Abdelrahim Zoued

Eric Durand
email: edurand@imm.cnrs.fr

Yannick R Brunet

Silvia Spinelli

Badreddine Douzi

Mathilde Guzzo

Nicolas Flaugnatti

Pierre Legrand

Laure Journet

Rémi Fronzes

" Badreddine Douzi

" Journet

Tâm Mignot

Christian Cambillau
email: cambillau@afmb.univ-mrs.fr

Eric Cascales
email: cascales@imm.cnrs.fr

Priming and polymerization of a bacterial contractile tail structure. 1" 2"

these results, we propose that TssA primes and coordinates tail tube and sheath 2" biogenesis.

3"

Contractile injection machines are fascinating nano-structures evolved to deliver 4" macromolecules into target cells [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF] . These machines have been elaborated for different 5"

purposes such as the injection of DNA into host cells in the case of bacteriophages, for the 6" delivery of protein effectors into bacterial or eukaryotic cells in the case of R-pyocins, 7"

Photorhabdus Virulence Cassettes, Anti-feeding prophages or Type VI secretion systems 8" (T6SS) or for inducing metamorphosis in invertebrates [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Ge | Atomic structures of a bactericidal contractile nanotube in its pre-and 22" postcontraction states[END_REF][START_REF] Bönemann | Tubules and donuts: a type VI secretion story[END_REF][START_REF] Yang | Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. 27[END_REF][START_REF] Shikuma | Marine tubeworm metamorphosis induced by arrays of bacterial phage 29" tail-like structures[END_REF][START_REF] Heymann | Three-dimensional structure of the toxin-delivery particle antifeeding 31" " 10" prophage of Serratia entomophila[END_REF] . These machines are constituted of a 9" tubular edifice called a tail [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Kube | Structural comparison of contractile nanomachines[END_REF][START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF] . The tail is essentially composed of a rigid inner tube wrapped 10" by a contractile structure -the sheath -that is assembled in an extended conformation that 11" stores mechanical energy necessary for its contraction and to propel the inner tube towards the 12" target [START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF] . The tail is assembled on the baseplate that varies in terms of composition and number 13" of subunits; however, a minimal baseplate is constituted of the hub protein surrounded by 14" wedges [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Kube | Structural comparison of contractile nanomachines[END_REF][START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF] . The baseplate is not only the platform for the assembly of the tube/sheath but also 15" an important component of the signaling cascade that triggers sheath contraction [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF] . Tails are 16" usually completed by terminator proteins that stabilize the sheath and maintain tube and 17" sheath together at the distal end to prevent energy dissipation during sheath contraction and to 18" permit proper ejection of the inner tube [START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF][START_REF] Ferguson | Pulse-chase analysis of the in vivo assembly of the 5" bacteriophage T4 tail[END_REF][START_REF] Rybakova | Role of antifeeding prophage (Afp) protein Afp16 in terminating the 7" length of the Afp tailocin and stabilizing its sheath[END_REF] .

19"

The T6SS is composed of a contractile structure anchored to the cell envelope by the 20"

TssJLM membrane complex that serves as docking station as well as channel for the passage 21" of the inner tube during sheath contraction [START_REF] Aschtgen | The SciZ protein anchors 9" the enteroaggregative Escherichia coli Type VI secretion system to the cell wall[END_REF][START_REF] Felisberto-Rodrigues | Towards a structural comprehension of bacterial type VI 12" secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. 13[END_REF][START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF][START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF] (Extended Data Fig. 1a). The contractile 22" structure is composed of the tail tube made by stacks of Hcp hexameric rings, wrapped by a 23" sheath-like structure constituted of the TssB and TssC subunits (Extended Data Fig. 1a) [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF] .

24"

During T6SS biogenesis, the assembly of the tube and sheath are coordinated: the insertion of 25" a tube ring immediately preceding that of a sheath block [START_REF] Brunet | Type VI secretion and bacteriophage tail 19" tubes share a common assembly pathway[END_REF] . This tail polymerizes on a 26" baseplate-like complex composed of the VgrG hub, and the TssE, TssF, TssG and TssK 27" subunits [START_REF] Leiman | Type VI secretion apparatus and phage tail-associated protein complexes 21" share a common evolutionary origin[END_REF][START_REF] Basler | Type VI secretion 23" requires a dynamic contractile phage tail-like structure[END_REF][START_REF] English | analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct 26" oligomeric states of TssK and identifies a TssK-TssFG subcomplex[END_REF][START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF] (Extended Data Fig. 1a). The TssBC sheath polymerizes in tens of seconds to 28" build a ~ 600-nm long structure that contracts in a few milliseconds [START_REF] Basler | Type VI secretion 23" requires a dynamic contractile phage tail-like structure[END_REF] . Contraction of the 29" sheath propels the Hcp inner tube towards the target cell, such as a nano-crossbow, [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF] and is 30" responsible for the delivery of toxin effectors as it correlates with lysis of the competitor 31" bacterium [START_REF] Basler | Tit-for-tat: type VI secretion system counterattack 5" during bacterial cell-cell interactions[END_REF][START_REF] Brunet | Imaging type VI 7" secretion-mediated bacterial killing[END_REF] . Recent cryo-electron microscopy (cryo-EM) studies have revealed the atomic 32" structure of the T6SS sheath in its contracted conformation [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-9" state-specific recycling mechanism[END_REF][START_REF] Kudryashev | Structure of the Type VI secretion system contractile sheath[END_REF] . The sheath is a helical 33" " 3" structure composed of 6-TssB/TssC heterodimer strands, each heterodimer being stabilized by 1"

an intra-and inter-strand handshake domain [START_REF] Kudryashev | Structure of the Type VI secretion system contractile sheath[END_REF] . In addition, a cryo-EM study of the pyocin R2 2" has provided information regarding the atomic structure of this contractile nanotube in its 3" extended conformation and on how it interacts with the inner tube [START_REF] Ge | Atomic structures of a bactericidal contractile nanotube in its pre-and 22" postcontraction states[END_REF] . Although the general 4" mechanism of T6SS assembly and the structure of the T6SS sheath are now well documented, 5" critical details are missing such as how the polymerization of the sheath is controlled, how 6" tube and sheath assembly is coordinated and how tail polymerization is stopped.

7"

TssA initiates tail tube/sheath polymerization.

8"

During T6SS tail biogenesis, (i) the recruitment and assembly of Hcp hexamers and TssBC 9" sheath blocks should be coordinated and (ii) the tail tube and sheath should be firmly attached 10" together at the distal end to allow proper tube throwing during contraction. We therefore 11"

hypothesized that at least one of the T6SS core proteins must be required to coordinate and/or 12"

terminate Hcp/TssBC tail assembly. Such candidate subunit(s) should interact with both the 13" tube protein (Hcp) and with at least one component of the sheath (TssB and/or TssC). We 14" therefore performed a systematic bacterial two-hybrid analysis in which Hcp, TssB and TssC 15"

were used as baits to identify prey partners within T6SS subunits. Extended Data Fig. 1b 16"

shows that a number of baseplate components (TssE, TssF, TssG and VgrG) interact with 17"

either Hcp or TssC. However, a unique protein, TssA (gene accession number: 284924261), 18" interacts with both tube and sheath components. Recent data showed that TssA is required for 19"

proper formation of Hcp tube [START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF] , arguing against a role of TssA as a terminator-only protein. In 20" agreement with this conclusion, fluorescence microscopy experiments show that T6SS 21" sheathes do not assemble in tssA cells (Extended Data Fig. 1c). In addition, Hcp tube proteins 22" are not released in tssA cells (Extended Data Fig. 1d). Collectively, these data suggest a 23" critical role of TssA as a regulator of T6SS tail biogenesis.

24"

Given that T6SS tube/sheath assembly is initiated on the baseplate complex which is 25" docked at the cytoplasmic side of the TssJLM complex [START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF] , we hypothesized that TssA interacts 26" with baseplate or membrane complex components. First, fractionation experiments showed 27" that the TssA protein mainly localizes in the cytoplasm but a significant amount of the protein 28" is associated with the membrane fraction (Extended Data Fig. 1e). TssA association with the 29" inner membrane is likely dependent on the T6SS membrane complex as co-purification and 30"

gel filtration experiments showed that TssA binds to the detergent-solubilized TssJLM 31" complex (Extended Data Fig. 2a-b). Negative-stain electron microscopy (EM) of the TssJLM-

32"

TssA complex further demonstrated the presence of a ~300 Å-large complex associated to the 33" " 4" cytoplasmic base of the TssJLM rocket-like structure (Extended Data Fig. 2c-d). To gain 1" further information on TssA localization and dynamics, we fused TssA to a fluorescent 2" reporter, the superfolder-Green Fluorescent Protein (sfGFP). sfGFP was inserted at the tssA 3" locus on the chromosome, to engineer cells producing a functional sfGFP-TssA chimera 4" protein. Time-lapse microscopy recordings showed that TssA does not distribute randomly 5" but rather assembles 1-3 discrete foci, located close to the membrane (Fig. 1a). Most of these 6" foci are not fixed and show directional movement (Extended Data Fig. 3a). Kymographic

7"

analyses confirmed that TssA foci move with an unidirectional trajectory, at a constant 8" velocity (see the schematic representation in the inset of the Fig. 1a panel, and the kymograph 9"

in Extended Data Fig. 3b). Based on these trajectory, we wondered whether TssA foci might 10" be pushed by the elongation of the sheath. In ∆tssBC cells, TssA foci were still assembled at 11" close proximity to the membrane, but their dynamics was completely abolished: the TssA foci 12" remained static (Fig. 1b and Extended Data Fig. 3a). This experiment defined that the TssBC 13" sheath does not interfere with TssA recruitment and primary localization but rather pushes the 14"

TssA cluster during its elongation. We further monitored TssB-mCherry and sfGFP-TssA 15" dynamics. Time-lapse recordings and kymographic analyses confirmed that TssA-containing 16" complexes assemble first close to the membrane and are then pushed by the sheath towards 17" the opposite side of the bacterium (Fig. 1c and Extended Data Fig. 3b). In addition,

18"

Fluorescence Lifetime Imaging Microscopy (FLIM) assays demonstrated that TssA molecules 19"

do not appear to turn over between the foci and the intracellular pool therefore suggesting that 20" the same TssA or TssA-containing complex remains at the distal end of the sheath (Extended 21" Data Fig. 3c-d). However, because sheath contraction is a very fast event that occurs in a few 22" msec [START_REF] Basler | Type VI secretion 23" requires a dynamic contractile phage tail-like structure[END_REF] and is immediately followed by ClpV-mediated disassembly [START_REF] Kapitein | ClpV recycles VipA/VipB tubules and prevents non-productive tubule 13" formation to ensure efficient type VI protein secretion[END_REF] our experiments could 23" not define whether TssA remains associated to the sheath during contraction.

24"

To provide insights onto the assembly of the T6SS and particularly on the early events 25"

prior to tube/sheath elongation, we tested the localization and dynamics of sfGFP-TssA in 26" various mutant backgrounds. The biogenesis of the T6SS begins with the initial positioning of 27"

the TssJ outer membrane lipoprotein and progresses with the sequential recruitment of the 28" inner membrane TssM and TssL proteins [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF] , and then the TssEFGK-VgrG baseplate [START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF] .

29"

Deletion of baseplate components or Hcp did not affect TssA localization but abolished its 30" dynamics (Fig. 1d and Extended Data Fig. 3e-h). By contrast, the sfGFP-TssA fluorescence 31" was diffuse in tssM cells but remained clustered in tssL cells (Fig. 1d and Extended Data Fig.

32"

3e-h). Taken together, these results showed that TssA is recruited in the early stages of T6SS 33" " 5" biogenesis, after formation of the TssJ-TssM complex (Extended Data Fig. 3i). The 1" interaction of TssA with the TssJM complex was further confirmed by co-purification 2" experiments (Extended Data Fig. 2a).

3"

To identify TssA additional partners, the interaction of TssA with all the T6SS soluble 4" core components and the soluble domains of the TssL, TssM and TssJ proteins, was first 5" tested by a bacterial two-hybrid approach. Extended Data Fig. 4a shows that in addition to 6"

Hcp and TssC, and the previously described TssA-TssK interaction [START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] , TssA interacts with the 7" TssE and VgrG baseplate subunits. To validate these results by an alternative approach, native 8"

TssA, Hcp, TssE, VgrG and the TssBC complex were purified and the interactions were 9" assessed by Surface Plasmon Resonance (Extended Data Fig. 4b-e). In the four cases, we 10" observed interactions between the two partners, with dissociation constants ranging from ~ 2 11" to ~ 50 µM (Extended Data Fig. 4b-e). The affinities of these complexes are rather low but 12"

have to be replaced in the context of multiple interactions that probably act synergistically.

13"

The interaction of TssA with Hcp or TssBC are of better affinities compared to that of TssA 14"

with VgrG and TssE, whereas TssA dissociates more rapidly from VgrG or TssE than from 15"

Hcp or TssBC (Extended Data Fig. 4b-e). This suggests that the TssE and VgrG baseplate 16" proteins may first interact to TssA and that these interactions will be displaced by the tube 17" and sheath components (Hcp, TssBC).

18"

Based on the interaction network and on the fluorescence microscopy recordings, we 19" conclude that positioning of TssA on the membrane complex recruits the baseplate and 20"

initiates polymerization of the tube and sheath. However, TssA remains associated with the 21" distal end of the polymerized structure during the elongation process (see closing remarks).

22"

Structural organization of the TssA protein 23"

The EM analyses of the TssJLM-TssA complex (Extended Data Fig. 2d) suggest that 24"

TssA assembles a ~ 300 Å-large complex. A 6×His-tagged thioredoxin-TssA fusion (TRX-25"

TssA) was produced, purified to homogeneity by ion-metal affinity and size exclusion 26" chromatographies (Extended Data Fig. 5a). Gel filtration (Extended Data Fig. 5b) and on-line 27" multi-angle laser light scattering/quasi-elastic light scattering/absorbance/refractive index 28"

(MALS/QELS/UV/RI, Extended Data Fig. 5c)" analyses defined a mass of 891 kDa, which 29" corresponds to the mass of a TRX-TssA dodecamer (theoritical mass = 888 kDa).

30"

Crystallization attempts with the purified full-length TssA protein obtained after tag cleavage 31" by the TEV protease failed. We therefore examined the protein sample using small angle X-32" rays scattering (SAXS, Extended Data Fig. 5d-j) and EM after negative staining (EM, 33" " 6"

Extended Data Fig. 5k-r). The ~ 19-Å resolution single particle reconstruction of TssA from 1" the electron micrographs (Extended Data Fig. 5k-n) showed that TssA assembles two stacked 2" hexamers with arm-like short extensions (Extended Data Fig. 5p-r). However, we noted that 3" the EM density does not account for the complete mass of a TssA dodecamer and therefore 4" suspected that the arms may represent a flexible domain, shortened by the averaging 5"

procedure. This was confirmed by SAXS studies demonstrating that TssA is composed of a 6" central hexameric core bearing six long arms yielding a starfish-like structure (Extended Data 7" Fig. 5h-j and Extended Data Table 1). Whereas the SAXS model allows to better visualize the 8" arm length compared to the EM reconstruction, its low resolution impairs the visual 9" separation of the dimeric arms.

10"

Limited proteolysis of full-length TssA using proteinase K yielded three stable 11" fragments of ~ 45, ~ 33 and ~ 19 kDa (Extended Data Fig. 6a) corresponding to the N-12" terminal 1-392 and C-terminal 221-531 and 393-531 regions of TssA respectively (Extended 13" Data Fig. 6b). Based on this result, two domains corresponding to fragments 1-400 (TssA Nt , 14" theoretical molecular weight: 44.2 kDa) and 395-531 (TssA Ct , theoretical molecular weight 15"

15.1 kDa) were designed. Bacterial two hybrid experiments showed that these two domains 16" oligomerize independently (Extended Data Fig. 6c). MALS/QELS/UV/RI analyses of the 17" purified TssA Nt and TssA Ct domains revealed that the N-terminal fragment is a dimer in 18" solution (Extended Data Fig. 6d) whereas the C-terminal domain is dodecameric (Extended 19" Data Fig. 6e).

20"

The N-terminal and C-terminal domain crystallized, and 3.37-Å and 3.35-Å resolution 21" datasets were collected respectively (Extended Data Table 2). In agreement with the 22" MALS/QELS/UV/RI data, TssA Nt is a dimer in the crystal. The peptidic chain starts at 23" residue 221 (instead of 1) and stops at residue 377 (instead of 401). This fragment, named 24" TssA Nt2 , results from protein cleavage during crystallization. The TssA Nt2 domain is 25" constituted of 3 pairs of helices (1-2, 3-4 and 5-6) structured as a bundle followed by a 7th 26" helix perpendicular to the others (Extended Data Fig. 6f-g). The TssA Ct structure features two 27" stacked head-to-head hexamers 30-Å thick, with an external diameter of 100 Å (Fig. 2a). The 28" overall form of the hexamer is unique and resembles that of a kaleidocycle with 6 triangular 29" wedges that contact each other via α-helix hinges (Fig. 2a). Docking of these X-ray structures 30" onto the starfish-shaped EM and SAXS reconstructions (Fig. 2b and Extended Data Fig. 7a-n

) 31"
showed that the TssA Ct diameter and width coincide exquisitely with the central core of TssA 32"

whereas TssA Nt2 dimers fit in the arms at close proximity to the TssA Ct central core. Large 33" " 7" density regions remain available at the extremity of the arms and likely correspond to TssA Nt1 1" domains.

2"

To understand the contribution of the TssA central core and arm domains to the T6SS 3" assembly mechanism, we tested interactions between these domains and TssA partners.

4"

Bacterial two-hybrid analyses demonstrated that TssA Nt interacts with TssBC and TssE 5"

whereas TssA Ct is sufficient to make contacts with Hcp and VgrG (Extended Data Fig. 6h).

6"

SPR analyses further confirmed the TssA Nt -TssBC and TssA Ct -Hcp interactions with K D s of ~ 7"

4.2 and ~ 48 µM respectively (Extended Data Fig. 6i-j).

8"

Closing remarks 9"

In this work we combined structural, functional and microscopy analyses to reveal the 10" specific role of TssA subunit during T6SS biogenesis. TssA is recruited to an early stage of 11" T6SS biogenesis, positions the baseplate, and initiates and guides tail tube/sheath 12" polymerization.

13"

A TssA dodecamer is recruited to the TssJM complex and clusters in discrete foci that 14" represent the site of assembly of the T6SS tail tube and sheath. Previous studies have shown 15" that TssA is not required for proper recruitment of TssL to the TssJM complex [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF] , and hence 16" the T6SS assembly pathway is branched on TssM (Extended Data Fig. 3i). Protein-protein 17" interaction and fluorescence microscopy analyses defined that TssA recruits two components 18" of the baseplate (VgrG and TssE). Once the baseplate is positioned, the polymerization of the 19" tube/sheath structure is initiated. Further co-localization studies demonstrated that the initial 20" static TssA clusters are then pushed towards the opposite side of the cell by the elongation of 21" the sheath structure. FLIM experiments suggested that the same TssA particle remains 22" associated to the distal extremity of the sheath. This result is also in agreement with (i) the 23" interaction of TssA with both tube (Hcp) and sheath (TssC) components, and with the higher 24" affinity of TssA for these two partners compared to the two baseplate components, suggesting 25" that the recruitment of Hcp and TssBC displaces TssA from the baseplate.

26"

TssA exhibits fascinating structural characteristics. Twelve TssA proteins assemble a 27" 6-fold symmetry starfish-like particle composed of a central core bearing six elongated 170-Å 28" long arms. The TssA central core, corresponding to the C-terminal domain, has a size and a 29" shape comparable to that of an Hcp hexamer (Extended Data Fig. 7o) and interacts with the 30" N-terminal gp27-like VgrG module and Hcp, two proteins whose structures have been shown 31"

to be superimposable [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-9" state-specific recycling mechanism[END_REF] , suggesting that TssA recognizes the same fold. The TssA arms 32" " 8" interact with TssE and TssC. TssE is the homologue of gp25, a component of the 1" bacteriophage baseplate wedges that assemble around the gp27 hub [START_REF] Leiman | Contractile tail machines of bacteriophages[END_REF][START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF] . Therefore the 2"

positioning of the TssA central core on VgrG or Hcp allows the arms to contact the outer 3" wedges or sheath rings. Indeed, molecular docking of the TssA EM volume to the sheath 4" model in the extended conformation shows that the TssA arms are interdigitated with sheath 5" subunits resulting in very complementary shapes and tight contacts (Extended Data Fig. 8a-b).

6"

Such an efficient complementarity cannot be modelled with the contracted tail sheath, 7"

suggesting that TssA does not bind, or more weakly, to this conformation (Extended Data Fig. 8" 8c).

9"

The TssA C-terminal domain is a 6-wedge kaleidocycle in which the six wedges are 10" contacting each other by hinge-like helices at the outward of the structure. We hypothesize 11" that large conformational modifications happen, such as the displacement of these wedges to 12" the exterior, opening a large central lumen. This lumen will have a diameter (~ 90 Å) 13" sufficient to accommodate a hexameric Hcp ring. We therefore propose a functional model in 14"

which TssA controls the coordinated polymerization of the tube and sheath structure (Fig. 3 15"

and Supplementary Video file): the recruitment of an Hcp ring by TssA will open the lumen 16"

allowing the incorporation of this Hcp ring to the growing structure. Then the TssA arms 17" might be involved in the recruitment and proper positioning of the TssBC strands around this 18"

Hcp ring, before the insertion of a new Hcp ring, etc (Fig. 3a). This model implies that the 19" Hcp hexameric unit is incorporated immediately before the TssBC sheath building block, a 20" hypothesis consistent with data showing that in both bacteriophage T4 and T6SS, tube and 21" sheath grow up from the baseplate together, with the tube protein leading and directing sheath 22" assembly [START_REF] Brunet | Type VI secretion and bacteriophage tail 19" tubes share a common assembly pathway[END_REF][START_REF] King | Assembly of the tail of bacteriophage T4[END_REF] . However, the current data cannot rule out that a pre-formed Hcp-TssBC 23" complex is recruited to the growing structure (Fig. 3b). In these models, the TssA arms secure 24" the sheath under an extended state by connecting it to the rigid Hcp tube, explaining how the 25"

sheath is maintained in a metastable conformation during elongation. In bacteriophages,

26"

sheath contraction is initiated at the baseplate and progresses to the head. We therefore 27"

propose that TssA remains attached to the distal end during T6SS sheath contraction until the 28" last TssBC row contracts to prevent energy dissipation and permit proper propulsion of the 29"

Hcp tube, before being released. This model is in agreement with the docking simulations 30"

showing tight contacts of TssA with the extended sheath only (Extended Data Fig. 8a-b). The 31" function of the TssA protein is therefore different from that of the distal tail proteins (Dit) of 32"

Siphoviridae that prime tube/sheath polymerization but remain attached to the baseplate [START_REF] Vegge | Structural characterization and assembly of the distal tail structure of the 19" temperate lactococcal bacteriophage TP901-1[END_REF] . It 33" " 9" is however closely related to the function of the gp3/gp15 proteins of Myoviridae. Although 1"

the TssA and gp15 folds are highly divergent, the overall architecture of the central core of 2"

TssA is similar in terms of size and diameter to gp15 or gp3 [START_REF] Pell | The X-ray crystal structure of the phage lambda tail terminator protein reveals 21" the biologically relevant hexameric ring structure and demonstrates a conserved mechanism of 22" tail termination among diverse long-tailed phages[END_REF][START_REF] Fokine | The molecular architecture of the bacteriophage T4 neck[END_REF] (Extended Data Fig. 8d-f).

3"

Interestingly, the gp3/gp15 proteins, also known as tail terminators, do not only complete tail 4" assembly: pulse-chase studies of bacteriophage T4 biogenesis demonstrated that gp15 is not 5" recruited once the tail tube/sheath has been polymerized but, conversely, that it is assembled 6" on the baseplate prior to the gp19 tube and gp18 sheath proteins [START_REF] Ferguson | Pulse-chase analysis of the in vivo assembly of the 5" bacteriophage T4 tail[END_REF] . However, it is found at the 7" opposite extremity of the baseplate once the tail is completed and it stabilizes the sheath [START_REF] King | Assembly of the tail of bacteriophage T4[END_REF] .

8"

This dynamic is very similar to that observed for TssA, that is first bound to the baseplate but 9"

found at the distal end once the tail is completed. Taken together these data suggest that TssA 10" and gp15 may both prime, control the polymerization, complete and stabilize the T6SS and 11" bacteriophage T4 tails respectively. Finally, according to this model, the T6SS tail tube/sheath 12" is growing by the incorporation of building blocks at the distal end of the structure, a point 13" that remains to be experimentally addressed. Although the newly incorporated subunits do not 14" transit through the Hcp lumen, this mechanism is similar to that of the flagellar cap complex, 15" which binds to the hook and incorporates new flagellin subunits at the distal end by a rotary 16" mechanism [START_REF] Yonekura | The bacterial flagellar cap as the rotary promoter of flagellin self-assembly. 26[END_REF] . Defining the TssA conformational modifications that occur during tail 17" elongation will shed light on the molecular mechanism of T6SS tail assembly and how the 18" sheath is maintained in the extended state. working model is shown in the Supplementary Video. 6"

7"

METHODS

8"

Bacterial strains, growth conditions and chemicals. The strains, plasmids and oligonucleotides used in this 9" study are listed in the Supplementary Table . The enteroaggregative E. coli EAEC strain 17-2 and its ∆tssA, 10" ∆tssBC, ∆tssE, ∆tssF, ∆tssG, ∆tssK, ∆tssL, ∆tssM, ∆hcp, ∆vgrG, and tssB-mCherry isogenic derivatives were

11"

used for this study [START_REF] Aschtgen | The SciZ protein anchors 9" the enteroaggregative Escherichia coli Type VI secretion system to the cell wall[END_REF][START_REF] Brunet | Type VI secretion and bacteriophage tail 19" tubes share a common assembly pathway[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] . The E. coli K-12 DH5α, W3110, BTH101 and T7 Iq pLys strains were used for cloning

12"

steps, co-immunoprecipitation, bacterial two-hybrid and protein purification respectively. Strains were routinely

13"

grown in LB rich medium (or Terrific broth medium for protein purification) or in Sci-1 inducing medium (SIM;

14"

M9 minimal medium, glycerol 0.2%, vitamin B1 1 µg/mL, casaminoacids 100 µg/mL, LB 10%, supplemented

15"

or not with bactoagar 1.5%) with shaking at 37°C [START_REF] Brunet | An epigenetic switch involving 18" overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster[END_REF] . Plasmids were maintained by the addition of ampicillin ( 10016" µg.mL -1 for E. coli K-12, 200 µg/mL for EAEC), kanamycin (50 µg.mL -1 ) or chloramphenicol (30 µg.mL -1 ).

17"

Expression of genes from pBAD, pETG20A/pRSF or pASK-IBA vectors was induced at A 600nm ~ 0.6 with

18"

0.02% of L-arabinose (Sigma-Aldrich) for 45 min, 0.5-1 mM of isopropyl-β-D-thio-galactopyrannoside (IPTG,

19"

Eurobio) for 14 hours or 0.02 µg/mL of anhydrotetracyclin (AHT, IBA Technologies) for 45 min. respectively.

20"

For BACTH experiments, plates were supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

21"

(X-Gal, Eurobio, 40 µg/mL).

22"

Strain construction. The tssA gene was deleted into the enteroaggregative E. coli 17-2 strain using a modified

23"

one-step inactivation procedure [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 21" using PCR products[END_REF] as previously described [START_REF] Aschtgen | The SciZ protein anchors 9" the enteroaggregative Escherichia coli Type VI secretion system to the cell wall[END_REF] using plasmid pKOBEG [START_REF] Chaveroche | A rapid method for efficient gene replacement in the 23" filamentous fungus Aspergillus nidulans[END_REF] . Briefly, a kanamycin

24"

cassette was amplified from plasmid pKD4 [START_REF] Franke | a program for rapid ab-initio shape determination in small-angle 50[END_REF] using oligonucleotides carrying 50-nucleotide extensions

25"

homologous to regions adjacent to tssA. After electroporation of 600 ng of column-purified PCR product,

26"

kanamycin resistant clones were selected and verified by colony-PCR. The kanamycin cassette was then excised

27"

using plasmid pCP20 [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 21" using PCR products[END_REF] . The deletion of tssA was confirmed by colony-PCR. The same procedure was used to

28"

introduce the mCherry-coding sequence upstream the stop codon of the tssB gene (vector pmCh-KD4 as

29"

template for PCR amplification) or the super-folder GFP-coding sequence downstream the start codon (vector

30"

pKD4-sfGFP as template) or upstream the stop codon (vector psfGFP-KD4 as template) of the tssA gene to yield

31"

strains producing TssB-mCherry, sfGFP-TssA or TssA-sfGFP from their original chromosomal loci.

32"

Plasmid construction. All bacterial two-hybrid plasmids and the plasmid producing the TssJLM membrane

33"

core complex (pRSF-TssJ ST -FL TssL-6His TssM, pRSF-TssJLM) have been described previously [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] . Polymerase

34"

Chain Reactions (PCR) were performed using a Biometra thermocycler using the Q5 (New England Biolabs) or

35"

Pfu Turbo (Agilent Technologies) DNA polymerases. Restriction enzymes were purchased from New England

36"

Biolabs and used according to the manufacturer's instructions. Custom oligonucleotides were synthesized by

37"

Sigma Aldrich and are listed in the Supplementary Table . Enteroaggregative E. coli 17-2 chromosomal DNA

38"

was used as a template for all PCRs. E. coli strain DH5α was used for cloning procedures. All the plasmids

39"

(except for pETG20A and pDEST17 derivatives) have been constructed by restriction-free cloning 34 as

40"

previously described [START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] . Briefly, the gene of interest was amplified using oligonucleotides introducing extensions

41"

annealing to the target vector. The double-stranded product of the first PCR has then been used as

42"

oligonucleotides for a second PCR using the target vector as template. PCR products were then treated with

43"

DpnI to eliminate template plasmids and transformed into DH5α-competent cells. For protein purification, the

44"

sequences encoding the full-length TssA (residues 1-542), the TssA N-terminal (residues 1-392), the TssA Nt2

45"

(residues 221-377) and C-terminal (residues 393-542) domains, the N-terminal domain of VgrG (residues 1-

46"

490), the full-length TssE or both TssB and TssC were cloned into the pETG-20A (TssA, TssA Ct , VgrG Nt , TssE)

47"

or pDEST17 (TssA Nt , TssBC) expression vector (kind gifts from Dr Arie Geerlof, EMBL, Hamburg) according

48"

to standard Gateway protocols. Proteins produced from pETG20A derivatives are fused to an N-terminal 6×His-

49"

tagged thioredoxin (TRX) followed by a cleavage site for the Tobacco etch virus (TEV) protease whereas

50"

proteins produced from pDEST17 are fused to an N-terminal 6×His tag followed by a TEV protease cleavage

51"

site. All constructs have been verified by restriction analyses and DNA sequencing (Eurofins or MWG).

52" " 15"

Bacterial two-hybrid assay (BACTH). The adenylate cyclase-based bacterial two-hybrid technique [START_REF] Karimova | A bacterial two-hybrid system based on a 27" reconstituted signal transduction pathway[END_REF] was used

1"

as previously published [START_REF] Battesti | The bacterial two-hybrid system based on adenylate cyclase reconstitution 29" in Escherichia coli[END_REF] . Briefly, the proteins to be tested were fused to the isolated T18 and T25 catalytic

2"

domains of the Bordetella adenylate cyclase. After introduction of the two plasmids producing the fusion

3"

proteins into the reporter BTH101 strain, plates were incubated at 30°C for 48 hours. Three independent colonies

4"

for each transformation were inoculated into 600 µl of LB medium supplemented with ampicillin, kanamycin

5"

and IPTG (0.5 mM). After overnight growth at 30°C, 10 µL of each culture were dropped onto LB plates

6"

supplemented with ampicillin, kanamycin, IPTG and X-Gal and incubated for 16 hours at 30 °C. The

7"

experiments were done at least in triplicate and a representative result is shown.

8"

Fluorescence microscopy and image treatment. Fluorescence microscopy experiments have been performed

9"

essentially as described [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF][START_REF] Brunet | Type VI secretion and bacteriophage tail 19" tubes share a common assembly pathway[END_REF][START_REF] Brunet | Imaging type VI 7" secretion-mediated bacterial killing[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] . Briefly, cells were grown overnight in LB medium and diluted to an A 600nm ~ 10" 0.04 into Sci-1 inducing medium (SIM). Exponentially growing cells (A 600nm ~ 0.8-1) were harvested, washed in

11"

phosphate buffered saline buffer (PBS), resuspended in PBS to an A 600nm ~ 50, spotted on a thin pad of 1.5%

12"

agarose in PBS, covered with a cover slip and incubated for one hour at 37°C prior to microscopy acquisition.

13"

For each experiment, ten independent fields were manually defined with a motorized stage (Prior Scientific) and

14"

stored (X, Y, Z, PFS-offset) in our custom automation system designed for time-lapse experiments. Fluorescence

15"

and phase contrast micrographs were captured every 30 sec. using an automated and inverted epifluorescence

16"

microscope TE2000-E-PFS (Nikon, France) equipped with Perfect Focus System (PFS). PFS automatically

17"

maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times despite 18" mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper

19"

Scientific SARL, France) and a 100×/1.4 DLL objective. The excitation light was emitted by a 120 W metal 20" halide light. All fluorescence images were acquired with a minimal exposure time to minimize bleaching and

21"

phototoxicity effects. The sfGFP images were recorded by using the ET-GFP filter set (Chroma 49002) using an

22"

exposure time of 200-400ms. The mCherry images were recorded by using the ET-mCherry filter set (Chroma

23"

49008) using an exposure time of 100-200ms. Slight movements of the whole field during the time of the

24"

experiment were corrected by registering individual frames using StackReg and Image Stabilizer plugins for

25"

ImageJ. sfGFP and mCherry fluorescence channels were adjusted and merged using ImageJ

26"

(http://rsb.info.nih.gov/ij/). For statistical analyses, fluorescent foci were automatically detected. First, noise and

27"

background were reduced using the 'Subtract Background' (20 pixels Rolling Ball) plugin from Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] . The

28"

sfGFP foci were automatically detected by a simple image processing: (1) create a mask of cell surface and

29"

dilate (2) detect the individual cells using the "Analyse particle" plugin of Fiji (3) sfGFP foci were identified by 30" the "Find Maxima" process in Fiji. To avoid false positive, each event was manually controlled in the original

31"

data. Box-and-whisker representations of the number of foci per cell were made with R software. T-tests were

32"

performed on R to statistically compare each population. Kymographs were obtained after background

33"

fluorescence substraction and sectioning using the Kymoreslicewide plug-in under Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] . Sub-pixel resolution

34"

tracking of fluorescent foci: Fluorescent foci were detected using a local and sub-pixel resolution maxima

35"

detection algorithm and tracked over time with a specifically developed plug-in for ImageJ. The X and Y

36"

coordinates were obtained for each fluorescent focus on each frame. The Mean Square Displacement (MSD) was

37"

calculated as the distance of the foci from its location at t=0 at each time using R software and plotted over time.

38"

For each strain tested, the MSD of at least 10 individual focus trajectories was calculated. For statistical analyses

39"

of mobile trajectories, kymograph analyses were performed and the percentage of fixed, mobile with random

40"

dynamics and mobile with unidirectional trajectory foci are reported.

41"

Fluorescence Lifetime Imaging (FLIM). FLIM experiments were carried on the same microscope device used

42"

for the time-lapse microscopy experiments except with a laser of 488 nm. For each cell a region of interest that

43"

corresponds to the size of the laser beam was focused away from TssB mCh sheath-labelled sfGFP TssA for a time of

44"

3 seconds at a maximum intensity of 100%. The extinction of the complete sfGFP TssA pool was checked by (i) the

45"

absence of recovery of bleached sfGFP TssA-membrane clusters and (ii) by the overall drop and lack of recovery in

46"

intracellular intensity.

47"

Protein purification. E. coli T7 Iq pLysS cells bearing pETG20A or pDEST17 derivatives were grown at 37°C

48"

in Terrific Broth to an A 600 ~ 0.9 and gene expression was inducted with 0.5 mM IPTG for 16 hours at 17°C.

49"

Cells were harvested, resuspended in Tris-HCl 20 mM pH8.0, NaCl 150 mM and lysozyme (0.25 mg/mL) and

50"

broken by sonication. Soluble proteins were separated from inclusion bodies and cell debris by centrifugation 30 51" min at 20,000 × g. The His-tagged fusions were purified using ion metal Ni 2+ affinity chromatography (IMAC)

52"

using a 5-mL HisTrap column (GE Healthcare) and eluted with a step gradient of imidazole. The fusion proteins

53"

were further digested overnight at 4°C by a Hexahistidine-tagged TEV protease using a 1:10 (w/w)

54"

protease:protein ratio. The TEV protease and contaminants were retained by a second IMAC and the purified

55"

proteins were collected in the flow through. Proteins were further separated on preparative Superdex 200 or

56"

Superose 6 gel filtration column (GE Healthcare) equilibrated in Tris-HCl 20 mM pH 8.0, NaCl 150 mM. The

57"

fractions containing the purified protein were pooled and concentrated by ultrafiltration using the Amicon

58"

technology (Millipore, California, USA)."The seleno-Methionine (SeMet) derivatives of TssA Nt and TssA Ct were

59"

produced in minimal medium supplemented with 100 mg/L of Lysine, Phenylalanine and Threonine, 50 mg/L of 60" " 16"

Isoleucine, Leucine, Valine and Seleno-Methionine. Gene induction and protein purification were performed as 1" described above.

2"

Limited proteolysis. The full-length TssA protein was subjected to Proteinase K limited proteolysis (1/10

3"

protease:protein ratio). The reaction was quenched at different time points by the addition of 1mM PMSF and

4"

further boiling for 10 min at 96°C. Samples were analyzed by SDS-PAGE and Coomassie blue staining.

5"

Digested products were identified by Edman N-terminal sequencing and electrospray mass sprectrometry

6"

(Proteomic platform, Institut de Microbiologie de la Méditerranée, Marseille, France).

7"

Analytical gel filtration analysis and MALS/QELS/UV/RI-coupled size exclusion chromatography. Size

8"

exclusion chromatography (SEC) was performed on an Alliance 2695 HPLC system (Waters) using KW803 and

9"

KW804 columns (Shodex) run in Tris-HCl 20 mM pH 8.0, NaCl 150 mM at 0.5 mL/min. MALS, UV 10" spectrophotometry, QELS and RI were monitored with MiniDawn Treos (Wyatt Technology), a Photo Diode

11"

Array 2996 (Waters), a DynaPro (Wyatt Technology) and an Optilab rEX (Wyatt Technology), respectively, as 12" described [START_REF] Felisberto-Rodrigues | Towards a structural comprehension of bacterial type VI 12" secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. 13[END_REF] . Mass and hydrodynamic radius calculation were performed with the ASTRA software (Wyatt

13"

Technology) using a dn/dc value of 0.185 mL/g.

14"

Surface Plasmon Resonance analysis. Steady state interactions were monitored using a BIAcore T200 at 15" 25°C [START_REF] Felisberto-Rodrigues | Towards a structural comprehension of bacterial type VI 12" secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. 13[END_REF] . All the buffers were filtered on 0.2 µm membranes before use. The HC200m sensor chip (Xantech) was 16" coated with purified Hcp, VgrG, TssE or TssBC complex, immobilized by amine coupling (∆RU=4000-4300). A

17"

control flow-cell was coated with thioredoxin immobilized by amine coupling at the same concentration 18" (∆RU=4100). Purified TssA, TssA Nt and TssA Ct (six concentrations ranging from 3.125 to 100 µM) were

19"

injected and binding traces were recorded in duplicate. The signal from the control flow cell and the buffer

20"

response were subtracted from all measurements. The dissociation constants (K D ) were estimated using the

21"

GraphPad Prism 5.0 software on the basis of the steady state levels of ∆RU, directly related to the concentration

22"

of the analytes. The K D were estimated by plotting on X axis the different concentration of analytes and the

23"

differents ∆RU at a fixed time (5 s before the end of the injection step) on the Y axis. For K D calculation,

24"

nonlinear regression fit for XY analysis was used and one site (specific binding) as a model which corresponds

25"

to the equation : Y = Bmax*X/(Kd + X).

26"

Co-purification experiments. Different combinations of plasmids were transformed in BL21(DE3): (i) pRSF-

27"

TssJLM + pIBA37(+), (ii) pRSF + pIBA37-FL TssA, (iii) pRSF-TssJLM + pIBA37-FL TssA and (iv) pRSF-TssJM

28"

+ pIBA37-FL TssA. Transformed BL21(DE3) cells were grown at 37ºC in 200 mL LB medium supplemented

29"

with kanamycin and ampicillin until A 600 ~ 0.6 and gene induction was achieved by the addition of IPTG (1 mM)

30"

and anhydrotetracycline (0.02 µg/mL) during 15 hours at 16ºC. After cell lysis through three passages at the

31"

French press, total membranes were isolated as described previously [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF] . Membranes were solubilized by the

32"

addition of 1% Triton X100 (Affimetrix). Solubilized membrane fractions were purified on a 1 mL Streptactin

33"

column (GE Healthcare). The column was washed with buffer S (Hepes 50 mM pH 7.5, NaCl 50 mM, Triton 34" X100 0.075%) and bound proteins were eluted with buffer S supplemented with desthiobiotin (2.5 mM) and

35"

visualized by Coomassie blue staining and immunoblotting. For EM analyses, BL21(DE3) cells producing

36"

TssJLM and FLAG-tagged TssA were grown and the TssJLM-A complex was purified as described for the

37"

TssJLM membrane core complex [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF] . After the two consecutive affinity columns (His-and Strep-Trap-HP), the

38"

pooled fractions were injected onto a Superose 6 10:300 column equilibrated in Hepes 50 mM pH 7.5, NaCl 50

39"

mM supplemented with 0.025% DM-NPG.

40"

Electron microscopy observation of the TssJLMA complex. Nine microlitres of the purified TssJLMA

41"

complex (~ 0.01 mg/mL) were incubated to glow-discharged carbon-coated copper grids (Agar Scientific) for 30

42"

sec. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl

43"

acetate. Images were collected on an FEI Tecnai F20 FEG microscope operating at a voltage of 200 kV,

44"

equipped with a direct electron detector (Falcon II) at 50,000 magnification.

45"

Transmission electron microscopy, single particle analysis and image processing. Nine microliters of the

46"

purified full-length TssA protein (~ 0.01 mg/mL) were incubated on a glow-discharged carbon-coated copper

47"

grid (Agar Scientific) for 30 sec. After absorption, the sample was blotted, washed with three drops of water and

48"

then stained with 2% uranyl acetate. Images were recorded automatically using the EPU software on a FEG

49"

microscope operating at a voltage of 200 kV and a defocus range of 0.6-25 nm, using a FEI Falcon-II detector

50"

(Gatan) at a nominal magnification of 50,000, yielding a pixel size of 1.9 Å. A dose rate of 25 electrons per Å [START_REF] Ge | Atomic structures of a bactericidal contractile nanotube in its pre-and 22" postcontraction states[END_REF]

51"

per second, and an exposure time of 1 sec were used. A total of 100,000 particles were automatically selected

52"

from 500 independent images and extracted within boxes of 180 pixels × 180 pixels using EMAN2/BOXER [START_REF] Tang | EMAN2: an extensible image processing suite for electron microscopy[END_REF] .

53"

The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two-and three-dimensional

54"

(2D and 3D) classifications and refinements were performed using RELION 1.3 [START_REF] Scheres | implementation of a Bayesian approach to cryo-EM structure determination[END_REF][START_REF] Scheres | Semi-automated selection of cryo-EM particles in RELION-1.3[END_REF] . The automatically selected

55"

dataset was clean up by three rounds of reference-free 2D class averaging, and highly populated classes

56"

displaying high-resolution features were conserved and a final dataset of 20,000 particles was assembled. An

57"

initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in

58"

Relion with 5 classes. The particles corresponding to most populated class (~ 18,000) were used for refinement.

59"

The Relion auto-refine procedure was used to obtain a final reconstruction at ~ 19-Å resolution after masking 60" " 17"

and with D6 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation

1"

(FSC) 0.143 criterion, and FSC curve were corrected for the effects of a soft mask on the FSC curve using high-

2"

resolution noise substitution (Extended Data Fig. 5o) [START_REF] Chen | High-resolution noise substitution to measure overfitting and validate resolution in 3D 39" structure determination by single particle electron cryomicroscopy[END_REF] . All density maps were corrected for the modulation

3"

transfer function of the detector and then sharpened by applying a negative B-factor (-1000) that was estimated

4"

using automated procedures. The electron microscopy map of the EAEC TssA full-length protein has been

5"

deposited in the Electron Microscopy Data Bank under accession number EMD-3282.

6"

Small-Angle X-ray Scattering analysis and ab initio three-dimensional shape reconstruction. Small-Angle

7"

X-ray Scattering (SAXS) analyses were performed at the ID29 beamline (European Synchrotron Radiation

8"

Facility, Grenoble, France) at a working energy of 12.5 keV (λ= 0.931 Å). Thirty microlitres of protein solution 9" at 1.6, 3.7, 7.1, 9.8 and 14.9 mg/mL in Tris-HCl 20 mM pH 8.0, NaCl 150 mM were loaded by a robotic system 10" into a 2-mm quartz capillary mounted in a vacuum and ten independent 10-sec exposures were collected on a

11"

Pilatus 6M-F detector placed at a distance of 2.85 m for each protein concentration. Individual frames were

12"

processed automatically and independently at the beamline by the data collection software (BsxCUBE), yielding

13"

radially averaged normalized intensities as a function of the momentum transfer q, with q=4πsin(θ)/λ, where 2θ

14"

is the total scattering angle and λ is the X-ray wavelength. Data were collected in the range q= 0.04-6 nm -1 . The

15"

ten frames were combined to give the average scattering curve for each measurement. Data points affected by 16" aggregation, possibly induced by radiation damage, were excluded. Scattering from the buffer alone was also 17" measured prior to and after each sample analysis and the average of these two buffer measures was used for

18"

background subtraction using the program PRIMUS [START_REF] Konarev | PRIMUS: a Windows PC-42" based system for small-angle scattering data analysis[END_REF] from the ATSAS package [START_REF] Konarev | ATSAS 2.1, a program package for 44" small-angle scattering data analysis[END_REF] . PRIMUS was also used to

19"

perform Guinier analysis [START_REF] Guinier | La diffraction des rayons X aux très petits angles; application à l'étude de phénomènes 46[END_REF] of the low q data, which provides an estimate of the radius of gyration (R g ).

20"

Regularized indirect transforms of the scattering data were carried out with the program GNOM [START_REF] Svergun | Determination of the regularization parameter in indirect-transform methods using 48" perceptual criteria[END_REF] to obtain P(r)

21"

functions of interatomic distances. The P(r) function has a maximum at the most probable intermolecular

22"

distance and goes to zero at Dmax, the maximum intramolecular distance. The values of Dmax were chosen to

23"

fit with the experimental data and to have a positive P(r) function. Tridimensionnal (3D) bead models that fit

24"

with the scattering data were built with the program DAMMIF [START_REF] Franke | a program for rapid ab-initio shape determination in small-angle 50[END_REF] . Ten independent DAMMIF runs were

25"

performed using the scattering profile of TssA, with data extending up to 0.35 nm -1 , using slow mode settings,

26"

assuming P6 symmetry and allowing for a maximum 500 steps to grant convergence. The models resulting from

27"

independent runs were superimposed using the DAMAVER suite [START_REF] Volkov | Uniqueness of ab initio shape determination in small-angle scattering[END_REF] yielding an initial alignment of structures

28"

based on their axes of inertia followed by minimisation of the normalized spatial discrepancy (NSD) [START_REF] Kozin | Automated matching of high-and low-resolution structural models[END_REF] . The NSD

29"

was therefore computed between a set of ten independent reconstructions, with a range of NSD from 0.678 to 30" 0.815. The aligned structures were then averaged, giving an effective occupancy to each voxel in the model, and

31"

filtered at half-maximal occupancy to produce models of the appropriate volume that were used for all

32"

subsequent analyses. All the models were similar in terms of agreement with the experimental data, as measured

33"

by DAMMIF χ parameter and the quality of the fit to the experimental curve (calculated SQRT Chi value =

34"

1.774). The SAXS data parameters are provided in Extended Data Table 1.

35"

Crystallization, data collection, processing and refinement. Seleno-methionine (SeMet)-labelled TssA Nt2 and

36"

TssA Ct crystallization trials were carried out by the sitting-drop vapor diffusion method in 96-well Greiner

37"

crystallization plates at 20°C, using a nanodrop-dispensing robot (Cartesian Inc.). Crystals of SeMet-labelled

38"

TssA Ct grew in a few days after mixing 300 nL of protein at 4.7 mg/mL with 100 nL of 20% PEG 8000, 0.2M

39"

Calcium Acetate, 0.1 M MES pH 6.8. Crystals of SeMet-labelled TssA Nt grew in a few days after mixing 300 nL

40"

of protein at 4.7 mg/mL with 100 nL of 29% PEG 3350, 0.1 M Hepes pH 7.5. Crystals were cryoprotected with

41"

mother-liquor supplemented with 20 % polyethylene glycol and flash frozen in liquid nitrogen. Datasets were

42"

collected at the SOLEIL Proxima 1 beamline (Saint-Aubin, France). After processing the data with XDS 49 , the

43"

scaling was performed with SCALA and the structures were solved using the SHELXD program [START_REF] Schneider | Substructure solution with SHELXD[END_REF] . The structure

44"

was refined with AutoBUSTER 51 alternated with model rebuilding using COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] . The final data collection and

45"

refinement statistics are provided in Extended Data Table 2. The Ramachadran plots of the TssA Nt and TssA Ct

46"

structures exhibit 90.7/3.3 and 91.8/2.9 residues in the favored and outlier areas, respectively. Figures were made

47"

with PyMOL 53 .

48"

Tail sheath modeling and TssA docking to contracted and extended sheaths. The tail sheath modelling was

49"

performed using the Vibrio cholerae VipAB (TssBC) complex as starting structure [START_REF] Kudryashev | Structure of the Type VI secretion system contractile sheath[END_REF] (PDB: 3J9G) and the

50"

contracted tail sheath structures of Vibrio cholerae [START_REF] Kudryashev | Structure of the Type VI secretion system contractile sheath[END_REF] . To date, however, the molecular structure of the extended

51"

(non contracted) sheath is not available. In a recent paper a low resolution model of the extended VipAB sheath

52"

was modelled using the low resolution EM map of the extended T4 phage tail sheath [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-9" state-specific recycling mechanism[END_REF] . By superimposing the

53"

VipAB EM map to the gp18 bacteriophage T4 sheath protein structure, gross features of the sheath structure

54"

were obtained [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-9" state-specific recycling mechanism[END_REF] . A similar approach was applied with Chimera [START_REF] Pettersen | UCSF Chimera -a visualization system for exploratory research and analysis[END_REF] using the VipAB molecular model in the

55"

extended T4 phage tail sheath instead of using the low resolution VipAB EM map, yielding a model similar to

56"

that of Kube et al. [START_REF] Kube | Structure of the VipA/B type VI secretion complex suggests a contraction-9" state-specific recycling mechanism[END_REF] , but with molecular details. The sheath internal channel diameter shrinks from 110 to ~95 Å

57"

diameter, and the external diameter from ~290 Å to ~190 Å. The internal diameter of the tail sheath makes it

58"

possible to fit stacked Hcp hexamers that are in contact with the tail sheath internal wall. Both extended and

59"

contracted tail sheath conformations were used to explore the faisability of sheath complexes with TssA using its 60" " 18" EM map. TssA being at the distal end of the sheath, the polarity of the sheath was taken into consideration. It

1"

was suggested that the polarity of T6SS tail sheath is similar to that of bacteriophage T4 and therefore that the

2"

VipA (TssB) N-terminal and VipB (TssC) C-terminal helices point to and contact the baseplate [START_REF] Brunet | An epigenetic switch involving 18" overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster[END_REF] . TssA was

3"

therefore docked at the opposite extremity of the tail sheath using Chimera 54 .

4"

Miscellaneous. Hcp release [START_REF] Aschtgen | The SciZ protein anchors 9" the enteroaggregative Escherichia coli Type VI secretion system to the cell wall[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] and fractionation assays [START_REF] Aschtgen | The SciZ protein anchors 9" the enteroaggregative Escherichia coli Type VI secretion system to the cell wall[END_REF][START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF] have been performed as previously described.

5"

SDS-Polyacrylamide gel electrophoresis was performed using standard protocols. For immunostaining, proteins

6"

were transferred onto 0.2 µm nitrocellulose membranes (Amersham Protran), and immunoblots were probed

7"

with primary antibodies, and goat secondary antibodies coupled to alkaline phosphatase, and developed in

8"

alkaline buffer with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium." The anti-TolB polyclonal

9"

antibodies are from our laboratory collection, while the anti-FLAG (M2 clone, Sigma Aldrich) and anti-EFTu

10"

(Roche) monoclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse secondary

11"

antibodies (Beckman Coulter) have been purchased as indicated.

12" Accession numbers. Coordinates and structure factors have been deposited in the Protein Data Bank under

13"

accession numbers 4YO3 and 4YO5 for TssA Nt2 and TssA Ct respectively. Electron microscopy map for full-

14"

length TssA has been deposited in the Electron Microscopy Databank (EMDB) under accession code EMD-3282.

15" 16" 15"

Cascales, E. Structural characterization and oligomerization of the TssL protein, a component shared

16"

by bacterial type VI and type IVb secretion systems. J Biol Chem. 287, 14157-68 (2012).

17" 18"

Legend to Extended Data 19"

20"

Extended Data Table 1 | SAXS parameters of TRX-TssA.

21"

Extended Data Table 2 | Data collection, phasing and refinement statistics for SAD (SeMet)

22"

structures.

23"

Extended Data Figure 1 | Hcp and TssC interact with TssA, a cytoplasmic protein required for 24" sheath assembly and Hcp release. a, Schematic representation of the architecture of the bacterial

25"

Type VI secretion system. The scheme highlights the membrane complex anchoring the tail structure

26"

composed of the assembly baseplate, the spike, the tube and the sheath (cyto, cytoplasm; IM, inner

27"

membrane; PG, peptidoglycan layer; OM, outer membrane). b, Bacterial two-hybrid assay. BTH101

28"

reporter cells producing the indicated proteins or domains (TssLc, cytoplasmic domain of the TssL

29"

protein; TssMc and TssMp, cytoplasmic and periplasmic domain of the TssM protein respectively)

30"

fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on plates

31"

supplemented with IPTG and the chromogenic substrate X-Gal. Interaction between the two fusion

32"

proteins is attested by the dark blue color of the colony. The TolB-Pal interaction serves as a positive

33"

control. c, The absence of TssA prevents T6SS sheath dynamics. Fluorescence microscopy time-lapse

34"

recordings showing sheath dynamics using the chromosomally-encoded tssB-mCherry fusion in WT

35"

(tssB mCherry pBAD33), ∆tssA (∆tssA tssB mCherry pBAD33) and complemented ∆tssA (∆tssA tssB mCherry

36"

pBAD33-TssA VSV-G ) cells. Individual images were taken every 30 sec. Assembly and

37"

contraction/disassembly events are indicated above the time-lapse images. The scale bars are 1 µm.""d,

38"

The absence of TssA prevents Hcp release. Hcp release was assessed by separating whole cells (C) 39" " 20"

and supernatant (SN) fractions from 17-2 (WT), ∆tssA (∆tssA pBAD33, tssA) and complemented

1"

∆tssA (∆tssA pBAD33-TssA VSV-G , tssA WT ) cells producing FLAG epitope-tagged Hcp. A total of 1×10 [START_REF] Leiman | Morphogenesis of the T4 tail and tail fibers[END_REF]

2"

cells and the TCA-precipitated material from the supernatant of 2×10 8 cells were analysed by Western

3"

blot using anti-FLAG monoclonal antibody (lower panel) and anti-TolB polyclonal antibodies as a

4"

lysis control (upper panel). The molecular weight markers (in kDa) are indicated on the left. The

5"

uncropped scans of the western blots are provided in the Supplementary Figure ." e, TssA co-

6"

fractionates with cytoplasmic and membrane proteins. A fractionnation procedure was applied to

7"

EAEC ∆tssA cells producing FLAG-tagged TssA. Whole cells (T) were fractionated to isolate the

8"

supernatant (SN), periplasmic (P), cytoplasmic (C) and total membrane (M) fractions. Extracts from 9" 10 9 (T) or 2×10 [START_REF] Ferguson | Pulse-chase analysis of the in vivo assembly of the 5" bacteriophage T4 tail[END_REF] (SN, P, C, M) cells were separated by SDS-PAGE and immunodetected with anti-

10"

FLAG monoclonal (TssA), anti-EF-Tu (cytoplasmic marker) and TolB (periplasmic marker)

11"

antibodies. The molecular weight markers (in kDa) are indicated on the left. The uncropped scans of

12"

the western blots are provided in the Supplementary Figure.

20"

TssJLM-TssA complex. The asymmetry of the peak probably reflects the co-purification of different

21"

complexes or the dissociation of TssA from the TssJLM complex. c, Examples of representative raw

22"

particles observed for the purified TssJLM-TssA complex sample using negative-stain electron

23"

microscopy. A typical TssJLM complex is shown in red (number of particles observed n=240)

24"

whereas a TssA-bound TssJLM complex is shown in white (n=95). Scale bar is 10 nm. d,

25"

Magnification of the two complexes shown in panel c. Scale bar is 10 nm.

26"

Extended Data Figure 3

32"

sfGFP TssA cluster was chosen to define the bleached area (red circle). The laser (488 nm) was set to

33"

maximum power and focused for 3 s to ensure complete bleaching of the GFP diffusible pool. Images

34"

were taken every 30 s to follow recovery dynamics. The scale bar is 1 µm. d, Quantification of shown over time for n=10 independent sfGFP TssA foci after FLIM (blue line). The fluorescence

2"

intensity of the bleached focus was also followed over time (FRAP, red line). As a control for laser

3"

focussing and intensity, membrane-associated clusters were systematically bleached in these

4"

experiments and showed no recovery suggesting the total intracellular sfGFP TssA has been bleached by

5"

the laser. e, Representative fluorescence microscopy time-lapse recordings of the indicated ∆tssK,

6"

∆tssE, ∆tssF, ∆tssG or ∆hcp cells producing sfGFP TssA. Individual images were taken every 30 sec.

7"

Red arrowheads indicate the localizations of TssA foci. The scale bar is 1 µm. f, Representative large

8"

fields of fluorescence microscopy analyses showing localization of sfGFP TssA in the indicated strains.

9"

The scale bars are 1 µm. g, Box-and-whisker plots of the measured number of sfGFP TssA foci per cell 10"

for each indicated strain. The lower and upper boundaries of the boxes correspond to the 25% and

11"

75% percentiles respectively. The black bold horizontal bar represents the median values for each 12" strain and the whiskers represent the 10% and 90% percentiles. Outliers are shown as open circles. A

13"

Student t-test was used to report significant differences (ns, non significative; ***, p<0.0001). The

14"

number of cells studied per strain (n) is indicated on top. h, Statistical analyses of sfGFP TssA dynamics.

15"

sfGFP TssA dynamics were categorized as 'fixed', 'mobile with unidirectional trajectory' and 'mobile 16" with random dynamics' and the number of sfGFP TssA (n, on top) foci in each category is represented as

17"

percentage for each indicated strain. Kymographs for the two first categories are shown on bottom. i,

18"

Schematic representation of the assembly pathway of the T6SS based on this study and available

19"

data [START_REF] Durand | Biogenesis and structure of the Type VI secretion membrane core complex. 15[END_REF][START_REF] Brunet | Type VI secretion and bacteriophage tail 19" tubes share a common assembly pathway[END_REF][START_REF] Brunet | The Type VI secretion 1" TssEFGK-VgrG phage-like baseplate is recruited to the TssJLM membrane complex via 2" multiple contacts and serves as assembly platform for tail tube/sheath polymerization[END_REF][START_REF] Kapitein | ClpV recycles VipA/VipB tubules and prevents non-productive tubule 13" formation to ensure efficient type VI protein secretion[END_REF][START_REF] Zoued | TssK is a trimeric cytoplasmic protein interacting with components of both 15" phage-like and membrane anchoring complexes of the type VI secretion system[END_REF][START_REF] Gerc | Visualization of the Serratia Type VI secretion system reveals unprovoked attacks and 6" dynamic assembly[END_REF] . The biogenesis starts with the initial positioning of the TssJ outer membrane

20"

lipoprotein and the sequential recruitment of the indicated subunits (from left to right). The

21"

recruitment of TssA is dependent on TssM, and that of TssK is dependent on both TssL and TssA. The

22"

exact positions of VgrG and TssE (blue) in the pathway are not known but these two subunits are not

23"

required for TssA recruitment but necessary for Hcp and TssBC polymerization.

24"

Extended Data Figure 4 | TssA interacts with tail and baseplate components. a, TssA interaction

25"

network identified by bacterial two-hybrid analysis (see legend to Extended Data Fig. 1b). (b-e)

26"

Surface Plasmon Resonance interaction study of TssA with its partners identified by BACTH.

27"

Sensorgrams (variation of Plasmon Resonance in arbitrary unit (∆RU) as a function of reaction time

28"

(in sec)) were recorded upon injection of the purified native TssA protein (concentrations of 3.125

29"

(dark grey), 6.25, 12.5, 25, 50 and 100 (light grey) µM) on HC200m chips coated with the purified N-

30"

terminal domain of VgrG (b), purified TssE (c), Hcp (d) or TssBC complex (e) (upper panels). The

31"

graph reporting ∆RU as a function of the TssA concentration (lower panel) was used to estimate the

32"

indicated apparent dissociation constants (K D ). Off-rates (percentage of dissociation 400 s after ligand

33"

injection) are indicated.

9"

SAXS envelope of the "best representative" model of TssA, with top (h), side (i) and tilted (j) views.

10"

The scale bar is 100 Å. k-r, Low resolution EM model of TssA. k, Representative micrograph of the

11"

dataset used for image processing. White circles indicate isolated TssA dodecamers. l. Representative

12"

selected TssA particles. m-n, Gallery of representative top (m) and side (n) class averages generated

13"

after reference free 2D classification using Relion [START_REF] Scheres | implementation of a Bayesian approach to cryo-EM structure determination[END_REF] . o, Fourier Shell Correlation (FSC) curve of the

14"

TssA reconstruction. The Gold standard FSC curve was calculated in Relion 39 using the masked

15"

reconstruction of TssA. p-r, Top (p), side (q) and tilted (r) views of the tri-dimensional reconstruction

16"

model of the TssA dodecamer obtained by electron microscopy (accession number: EMD-3282). The

17"

scale bar is 50 Å. Whereas the SAXS model allows to better visualize the arm length compared to the

18"

EM reconstruction, its low resolution impairs the visual separation of the dimeric arms.

19"

Extended Data Figure 6 | Identification, oligomerisation and interaction analysis of TssA 20" domains. a, Limited proteolysis of purified TssA. The purified full-length TssA protein (first lane)

21"

was submitted to proteinase K limited proteolysis for the time indicated on top of each lane and

22"

analysed by SDS-PAGE and Coomassie blue staining. Stable fragments are indicated on the right with

23"

their boundaries (numbers identified in the sequence in panel b) and the corresponding fragment. The

24"

uncropped scan of the Coomassie blue stained gel is provided in the Supplementary Figure. b, TssA

25"

protein sequence. The localisation of the boundaries of the stable fragments obtained after Proteinase

26"

K limited proteolysis and electrospray mass spectrometry analyses are arrowed. The secondary

27"

structures observed in the crystal structures (Figure 2a and Extended Data Fig. 6f-g) are indicated on

28"

top of the corresponding sequence. c, Bacterial two-hybrid analysis of TssA Nt and TssA Ct interactions

29"

(see legend to Extended Data Fig. 1b). d-e, MALS/QELS/UV/RI analysis of the purified TssA Nt (d)

30"

and TssA Ct (e) fragments. f-g, X-ray structure of the TssA Nt2 domain (PDB: 4YO3). The rainbow

31"

colored ribbon representation of the TssA Nt2 monomer is shown (f, consecutive α-helices numbered

32"

α1 to α7) whereas the dimeric structure (g) highlights the helices at the interface (α1, α2 and α6). h,

33"

The TssA central core interacts with Hcp and VgrG whereas the TssA arms interact with TssE and

34"

TssC. Bacterial two-hybrid analysis of TssA Nt and TssA Ct interactions (see legend to Extended Data

35"

Fig. 1b). (i-j) Surface Plasmon Resonance interaction study of the purified TssA Ct (i) or TssA Nt (j) 36" "

23"

domains with the Hcp protein (i) or the TssBC complex (j). Sensorgrams (variation of Plasmon

1"

Resonance in arbitrary unit (∆RU) as a function of reaction time (in sec)) were recorded upon

2"

injection of the purified TssA Ct (i) or TssA Nt (j) domains (concentrations of 3.125 (dark grey), 6.25,

3"

12.5, 25, 50 and 100 (light grey) µM) on HC200m chips coated with the purified Hcp protein (i) or the

4"

TssBC complex (j) (upper panels). The graph reporting ∆RU as a function of the TssA domain

5"

concentration (lower panel) was used to estimate the indicated apparent dissociation constants (K D ).

6"

Extended Data Figure 7 | Comparison of the SAXS, EM and X-ray structures of TssA. a,

7"

Schematic representation and color code of the constructs used for SAXS (grey), electron microscopy

8"

(light blue) and X-ray (TssA Nt2 , dark blue; TssA Ct , red) analyses. The epitopes and theoretical

9"

molecular masses of the domains are indicated. TRX, thioredoxine; N, N-terminus; C, C-terminus. b,

10"

Fit between the experimental data (green dots) and the calculated scattering curves for TssA Nt2 and

11"

TssA Ct generated by CRYSOL (red line). c-f, SAXS/X-ray comparison. Top (c), side (d) and bottom

12"

(f) views of the fitting of TssA Nt2 (bleu ribbon) and TssA Ct (red ribbon) X-ray structures into the TssA

13"

SAXS envelope (transparent grey surface). Scale bars are 10 nm. Panel e is a magnification of a cut-

14"

away section of the fitting shown in d. Scale bar is 5 nm. g-i, SAXS/EM/X-ray comparison. Top (g)

15"

and side (h) views of the superimposition of SAXS (grey surface), EM (transparent light-bleu surface)

16"

and X-ray structures of TssA. Scale bars are 10 nm. Panel i is a magnification of a cut-away section of

17"

the superimposition shown in h. j-n, EM/X-ray comparison. Top (j), side (k) and bottom (l) views of

18"

the fitting of TssA Nt2 (bleu ribbon) and TssA Ct (red ribbon) X-ray structures into the TssA EM

24"

(green ribbon, PDB 4HKH 56 ) into the TssA SAXS envelope (grey surface). The scale bar is 10 nm.

25"

Extended Data Figure 8 T4 tail distal end comprising the gp19 tube (grey) and gp18 sheath (blue) proteins and the gp3 (green)

1"

and gp15 (red) neck proteins (d) and the T6SS tail distal end comprising the Hcp tube (grey) and

2"

TssBC sheath (blue) proteins and the TssA dodecamer (red) (e). The possibility that a functional

3"

homologue of bacteriophage T4 gp3 exists is shown by the question mark. f, Fitting of the model of

4"

the gp15 structure in complex with the last row of the gp18 sheath (in purple) [START_REF] Fokine | The molecular architecture of the bacteriophage T4 neck[END_REF] in the TssA SAXS

5"

envelope (grey surface).

6"

" 2 "

 2 the tip of the growing structure and incorporates new tube and sheath blocks. Based on 1"

19 "Figure 2 |Figure 3 |

 1923 Figure 2 | High-resolution structures of TssA domains. a, X-ray structure of the C-terminal 18" domain of TssA (TssA Ct ) (PDB: 4YO5). Top (left panel) and side (right panel) views of the 19" TssA Ct dodecamer structure, shown in ribbon representation with each monomer differently 20" colored. The inset of the right panel shows the rainbow colored (blue to red from the N-21" terminus) structure of one TssA Ct monomer. The consecutive α-helices are numbered α1 to 22" α7. The crystal structure of TssA Nt2 is shown in Extended Data Fig. 6f-g. b, Fitting of the 23" TssA Ct (red ribbon) and TssA Nt2 (blue ribbon) X-ray structures into the TssA EM 24" reconstruction top (top panel) or side (bottom panel) views (EMDB-3282; grey volume). The 25" scale bar is 10 nm. SAXS and negative-stain EM models of TssA are shown in Extended Data 26" Fig. 5h-j and 5p-r, respectively. The scale bar is 5 nm. 27"

13 "

 13 Extended Data Figure 2 | Purification and negative-stain electron microscopy analyses of the 14" TssJLM-TssA complex. a, TssA interacts with the TssJM complex. The total solubilized membrane 15" extract (T) of 4×10 9 cells producing the indicated proteins was subjected to affinity chromatography 16" using Streptactin resin. Bound proteins (E) were separated by SDS-PAGE and immunodetected with 17" anti-FLAG (TssA and TssL), anti-Streptag (TssJ) and anti-5His (TssM) monoclonal antibodies. The 18" molecular weight markers are indicated on the left. The uncropped scans of the western blots are 19" provided in the Supplementary Figure. b, Superose 6 10/300 gel filtration profile of the purified

|

  TssA localization and dynamics. a, Mean square displacement (MSD; in27"arbitrary units (a.u)) of a representative sfGFP TssA clusters in a wild-type strain (red line) or its ∆tssBC28"isogenic derivative (black line) were measured by sub-pixel tracking of fluorescent foci and plotted29"over time (in sec). b, Kymographic analysis reporting representative sfGFP TssA (green) and TssB mCherry30"(red) positions within the cell as a function of time. c, Representative Fluorescence lifetime imaging31"microscopy (FLIM) of sfGFP TssA clusters in the sfGFP TssA-TssB mCh strain. A membrane-associated

sfGFP

  TssA fluorescence dynamics over time after bleaching. The dynamics of fluorescence intensity is 1"

5 | 5 "

 55 TssA oligomerization and SAXS and EM structural models. a, 10 µg of 1" purified TssA were analysed by SDS-PAGE and Coomassie blue staining. The molecular weight 2" markers (in kDa) are indicated on the left, and TssA and its theoretical size are indicated on the right. 3" b, Superose 6 10/300 gel filtration profile of purified TssA (black line) and protein markers of known 4" size (colored lines). c, MALS/QELS/UV/RI analysis of purified TssA. The molecular mass of the TssA complex is indicated. d-j, Low-resolution SAXS model of TssA. d, Experimental scattering data 6" calculated from an ab initio model of TssA. The square root χ value of the 'best representative' model 7" is indicated. e, Representation of the Guinier plot calculated from the experimental curve. f, Pair 8" distance distribution. g, Kratky plot representative of a multi-domain protein with flexible linkers. h-j,

19 "

 19 envelope (transparent grey surface). Scale bars are 10 nm. Panels m and n are magnifications of the 20" top and bottom views of the docking of the TssA domain X-ray structures into the TssA EM map 21" highlighting the interface between the TssA central core (TssA Ct , red ribbon) and arms (TssA Nt2 , blue 22" ribbon). The C-terminal helix of TssA Nt2 (ends at position 377) and N-terminal helix of TssA Ct (starts 23" at position 395) are shown in yellow. o, Top view of the fitting of the X-ray structure of EAEC Hcp

|

  Models of tail sheath-TssA complexes and comparison between the 26" bacteriophage T4 gp15 and T6SS TssA subunits. a-b, Surface (a) and cross-section (b) views of the 27" complex of TssA (EM map, blue) with the extended tail sheath model (the last four rows shown in28"different colors). In the cut-away section, four stacks of Hcp rings are visible. As shown by bacterial29"two-hybrid and SPR analyses, Hcp contacts the TssA Ct central core whereas TssBC contacts the30"TssA Nt arms. The TssA arms fit between the TssBC monomers of the last row. c, Surface view of the31"complex of TssA (EM map, blue) with the contracted tail sheath model (the last four rows shown in32"different colors), highlighting the loose packing between TssA and the tail sheath in this conformation,33"suggesting that TssA might dissociate after sheath contraction. d-f, comparison between the34"bacteriophage T4 gp15 and T6SS TssA subunits. d-e, Schematic representations of the bacteriophage

  Hcp ! TssA Nt : TssBC ! K D = 54 ± 6.3 µM ! K D = 4.2 ± 0.4 µM ! time (sec) ! time (sec) !

Extended data

Fig. 1! TssA! TssB! TssC ! Hcp! TssE! TssF! TssG! VgrG! TssJ! TssK! TssL c! TssM c! TssM p! Pal! T18 fusions! TolB-T25! T25-TssC! T25-Hcp! TssB-T25! Extended data Fig. 2! His TssM! FL TssA! FL TssL! TssJ ST ! +! -! +! -! -! -! +! +! +! +! -! TssJ ST! FL TssL! His TssM! FL TssA! +! +! +! +! E T ! E T ! E T ! E T !

2"

Acknowledgments This work was funded by the Centre National de la Recherche Scientifique, the Aix-

3"

Marseille Université, and grants from the Agence Nationale de la Recherche to E.C. (ANR-10-JCJC-1303-03),

7"

Research. A.Z. and E.D. were supported by end-of-thesis (FDT20140931060) and post-doctoral

8"

(SPF20101221116) fellowships from the Fondation pour la Recherche Médicale respectively. We gratefully

9"

acknowledge the Soleil synchrotron radiation facility for beamtime allocation. We thank the members of the

10"

Cascales, Cambillau, Lloubès, Sturgis and Bouveret research groups for helpful discussions, E. Bouveret for

11"

providing vectors, protocols and advices for the bacterial two-hybrid assay, C. Bebeacua for preliminary EM

12"

analyses, L. Espinosa for the help regarding statistical analyses, R. Lebrun and S. Lignon (proteomic platform,

13"

IMM) for mass spectrometry analyses, Y. Cully for the Supplementary Video, O. Uderso, I. Bringer and A. Brun

14"

for technical assistance, and J.D. Barras-Elatable for encouragements.

15"

Authors Contributions

21"

Author Information Coordinates and structure factors have been deposited in the Protein Data Bank under

22"

accession numbers 4YO3 and 4YO5 for TssA Nt2 and TssA Ct respectively. Electron microscopy map for full-

23"

length TssA has been deposited in the Electron Microscopy Databank (B) under accession code EMD-3282. The

24"

authors declare no competing financial interests. Correspondence and requests for materials should be addressed

25"

to E.D. (edurand@imm.cnrs.fr), C.C. (cambillau@afmb.univ-mrs.fr) or E.C. (cascales@imm.cnrs.fr).

26"