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TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System*

  • Abdelrahim Zoued
    Footnotes
    Affiliations
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, UMR 7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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  • Eric Durand
    Footnotes
    Affiliations
    Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Case 932, Aix-Marseille Université, CNRS UMR 6098, 13288 Marseille Cedex 09, France
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  • Cecilia Bebeacua
    Footnotes
    Affiliations
    Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Case 932, Aix-Marseille Université, CNRS UMR 6098, 13288 Marseille Cedex 09, France
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  • Yannick R. Brunet
    Footnotes
    Affiliations
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, UMR 7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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  • Badreddine Douzi
    Affiliations
    Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Case 932, Aix-Marseille Université, CNRS UMR 6098, 13288 Marseille Cedex 09, France
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  • Christian Cambillau
    Affiliations
    Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Case 932, Aix-Marseille Université, CNRS UMR 6098, 13288 Marseille Cedex 09, France
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  • Eric Cascales
    Affiliations
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, UMR 7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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  • Laure Journet
    Correspondence
    To whom correspondence should be addressed: Laboratoire d'Ingénierie des Systèmes Macromoléculaires (UMR7255), CNRS, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Tel.: 33491164156; Fax: 33491712124;
    Affiliations
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, UMR 7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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  • Author Footnotes
    * This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Aix-Marseille University, and a grant from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03) (to E. C.). This work was also supported by the CNRS, the Aix-Marseille University, and by grants from the Marseille-Nice Genopole, IBiSA, and the Fondation de la Recherche Médicale (FRM DEQ2011-0421282) (to C. C.).
    This article contains supplemental Tables S1-S2 and Fig. S1.
    1 Supported by doctoral fellowships from the French Ministère de la Recherche.
    2 Supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116).
    3 Present address: Structural and Computational Biology & Cell Biology and Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Open AccessPublished:August 06, 2013DOI:https://doi.org/10.1074/jbc.M113.499772
      The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
      Background: The T6SS assembles from 13 proteins that form two sub-assemblies.
      Results: TssK is a cytoplasmic protein that interacts with Hcp, TssC, TssL, and TssA.
      Conclusion: The TssK complex is three-arm shaped and links the membrane and phage-like complexes in T6SS.
      Significance: The structural and functional characterization of TssK leads to a better understanding of T6SS assembly.

      Introduction

      Bacteria have developed macromolecular nanomachines to inject toxins to either prokaryotic or eukaryotic cells to clear an environmental niche or to cause diseases (
      • Hayes C.S.
      • Aoki S.K.
      • Low D.A.
      Bacterial contact-dependent delivery systems.
      ). One of the systems able to deliver toxic effectors to both prokaryotic and eukaryotic cells is the Type VI secretion system (T6SS)
      The abbreviations used are: T6SS, Type VI secretion system; IPTG, isopropyl-β-d-thio-galactopyrannoside; BACTH, bacterial two-hybrid assay; X-Gal, bromo-chloro-indolyl-galactopyrannoside; TEV, Tobacco Etch Virus; ML, maximum likelihood; PCA, principal component analysis; FSC, Fourier Shell Correlation; SAXS, small-angle x-ray scattering; NSD, normalized spatial discrepancy; Tss, Type 6 subunit; Hcp, hemolysin co-regulated protein.
      (
      • Silverman J.M.
      • Brunet Y.R.
      • Cascales E.
      • Mougous J.D.
      Structure and regulation of the type VI secretion system.
      ). The current model regarding T6SS function proposes that it acts similarly to the cell puncturing device of contractile bacteriophages (
      • Bönemann G.
      • Pietrosiuk A.
      • Mogk A.
      Tubules and donuts: a type VI secretion story.
      ,
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Cascales E.
      • Cambillau C.
      Structural biology of type VI secretion systems.
      ).
      Although several accessory proteins are usually necessary for its function, the T6SS assembles from 13 Tss (Type six subunits) proteins whose genes are conserved in all T6SS gene clusters and are thus called “core components” (
      • Bingle L.E.
      • Bailey C.M.
      • Pallen M.J.
      Type VI secretion: a beginner's guide.
      ,
      • Cascales E.
      The type VI secretion toolkit.
      ,
      • Boyer F.
      • Fichant G.
      • Berthod J.
      • Vandenbrouck Y.
      • Attree I.
      Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.
      ). A number of these conserved subunits share structural and functional similarities with contractile bacteriophage tail proteins including components of the tail tube, sheath, hub, and baseplate (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Leiman P.G.
      • Basler M.
      • Ramagopal U.A.
      • Bonanno J.B.
      • Sauder J.M.
      • Pukatzki S.
      • Burley S.K.
      • Almo S.C.
      • Mekalanos J.J.
      Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
      ,
      • Pell L.G.
      • Kanelis V.
      • Donaldson L.W.
      • Howell P.L.
      • Davidson A.R.
      The phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system.
      ,
      • Bönemann G.
      • Pietrosiuk A.
      • Diemand A.
      • Zentgraf H.
      • Mogk A.
      Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.
      ,
      • Lossi N.S.
      • Manoli E.
      • Förster A.
      • Dajani R.
      • Pape T.
      • Freemont P.
      • Filloux A.
      The HsiB1C1 (TssB-TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheathlike structure.
      ). For these reasons the structure assembled by these proteins and visualized by cryo-electron tomography and fluorescence microscopy has been called the “phage-like complex.” A second group of core components are the TssJ, TssM, and TssL membrane proteins that assemble a trans-envelope spanning complex. Aside these two groups, the TssA, TssF, TssG, and TssK core components have not been characterized yet.
      As briefly described above, the phage-like sub-complex is structurally and functionally similar to the contractile tail of Myoviridae. The crystal structures of the Hcp (hemolysin co-regulated protein) proteins present high similarity with the bacteriophage λ tail tube gpV protein (
      • Pell L.G.
      • Kanelis V.
      • Donaldson L.W.
      • Howell P.L.
      • Davidson A.R.
      The phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system.
      ,
      • Mougous J.D.
      • Cuff M.E.
      • Raunser S.
      • Shen A.
      • Zhou M.
      • Gifford C.A.
      • Goodman A.L.
      • Joachimiak G.
      • Ordoñez C.L.
      • Lory S.
      • Walz T.
      • Joachimiak A.
      • Mekalanos J.J.
      A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
      ,
      • Osipiuk J.
      • Xu X.
      • Cui H.
      • Savchenko A.
      • Edwards A.
      • Joachimiak A.
      Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa.
      ,
      • Jobichen C.
      • Chakraborty S.
      • Li M.
      • Zheng J.
      • Joseph L.
      • Mok Y.K.
      • Leung K.Y.
      • Sivaraman J.
      Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.
      ). Six Hcp assemble into rings of about 80–90 Å wide with an internal diameter of about 30–40 Å (
      • Mougous J.D.
      • Cuff M.E.
      • Raunser S.
      • Shen A.
      • Zhou M.
      • Gifford C.A.
      • Goodman A.L.
      • Joachimiak G.
      • Ordoñez C.L.
      • Lory S.
      • Walz T.
      • Joachimiak A.
      • Mekalanos J.J.
      A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
      ,
      • Osipiuk J.
      • Xu X.
      • Cui H.
      • Savchenko A.
      • Edwards A.
      • Joachimiak A.
      Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa.
      ,
      • Jobichen C.
      • Chakraborty S.
      • Li M.
      • Zheng J.
      • Joseph L.
      • Mok Y.K.
      • Leung K.Y.
      • Sivaraman J.
      Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.
      ) that stack on each other to form tubes in vitro (
      • Leiman P.G.
      • Basler M.
      • Ramagopal U.A.
      • Bonanno J.B.
      • Sauder J.M.
      • Pukatzki S.
      • Burley S.K.
      • Almo S.C.
      • Mekalanos J.J.
      Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
      ,
      • Mougous J.D.
      • Cuff M.E.
      • Raunser S.
      • Shen A.
      • Zhou M.
      • Gifford C.A.
      • Goodman A.L.
      • Joachimiak G.
      • Ordoñez C.L.
      • Lory S.
      • Walz T.
      • Joachimiak A.
      • Mekalanos J.J.
      A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
      ,
      • Osipiuk J.
      • Xu X.
      • Cui H.
      • Savchenko A.
      • Edwards A.
      • Joachimiak A.
      Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa.
      ,
      • Jobichen C.
      • Chakraborty S.
      • Li M.
      • Zheng J.
      • Joseph L.
      • Mok Y.K.
      • Leung K.Y.
      • Sivaraman J.
      Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.
      ,
      • Ballister E.R.
      • Lai A.H.
      • Zuckermann R.N.
      • Cheng Y.
      • Mougous J.D.
      In vitro self-assembly of tailorable nanotubes from a simple protein building block.
      ). The VgrG (valine glycine repeat protein) trimer is structurally homologous to the bacteriophage T4 gp27/gp5 spike complex which is used as a puncturing device to perforate the host cell envelope (
      • Leiman P.G.
      • Basler M.
      • Ramagopal U.A.
      • Bonanno J.B.
      • Sauder J.M.
      • Pukatzki S.
      • Burley S.K.
      • Almo S.C.
      • Mekalanos J.J.
      Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
      ,
      • Pukatzki S.
      • Ma A.T.
      • Revel A.T.
      • Sturtevant D.
      • Mekalanos J.J.
      Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.
      ). TssE is an homolog of gp25, a structural component of the phage baseplate (
      • Bingle L.E.
      • Bailey C.M.
      • Pallen M.J.
      Type VI secretion: a beginner's guide.
      ,
      • Lossi N.S.
      • Dajani R.
      • Freemont P.
      • Filloux A.
      Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa.
      ) while the TssB and TssC subunits (also called VipA and VipB in Vibrio cholerae) interact with each other and assemble cogwheel-like tubular structures of around 300 Å in diameter with an internal 100 Å-diameter channel resembling the bacteriophage contractile sheath (
      • Bönemann G.
      • Pietrosiuk A.
      • Diemand A.
      • Zentgraf H.
      • Mogk A.
      Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.
      ,
      • Lossi N.S.
      • Manoli E.
      • Förster A.
      • Dajani R.
      • Pape T.
      • Freemont P.
      • Filloux A.
      The HsiB1C1 (TssB-TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheathlike structure.
      ). Cryo-electron tomography and immuno-electron microscopy experiments revealed the existence of such “sheath-like” structures in the cytoplasm of T6SS+ cells (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Basler M.
      • Mekalanos J.J.
      Type 6 secretion dynamics within and between bacterial cells.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ). Interestingly, these structures exist in two distinct conformations. Indeed, by using TssB fusion to the green fluorescent protein (GFP), time-lapse fluorescence microscopy experiments demonstrated that these structures are highly dynamic and cycle between extended and contracted conformations through elongation, contraction, and disassembly (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Basler M.
      • Mekalanos J.J.
      Type 6 secretion dynamics within and between bacterial cells.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ,
      • Brunet Y.R.
      • Espinosa L.
      • Harchouni S.
      • Mignot T.
      • Cascales E.
      Imaging type VI secretion-mediated bacterial killing.
      ). Disassembly of contracted T6SS sheaths is catalyzed by the ClpV AAA+ ATPase (
      • Bönemann G.
      • Pietrosiuk A.
      • Diemand A.
      • Zentgraf H.
      • Mogk A.
      Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.
      ,
      • Basler M.
      • Mekalanos J.J.
      Type 6 secretion dynamics within and between bacterial cells.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ,
      • Pietrosiuk A.
      • Lenherr E.D.
      • Falk S.
      • Bönemann G.
      • Kopp J.
      • Zentgraf H.
      • Sinning I.
      • Mogk A.
      Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion.
      ). By analogy with the bacteriophage infection process, the current model suggests that the T6SS functions as an inverted bacteriophage and that TssB/C sheath contraction propels the Hcp tube and VgrG outside the cell to puncture the target cell and to deliver toxin effectors (
      • Silverman J.M.
      • Brunet Y.R.
      • Cascales E.
      • Mougous J.D.
      Structure and regulation of the type VI secretion system.
      ,
      • Bönemann G.
      • Pietrosiuk A.
      • Mogk A.
      Tubules and donuts: a type VI secretion story.
      ,
      • Kanamaru S.
      Structural similarity of tailed phages and pathogenic bacterial secretion systems.
      ). In agreement with this model, the Hcp and VgrG proteins are usually found in the supernatant of T6SS-producing cells (
      • Mougous J.D.
      • Cuff M.E.
      • Raunser S.
      • Shen A.
      • Zhou M.
      • Gifford C.A.
      • Goodman A.L.
      • Joachimiak G.
      • Ordoñez C.L.
      • Lory S.
      • Walz T.
      • Joachimiak A.
      • Mekalanos J.J.
      A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
      ,
      • Pukatzki S.
      • Ma A.T.
      • Sturtevant D.
      • Krastins B.
      • Sarracino D.
      • Nelson W.C.
      • Heidelberg J.F.
      • Mekalanos J.J.
      Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.
      ,
      • Zheng J.
      • Leung K.Y.
      Dissection of a type VI secretion system in Edwardsiella tarda.
      ) and contraction of the sheath is correlated with lysis of the prey cell (
      • Brunet Y.R.
      • Espinosa L.
      • Harchouni S.
      • Mignot T.
      • Cascales E.
      Imaging type VI secretion-mediated bacterial killing.
      ).
      Based on cryo-electron microscopy images, this phage-like complex has been proposed to be anchored to the membrane (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ). An immunoprecipitable complex composed of TssM, TssL, and TssJ was identified in EAEC (
      • Aschtgen M.S.
      • Gavioli M.
      • Dessen A.
      • Lloubès R.
      • Cascales E.
      The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall.
      ). Biochemical, structural, and protein-protein interaction studies have provided insights onto the characteristics of these subunits (
      • Aschtgen M.S.
      • Gavioli M.
      • Dessen A.
      • Lloubès R.
      • Cascales E.
      The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall.
      ,
      • Aschtgen M.S.
      • Bernard C.S.
      • De Bentzmann S.
      • Lloubès R.
      • Cascales E.
      SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli.
      ,
      • Ma L.S.
      • Lin J.S.
      • Lai E.M.
      An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.
      ,
      • Felisberto-Rodrigues C.
      • Durand E.
      • Aschtgen M.S.
      • Blangy S.
      • Ortiz-Lombardia M.
      • Douzi B.
      • Cambillau C.
      • Cascales E.
      Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar.
      ,
      • Aschtgen M.S.
      • Zoued A.
      • Lloubès R.
      • Journet L.
      • Cascales E.
      The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC.
      ,
      • Durand E.
      • Zoued A.
      • Spinelli S.
      • Watson P.J.
      • Aschtgen M.S.
      • Journet L.
      • Cambillau C.
      • Cascales E.
      Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems.
      ). While TssL is a bitopic membrane protein with its N terminus located in the cytoplasm (
      • Ma L.S.
      • Lin J.S.
      • Lai E.M.
      An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.
      ,
      • Aschtgen M.S.
      • Zoued A.
      • Lloubès R.
      • Journet L.
      • Cascales E.
      The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC.
      ,
      • Durand E.
      • Zoued A.
      • Spinelli S.
      • Watson P.J.
      • Aschtgen M.S.
      • Journet L.
      • Cambillau C.
      • Cascales E.
      Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems.
      ), TssM has three transmembrane helices (TMH) with a large C-terminal periplasmic domain interacting with the TssJ outer membrane lipoprotein (
      • Zheng J.
      • Leung K.Y.
      Dissection of a type VI secretion system in Edwardsiella tarda.
      ,
      • Aschtgen M.S.
      • Bernard C.S.
      • De Bentzmann S.
      • Lloubès R.
      • Cascales E.
      SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli.
      ,
      • Felisberto-Rodrigues C.
      • Durand E.
      • Aschtgen M.S.
      • Blangy S.
      • Ortiz-Lombardia M.
      • Douzi B.
      • Cambillau C.
      • Cascales E.
      Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar.
      ). A cytoplasmic loop located between TMH2 and TMH3 contains an ATP binding and hydrolysis Walker A motif that was shown to be required for T6SS function in Agrobacterium tumefaciens (
      • Ma L.S.
      • Lin J.S.
      • Lai E.M.
      An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.
      ). The trans-envelope complex is usually stabilized by a peptidoglycan-binding motif localized at the C terminus of the TssL subunit or on an accessory component (
      • Aschtgen M.S.
      • Gavioli M.
      • Dessen A.
      • Lloubès R.
      • Cascales E.
      The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall.
      ,
      • Aschtgen M.S.
      • Thomas M.S.
      • Cascales E.
      Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP. what else?.
      ).
      Regarding this membrane complex, comparative bioinformatic analyses reported a conserved genomic organization between the tssJ, tssK, tssL, and tssM genes (
      • Boyer F.
      • Fichant G.
      • Berthod J.
      • Vandenbrouck Y.
      • Attree I.
      Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.
      ). Among these genes, tssK encodes an uncharacterized putative soluble protein. The tssK gene is organized in tandem with tssL in 74% of the T6SS gene clusters suggesting a functional link or interaction between these two proteins (
      • Boyer F.
      • Fichant G.
      • Berthod J.
      • Vandenbrouck Y.
      • Attree I.
      Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.
      ). The association of TssK with the membrane complex is also strongly suggested by the observation that this protein has been found associated with the inner membrane proteome in Pseudomonas aeruginosa (
      • Casabona M.G.
      • Vandenbrouck Y.
      • Attree I.
      • Couté Y.
      Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane.
      ). Interestingly, although TssK is potentially linked to the membrane complex, the Vibrio cholerae TssK protein was recently shown to co-purify with isolated sheaths (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ). TssK is therefore a good candidate to link both the T6SS membrane and phage complexes. In this study, we report the characterization of the TssK protein encoded by the sci-1 T6SS gene cluster in enteroaggregative Escherichia coli (accession numbers: EC042_4526, YP_006098803.1). We found that TssK1 interacts with the cytoplasmic domain of the two inner membrane proteins TssL1 and TssM1. We also identified direct contacts between TssK1 and the T6SS tail components TssC1 and Hcp1. Further biochemical and structural characterization of the TssK1 purified protein using size exclusion chromatography, electron microscopy and small angle x-ray scattering showed that TssK1 is trimeric in solution and forms three arm-shaped particles.

      DISCUSSION

      Important advances have recently been made regarding the assembly of the phage-related T6SS complex and the identification of the effector proteins delivered by this apparatus. However, the roles of several T6SS core components remain enigmatic. In this study we report the characterization of the TssK protein encoded within the sci-1 T6SS gene cluster of enteroaggregative E. coli. A schematic representation of the main results of this work is shown in Fig. 9. We first found that this conserved protein is necessary for T6SS function, a result in agreement with the observations that the tssK genes from E. tarda, V. cholera, and A. tumefaciens have been demonstrated to be required for T6SS function in these microorganisms (
      • Zheng J.
      • Leung K.Y.
      Dissection of a type VI secretion system in Edwardsiella tarda.
      ,
      • Zheng J.
      • Ho B.
      • Mekalanos J.J.
      Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae.
      ,
      • Lin J.S.
      • Ma L.S.
      • Lai E.M.
      Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation.
      ). Time-lapse fluorescence microscopy experiments using a TssB-sfGFP fusion protein further showed that T6SS sheath dynamics is abrogated in tssK1 cells. By contrast to V. cholerae, P. aeruginosa, and EAEC WT cells in which T6SS sheaths undergo cycles of assembly and contraction (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Basler M.
      • Mekalanos J.J.
      Type 6 secretion dynamics within and between bacterial cells.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ,
      • Brunet Y.R.
      • Espinosa L.
      • Harchouni S.
      • Mignot T.
      • Cascales E.
      Imaging type VI secretion-mediated bacterial killing.
      ) or to V. cholerae ΔclpV cells in which contracted sheaths are not disassembled and form foci (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ), the TssB-sfGFP fluorescence was diffuse, and no punctuate foci could be observed in tssK1 cells. This suggests that no tubular assembly of TssB/TssC occurs in the absence of TssK, a phenotype similar to that observed in V. cholerae tssM, fha, hcp, and tssE or EAEC tssE mutant cells (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ,
      • Kapitein N.
      • Bönemann G.
      • Pietrosiuk A.
      • Seyffer F.
      • Hausser I.
      • Locker J.K.
      • Mogk A.
      ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
      ,
      • Brunet Y.R.
      • Espinosa L.
      • Harchouni S.
      • Mignot T.
      • Cascales E.
      Imaging type VI secretion-mediated bacterial killing.
      ). TssK1 thus appears to be involved at a stage upstream ClpV during T6SS sheath assembly. Attempts to localize the TssK1 protein using a chromosomal tssK1-sfGFP gene fusion were unsuccessful so far, due to undetectable fluorescence levels suggesting a low representation of the protein.
      Figure thumbnail gr9
      FIGURE 9Schematic representation of the TssK protein interaction network. The localization and topologies of several type VI secretion subunits are represented. Arrows show interactions detected in this study by biochemical (red) or two-hybrid approaches (blue) (summarized in ). The membrane-associated and bacteriophage-like subassemblies are framed in green and blue, respectively. OM: outer membrane, IM: inner membrane.
      Bacterial two-hybrid and co-immunoprecipitation assays revealed that TssK1 forms oligomers. Further gel filtration experiments showed that the TssK1 protein elutes with an apparent molecular weight corresponding to that of a trimer. The 3-fold organization of TssK1 was validated by visualization of the purified protein using electron microscopy. Image reconstruction of TssK1 particles showed that it forms a three-armed structure with an overall pyramidal shape. In electron microscopy, the three arms are bent under the structure while are in an open conformation in solution as shown by SAXS analysis. Although a conformational flexibility of the protein might exist, our data cannot rule out that (i) the length of the arms has been underestimated due to the average between “bent” and “elongated” conformations on EM grids or (ii) the TssK1 conformation on EM grids is induced by preparation of the specimen (fixation or staining procedures).
      In this study, we determined the TssK1 interaction network (a summary of the interactions is presented in supplemental Table S2). Using a bacterial two-hybrid screen, we revealed that TssK1 interacts with (i) the cytoplasmic domains of TssL1 and TssM1, two inner membrane subunits of the T6SS membrane complex, (ii) Hcp1 and TssC1, two components of the phage tail-related sub-complex and (iii) TssA1, a core-component of unknown function. These interactions are in agreement with the cytoplasmic localization of TssK1 as shown by fractionation, since both Hcp1 and TssC1 are part of the cytosolic tubular tail-like structure. The TssK1-TssM1c interaction detected by BACTH analysis was the only interaction we could not validate by co-immunoprecipitation. TssM1c may not have the same conformation upon fusion with T18/T25 domains and further analysis will be required to confirm this interaction. The interactions of TssK1 with TssL1c, Hcp1, TssA1, and TssC1 were all confirmed using co-immunoprecipitations. The TssK1-Hcp1 interaction was also tested in vitro using SPR, and was shown to have a KD of 26 μm. Overall, these results demonstrate that TssK1 interacts with components of both membrane and phage sub-complexes at the cytoplasmic face of the T6SS apparatus. The link between TssK1 and the membrane complex is in agreement with recent publications. First, a bioinformatic approach showed that the tssK gene co-occurs with the tssL, tssM, and tssJ genes, and a strong link between TssK and TssL in co-occurrence and genomic organization studies has been demonstrated (
      • Boyer F.
      • Fichant G.
      • Berthod J.
      • Vandenbrouck Y.
      • Attree I.
      Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.
      ). More recently, the TssK protein was shown to co-fractionnate with inner membrane proteins in the P. aeruginosa membrane proteome (
      • Casabona M.G.
      • Vandenbrouck Y.
      • Attree I.
      • Couté Y.
      Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane.
      ). Regarding the link between TssK and the T6SS phage tail-like structure, no co-occurrence can be found but TssK was co-purified with T6SS sheaths (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ), a result in agreement with its interaction with TssC. Thus, one may hypothesize that TssK is a structural component that connects the phage-like structure and the membrane complex at the cytoplasmic side of the inner membrane. What can be its function? The observation that no T6SS sheath-like structures assemble in tssK1 mutant cells suggests that TssK may (i) initiate Hcp tube or TssBC sheath polymerization or (ii) recruit phage-like components to the site of assembly/secretion, i.e. the membrane complex. By analogy with contractile bacteriophages, T6SS likely require a baseplate-like complex. As noted by Basler et al. cryo-electron tomographs showed that the T6SS sheath-like tubular structures are connected to the inner membrane through a flared bell-like density that may correspond to the baseplate (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ). The TssE protein, which shares clear homologies with the bacteriophage gp25 wedge protein, is obviously one of the baseplate components (
      • Cascales E.
      • Cambillau C.
      Structural biology of type VI secretion systems.
      ,
      • Bingle L.E.
      • Bailey C.M.
      • Pallen M.J.
      Type VI secretion: a beginner's guide.
      ,
      • Cascales E.
      The type VI secretion toolkit.
      ,
      • Lossi N.S.
      • Dajani R.
      • Freemont P.
      • Filloux A.
      Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa.
      ). Although no interaction between TssK1 and TssE1 has been detected in our two-hybrid screen and no co-occurrence between the tssE and tssK genes has been inferred from bioinformatic studies, the cytoplasmic localization of TssK1, its interaction network and its requirement for sheath elongation suggest that it could be part of this baseplate complex. Interestingly, it is worth to note that the three-armed shape of TssK1 has an external diameter of ∼155 Å, a size similar to the diameter of the sheath (∼110 and 145 Å in the extended and contracted conformations respectively,
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ). Although our chromosomally encoded TssK1-sfGFP fusion was not sufficiently produced to localize TssK1, defining its position within the T6SS will help to further understand its function.

      Acknowledgments

      We thank the members of the Cascales, Cambillau, Lloubès, Bouveret, and Sturgis research groups for discussion, Tâm Mignot for fluorescence microscopy, Charlotte Gaviard, Valentin Tutagata, Annick Brun, Isabelle Bringer, and Olivier Uderso for technical assistance and Mylène Micautond for encouragement.

      REFERENCES

        • Hayes C.S.
        • Aoki S.K.
        • Low D.A.
        Bacterial contact-dependent delivery systems.
        Annu. Rev. Genet. 2010; 44: 71-90
        • Silverman J.M.
        • Brunet Y.R.
        • Cascales E.
        • Mougous J.D.
        Structure and regulation of the type VI secretion system.
        Annu. Rev. Microbiol. 2012; 66: 453-472
        • Bönemann G.
        • Pietrosiuk A.
        • Mogk A.
        Tubules and donuts: a type VI secretion story.
        Mol. Microbiol. 2010; 76: 815-821
        • Basler M.
        • Pilhofer M.
        • Henderson G.P.
        • Jensen G.J.
        • Mekalanos J.J.
        Type VI secretion requires a dynamic contractile phage tail-like structure.
        Nature. 2012; 483: 182-186
        • Cascales E.
        • Cambillau C.
        Structural biology of type VI secretion systems.
        Phil. Trans. R. Soc. B. 2012; 367: 1102-1111
        • Bingle L.E.
        • Bailey C.M.
        • Pallen M.J.
        Type VI secretion: a beginner's guide.
        Curr. Opin. Microbiol. 2008; 11: 3-8
        • Cascales E.
        The type VI secretion toolkit.
        EMBO Rep. 2008; 9: 735-741
        • Boyer F.
        • Fichant G.
        • Berthod J.
        • Vandenbrouck Y.
        • Attree I.
        Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?.
        BMC Genomics. 2009; 10: 104
        • Leiman P.G.
        • Basler M.
        • Ramagopal U.A.
        • Bonanno J.B.
        • Sauder J.M.
        • Pukatzki S.
        • Burley S.K.
        • Almo S.C.
        • Mekalanos J.J.
        Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4154-4159
        • Pell L.G.
        • Kanelis V.
        • Donaldson L.W.
        • Howell P.L.
        • Davidson A.R.
        The phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4160-4165
        • Bönemann G.
        • Pietrosiuk A.
        • Diemand A.
        • Zentgraf H.
        • Mogk A.
        Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion.
        EMBO J. 2009; 28: 315-325
        • Lossi N.S.
        • Manoli E.
        • Förster A.
        • Dajani R.
        • Pape T.
        • Freemont P.
        • Filloux A.
        The HsiB1C1 (TssB-TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheathlike structure.
        J. Biol. Chem. 2013; 288: 7536-7548
        • Mougous J.D.
        • Cuff M.E.
        • Raunser S.
        • Shen A.
        • Zhou M.
        • Gifford C.A.
        • Goodman A.L.
        • Joachimiak G.
        • Ordoñez C.L.
        • Lory S.
        • Walz T.
        • Joachimiak A.
        • Mekalanos J.J.
        A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
        Science. 2006; 312: 1526-1530
        • Osipiuk J.
        • Xu X.
        • Cui H.
        • Savchenko A.
        • Edwards A.
        • Joachimiak A.
        Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa.
        J. Struct. Funct. Genomics. 2011; 12: 21-26
        • Jobichen C.
        • Chakraborty S.
        • Li M.
        • Zheng J.
        • Joseph L.
        • Mok Y.K.
        • Leung K.Y.
        • Sivaraman J.
        Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda.
        PLoS One. 2010; 5: e12910
        • Ballister E.R.
        • Lai A.H.
        • Zuckermann R.N.
        • Cheng Y.
        • Mougous J.D.
        In vitro self-assembly of tailorable nanotubes from a simple protein building block.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3733-3738
        • Pukatzki S.
        • Ma A.T.
        • Revel A.T.
        • Sturtevant D.
        • Mekalanos J.J.
        Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15508-15513
        • Lossi N.S.
        • Dajani R.
        • Freemont P.
        • Filloux A.
        Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa.
        Microbiology. 2011; 157: 3292-3305
        • Basler M.
        • Mekalanos J.J.
        Type 6 secretion dynamics within and between bacterial cells.
        Science. 2012; 337: 815
        • Kapitein N.
        • Bönemann G.
        • Pietrosiuk A.
        • Seyffer F.
        • Hausser I.
        • Locker J.K.
        • Mogk A.
        ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion.
        Mol. Microbiol. 2013; 87: 1013-1028
        • Brunet Y.R.
        • Espinosa L.
        • Harchouni S.
        • Mignot T.
        • Cascales E.
        Imaging type VI secretion-mediated bacterial killing.
        Cell Rep. 2013; 3: 36-41
        • Pietrosiuk A.
        • Lenherr E.D.
        • Falk S.
        • Bönemann G.
        • Kopp J.
        • Zentgraf H.
        • Sinning I.
        • Mogk A.
        Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion.
        J. Biol. Chem. 2011; 286: 30010-30021
        • Kanamaru S.
        Structural similarity of tailed phages and pathogenic bacterial secretion systems.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4067-4068
        • Pukatzki S.
        • Ma A.T.
        • Sturtevant D.
        • Krastins B.
        • Sarracino D.
        • Nelson W.C.
        • Heidelberg J.F.
        • Mekalanos J.J.
        Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1528-1533
        • Zheng J.
        • Leung K.Y.
        Dissection of a type VI secretion system in Edwardsiella tarda.
        Mol. Microbiol. 2007; 66: 1192-1206
        • Aschtgen M.S.
        • Gavioli M.
        • Dessen A.
        • Lloubès R.
        • Cascales E.
        The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall.
        Mol. Microbiol. 2010; 75: 886-899
        • Aschtgen M.S.
        • Bernard C.S.
        • De Bentzmann S.
        • Lloubès R.
        • Cascales E.
        SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli.
        J. Bacteriol. 2008; 190: 7523-7531
        • Ma L.S.
        • Lin J.S.
        • Lai E.M.
        An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens.
        J. Bacteriol. 2009; 191: 4316-4329
        • Felisberto-Rodrigues C.
        • Durand E.
        • Aschtgen M.S.
        • Blangy S.
        • Ortiz-Lombardia M.
        • Douzi B.
        • Cambillau C.
        • Cascales E.
        Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar.
        PLoS Pathog. 2011; 7: e1002386
        • Aschtgen M.S.
        • Zoued A.
        • Lloubès R.
        • Journet L.
        • Cascales E.
        The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC.
        Microbiologyopen. 2012; 1: 71-82
        • Durand E.
        • Zoued A.
        • Spinelli S.
        • Watson P.J.
        • Aschtgen M.S.
        • Journet L.
        • Cambillau C.
        • Cascales E.
        Structural characterization and oligomerization of the TssL protein, a component shared by bacterial type VI and type IVb secretion systems.
        J. Biol. Chem. 2012; 287: 14157-14168
        • Aschtgen M.S.
        • Thomas M.S.
        • Cascales E.
        Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP. what else?.
        Virulence. 2010; 1: 535-540
        • Casabona M.G.
        • Vandenbrouck Y.
        • Attree I.
        • Couté Y.
        Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane.
        Proteomics. 2013; 13: 2419-2423
        • Brunet Y.R.
        • Bernard C.S.
        • Gavioli M.
        • Lloubès R.
        • Cascales E.
        An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster.
        PLoS Genet. 2011; 7: e1002205
        • Datsenko K.A.
        • Wanner B.L.
        One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645
        • van den Ent F.
        • Löwe J.
        RF cloning: a restriction-free method for inserting target genes into plasmids.
        J. Biochem. Biophys. Methods. 2006; 67: 67-74
        • Karimova G.
        • Pidoux J.
        • Ullmann A.
        • Ladant D.
        A bacterial two-hybrid system based on a reconstituted signal transduction pathway.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 5752-5756
        • Battesti A.
        • Bouveret E.
        The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli.
        Methods. 2012; 58: 325-334
        • Sciara G.
        • Blangy S.
        • Siponen M.
        • Mc Grath S.
        • van Sinderen D.
        • Tegoni M.
        • Cambillau C.
        • Campanacci V.
        A topological model of the baseplate of lactococcal phage Tuc2009.
        J. Biol. Chem. 2008; 283: 2716-2723
        • Tang G.
        • Peng L.
        • Baldwin P.R.
        • Mann D.S.
        • Jiang W.
        • Rees I.
        • Ludtke S.J.
        EMAN2: an extensible image processing suite for electron microscopy.
        J. Struct. Biol. 2007; 157: 38-46
        • Shaikh T.R.
        • Gao H.
        • Baxter W.T.
        • Asturias F.J.
        • Boisset N.
        • Leith A.
        • Frank J.
        SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs.
        Nat. Protoc. 2008; 3: 1941-1974
        • Scheres S.H.
        Classification of structural heterogeneity by maximum-likelihood methods.
        Methods Enzymol. 2010; 482: 295-320
        • Scheres S.H.
        • Núñez-Ramirez R.
        • Sorzano C.O.
        • Carazo J.M.
        • Marabini R.
        Image processing for electron microscopy single-particle analysis using XMIPP.
        Nat. Protoc. 2008; 3: 977-990
        • van Heel M.
        • Schatz M.
        Fourier shell correlation threshold criteria.
        J. Struct. Biol. 2005; 151: 250-262
        • Konarev P.V.
        • Volkov V.V.
        • Sokolova A.V.
        • Koch M.H.J.
        • Svergun D.I.
        PRIMUS: a Windows PC-based system for small-angle scattering data analysis.
        J. Appl. Crystallogr. 2003; 36: 1277-1282
        • Konarev P.V.
        • Petoukhov M.V.
        • Volkov V.V.
        • Svergun D.I.
        ATSAS 2.1, a program package for small-angle scattering data analysis.
        J. Appl. Crystallogr. 2006; 39: 277-286
        • Guinier A.
        La diffraction des rayons X aux très petits angles: application à l'étude de phénomènes ultramicroscopiques.
        Ann. Phys. 1939; 12: 161-237
        • Svergun D.I.
        Determination of the regularization parameter in indirect transform methods using perceptual criteria.
        J. Appl. Crystallogr. 1992; 25: 495-503
        • Franke D.
        • Svergun D.I.
        DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering.
        J. Appl. Crystallogr. 2009; 42: 342-346
        • Volkov V.V.
        • Svergun D.I.
        Uniqueness of ab initio shape determination in small-angle scattering.
        J. Appl. Crystallogr. 2003; 36: 860-864
        • Kozin M.B.
        • Svergun D.I.
        Automated matching of high- and low-resolution structural models.
        J. Appl. Crystallogr. 2001; 34: 33-41
        • Zheng J.
        • Ho B.
        • Mekalanos J.J.
        Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae.
        PLoS One. 2011; 6: e23876
        • Lin J.S.
        • Ma L.S.
        • Lai E.M.
        Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation.
        PLoS One. 2013; 8: e67647