dear Editor,E. coli RND transporter AcrB (EcAcrB) forms a homo-trimer  exporters are widely expressed across the lineage of Gram-negative bacteria Fig. S1). Their functional role is to utilize the proton-motive force of the cell to drive the expulsion of cytotoxic substances from the outer leaflet of the inner membrane as well as the periplasm to extracellular space  and CTM (TMs 7–12). A group of conserved residues, including D407TM4 , D408TM4, and K940TM10, form a water molecule-mediated hydrogen-bond (H-bond) network in the subdomain interface , thus affecting proton transport  EcAcrB expressed in the Δacrb cell by using a pET21a-based plasmid was able to rescue the cell growth in the presence of 5-ng/mL concentration of ciprofloxacin  consisting of one strand (strand-N1: residues P31–P36) connecting the helix TM1 with the PN1 subdomain of the porter domain and another strand (strand-N2: residues I390–T392) connecting TM3 with TM4. Similarly, the subdomain CTM contains a β-sheet , we first replaced this helix with 12 repeats of Gly-Ser pair (12-GS). As shown in Fig. TM4 and L944TM10, to prevent proton leakage . This hydrophobic blockage is presumably removed in the deprotonated status due to the relative movement between TMs 4 and 10. In agreement with their putative roles in the hydrophobic blockage, point mutations of V411T (low expression), L944N, and L944T resulted in loss of transport activity . Together with hydrophobic mismatch forces . Thus, this helix is expected to serve as a pivotal point to generate the rotation torque required for tilting of the TM domain when protonation occurs. Our mutagenesis data showed that disrupting the amphipathic property of α6-7 or its interaction with the TM domain reduces or abolishes the transport activity of EcAcrB  (No. 2015CB910104 to XCZ) and National Natural Science Foundation of China (Grant No. 31470745 to XCZ).The authors thank Dr. Yong Tao of the Institute of Microbiology, CAS, for providing the ∆Xuejun C. Zhang and Min Liu declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.Supplementary material 1 (DOCX 940 kb)Below is the link to the electronic supplementary material.


Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus.The causal agent of pine wilt disease, pine wood nematode (PWN) ( Native species can live in their natural niche, yet invasive spices are more aggressive. Invasive species have a wide range of adaptation to various environmental factors . Among tB. xylophilus is the causal agent of pine wilt disease, which causes enormous ecological and financial losses by damaging the pine forest resources in Asia, North America, and Europe [B. xylophilus before [B. xylophilus have not been studied.As one of the most devastating invasive species in the world, d Europe ,7,8. It d Europe ,10,11. Rs before . HoweverCaenorhabditis elegans (C. elegans) is an excellent model organism in neurobiology research. The mechanism of temperature sensing has been widely studied in this organism [C. elegans, which gives considerable guidance in the research of B. xylophilus. We hypothesize that there are similar temperature-detecting molecular mechanisms between C. elegans and B. xylophilus. It has been reported that the cGMP pathway is involved in regulating the process of low temperature signal transduction in C. elegans [C. elegans, daf-11, tax-2, and tax-4, encode transmembrane guanylyl cyclase, cyclic nucleotides-gated channels β subunits, and cyclic nucleotides-gated channels α subunits, respectively [daf-11, tax-4, and tax-2 showed increments of survival rates in low temperature [B. xylophilus genome [B. xylophilus orthologous genes named Bx-daf-11, Bx-tax-2, and Bx-tax-4, which may have similar functions with daf-11, tax-2, and tax-4 in defending against low temperature. This article is aimed to further research the function and expression patterns of Bx-daf-11, Bx-tax-2, and Bx-tax-4 from B. xylophilus in response to low temperature.organism ,15,16,17organism , the estorganism , the insectively ,20,21. Ms genome , we idenBx-daf-11 was 3216 bp encoding 1071 amino acids. CDS of Bx-tax-2 was 2253 bp encoding 750 amino acids. CDS of Bx-tax-4 was 1872 bp encoding 623 amino acids. Blastp results showed that the deduced amino acid sequence of Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 have a relatively high level of identity with the DAF-11, TAX-2, and TAX-4 protein of several nematodes. On this basis, selected homologous amino acid sequences were downloaded from NCBI. Multiple sequence alignments  of ignments A–C were ignments A–C.5372H8369N1435O1631S64, C3877H6104N1058O1139S27, and C3219H5078N860O929S25, respectively. Their deduced isoelectric points were 5.07, 8.72, and 6.76, respectively. The putative molecular masses were 121, 87, and 71 kDa, respectively. No signal peptide sequence was found by SignalP analysis. Results of TMHMM analysis showed that there were three transmembrane regions in both Bx-TAX-2 and Bx-TAX-4, one transmembrane region in Bx-DAF-11. This illustrated that Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 were membrane-binding proteins. Results of MotifFinder analysis showed that there was an adenylate and guanylate cyclase catalytic domain, an atrial natriuretic factor receptor domain, a protein tyrosine kinase domain, and an NO-binding-associated domain in Bx-DAF-11. There was a cyclic nucleotide-binding domain and an ion transport protein domain in Bx-TAX-2. There was also a C-terminal leucine zipper domain of cyclic nucleotide-gated channels, an ion transport protein, and a cyclic nucleotide-binding domain in Bx-TAX-4.The protein molecular formula of Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 were CB. xylophilus were calculated at 5 °C (low temperature) and 25 °C (regular temperature) every 2 days over 15 days. We found that the lifespan of B. xylophilus was extended at 5 °C . Surprisingly, three cGMP genes of B. xylophilus revealed different transcript patterns from C. elegans cGMP genes [Bx-daf-11, Bx-tax-2 and Bx-tax-4 revealed higher transcript levels at low temperature than regular temperature over 7 days  [Bx-daf-11, Bx-tax-2, and Bx-tax-4 RNAi in B. xylophilus. The Bx-daf-11, Bx-tax-2, and Bx-tax-4 dsRNA had no obvious effect on the transcript level of Actin (B. xylophilus.The RNAi of . (2012)  to allow. (2012)   at 25 °C . This inB. xylophilus. The past decade has witnessed a rapid progress in the understanding of how high temperature influences lifespan of B. xylophilus [It is commonly known that both homeotherms and poikilotherms can have extended lifespans at lower body temperatures. Cold-dependent lifespan extension is not a passive thermodynamic process but an active one that can be promoted by genetic programs at low temperatures . In thislophilus ,24,25. HBx-daf-11, Bx-tax-2, and Bx-tax-4 were cloned and analyzed from B. xylophilus. Sequence analyzing indicated that the deduced amino acid sequences of Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 showed a high level of identity with other nematodes. Motif analysis revealed that Bx-daf-11 may encode a membrane-bound form of guanylate cyclase, which has an ability to bind to extracellular ligands and synthesizing cGMP. Bx-tax-2 and Bx-tax-4 may encode a membrane-bound ion-gated channel. In addition, Bx-daf-11, Bx-tax-2, and Bx-tax-4 revealed a higher transcript abundance under low temperature. Furthermore, the RNAi method was utilized to study the functions of Bx-daf-11, Bx-tax-2, and Bx-tax-4 against low temperature. The results indicated that low temperature extended lifespan of B. xylophilus through three genes of the cGMP pathway. Considering all these factors, we hypothesized that low temperature upregulated the transcription of Bx-daf-11 by binding with extracellular ligand. Bx-daf-11 synthesized cGMP in the cytoplasm, which promoted the transcript level of Bx-tax-2 and Bx-tax-4, encoding the membrane-bound ion-gated channel. Then, the ion concentration changed so that other physiological and biochemical activities were regulated to extend the lifespan of B. xylophilus.In this article, the CDS of three cGMP pathway genes 2) turn into specialized third-stage dauer larva (DL3). DL3 accumulate around chambers of its vector beetles Monochamus alternatus [4) enter the tracheae of the beetle. These DL4 will be vectored to other pine trees by the beetle [3 [B. xylophilus, and these three genes are potential targets for the control of this destructive plant parasite nematode. This conclusion provides considerable guidance to the research about low-temperature-induced dauer formation of B. xylophilus in late summer and early autumn. However, future efforts are needed to discover other unknown genes and pathways related to low-temperature-induced lifespan extension in B. xylophilus.Few articles about genetic mechanisms of low-temperature-induced lifespan extension in this devastating nematode have been published. In early autumn, low temperature as an environmental signal induces adult PWN to enter winter diapause in the host. Second-stage propagative juveniles  as described by Wang, 2012 . Then, Rlophilus . The homsequence . PCR pro2O were mixed evenly. A suspension (0.1 mL) containing about 80–120 nematodes were transferred to 1.5 mL centrifuge tubes with 1 mL of distilled water. The tubes were separated into two groups: one group was cultured at 5 °C and another at 25 °C. The survival rates of nematodes were calculated by assessing movement response to mechanical stimulation every 2 days over 15 days for each group by transferring nematodes to a microtiter plate, then observed through a dissecting microscope. Temperatures (5 °C and 25 °C) were maintained by two climatic cabinets .About 10,000 nematodes in 10 mL ddHhttps://www.ncbi.nlm.nih.gov/orffinder/) was used to translate the complete protein CDS sequence into amino acids. Homologous Bx-daf-11, Bx-tax-2, and Bx-tax-4 amino acid sequences of other organisms were obtained from National Center for Biotechnology Information with BLASTP (available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments of amino sequences were carried out using Geneious Basic 3.6.1 and phylogenetic analyses of Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 were performed with Mega 7.0 software using a maximum likelihood tree. Deduced protein molecular formula, molecular weights, and isoelectric point calculations were studied in Expasy (available online: http://web.expasy.org/protparam/). The GenomeNet Database (available online: http://www.genome.jp/tools/motif/) was used to search the motifs of Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4. The bioinformatics website Center for Biological Sequence was used to analyze Predictions of Transmembrane helices (available online: http://www.cbs.dtu.dk/services/TMHMM/) and signal peptide .The ORF Finder . We used the cycle threshold data to calculate the fold of relative transcript abundance (each treated time point/no treated group). -tax-4-R  were uti-tax-4-R . qPCR waBx-daf-11, Bx-tax-2, and Bx-tax-4 as outlined in Feng Wang et al. [B. xylophilus  was used in this research. dsRNA was obtained using the MAXIscript T7/T3 RNA Synthesis Kit  with the following primers: i-Bx-daf-11-F, i-Bx-daf-11-R, i-Bx-tax-2-F, i-Bx-tax-2-R, i-Bx-tax-4-F, and i-Bx-tax-4-R . The CK nematodes were soaked in ddH2O only. After intermittent stirring for 24 h at 25 °C, the nematodes were washed with ddH2O to remove the external dsRNA and then observed fluoroscopically to detect the uptake of FITC.In this research, RNAi method was utilized to study the functions of g et al. . Mixed-s-tax-4-R . T7 prom-tax-4-R . The nem2O within each 1.5 mL centrifuge tube performed in triplicate were used to calculate the survival rates of nematodes every 2 days over 16 days.The RNAi-treated nematodes were divided into two groups: The first group was used to examine the efficiency of RNAi with qPCR, and the second one was used to calculate survival rate under 5 and 25 °C environments. Temperature was provided by two climatic cabinets as mentioned above. Total RNA was extracted from the CK nematodes and the dsRNA-treated nematodes after intermittent stirring for 24 h. qPCR was then performed as mentioned above with q-Bx-daf-11-F, q-Bx-daf-11-R, q-Bx-tax-2-F, q-Bx-tax-2-R, q-Bx-tax-4-F, q-Bx-tax-4-R, q-Actin-F, and q-Actin-R. About 100 nematodes in 1 mL of ddHt-tests. Statistically significant differences are indicated with asterisks .All assays were studied with three replicates as three independent trials. Microsoft Excel was utilized to calculate the mean ± SD of three independent experiments. SPSS Statistics 20.0.0 software  was applied to determine the statistical significance with the paired B. xylophilus by upregulating Bx-daf-11, Bx-tax-2, and Bx-tax-4. This is different from the expression pattern of C. elegans [Bx-daf-11, Bx-tax-2, and Bx-tax-4 have similar molecular characteristics with orthologous genes daf-11, tax-2, and tax-4 [B. xylophilus at low temperatures. The three genes are the potential targets for the control of this devastating plant parasite nematode.A low temperature extends the lifespan of  elegans . Bioinfond tax-4 ,19,20,21


S, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker  and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3’ trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase Z SequencE. coli, in which -CCA76 is transcriptionally encoded. In eukaryotes, -CCA76 is not transcriptionally encoded; CCA-addition is thus required in eukaryotic nuclei and plastids. tRNase Z provides the principal mechanism for endonucleolytic removal of eukaryotic 3’ trailers, leaving the discriminator base (N73) with a 3’-OH ready for CCA addition. This pathway may be complemented by exonucleases in S. cerevisiae . S). S3]). evisiae .S and tRNase ZL, respectively) both endonucleolytically cleave pre-tRNA 3’ end trailers, however the two forms are unevenly distributed among the domains of life. Bacteria and archae exclusively possess tRNase ZS. While tRNase ZS is found in some eukaryotes, tRNase ZL is more widespread .The tRNase Z function and a gene encoding the enzyme are widely conserved . A short++ ions which direct H2O in general in-line acid-base catalysis, four of them in the signature His cluster .tRNase Z is a member of the β-lactamase family of metal-dependent hydrolases, characterized by an αβ/βα sandwich fold with the active site located at the interface between the domains . Motifs S functions as a homodimer of identical subunits. In addition to Motifs I–V, a unique flexible arm  pro proS funL emerged as a tandem duplication of tRNase ZS with subsequent divergence of the amino and carboxy domains (first suggested by [H. sapiens tRNase ZL than that of tRNase ZS [L is a monomer in solution based on size exclusion chromatography [S. cerevisiae tRNase Z [L).Sequence and structural studies show that tRNase Zested by  and subsested by ). The amRNase ZS . tRNase tography  and in ttRNase Z   t) t13]) ttructure .st inframe methionine (MYLV…) of D. melanogaster tRNase Z (NCBI NP_724916.1) and the following 19 residues are interpreted to be a mitochondrial targeting sequence , then converted to V X 10−11 M/min by multiplying by nM [S], and further analyzed using the single ligand binding function in SigmaPlot. kcat was obtained by dividing Vmax by [E]. Concentration of each variant enzyme was adjusted as necessary depending on the impairment factor observed in previous kinetic experiments. The processing experiments with each variant were repeated until acceptable standard errors were achieved.Nuclear encoded pre-tRNAescribed . Kinasinst dimension tube gels and SDS electrophoresis in the 2nd dimension as previously described . B. BdomTprtructure , the 1stdomTprox and 13 residues in Tflex. A short sequence further into the linker is characterized by contiguous glutamates (PEEY region). The goal of an Ala scan is to discover residues of sufficient importance that, when replaced by Ala, cause a significant effect on enzyme activity. Such effects were not observed within the PEEY region, which will therefore not be discussed further. The N-T region is generally conserved in location and hydrophilicity but does not align well and was therefore not examined. Once results of processing kinetics were available, flexibility of selected variants with suggestive functional impairments were studied by limited proteolysis with trypsin and protein gel electrophoresis as in [The regions subjected to single residue Ala substitution and kinetic analysis include 19 residues in Nst half of NdomTprox, suggested by PsiPred to be in α-helix, are unremarkable. Alanine substitutions in two bulky hydrophobics spaced three residues apart close to the carboxy end of NdomTprox, Phe329Ala and Leu332Ala, strikingly impair processing with impairment factors of 2,700X and 700X relative to wild type . The NdomTprox region is proximal to the preferred trypsin N-T cleavage site at K348/K351 which produces stable Cdom1species  toward the carboxy end of the region  in D. melanogaster tRNase Z which gives rise to the Cdom1 species ; the bulky hydrophobic character of Y361 is, however, probably more important than the polarity of its OH group.Internal subdomains are apparently created by juxtaposition of several bulky hydrophobic R groups shielded from solvent, producing a micellar spherule inflated like a beach ball . Substitwhite in  leads todomTprox and Tflex regions exert their effects through interactions with the skeleton of two twisted β sheets that organize the amino domain (S. cerevisiae tRNase Z (5MTZ) with the amino domain light grey, carboxy domain dark grey, twisted β sheet 15, 14, 1–6 green and 13–7 blue. The hydrophobicity collapse in α8/β13 could thus be transmitted into the twisted β sheet in the amino domain on the carboxy side of the flexible arm, as suggested by the enlarged view in Substitutions in both No domain . Fig 10A392 makes backbone H-bonds with the carboxy group of H315 and the amino group of H317 in β12 , one of two short antiparallel β strands in the linker (β15–14) which join a twisted sheet (β1—β6) on the amino side of the flexible arm through backbone H-bonds between β14—β1 . The flexible linker clearly associates here with the twisted β sheet which forms half the skeleton of the amino domain, on the amino side of the flexible arm  associated with mitochondrially based cardiac hypertrophy [Based on the alignment in e Z Figs  and 7 coertrophy  is locatD. melanogaster tRNase Z through Ala scanning mutagenesis followed by processing kinetics was aided by analysis of flexibility using limited proteolysis and two-dimensional protein electrophoresis. This approach, informed by interpretation of a recent crystal structure of the S. cerevisiae homolog, uncovered a previously unknown hydrophobic subdomain formed across the amino domain—linker boundary, leading us to suggest that peripheral substitutions affect the skeleton of twisted β sheets in the amino domain on both sides of the flexible arm.A biochemical exploration of little-understood regions of


Intense biological conflicts between prokaryotic genomes and their genomic parasites have resulted in an arms race in terms of the molecular “weaponry” deployed on both sides. Using a recursive computational approach, we uncovered a remarkable class of multidomain proteins with 2 to 15 domains in the same polypeptide deployed by viruses and plasmids in such conflicts. Domain architectures and genomic contexts indicate that they are part of a widespread conflict strategy involving proteins injected into the host cell along with parasite DNA during the earliest phase of infection. Their unique feature is the combination of domains with highly disparate biochemical activities in the same polypeptide; accordingly, we term them polyvalent proteins. Of the 131 domains in polyvalent proteins, a large fraction are enzymatic domains predicted to modify proteins, target nucleic acids, alter nucleotide signaling/metabolism, and attack peptidoglycan or cytoskeletal components. They further contain nucleic acid-binding domains, virion structural domains, and 40 novel uncharacterized domains. Analysis of their architectural network reveals both pervasive common themes and specialized strategies for conjugative elements and plasmids or (pro)phages. The themes include likely processing of multidomain polypeptides by zincin-like metallopeptidases and mechanisms to counter restriction or CRISPR/Cas systems and jump-start transcription or replication. DNA-binding domains acquired by eukaryotes from such systems have been reused in XPC/RAD4-dependent DNA repair and mitochondrial genome replication in kinetoplastids. Characterization of the novel domains discovered here, such as RNases and peptidases, are likely to aid in the development of new reagents and elucidation of the spread of antibiotic resistance.IMPORTANCE This is the first report of the widespread presence of large proteins, termed polyvalent proteins, predicted to be transmitted by genomic parasites such as conjugative elements, plasmids, and phages during the initial phase of infection along with their DNA. They are typified by the presence of multiple domains with disparate activities combined in the same protein. While some of these domains are predicted to assist the invasive element in replication, transcription, or protection of their DNA, several are likely to target various host defense systems or modify the host to favor the parasite's life cycle. Notably, DNA-binding domains from these systems have been transferred to eukaryotes, where they have been incorporated into DNA repair and mitochondrial genome replication systems. Cellular genomes are pitted in multilevel conflicts involving a diverse array of nonself genomes, including bacteriophages, plasmids, and conjugative transposons , 2. At o3–6–9–While conflict and damage repair systems encoded by cellular genomes have been subject to intense scrutiny in several recent studies , 11, muc13–18–Bacillus phage SPP1 gp7), which are structural components of the head in phages utilizing the portal terminase packaging system  DNA processing and transfer proteins are seen primarily in conjugative transposons and plasmids and are intimately involved in the mechanics of conjugation. The key protein here is a relaxase with a catalytic domain of the rolling-circle replication (RCR) superfamily that nicks the element's DNA, forming a covalent linkage with a single strand of the element's DNA. The DNA is then unwound, transferred along with the linked relaxase through the T4SS, and religated by the relaxase to form a single-stranded circle in the new host , 15. ii, with thg system , 25. Theg system , 26. ly genes , 36. Exaly genes , 38, whiGiven our interest in biological conflicts during invasion by nonself elements, we used comparative genomic analysis to better understand the components and diversity of systems such as those described above. In the course of this analysis, we uncovered a remarkable class of proteins that are widely distributed in phages, prophages, plasmids, and conjugative transposons and characterized by the fusion in the same polypeptide of multiple protein domains with a striking diversity of biochemical activities. Accordingly, we term them polyvalent proteins. They encompass at least 131 distinct domain types, many of which we predict to mediate an array of functions needed to establish the invasive elements in host cells, promote their replication, and overcome host defenses directed against them. We propose that these polyvalent proteins represent a hitherto undescribed general strategy used by invasive genomes in the face of the ongoing arms race with their prokaryotic hosts and rival invasive elements.large polyvalent-protein-associated domain) prefix followed by a number. By this iterative method, we assembled a comprehensive inventory of >9,928 polyvalent proteins with 131 domains  whose domain architectural and gene neighborhood linkages are presented as a contextual network , we first initiated sequence profile searches by using a comprehensive library of previously identified exemplars of such proteins as queries . These i network . The 100 network .YP_950528.1) is a multidomain protein with an N-terminal alpha-helical region (998 aa) involved in protein injection into the host, a middle polymerase domain that provides the core DNA-dependent RNAP activity , and a C-terminal 1,400-aa region required for encapsidation . Domain context analysis showed that the N4-like RNAPs are always fused to one or more MPTase domains and can additionally be fused in comparable large proteins to a lysozyme/transglycosylase domain , a novel predicted RNase of the barnase/EndoU/colicin E5/D-RelE (BECR)-like fold , and a NAD+-dependent ART domain     . Thus, a87992.1)  and 2.1549-like elements of Firmicutes  The first is the presence in a plasmid or conjugative transposon encoded alongside T4SS-related components . Examplermicutes  and the eumoniae , and seveumoniae . This iseumoniae   (Acetonema longum (accession no. WP_004096050.1) containing at least nine domains, of which the known enzymatic domains include the cyclic-diguanylate-generating GGDEF, SWI2/SNF2 ATPase, DNA adenine methyltransferase (MTase), zincin-like MPTase, and ART domains  . Althoug × 10−9) , 45. The × 10−9) , 47. Mos domains . This obWe next present a systematic analysis and functional prediction (where possible) of the domains that are commonly found in polyvalent proteins, followed by an analysis of their contextual associations.One of the most frequently found classes of catalytic domains observed in polyvalent proteins are predicted to operate on proteins and peptides. These include peptidases and four families of protein-modifying domains, namely, the ARTs, serine/threonine/tyrosine-type protein kinases, GCN5-like acetyltransferases (GNATs), and the polymerase β (Pol-β) superfamily nucleotidyltransferases (NTases). These domains are also encountered in diverse conflict systems such as polymorphic toxin, toxin-antitoxin, antibiotic resistance, and bacterial effector systems targeting eukaryotic host proteins , 48, 49.Polyvalent proteins possess four distinct superfamilies of peptidases. Of these, the zincin-like MPTase is by far the most widespread and is overall the most common type of domain in polyvalent proteins. It is also the most prominent hub domain in the overall domain network of polyvalent proteins . About 32+ ion in the active-site pocket, whereas the glutamate in the HEXXH motif is predicted to function as the general base that activates a water molecule for proteolysis . None of these clades are specifically related to other previously defined clades of zincin-like MPTases, suggesting that they are likely to constitute a distinct group that diversified in the context of polyvalent proteins.The core fold of the zincin-like MPTase domain is characterized by the presence of a three-stranded β sheet flanked by an N-terminal helix and two C-terminal helices , 50. Theteolysis . For exateolysis . In yet darA operon. This protein has been shown to be proteolytically processed into a 76-kDa (∼700-aa) polypeptide before being packaged into the phage head  along with a phage N4-like RNAP module , serine (S), and glutamate (E) residues and are grouped in the larger R-S-E-2 clade of ARTs ; neverthftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html). They also contain unique distinguishing features such as a glutamate N terminal to strand 2 associated with the ATP-binding site, a conserved glutamine in the last strand of the N-terminal α+β subdomain, and an insert between the conserved histidine and aspartate residues involved in phosphotransfer (see File S2 at the URL mentioned above). This type of kinase domain is present primarily in polyvalent proteins of (pro)phages of the Verrucomicrobia-Chlamydia and Bacteroidetes lineages of bacteria. The latter additionally contain stand-alone versions of this distinct kinase domain. In Verrucomicrobiumspinosum, the kinase-containing polyvalent proteins are clustered in two loci. In the vicinity of the gene encoding the large polyvalent protein, which is likely to be the primary locus, there are several smaller open reading frames coding for other domains typical of polyvalent proteins  , suggesting deployment in the capsid like the polyvalent proteins. Both of these families, though not sister clades, belong to the larger clade of RimI-like GNATs  . Solo mensferase , 62), suThese NTase domains are found primarily in polyvalent proteins from firmicute mobile elements. They are usually fused to an ArdC-N domain (see below) and found in the context of genes coding for T4SS-like DNA transfer systems of conjugative transposons or plasmids . In addiGiven the effectiveness of damage to genomic DNA and various RNAs, particularly those associated with translation, in crippling a biological system, enzymes targeting nucleic acids are the mainstay in biological conflicts on both the side of the host and that of invasive elements , 10, 41.ftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html). We found that the N-terminal LPD36 domain is likely to adopt a fold with four α helices and is distinguished by a characteristic GXGU motif  between the second and third conserved helices. Although profile searches did not retrieve any relationship to known domains, its position is reminiscent of the α-helical HdsM-N domain that is found N terminal to the EcoKI-like methylase found in type I restriction enzymes. This observation, together with the above-noted motif, which is suggestive of nucleotide binding, suggests that the LPD36 domain might be involved in base recognition and flipping, as seen in equivalent N-terminal domains of other adenine MTases  helicase and at its N terminus to a previously uncharacterized domain, LPD36, found only in polyvalent proteins. The archetypal member is the DarB protein from phage P1/P7, which is involved in antirestriction  Fig. 4FFig. 4F. e MTases , 68.Methylomonas SnoC-wHTH; accession no. WP_020485828.1; The SF2 helicase-like module associated with the C terminus of the adenine MTase belongs to a vast group of enzymes performing ATP-dependent helicase or translocation activity on various nucleic acid polymers. Analysis of the sequence conservation patterns revealed that there are two major families of SF2 helicase-like domains in polyvalent proteins, both of which are specifically related to the SWI2/SNF2 ATPase clade. Like the classical SWI2/SNF2 ATPases, these families from polyvalent proteins also possess a multihelical insert after strand 4  of the first of the two nucleoside triphosphatase (NTPase) domains typical of helicase-like modules . They alcis against attack by EcoK1-like type I restriction enzymes  and and51) a enzymes . The str enzymes , 76–78. zymes 76–. A similhelicase  cooccurshelicase . These Uhelicase . On the Acinetobacter  the DdrB-like ParB domain is fused to a lysozyme domain in the context of late phage genes, supporting the idea that it is injected along with breaching of the host peptidoglycan by the latter domain. In the exceptional instance of Gracilimonas (accession no. WP_020402799), a DdrB-like ParB domain is associated with an ArdC-N domain that is normally associated with T4SS-like DNA transfer systems (see below). However, the genomic contexts do not reveal any further connections to mobile elements or phages, suggesting that it might have been acquired by the host as defense against invading elements (see below).We recovered a distinct family of domains belonging to the ParB/sulfiredoxin (ParB/Srx) superfamily in polyvalent proteins, which are prototyped by the version found in the phage P1 DdrB protein . DdrB-like ParB domains have an α+β fold with four strands and two conserved helices in common with other members of the ParB/Srx superfamily . They alBacillus cereus Bce_0392 (ParB-methylase), the ParB domain, which is fused to an adenine methylase, was shown to possess nonspecific nicking endonuclease activity  is fused to an HNH domain and is likely to function as a UTPase and a nonspecific nuclease , 83. In activity . In the activity . Similaractivity , 85, 86.activity . Howeveractivity . In a siactivity . While PEscherichia coli RadC has been implicated in DNA repair   and are limited to only the Firmicutes and Fusobacteria , suggesting a phage source for this domain in the conjugative elements. The above patterns suggest that the primase domains have been independently incorporated into polyvalent proteins on multiple occasions. This provided us with evidence of a strong selective pressure that channels the emergence of these multidomain polyvalent proteins across diverse elements.Two unrelated primase domains, namely, the Toprim-type e.g., DnaG primases) and the archaeoeukaryotic primase fold Primpol , and the coliphage N4-like virion RNAP modules are widely found across polyvalent proteins , and G.  primasesnt.html) . The Topnt.html) . Further domains  in polyvbacteria  and 4J. In contrast, the N4-like virion RNAPs are found only in N4-like phages or prophages in stereotypic gene neighborhoods with late genes coding for virion components  Fig. 1DFig. 1D. NP_050634.1), a major component of the phage head contains a C-terminal PBECR domain  across a group of Helicobacter (pro)phages. The basic collection of domains in this polyvalent protein includes four distinct PBECR domains and DdrB-like ParB    and 5A aThe 2H phosphoesterase domains catalyze a related set of reactions, namely, as processing enzymes of ends of RNAs with 2′-3′ cyclic phosphate linkages produced by metal-independent RNase attacks, cyclic nucleotides with 2′-3′ cyclic linkages, and polynucleotides with 2′-5′ linkages, and as RNases which generate ends with cyclic 2′-3′ phosphates , 106. WhRecent studies have shown that nucleotides and other small molecules derived from them are signaling agents at the center of several intergenomic conflicts in both prokaryotes and eukaryotes , 4, 110.Spirochaetes and epsilon Proteobacteria. RelA/SpoT domains are also fused to the MuF domain in phage/prophages and in MuF-based toxin delivery systems . Analysis of the genome contexts of these proteins suggests that they are present mainly in (pro)phage polyvalent proteins and are likely to be packaged into the virion (Burkholderia virus BcepF1), suggesting that they might be injected into the host during infection. Related InPase domains are encoded in the neighborhood of or are fused to the NUDIX and GNAT domains , while others from actinobacteria  are linked in neighborhoods with the tunicamycin  domains belonging to the so-called type I InPase family  and, cone virion , and G. rivative ) resistacription . This isption  with a purine or pyrimidine base or an NH2 group along with anomeric inversion of the ribose ring (Proteobacteria and Verrucomicrobiae. It is predominantly found in (pro)phage polyvalent proteins, with the simplest multidomain architectures showing fusions to the MuF domain . Moreover, they share a highly conserved arginine in the loop after the second strand with the ComFC-like PRTases, which have been previously implicated in competence-related DNA uptake , which also cleaves peptidoglycan but belongs to a fold distinct from the lysozyme domain  various noncatalytic domains with predictable function, (ii) previously defined domains with poorly characterized function, and (iii) newly defined large polyvalent-protein-associated domains (LPDs).AAD52160.1). It is overall the second most common domain in polyvalent proteins. Previous studies have suggested that the ArdC-N domain binds ssDNA  indicates that the nested hairpin structure is critical for binding of ssDNA, suggesting that this might be a conserved feature of ArdC-N domains  is one prototyped by the N terminus of the plasmid pSa antirestriction protein ArdC , the Rho-N/SAP HEH fold domain , other HTH domains, and a histone fold  domains . In phage P1, DarA is known to be proteolytically cleaved and incorporated into the phage head . In light of the above observations on the characterized domains in the polyvalent proteins, these potentially enzymatic domains might perform novel catalytic functions in some of the previously noted categories. Further contextual analysis shows that some of them are strongly associated with a limited number of domains indicating functional linkages between them: LPD10 and LPD24 with LPD11; LPD28 with an MPTase; LPD30 with LPD29, and LPD36, as noted above, with the adenine methylase  whose precise functions remain elusive, as most of them cannot be currently unified with known domains. Nevertheless, the conservation patterns and secondary structures provide tantalizing hints regarding some of them. For example, at least seven of these domains  are potentially enzymatic, as they show strongly conserved charged and polar residues typical of enzymes  , which means that the shortest path between any two nodes in the network passes through them  typical of several other biological networks, namely, a power law-like distribution of number of edges  . This meP = 1)  by a genugh them . In sociWe also examined other network parameters, such as hub and authority scores, which were network measures originally developed for the internet to rank highly connected and authoritative Web pages (see Table S3 at the URL mentioned above) . In the Cliques are the most densely connected subnetworks within networks where every node is connected to every other node of the clique. We accordingly detected the largest cliques in our network (size of seven or eight nodes) and examined the subnetwork obtained by merging these cliques. This produced a subnetwork of 37 nodes  that, whftp://ftp.ncbi.nih.gov/pub/aravind/polyvalent/polyvalent.html). However, they are more prevalent in the former, and currently, almost all examples of large proteins  come from bacteria. The most basic contextual linkages are related to the type of element deploying the polyvalent protein. (i) The association with flanking genes encoding RCR-related components such as relaxases and the Tra/Vir proteins that constitute the T4SS-like DNA pump and the rest of the DNA transfer apparatus . Repeated accretion from solo modules or smaller multidomain proteins also points to an arms race with the host that has selected for multipronged strategies to be deployed at once along with the transferred DNA. This potentially allows the invading elements to simultaneously present multiple alternative options against host defenses that might be directed at particular strategies of the invasive element. This is consistent with evidence of evolution of multipronged strategies on the host side that target different aspects of the element's biology. An analogy may be drawn between the polyvalent proteins described in this work and the polyproteins of eukaryotic RNA viruses . Both caFirmicutes, Proteobacteria, Bacteroidetes, Spirochaetes, and Fusobacteria . These lineages also show greater complexity in their architectures than other prokaryotic lineages (see File S7 at the URL mentioned above). Of the lineages with many representative genomes, the archaea and cyanobacteria show a particular paucity of polyvalent proteins. One possibility is that in these lineages, the primary mobile elements and phages infecting these hosts are likely to be distinct from those that deploy polyvalent proteins. In particular, phages deploying polyvalent proteins belong to the terminase portal class. These phages lack a membrane internal to the capsid typical of the viruses with the HerA/FtsK class of DNA pumps. This feature might expose their DNA to a more immediate attack by host systems. Finally, we found that at least one domain, the ArdC-N domain, which is specific to polyvalent proteins, has spread beyond the circle of prokaryotic mobile elements and their host genomes. This domain was acquired by eukaryotes on two independent occasions, once in the form of the DNA-binding domains of the XPC/Rad4 protein and once in the form of the DNA-binding domains of the Trypanosoma Tc38 family of proteins. In the first case, the transfer appears to have preceded the last eukaryotic common ancestor and the domain was incorporated into a protein that is part of the DNA repair network unrelated to the original role of the ArdC-N domain . This suggests that they were recruited primarily for their distinctive ssDNA recognition capability, which proved useful in the context of DNA mismatch recognition . The length (L) and score (S) threshold parameters were variably adjusted, depending on need. For example, the threshold parameters for clustering of nearly identical proteins were L = 0.9 and S = 1.2. The HHpred program was used for profile-profile searches  program. The in-house TASS package, which comprises a collection of Perl scripts, was used to automate aspects of large-scale analysis of sequences, structures, and genome context. Network analysis was performed in the R language with the igraph and circlize packages  , 158. Sisearches . Structusearches , 161. Musearches  and PCMAsearches  programssearches . For presearches  was usedrameters . Structupackages , 168.


Echinochloa crus-galli were investigated. The results showed that metamifop interacted with the CT domain of ACCase in E. crus-galli. The three-dimensional structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling, molecular docking and molecular dynamics (MD) simulations. Metamifop has a different mechanism of inhibiting the CT domain compared with other ACCase inhibitors as it interacted with a different region in the active site of the CT domain. The protonation of nitrogen in the oxazole ring of metamifop plays a crucial role in the interaction between metamifop and the CT domain. The binding mode of metamifop provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors.Acetyl-coenzyme A carboxylase (ACCase) plays crucial roles in fatty acid metabolism and is an attractive target for herbicide discovery. Metamifop is a novel ACCase-inhibiting herbicide that can be applied to control sensitive weeds in paddy fields. In this study, the effects of metamifop on the chloroplasts, ACCase activity and carboxyltransferase (CT) domain gene expression in  Metamifop exhibits high control efficacy against sensitive weeds, especially Echinochloa crus-galli in paddy fields. ACCase plays a crucial role in fatty acid biosynthesis in plants and is an attractive target for herbicide exploitationMetamifop (CAS number: 256412-89-2), developed by Dongbu Hannong Chemical Co., Ltd. , is a postemergence herbicide classified as a lipid synthesis inhibitor (inhibition of acetyl-coenzyme A carboxylase (ACCase)) according to the Herbicide Resistance Action Committee  CT domain in complex with haloxyfop1213Setaria italica CT domain in complex with ACCase inhibitors suggested that the orientation of the carboxyl group of the inhibitors bound to the binding pocket differed, but these ACCase inhibitors can form a hydrogen-bond with the Ser698 residueACCase-inhibiting herbicides, such as haloxyfop, diclofop, and sethoxydim, inhibit the CT domain of ACCase9E. crus-galli are described. The structure of the CT domain of E. crus-galli ACCase in complex with metamifop was examined by homology modelling and molecular dynamics (MD) simulations. The mechanism of CT domain inhibition by metamifop was rather different from that of other ACCase herbicides. This study provides a foundation for elucidating the molecular mechanism of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors. Moreover, molecular insight into the interactions between metamifop and the CT domain may aid in understanding the significance of the protonation of compounds.Although the binding mode of herbicides to the CT domain of yeast ACCase was previously established, these herbicides exhibited low inhibitory activity against the yeast CT domain. Some residues in the active region of the dimer interface between the plant and other ACCases exhibited significant variations that may lead to a change in the dimer organization for herbicide bindingTo investigate the effect of metamifop on the physical organization of chloroplast in plant cells, the ultrastructure was examined by transmission electron microscopy (TEM). Significant alteration in the chloroplast structure was observed in metamifop-sprayed plants compared with healthy control plants. Electron micrographs of chloroplasts from metamifop-sprayed plants, compared with control plants  at the sE. crus-galli is 1659 bp (accession number KU198448), encoding 553 amino acid residues corresponding to residues 1640 to 2193 of Alopecurus myosuroides (accession number AJ310767). The deduced amino acid sequence exhibited approximately 93% identity with the CT domain of plastidic ACCase from A. myosuroides, whereas only 78% amino acid identity was observed with cytosolic ACCase from A. myosuroides (accession number AJ632096). A phylogenetic tree of the CT domain in plant ACCase showed that the CT domain gene cloned from sensitive E. crus-galli was classified in the plastidic clades  at the 2-leaf stage  was increased approximately 2.0-fold compared with healthy control plants. In contrast, reduced expression was found at the 2-leaf stage in plants sprayed with a medium dose of metamifop  compared with control plants. As the plants grew to the 3-leaf stage, slightly significant differences in the expression of the CT domain gene were noted between metamifop-treated and healthy control plants. Interestingly, the expression of the CT domain was significantly upregulated from the 2-leaf stage to the 3-leaf stage at a medium dose of metamifop.The peak expression level of the CT domain gene from plants treated with a low dose of metamifop  value for metamifop was 41.1 nM. These results verified that metamifop efficiently inhibited ACCase activity.The effect of different doses of metamifop on ACCase activity was determined using a dose-response curve . The triE. crus-galli ACCase and yeast ACCase was 53%; thus, the information from the crystal structure of the yeast CT domain (PDB code: 1UYS and 1UYT) should be applied to homology modelling of the E. crus-galli CT domainE. crus-galli CT domain was good, with most of the residues (91.5%) in the most favoured region of the Ramachandran plot and only 0.2% in the disallowed region  plot of the Cα atoms for the CT domain and the total energy (ETOT) plot for the simulation system indicated that the system had achieved equilibrium after relaxation , and propanamide  and aspartates  were key residues for electrostatic interactions. These acidic amino acids generated an acidic environment for metamifop interactions with the active site of the dimer interface. However, the phenomenon of protonation of nitrogen on the oxazole ring in metamifop could not occur when these acidic amino acids were mutated to neutral or basic amino acid residues; thus, metamifop could not form a hydrogen-bond to link Thr194. More importantly, the binding free energy of metamifop in complex with the mutant CT domain was significantly increased compared with the wild type CT domain, indicating that metamifop cannot bind with mutated CT domain well. Based on the theory of the computational results, we hypothesized that the protonation of nitrogen in the oxazole ring of metamifop can occur spontaneously under acidic conditions panamide  were anapanamide . MoreovetamifopH . The resE. crus-galli and inhibited the CT domain activity of ACCase, thereby shutting down fatty acid biosynthesis. Metamifop may imitate the behaviour of the substrate biotin to protonate the nitrogen of the oxazole ring, form an NH- functional group and then interact with the CT domain.The molecular mechanism for the inhibitory action of metamifop has not been previously reported. In this study, we clearly demonstrated that metamifop was bound in the active site of the CT domain of chloroplast ACCase in Metamifop caused more severe damage to the ultrastructure of chloroplasts with increased doses. Increased amounts of plastoglobules in the chloroplasts of plants were noted with a relatively high dose of metamifop . Plastog17E. crus-galli. Additionally, the effect of different doses of metamifop on ACCase activity was determined using a dose-response curve, which demonstrated that metamifop can efficiently inhibit ACCase activity. A previous study also found the IC50 value for quizalofop-P-ethyl (an ACCase inhibitor) for the inhibition of E. crus-galli ACCase (Geqiushan S biotype) was 41 nME. crus-galli.ACCase played a crucial role in the lipid metabolism in plants; therefore, it was an attractive target for herbicide discovery71213E. crus-galli, the three-dimensional structure of CT domain of E. crus-galli ACCase in complex with metamifop was formed and examined by homology modelling, molecular docking and MD simulations. The structure clearly showed that metamifop was bound in the active site of the CT domain of E. crus-galli ACCase at its dimer interface. However, metamifop interacted with a different region from other ACCase inhibitors, as represented by haloxyfopA. myosuroides CT domain in complex with haloxyfop and clodinafop also revealed these key interactionsTo decipher the molecular mechanism of metamifop inhibition of the CT domain of acetyl-coenzyme A carboxylase in Mycobacterium tuberculosis ACCase in complex with haloxyfop revealed that Gly138 (corresponding to Gly171) hydrogen-bonded with the carboxylate oxygen of haloxyfop12131213E. crus-galli was crucial for binding with metamifop.More importantly, the protonation of nitrogen on the oxazole ring of metamifop played a key role in binding interactions between metamifop and the CT domain. The additional proton gained by protonation was hydrogen-bonded to Gly171 (corresponding to Gly1734 next to Ile1735 in the yeast CT domain). Similarly, the crystal structure of the sixth CT domain of A. myosuroides may play a crucial role in ACCase-inhibition herbicides binding to the CT domain of grass ACCase. Gramineous weeds resistant to ACCase-inhibition herbicides have increased rapidly, mostly caused by a point mutation or multiple mutations in the CT domain of chloroplastic ACCase. Interestingly, the Ile1781/Leu mutation has been identified in almost all resistant weeds25Toxoplasma gondii had a Leu residue at position 1705  but was still sensitive to the aryloxyphenoxypropionate herbicidesThe active site Ile172 that is equivalent to Ile1781 in the CT domain of The residue Thr194 also played a key role in the interaction with metamifop. Initially, the oxygen on amide was hydrogen-bonded to Thr194 . HoweverM. tuberculosis CT domain in complex with haloxyfop showed that the side chain oxygen atom of Tyr326 (similar to Phe1956’ in the yeast structure) was hydrogen-bonded to the backbone nitrogen of Thr160 through a water linkage, making the binding pocket stableE. crus-galli was consistent with the position of Thr160 in M. tuberculosis AccD6, indicating that Thr194 may stabilize the interaction of CT domain with metamifop. The crystallographic structure of Staphylococcus aureus pyruvate carboxylase in complex with coenzyme A suggested that Thr908 was hydrogen-bonded to the N1′ atom of biotin. Importantly, a general acid was needed to protonate the N1′ atom of biotinIn vitro, the protonation of the nitrogen on the oxazole ring was confirmed by our experiments. These results demonstrated that the protonation of the nitrogen would occur in the binding with the E. crus-galli CT domain.Metamifop imitated the behaviour of the substrate biotin to protonate the nitrogen of the oxazole ring and form an NH- functional group that was hydrogen-bonded to the CT domain. The crystal structure of Deciphering the mechanism of metamifop provides a foundation for elucidating the molecular basis of target resistance and cross-resistance among ACCase herbicides, and for designing and optimizing ACCase inhibitors against weeds. Moreover, this study may aid in understanding of the significance of the protonation of compound in interaction of compound with target enzyme.E. crus-galli seeds collected from an untilled field in Wuxue County, China in 2011 were self-crossed for three successive generations as a susceptible biotype (WX103). WX103 is susceptible to metamifop, bispyribac-sodium, penoxsulam and quinclorac. Seeds of WX103 were germinated on wet paper for 48 h at 28 °C under constant light (approx. 3000 lx). The seedlings were transplanted to pots filled with a mixture of sand and loam in a greenhouse at 25 °C with a 16-h light/8-h dark photoperiod.E. crus-galli plants grew to the 2-leaf stage and were treated with different doses of metamifop at 0, 12.5, 45 and 90 g a.i. ha−1. After three days, fresh leaves were harvested with a sharp razor blade and cut into small pieces. Then, the samples were fixed in 2.5% (v/v) glutaraldehyde and 1% (v/v) osmium tetroxide. The sections were treated as described by Wang, Y. et al.E. crus-galli using the RNAprep Pure Plant Kit . For RT-PCR, first-strand cDNA synthesis was reverse transcribed from the total RNA using the RevertAid RT Kit (Thermo Scientific). The amplification product obtained from at least 6 independent PCR reactions was purified and then sequenced on both strands using gene-specific primers. The isolated CT domain sequence was aligned with other plant CT domain sequences from cytosol and plastid ACCases, and then a phylogenetic tree was constructed by the neighbour-joining method using Mega 6Total RNA was isolated from leaves of The level of CT domain gene expression was analysed by real-time PCR using β-actin as the reference gene. Triplicate cDNA aliquots for each sample from individual plants were amplified by qPCR. The relative changes in gene expression were calculated by the et al.When plants grew to the 2- to 3-leaf stage, the shoots were cut at the base and then stored at −80 °C. ACCase isolation was performed according to the procedures described by Cocker et al.The enzyme activity assay method was modified based on Zhibo H. E. crus-galli ACCase (accession number KU198448) was numbered 1 to 553. Based on the templates of the B and C chains from the crystal structures of the yeast CT domain and its complexes with haloxyfop and CoA (PDB code: 1UYS and 1UYT), the theoretical structure of the CT domain from E. crus-galli was developed using Modeller 9.12http://services.mbi.ucla.edu/SAVES/. Finally, the most suitable model was selected for molecular docking with metamifop to build the theoretical model of the E. crus-galli CT domain in complex with metamifop (CT-metamifop complex) using the Surflex-docking program with Sybyl 7.3 The amino acid sequence of + ions.The CT-metamifop complex was subjected to MD simulations using the SANDER module of the AMBER 14 package37−1·Å2. Then, the model was relaxed in 4 discrete simulation steps of 250 ps at 300 K and constant pressure (1 atm) by decreasing the force constant of the imposed position restraint on all CA, C, N atoms of the complex from 3 to 0 kcal·mol−1·Å2. The MD simulations ran for an additional 3 ns after relaxation at a constant temperature of 300 K with a time constant for heat bath coupling of 5 ps using the weak-coupling method. The Particle Mesh Ewald methodThe system was minimized in 2000 steps steepest descent (SD) and 2000 steps conjugate gradient (CG) with the system free and heated gradually from 0 to 300 K using Langevin thermostats with a collision frequency of 1.0 in 50 ps with the force constant of a restraint on all CA, C, N atoms of the complex at 4 kcal·molBinding free energy and energy decomposition were calculated using the molecular mechanics-Generalized Born Surface Area (MM/GBSA) methodFor the first stage, metamifop binding at the active site of the CT dimer interface, structures were sampled from the MD trajectories from 10 to 380 ps. For the second stage, metamifop moving to the entrance of binding channel, CT-metamifop complexes were selected from 382 to 3000 ps for the binding free energy calculation.In vitro, 0.1 M HCl was added to a solution of metamifop in MeOH, and the reaction system was agitated at room temperature for 12 h. The reaction products were neutralized with 0.1 M NaOH, and the target product was extracted by ethyl acetate. The solvent was removed in vacuo, and the product was dried by vacuum drier at 50 °C. The change in the chemical shift of the hydrogen closest to the nitrogen in the oxazole ring of metamifop, determined by comparing 1H NMR spectrum diagrams of the product to the spectrum of metamifop, was used to confirm the protonation of the nitrogen.How to cite this article: Xia, X. et al. Mechanism of metamifop inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase in Echinochloa crus-galli. Sci. Rep.6, 34066; doi: 10.1038/srep34066 (2016).


Receptor-type protein tyrosine phosphatases (RPTPs) of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, dimerization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC) in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascular integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with controls. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a membrane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane domains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that removal of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family  also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and transmembrane domain of R3-PTPs in interaction with VE-cadherin. The  synapse –14.The R3 subgroup of RPTPs includes density-enhanced phosphatase 1 (DEP-1), vascular endothelial protein tyrosine phosphatase (VE-PTP), stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1), glomerular epithelial protein 1 (GLEPP1), and receptor-type protein tyrosine phosphatase Q (PTPRQ). They share a similar structure composed of a heavily glycosylated ectodomain containing a variable number of fibronectin III (FNIII)-like repeats, a transmembrane spanning domain, and a single intracellular phosphatase domain. The number of FNIII repeats ranges from 8 in GLEPP1, which also has a truncated isoform lacking the ectodomain, to 17 in VE-PTP and 18 in PTPRQ. The prototypical FNIII domain is found in the extracellular matrix protein fibronectin, and cells interact with this molecule via integrin α5β1 and αVβ3 binding to the RGD (Arg-Gly-Asp) sequence in exposed loop of FNIII-10 and the synergy site in FNIII-9 ; howeverin vitro and in vivo studies to maintain endothelial cell contact integrity via an interaction with the cell adhesion molecule VE-cadherin [th FNIII domain of VE-PTP and 5th cadherin domain of VE-cadherin [bona fide protein-protein interaction. Although more recent versions of the system have aimed to address this issue [R3-PTPs play many important physiological roles . VEcadherin –25, and cadherin . Co-immucadherin . In our cadherin . The tecis issue , it is ris issue . Using tMouse monoclonal anti-myc antibody (4A6) was purchased from Millipore. Anti-HA antibody (12CA5), anti-VE-cadherin antibody (BV9) and anti β-actin antibody were obtained from Abcam. Goat anti-mouse IgG-HRP linked secondary antibody was obtained from Sigma.Human cDNAs for VE-PTP (BC096364.3), GLEPP1 (BC126203), SAP-1 (BC111716), sialophorin (BC012350) and VE-cadherin (BC096364.3) were purchased from SourceBioscience. A synthetic sequence was inserted into the BC096364.3 clone to generate a full-length gene sequence that corresponds to P33151-1. A construct encoding human DEP-1 was kindly provided by A. Weiss . The pBiFC-VN155  and pBiFC-VC155  vectors encoding N- and C-terminal fragments of the Venus yellow fluorescent protein (YFP) respectively were from Addgene .ApaI/XhoI or ApaI/EcoRI restriction enzyme sites of the pBiFC vectors. The ectodomain and transmembrane coding sequences for the above proteins were amplified by PCR using Platinum Pfx DNA Polymerase (Invitrogen), and subsequently inserted into the XhoI site or EcoRI/XhoI sites of the vectors. Oligonucleotide primer sequences are listed in ApaI/KpnI of pBiFC-VC155. Construct sequences were confirmed by Sanger sequencing (GATC Biotech).A myc-tag (EQKLISEEDL) in pBiFC-VN155 or HA-tag (YPYDVPDYA) in pBiFC-VC155 was inserted after the signal peptide for each of the constructs by cloning a synthetic DNA sequence  purchaseXmaI restriction enzyme site. Following PCR amplification using the VE-PTP-VN construct as a template the product was digested with DpnI/XmaI restriction enzymes and ligated to create expression vectors for the deletion or truncation mutants indicated. Membrane anchored pBiFC-VN155 or pBiFC-VC155 constructs containing the Lck myristoylation sequence followed by a myc-tag were generated by cloning a synthetic adaptor DNA sequence (ApaI/EcoRI of the vector.Deletion and truncation mutants of VE-PTP were generated by PCR . Primerssequence  into the2 in a humidified incubator. Cells were trypsinized at 80% confluence and seeded 24 hours prior to transfection on 6-well (3 x 105 cells / well) plates.Human Embryonic Kidney 293T (HEK293T) cells (ATCC code CRL-11268) were maintained in Dulbecco's Modified Eagle's Medium , containing GlutaMAX, high glucose, sodium pyruvate and supplemented with 10% (v/v) fetal bovine serum at 37°C and 5% COTransient transfections were performed using Lipofectamine LTX Plus (Invitrogen) in 500 μl Optimem according to the manufacturer’s instructions with 0.5 μg of each construct. After 24 hours cells were stained with CellMask™ Deep Red plasma membrane stain (ThermoFisher Scientific) for 1 hour. The media was then removed, cells were rinsed with warm Hanks' Balanced Salt Solution (HBSS) and images acquired by sequential scanning using a Leica TCS SP2 confocal system with a 63× ceramic dipping objective. A 514 nm Argon ion laser was used to excite Venus YFP (Ex/Em 514/527 nm) with detection at 525–560 nm; and a 633 nm Helium Neon laser, intensity 35%, was used for the red stain (Ex/Em 649/666 nm) with detection at 660–685 nm, together with a dichroic beamsplitter DD458/514.https://imagej.net/Fiji) was used to trace the border of each cell  and determine the average pixel intensity in both the yellow and red channels of the selected region. A background value from a region with no cells was subtracted. Values for both the yellow/red ratio for each region of interest and mean BiFC fluorescent intensity for each pairing were then calculated. Data was compared using IBM SPSS Statistics 22 software, using the Mann-Whitney U test. A value of P<0.05 was considered statistically significant for any set of data.For each combination of BiFC constructs analyzed, three images were obtained from three or more independent experiments. Fiji image analysis software . The total cell lysate (20 μl) was heated to 95°C for 2 minutes and loaded onto a 4–20% Mini-Protean TGX precast SDS-PAGE gel (Bio-Rad) and subjected to electrophoresis at 110V for 60 minutes. Proteins were transferred to PVDF membranes  in transfer buffer  for 2 hours at room temperature. Membranes were blocked with 3% w/v non-fat dry milk in Tris-buffered saline (TBS) and incubated with specific primary antibodies for 1 hour at room temperature. Following washing with TBS containing 0.05% Tween-20 (TBS-T) membranes were incubated with peroxidase-linked secondary antibody and developed with SuperSignal West Femto Maximum Sensitivity ECL Substrate (ThermoScientific) and visualized using an imaging system (UVP) with Labworks 4.1.5 in 50 μl) were incubated for 30 mins with anti myc-tag antibody  at a 1:100 dilution and then washed with 900 μl of FACS buffer and incubated with goat anti-mouse Alexa fluor-488 (Invitrogen) at 1:200 dilution for 30 mins on ice in the dark. An isotype control antibody and non-transfected cells were used as controls to validate antibody specificity. Cells were diluted in 450 μl FACS buffer and analysed on a BD Accuri C6 Plus flow cytometer. Cells were gated using forward scatter (FS) and side scatter (SS) to exclude dead cells and cellular debris.Transiently transfected HEK293T cells were detached with Accutase, centrifuged for 5 mins at 400 g and resuspended in ice-cold FACS buffer (PBS supplemented with 0.2% BSA and 0.02% sodium azide). Cells   as a fusion with the N- or C-terminal vYFP sequence (designated SPN-VN and SPN-VC respectively) was used. The SPN protein is similar to R3-PTPs in that it has a large highly-glycosylated ectodomain and a single transmembrane spanning region; however, it has no homology to VE-PTP, or other R3-PTPs, and for these reasons has previously been used as a control in other studies of R3-PTP function . AnotherThe fluorescence complementation signal resulting from VE-PTP-VN and VE-cadherin-VC co-expression in HEK-293T versus the VE-PTP-VN and the control pairings is shown,th cadherin domain of VE-cadherin and 17th FNIII-like domain of VE-PTP) are sufficient to facilitate this interaction and other parts of the molecules  are not required [th FNIII-like domain has been removed) and VE-PTP constructs  in live cell BiFC assays to further investigate the molecular basis of the VE-PTP with VE-cadherin interaction (th FNIII-like domain (Δ17 FN) led to an almost complete loss of cell surface, although the total expression levels for this construct are also lower , co-localisation of the BiFC signal and plasma membrane staining was negligible, indicating that the interaction takes place in an intracellular structure (th domain (17 FN) reaches the cell surface, the BiFC signal is almost exclusively intracellular and is not apparent at the plasma membrane.Comparison of average BiFC fluorescence intensity obtained with the VE-PTP-VN truncation or deletion mutants paired with VE-cadherin, relative to control proteins paired with VE-cadherin, revealed that a specific interaction was observed with all of the VE-PTP constructs . A statitructure . These fThe molecular basis for the interaction of VE-cadherin with receptor-type phosphatases was explored further by examining whether other members of the R3-PTP family  have the potential to interact with this protein. Expression analysis of whole cell lysates from transfected HEK-293T cells indicated all three constructs  were expCo-expression of BiFC constructs for other R3-PTP family members  with VE-cadherin resulted in a significantly higher average BiFC signal relative to the R3-PTP construct paired with myr-VC . The BiFThe molecular basis for the interaction of VE-PTP with VE-cadherin has previously been studied using co-immunoprecipitation experiments; however, it has not to-date been studied in live cells, and it is not known whether other R3 subgroup RPTPs have the potential to interact with VE-cadherin. Here we used the bimolecular fluorescence complementation technique to investigate the basis for the interaction of VE-PTP and VE-cadherin in live cells. Quantitative assessment of the fluorescence images obtained on co-expression of VE-PTP and VE-cadherin constructs containing the ectodomain and transmembrane domain of these molecules indicated a BiFC signal that is due to a specific interaction, rather than self-assembly of constructs and background fluorescence.et al study [th FNIII-like domain from the ectodomain of VE-PTP would be sufficient to abolish the VE-PTP interaction with VE-cadherin; however, this was not observed in our BiFC studies. A similar BiFC signal was obtained with VE-PTP-VN, containing the full extracellular domain and transmembrane domain of VE-PTP, and VE-PTP-VN (Δ17FN), in which the 17th FNII-like domain proximal to the membrane has been deleted. This was surprising since VE-PTP-VN (Δ17FN) is expressed at significantly lower levels than VE-PTP-VN. Flow cytometry showed that expressed VE-PTP-VN (Δ17FN) does not reach the cell surface and line scan analysis across the cell membrane of images indicated that the observed BiFC signal was predominantly intracellular, most likely in the endoplasmic reticulum or Golgi.We had hypothesized, based on the Nawroth al study , that deet al study, there are differences in the constructs used. In the construct VE-PTP-VN (Δ17FN) the 17th FNIII domain of VE-PTP was deleted, which brings the 16th FNIII domain proximal to the transmembrane domain, while the earlier study used a construct in which FNIII domains 1–16 had been deleted and a construct which expressed only the 17th domain. Flow cytometry analysis of VE-PTP truncation constructs to determine cell surface expression was not reported in the Nawroth et al study, so it is possible that interactions observed in the co-immunoprecipitation reflect interaction of protein in the cytoplasm, as in our study. Our analysis of VE-PTP truncation mutants in which FNIII-like domains 1–4, 1–9 and 1–16 have been deleted demonstrated that this region is dispensable for interaction with VE-cadherin, and that a construct consisting of only the 17th FNIII-like domain and transmembrane domain can still interact with VE-cadherin. This observation suggests that the 17th FNIII-like domain and transmembrane domains of VE-PTP are important for the interaction with VE-cadherin and are in agreement with Nawroth et al; however, the interaction observed in our study was predominantly intracellular rather than at the plasma membrane.In addition to the differences in experimental approach (co-immunoprecipitation vs live cell BiFC) between our study and the Nawroth th FNIII-like domain of VE-PTP alone is not sufficient to support the interaction of VE-PTP and VE-cadherin at the plasma membrane.Our measurements of the cellular BiFC levels with various VE-PTP fusion constructs co-expressed with VE-cadherin fusion constructs reflect the signal from the whole cell and do not distinguish intracellular versus plasma membrane bound signals. Attempts to accurately quantitate the two fractions by various image analysis approaches are hampered by the fact that in some, but not all cells, a substantial fraction of the BiFC signal originates from the endoplasmic reticulum and Golgi which lies in close proximity to the plasma membrane and thereby gives an over-estimate of the plasma membrane BiFC signal. Nonetheless the combination of BiFC in live cells, flow cytometry and image analysis by line scanning has allowed us to confirm that the 17A key question is whether the BiFC interaction observed in intracellular structures represents a physiologically relevant interaction, or is a result of the heterologous expression system used. In other BiFC membrane protein studies it has been reported that expression of a non-specific partner protein, or a mutant that fails to form a complex, leads to loss of cell surface membrane protein expression, with retention of the BiFC signal inside intracellular compartments , 40. NonAnalysis of the potential of other R3-PTPs  to interact with VE-cadherin revealed that the series of 8–9 extracellular FNIII-like domains and transmembrane region from any of the R3-PTPs has the potential to mediate a direct interaction with VE-cadherin. This is of particular interest with regard to DEP-1 since it is expressed in endothelial cells together with VE-cadherin, its localization at inter-endothelial cell contacts overlaps with VE-cadherin, an interaction of DEP-1 with other endothelial cell tight junction proteins has been identified, and a role in regulation of endothelial cell permeability has been documented –48. We abona fide interaction between DEP-1 and SPN although due to the variability between the controls some caution must be exercised. Although both proteins are expressed in leukocytes we are not aware of any previous reports documenting this interaction. The finding highlights the importance of using several controls in BiFC studies with membrane proteins which we believe are more prone to artefactual results than studies of freely diffusing cytoplasmic proteins, since the sub-cellular location of these proteins is restricted to the plane of the membrane. The R3-PTP BiFC constructs used in this study were truncated following the transmembrane domain, with the intracellular sequence replaced by either the N- or C-terminal sequence of vYFP. Comparison of the fraction of cell-surface expression of the DEP-1-VN construct with the full-length DEP-1 indicated that the BiFC construct exhibited a similar level of cell-surface expression as DEP-1, suggesting that the generation of the fusion protein did not detract from effective trafficking of the protein.Our use of two different control constructs (SPN-VC and Myr-VC) in the BiFC experiments resulted in different average background fluorescent intensity values. In most cases use of the myr-VC control gave lower levels of BiFC intensity than the SPN-VC control underscoring the need to choose controls carefully. The observation that DEP-1 co-expressed with the SPN control was not significantly different from the DEP-1/VE-cadherin combination, but gave significantly higher levels of fluorescence than DEP-1 with the Myr-VC control could be interpreted as a In summary, association of VE-PTP with VE-cadherin is a key regulator of endothelial cell-cell interaction that maintains the integrity of blood vessels. Under conditions of inflammation or immune responses, these molecules dissociate allowing leukocytes to migrate through the endothelial cell lining into the extravascular tissue. Our results, using a live cell assay, demonstrate the importance of the ectodomain and transmembrane domain of VE-PTP in the interaction with VE-cadherin, and show that DEP-1 and other R3-PTPs have the potential to interact with VE-cadherin, raising questions as to the structural basis for the interaction of these molecules.S1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.