The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization
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{"title"=>"The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization", "type"=>"journal", "authors"=>[{"first_name"=>"Yannick R.", "last_name"=>"Brunet", "scopus_author_id"=>"36238714400"}, {"first_name"=>"Abdelrahim", "last_name"=>"Zoued", "scopus_author_id"=>"55193559500"}, {"first_name"=>"Frédéric", "last_name"=>"Boyer", "scopus_author_id"=>"35306340000"}, {"first_name"=>"Badreddine", "last_name"=>"Douzi", "scopus_author_id"=>"35091005400"}, {"first_name"=>"Eric", "last_name"=>"Cascales", "scopus_author_id"=>"6603332760"}], "year"=>2015, "source"=>"PLoS Genetics", "identifiers"=>{"pui"=>"606841305", "sgr"=>"84946605861", "issn"=>"15537404", "pmid"=>"26460929", "scopus"=>"2-s2.0-84946605861", "doi"=>"10.1371/journal.pgen.1005545"}, "id"=>"b37deda5-ce3a-3094-87d6-3d048b11ba25", "abstract"=>"The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins-TssA, TssE, TssF, TssG, TssK and VgrG-are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.", "link"=>"http://www.mendeley.com/research/type-vi-secretion-tssefgkvgrg-phagelike-baseplate-recruited-tssjlm-membrane-complex-via-multiple-con", "reader_count"=>61, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>4, "Student > Master"=>11, "Student > Bachelor"=>15, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>4, "Student > Master"=>11, "Student > Bachelor"=>15, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>25, "Agricultural and Biological Sciences"=>25, "Physics and Astronomy"=>2, "Immunology and Microbiology"=>6}, "reader_count_by_subdiscipline"=>{"Physics and Astronomy"=>{"Physics and Astronomy"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>6}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>25}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"New Zealand"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2357890"], "description"=>"<p>(A) Extracts from WT or Δ<i>sci-1</i> EAEC cells producing Hcp C38S (lane 1,-), Hcp C38S G96C S158C (lane 2, to probe head-to-tail packing, H-T), Hcp C38S Q24C A95C (lane 3, to probe head-to-head packing, H-H) or Hcp C38S G48C (lane 4, to probe tail-to-tail packing, T-T) after <i>in vivo</i> oxidative treatment with copper phenanthroline. (B) and (C) Extracts from EAEC Δ<i>tss</i> cells (B) or Δ<i>tss</i> cells producing the missing gene from pBAD33 complementation vectors (<i>tss</i>+) (C) and producing Hcp C38S (-), Hcp C38S G96C S158C (lane 2, H-T), Hcp C38S Q24C A95C (lane 3, H-H) or Hcp C38S G48C (lane 4, T-T) after <i>in vivo</i> oxidative treatment with copper phenanthroline. Samples were resolved on 12.5%-acrylamide SDS PAGE and Hcp and Hcp complexes were immunodetected with anti-FLAG monoclonal antibody. Molecular weight markers are indicated on the right.</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573920, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g001", "stats"=>{"downloads"=>8, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hcp_assembly_defects_in_vgrG_tssA_tssK_tssE_tssF_and_tssG_mutant_cells_/1573920", "title"=>"Hcp assembly defects in <i>vgrG</i>, <i>tssA</i>, <i>tssK</i>, <i>tssE</i>, <i>tssF</i> and <i>tssG</i> mutant cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357891"], "description"=>"<p>(A) Schematic representation of the <i>tssE</i>, <i>tssF</i> and <i>tssG</i> genes with their respective COG (clusters of orthologous groups [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.ref059\" target=\"_blank\">59</a>]) numbers. The homology between TssE and the bacteriophage T4 gp25 protein is also indicated. The number in the black bar between two genes indicates the level of co-occurrence of the two genes in bacterial genomes (adapted from [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.ref002\" target=\"_blank\">2</a>]). (B) Schematic representations of the PHA02553 and TIGR02243 protein families (with representative members the wedge proteins gp6 of bacteriophage T4 and gpJ of bacteriophage P2 respectively) and TssF. The number of amino-acid residues for each protein is indicated, as well as the fragment of gp6 for which the crystal structure is available (PDB 3H2T, [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.ref048\" target=\"_blank\">48</a>]). The regions of homology between the different proteins are indicated by the grey areas (amino-acid residue boundaries indicated). (C) and (D) Close-ups on the homology regions between TssF, PHA02553 and TIGR02243 and between TssG and TIGR02242 (representative member: wedge protein gpI from bacteriophage P2). The predicted secondary structures (green, -helix; red, -strand) are indicated. The grey areas indicate the regions of homology (amino-acid residue boundaries indicated).</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573921, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g002", "stats"=>{"downloads"=>6, "page_views"=>31, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_tssE_tssF_and_tssG_genes_co_occur_in_T6SS_genes_clusters_and_share_homologies_with_phage_baseplate_components_/1573921", "title"=>"<i>tssE</i>, <i>tssF and tssG</i> genes co-occur in T6SS genes clusters and share homologies with phage baseplate components.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357892"], "description"=>"<p>(A) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase were spotted on plates supplemented with IPTG and the chromogenic substrate X-Gal. Interaction between the two fusion proteins is attested by the blue colour of the colony. The TolB-Pal interaction serves as a positive control. (B, C, D) Co-immunoprecipitation assays. (B) TssF and TssG interact with the tube-forming protein Hcp. Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing FLAG-tagged TssF or TssG and HA-tagged Hcp were subjected to immunoprecipitation with anti-FLAG-coupled beads. The input (total soluble material, T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE, and immunodetected with anti-FLAG and anti-HA monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (C and D) TssG (C) and TssF (D) interact with the gp25-like subunit TssE. Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing HA-tagged TssF and FLAG-tagged TssE or VSV-G-tagged TssG and FLAG-tagged TssE (right panel) were subjected to immunoprecipitation with anti-FLAG-coupled beads. The input (total soluble material, T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE, and immunodetected with anti-FLAG, anti-HA and anti-VSV-G monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. The asterisks indicate light or heavy antibody chains. (E) Reconstitution experiments. Cleared lysates of cells producing VgrG-OB<sub>VSVG</sub>, TssE<sub>FL</sub>, TssF<sub>FL</sub> or TssG<sub>FL</sub> were mixed as indicated (+) and complexes were subjected to immunoprecipitation with anti-VSV-G-coupled beads. The immunoprecipitated materials were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with anti-VSV-G (upper panel) and anti-FLAG (lower panel) monoclonal antibodies. Mixes were TssE<sub>FL</sub>, TssF<sub>FL</sub> and TssG<sub>FL</sub> alone (lane 1) or VgrG-OB<sub>VSVG</sub> with TssE<sub>FL</sub>, TssF<sub>FL</sub> and TssG<sub>FL</sub> (lane 2) or with TssF<sub>FL</sub> and TssG<sub>FL</sub> (lane 3). The asterisks indicate light or heavy antibody chains. (F) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase, and the third partner were spotted on plates supplemented with IPTG and the chromogenic substrate X-Gal. The TolB-Pal interaction serves as a positive control.</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573922, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g003", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TssF_and_TssG_interact_with_phage_like_T6SS_components_/1573922", "title"=>"TssF and TssG interact with phage-like T6SS components.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357893"], "description"=>"<p>(A) TssF and TssG are stabilized upon co-production. The steady-state levels of TssF<sub>HA</sub> and TssG<sub>FL</sub> proteins produced alone or together were analyzed by Western blot immunodetections of whole cells using anti-HA or anti-FLAG monoclonal antibody. The stable Pal lipoprotein was used as a loading control. Incubation periods (min) after spectinomycin/chloramphenicol treatment are indicated. (B) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase were spotted on plates supplemented with IPTG and the chromogenic substrate Bromo-Chloro-Indolyl-β-D-galactopyrannoside. Interaction between the two fusion proteins is attested by the blue colour of the colony. The TolB-Pal interaction serves as a positive control. (C) TssF co-immunoprecipitates with TssG. Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing TssF<sub>HA</sub> and TssG<sub>FL</sub> were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total soluble material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE, and immunodetected with anti-HA and anti-FLAG monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (D) The genetic organization of the <i>tssF</i> and <i>tssG</i> genes in EAEC 17–2 and <i>E</i>. <i>coli</i> UPEC CFT073. Orthologous genes are schematically represented with the same color. A Clustal W alignment between EAEC TssF and TssG and the CFT073 NP_755275 protein is provided in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.s003\" target=\"_blank\">S2 Fig</a>. (E) The TssF-TssG fusion protein is functional. Hcp<sub>FLAG</sub> release was assessed by separating whole cells (C) and supernatant (S) fractions from WT, <i>tssFG</i> and complemented <i>tssFG</i> cell cultures. <i>tssFG</i> cells were complemented either with a plasmid bearing <i>tssF</i> and <i>tssG</i> genes contiguously organized (F+G), the <i>tssF’-‘tssG</i> fusion (F-G) or the <i>tssG’-‘tssF</i> fusion (G-F). A total of 2×10<sup>8</sup> cells and the TCA-precipitated material of the supernatant from 5×10<sup>8</sup> cells were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected using the anti-FLAG monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (control for cell integrity; upper panel).</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573923, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g004", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TssF_and_TssG_interact_and_stabilize_each_other_/1573923", "title"=>"TssF and TssG interact and stabilize each other.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357894"], "description"=>"<p>(A) A fractionation procedure was applied to EAEC cells producing TssF<sub>HA</sub> and TssG<sub>FL</sub> (T, Total fraction), allowing separation between the periplasm (P), the cytoplasm (C) and membrane fractions (M). Samples were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with antibodies directed against EFTu (cytoplasm), TolB (periplasm), OmpA (OM) proteins, and the HA epitope of TssF or the FLAG epitope of TssG. Molecular weight markers are indicated on the left. (B) Total membranes (T) from EAEC cells producing TssF<sub>HA</sub> and TssG<sub>FL</sub> were separated on a discontinuous sedimentation sucrose gradient. Collected fractions were analyzed for contents using the anti-TolA and anti-OmpA polyclonal antibodies or anti-HA and anti-FLAG monoclonal antibodies. In addition, NADH oxidase activity test (graph) was used as a reporter of the inner membrane protein containing fractions. NADH oxidase activity is represented relative to the total activity. The positions of the inner and outer membrane-containing fractions are indicated. (C) A fractionation procedure was applied to <i>E</i>. <i>coli</i> K-12 W3110 cells producing TssF<sub>HA</sub> and TssG<sub>FLAG</sub> (T, Total fraction), allowing separation between the periplasm (P), the cytoplasm (C) and membrane fractions (M). Samples were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with antibodies directed against EFTu (cytoplasm), TolB (periplasm), TolA (IM) proteins, and the HA epitope of TssF or the FLAG epitope of TssG. Molecular weight markers are indicated on the left. (D) Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase were spotted on plates supplemented with IPTG and the chromogenic substrate Bromo-Chloro-Indolyl-β-D-galactopyrannoside. Interaction between the two fusion proteins is attested by the blue colour of the colony. The TolB-Pal interaction serves as a positive control. (E) TssG co-immunoprecipitates with the cytoplasmic domain of TssM (TssMc, amino-acids 82–360). Soluble extracts of <i>E</i>. <i>coli</i> K-12 W3110 strain producing TssG<sub>VSVG</sub> only, or TssG<sub>VSVG</sub> and TssMc<sub>FL</sub> were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total soluble material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with anti-VSVG and anti-FLAG monoclonal antibodies (additional bands correspond or heavy antibody chains). Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (F) TssF and TssG remain cytoplasmic in a <i>tssM</i> deletion mutant. A fractionation procedure was applied to EAEC <i>tssM</i> cells co-producing TssF<sub>HA</sub> and TssG<sub>FL</sub> (T, Total fraction), allowing separation between the periplasm (P), the cytoplasm (C) and membrane fractions (M). Samples were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected with antibodies directed against EFTu (cytoplasm), TolB (periplasm), TolA (IM) proteins, and the HA epitope of TssF or the FLAG epitope of TssG. Molecular weight markers are indicated on the left.</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573924, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g005", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TssF_and_TssG_are_soluble_proteins_that_associate_with_the_IM_via_contacts_between_TssG_and_the_cytoplasmic_loop_of_TssM_/1573924", "title"=>"TssF and TssG are soluble proteins that associate with the IM via contacts between TssG and the cytoplasmic loop of TssM.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357895"], "description"=>"<p>(A) Time-lapse fluorescence microscopy recordings showing localization and dynamics of the functional <sub>sfGFP</sub>TssF fusion protein in wild-type (WT) cells (upper panel) or <i>tssBC</i> (second panel from top) <i>tssK</i> (third panel from top) or <i>tssM</i> (lower panel) derivatives. Individual images were taken every 30 sec. The positions of foci are indicated by white triangles. Scale bars are 1 m. Statistical analyses (number of foci/cell and dynamics) are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.s004\" target=\"_blank\">S3A and S3B Fig</a>. (B) Sheath polymerization events initiate on <sub>sfGFP</sub>TssF foci. Fluorescence microscopy time-lapse recording of wild-type EAEC cells producing <sub>sfGFP</sub>TssF and TssB<sub>mCh</sub>. The GFP channel (top panel), mCherry channel (middle panel), and merge channels (lower panel) are shown. Individual images (from left to right) were taken every 30 sec. Baseplate foci and distal ends of the sheaths are indicated by white arrowheads. A schematic diagram summarizing the observed events is drawn below. The scale bar is 1 μm. Statistical (initial positioning of <sub>sfGFP</sub>TssF clusters, percentage of sheaths with basal <sub>sfGFP</sub>TssF foci) and kymograph analyses are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.s004\" target=\"_blank\">S3C and S3D and S3E Fig</a>. (C) Time-lapse fluorescence microscopy recordings showing localization and dynamics of the functional TssK<sub>sfGFP</sub> fusion protein in wild-type (WT) cells (upper panel) or its <i>tssM</i> (lower panel) derivative. Individual images were taken every 30 sec. The positions of foci are indicated by the white triangles. Scale bars are 1 m. Statistical analyses (number of foci/cell and dynamics) are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.s004\" target=\"_blank\">S3B and S3F Fig</a>. (D) Time-lapse fluorescence microscopy recordings showing localization and dynamics of the functional <sub>sfGFP</sub>TssM fusion protein in <i>tssK</i> cells. Individual images were taken every 30 sec. The positions of foci are indicated by the white triangles. Scale bars are 1 m. Statistical analyses (number of foci/cell) are shown in <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005545#pgen.1005545.s004\" target=\"_blank\">S3G Fig</a>. (E) Assembly pathway between selected T6SS components. The membrane complex (MC) comprising the TssJLM protein (shown with the 11.6-A electron microscopy structure) is assembled first, and is used as docking station for TssK. TssK recruits the TssF/TssG complex to the apparatus to assemble the baseplate complex (BC, green rectangle) prior to assembly of the tail complex (TC, red rectangle) comprising the Hcp inner tube and the TssBC contractile sheath-like structure.</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573925, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g006", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TssF_and_TssK_assemble_into_static_foci_that_serve_as_platform_for_sheath_polymerization_/1573925", "title"=>"TssF and TssK assemble into static foci that serve as platform for sheath polymerization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357898"], "description"=>"<p>Schematic representation of the different stages of T6SS biogenesis. The TssJ-L-M membrane complex is first assembled in the cell envelope (IM, inner membrane; PG, peptidoglycan; OM, outer membrane) and recruits the baseplate-like assembly platform constituted of TssE, TssF, TssG, TssK, VgrG and possibly TssA (platform represented as a green disk with the VgrG green screw). Polymerization of the tail tube (Hcp rings, black rectangles) and sheath (TssBC strands, red rectangles) is initiated after completion of the platform.</p>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573927, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005545.g007", "stats"=>{"downloads"=>7, "page_views"=>101, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assembly_of_the_Type_VI_secretion_system_/1573927", "title"=>"Assembly of the Type VI secretion system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-10-13 03:49:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/2357916", "https://ndownloader.figshare.com/files/2357917", "https://ndownloader.figshare.com/files/2357918", "https://ndownloader.figshare.com/files/2357919", "https://ndownloader.figshare.com/files/2357920"], "description"=>"<div><p>The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins–TssA, TssE, TssF, TssG, TssK and VgrG–are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.</p></div>", "links"=>[], "tags"=>["fluorescence microscopy analyses", "13 T 6SS subunits", "Multiple Contacts", "TssG share", "type VI secretion system", "T 6SS tail tube", "tail components", "sequence homologies", "contractile bacteriophages", "toxin proteins", "understanding T 6SS assembly", "sheath polymerizes", "membrane TssM protein", "interaction network", "cytoplasmic contractile structure", "T 6SS baseplate", "TssJLM Membrane Complex", "phage components", "T 6SS", "assembly platform", "cell envelope", "Prokaryotic cells"], "article_id"=>1573934, "categories"=>["Uncategorised"], "users"=>["Yannick R. Brunet", "Abdelrahim Zoued", "Frédéric Boyer", "Badreddine Douzi", "Eric Cascales"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1005545.s001", "https://dx.doi.org/10.1371/journal.pgen.1005545.s002", "https://dx.doi.org/10.1371/journal.pgen.1005545.s003", "https://dx.doi.org/10.1371/journal.pgen.1005545.s004", "https://dx.doi.org/10.1371/journal.pgen.1005545.s005"], "stats"=>{"downloads"=>9, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_Type_VI_Secretion_TssEFGK_VgrG_Phage_Like_Baseplate_Is_Recruited_to_the_TssJLM_Membrane_Complex_via_Multiple_Contacts_and_Serves_As_Assembly_Platform_for_Tail_Tube_Sheath_Polymerization_/1573934", "title"=>"The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-10-13 03:49:49"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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