# Comparative anatomy of siphophage tails before and after interaction with their receptor

Alessio d'Acapito, Alice Decombe, Charles-Adrien Arnaud, Cécile Breyton

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Alessio d'Acapito, Alice Decombe, Charles-Adrien Arnaud, Cécile Breyton. Comparative anatomy of siphophage tails before and after interaction with their receptor. Current Opinion in Structural Biology, 2025, 92, pp.103045. 10.1016/j.sbi.2025.103045. hal-05088361

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# Comparative anatomy of siphophage tails before and after interaction with their receptor

Alessio d'Acapito<sup>a</sup>, Alice Decombe<sup>a</sup>,
Charles-Adrien Arnaud and Cécile Breyton

<figure><img src="image_2.png" /></figure>

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Siphophages are tailed bacteriophages characterised by their long noncontractile tails. In this review, we compare the recent electron cryo-microscopy structures of eight siphophage tails. We confirm and extend common building block organisation within the siphophage tails, particularly within the tail tip. Moreover, the description of the structure of siphophages T5 and λ tail after receptor binding, showing conformational changes only in the tail tip, explains how the siphophage tail opens, leading to DNA ejection. Conserved structural elements point to a general mechanism of infection for Gram-negative-infecting siphophages and allow considerations regarding the classification of the receptor-binding proteins into two different categories: host recognition receptors and membrane sensing receptors that trigger DNA ejection.

## Addresses

Univ. Grenoble Alpes, CNRS, CEA, IBS, F-38000, Grenoble, France

Corresponding author: Breyton, Cécile (cecile.breyton@ibs.fr)

**Current Opinion in Structural Biology** 2025, **92**:103045

This review comes from a themed issue on **Macromolecular Assemblies (2025)**

Edited by Chris Hill and Elena Orlova

For a complete overview see the **Issue** and the **Editorial**

Available online 24 April 2025

https://doi.org/10.1016/j.sbi.2025.103045

0959-440X/© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Siphophages represent a paraphyletic morphological family of bacteriophages, which accounts for the majority of known bacteriophages (~56%) and is overrepresented among tailed phages (~66% of *Caudoviricetes*) [1,2]. Coming from the Greek prefix “σίφων (siphōn)” meaning “tube”, their name refers to their long flexible and noncontractile tail that discriminates them from myophages (long contractile tail) and podophages (short tail). Siphophage tail assembly pathway is independent to that of the genome-

containing capsid. In the mature virion, the tail tube links the proximal capsid to the distal tail tip. The latter can either be elongated with a central straight fibre (e.g. T5 [3,4], λ [5,6]), or be a densely packed baseplate grafted with pods (e.g. JBD30, R4C, χ) or spikes (e.g. 80α, TP901-1, p2) (Figure 1a). The inherent tail tube flexibility of siphophages implies a different DNA delivery mechanism than that of myophages but also hinders achieving high resolution for such large and flexible structures. Yet, recent advances in electron cryo-microscopy (cryo-EM) and software efficiencies have led to the near-atomic characterisation of several siphophages or tails thereof [3–15] and the elucidation of the structural rearrangements after receptor binding [3,16], bringing significant advances to the field. Conserved and specialised protein domains of the tail tip act to specifically recognise the host (protein receptors or carbohydrate motifs), bring the tail tube extremity close to the bacterial surface and rearrange partially to provide a secure channel for DNA delivery into the host cytoplasm. In this review, we analyse and compare these siphophage tail structures in closed and opened conformations to draw a comprehensive understanding of their similarities and differences regarding their architecture and function at the molecular level.

## Native siphophage tails

### General considerations

The genes coding for phage structural proteins are typically arranged in a conserved order that mirrors the spatial arrangement of the coded proteins in the phage [17]. Another conserved feature among phages is the structure of their proteins despite no sequence homology [18,19]. Indeed, the tail is formed by the stacking of hexameric rings of a common tail tube domain (TTD), which is shared by all core proteins of both siphophage and myophages: the tail terminator, the tail tube protein (TTP), the distal tail protein (Dit) and the baseplate hub protein (BHP), the two latter proteins forming the core of the baseplate (reviewed in e.g. Refs. [20,21]). Two consecutive TTD domains can be borne by the same polypeptide, leading to either hexamers or trimers of proteins within the rings, which can be decorated by a variety of different domains (Table 1). We use the protein nomenclature proposed by Davidson et al. [17] to

<footnote><sup>a</sup>Contributed equally to the work.</footnote>
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

**Siphophage tail tips.** a. Density maps of all available siphophage tail tips structures, low-passed filtered at 10 Å, with RBDs identification, coloured as indicated in the inset. Baseplates are aligned on their respective Dit ring. Missing elements in the density maps are embodied as black dotted lines, two fibres only are depicted when missing (there are three in T5, four in λ and six in 80α). EMDB codes: T5, EMD-14869; λ, chimeric map of EMD-35824 and EMD-35826; JBD30, EMD-19263; R4C, EMD-34249; χ, EMD-43281; 80α, EMD-20872; p2, EMD-1699; TP901-1, EMD-1793. Both p2 and TP901-1 are from negative stain data. b. Tail tube cross-sections showing closure regions. Trimeric BHPs of different phage baseplate complexes were aligned on T5 BHP<sub>pb3</sub>. Upper panels show models as molecular surfaces and wires, while cross-sections are the respective cryo-EM maps coloured by corresponding domains except for p2 where a 3 Å calculated surface from the crystallographic data (PDB 2wzp) is shown. PDB and EMDB codes: T5, 7zhj, EMD-14869; λ, 8iyk, EMD-35826; JBD30, 8rk3, EMD-19263; R4C, 8gtc, EMD-34249; χ, 8vjh, EMD-43281; 80α, 6v8i, EMD-20872; p2, 2wzp. Tube diameter: 10 nm. Figure edited with ChimeraX [52]. BHP, baseplate hub protein; Dit, distal tail protein; RBD, receptor-binding domain.

emphasise these structural and genetic common features between siphophage and myophages.

### Tail tube

At the tail proximal extremity, the tail terminator protein ring connects the tail to the phage capsid. It shows

conserved structure among long-tailed phages [22] (Table 1). Additionally, some phages code a tail completion protein [22,23]. The tail tube of siphophages typically ranges from 50 to 200 nm but can reach up to 1 µm in length [13]. It is composed of the stacking of TTP hexameric rings, with acidic residues positioned to
guide and facilitate the rapid translocation of DNA through the tube lumen [11,12,21]. The tail length is determined by the tape measure protein (TMP), spanning the tube lumen as a coiled-coil [24–26]. This remains the most elusive protein of siphophage tails as its structure is rarely solved: its coiled-coil does not interact strongly with the tube lumen, probably to be spontaneously expelled upon infection [7]. In the baseplate of several siphophage tails structures, the TMP C-terminal extremity is resolved as a very conserved trimeric coiled-coil sitting on the bottom of the closed tail tube (Figure 2d). However, its stoichiometry within the tube could be hexameric [23]. The TMP is expected to be involved in DNA delivery to the host cytoplasm (see below) and is the target of some antiphage factors [27]. Both in T5 and λ, the TMP is cleaved during tail assembly, leading to a large N-terminal peptide and a small C-terminal peptide [3,25]. TMP cleavage in the other phages has not been investigated.

## Tail tip

### Baseplate module

At the tail distal extremity, the baseplate module is composed of a ring of the Dit, the BHP and associated proteins (Figure 1a). The Dit continues the tail tube as a hexamer or a trimer (Table 1). All solved Dit structures, apart that of λ, show a decoration domain that can serve as an anchor for receptor-binding protein (RBP) spikes or fibres (Figure 1a, Table 1, see also, e.g. Refs. [28,29]), which can also bear a hydrolytic activity (e.g. Refs. [30,31]). In T5, an additional protein forms an extra ring between the last TTP ring and that of Dit [3,4]. This decoration-less ring is surrounded by a collar that anchors the three lateral tail fibres. In λ, although the lab strain lost its lateral fibres and collar (mutation in the Stf gene), in the wild type strain these could be attached around the Dit, which is the only decoration-less ring of λ baseplate [6,12].

The BHP closes the tail tube. In the solved tail structures, it consistently displays C3 symmetry and can be a homotrimer as in Gram-positive-infecting siphophages (where it is also called Tal-Tail-associated lysin) and in T5, or heterotrimers for all other Gram-negative-infecting phages, where it is composed of two or three polypeptides (e.g. gpL, gpI and gpJ in λ) (Figures 1b, 2a and b, Table 1). Remarkably, their core architecture is the same as that of myophage BHPs: it is structured as four hub domains (hd). hdI and hdIV continue the tail tube, while hdII and hdIII form the distal part of the conserved tail tip core (Figure 2a and b, Table 1). In addition, Gram-negative-infecting phages bear two additional core domains: i) a small domain contributing to the interaction between subunits, which binds a Fe-S cluster in phages that bear a gpL-like subunit, and ii) a hdII insertion domain that contributes to closing the

tube (Figure 2a, Table 1). This core architecture can be decorated by putative receptor-binding domains (RBDs) that can bear hydrolysis activities (Figures 1a and 2a, Table 1). The tail tube is closed by either a plug domain or scattered loops and termini (Figure 1b and 2a), above which sits the resolved TMP C-terminus (Figure 2d). In R4C, as for the gene transfer agent [32], the tube is closed by the “Megatron” (BHP) N-terminus as an “iris” helix that superimposes remarkably with the TMP C-terminus position of the other phages (Figure 1b, slice 1 R4C and Figure 2d). The TMP C-terminus is not resolved in R4C. Interestingly, in all Gram-negative-infecting siphophage structures, a conserved linker tethers hdIV to a fibronectin type III domain (FNIII), linking the baseplate module, which closes the tube, to an extension module (Figure 2a and c). The linker is buried at the interface between two BHP subunits, stabilising the closed conformation of the tail tube (Figure 2c). In the case of Gram-positive-infecting phages 80α and TP901-1, there is no hdII insertion (Figure 2a, Table 1) and an α-helix stands as a link with an extension module in 80α, while in the case of p2, no equivalent of either of these structures is observed.

## Extension module

The extension module of T5 and λ forms the central fibre, which is composed of successive FNIIIs, a β-spike and the RBD (Figure 2a and b, Table 1). In T5, it is composed of three different proteins. In λ, only one protein, gpJ, bears these domains, and the FNIIIs are separated from the β-spike by an α-helical shaft (AHS) (Figure 2a and b, Table 1). Interestingly, T5 bears a unique RBP [33], while λ harbours three RBDs [5]. Consistently, T5 binds to FhuA, a monomeric outer membrane (OM) transporter [3], whereas λ recognises LamB, a trimeric porin [16]. In the other six phages, the extension module bears various putative RBD and/or harbours hydrolase activity (Figure 1a, Table 1). These phages, recognising carbohydrates, bear numerous RBPs and exhibit a bulky baseplate. This difference could arise from the weaker affinity of RBD-carbohydrate interactions compared with RBP-protein interactions, necessitating multiplying the valency of the phage-host interaction to secure adsorption [34].

## Opening mechanism of siphophage tails after interaction with their receptor

### General considerations

For myophages and other contractile tail-like bacterial machineries, contracted conformations are determined together with the native particles structure, the spontaneous or urea-treated contracted conformation being similar to that after interaction with the cell surface (see e.g. Refs. [35–38]). For siphophages however, obtaining the tail structure in a relevant “open” conformation is possible only after phage interaction with its receptor
<caption>Table 1</caption>

Protein composition and domain organisation of solved siphophage tail structures, with a focus on the tail tip. [48–51]

<table><thead><tr><th>Protein</th><th>Domain</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th><th>Name (Sym.) - residues</th></tr></thead><tbody><tr><td rowspan="2">Terminator</td><td>TTP fold</td><td>p142 (6)</td><td>gpU (6)</td><td>gp42 (6)</td><td>ORF18 (6)</td><td>gp21 (6)</td><td>gp52 (6)</td><td>ORF10</td><td>ORF41 (na)</td></tr><tr><td>TTP fold</td><td>pb6 (3)</td><td>gpV (6)</td><td>gp44 (6)</td><td>ORF10 (6)</td><td>gp22 (6)</td><td>gp53 (6)</td><td>ORF11 (6)</td><td>ORF42 (6)</td></tr><tr><td>TTP</td><td>Decoration</td><td>lg-like</td><td>lg-like</td><td>lg-like</td><td>-</td><td>lg-like<br>Globular β-sheet</td><td>-</td><td>na</td><td>-</td></tr><tr><td rowspan="2">TMP</td><td rowspan="2">Resolved Cter</td><td>pb2 (3)<br>1185-1219</td><td>gpH (3)<br>817-849</td><td>gp46 (3)<br>1126-1157</td><td>ORF12 (na)</td><td>gp25 (3)<br>1400-1431</td><td>gp57 (3)<br>1135-1154</td><td>ORF14 (na)</td><td>ORF45 (na)</td></tr><tr><td>1031-1079</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dit</td><td>TTP fold</td><td>pb9 (6)</td><td>gpM (6)</td><td>gp49 (3)</td><td>ORF13 (6)</td><td>gp26 (3)</td><td>gp58 (6)</td><td>ORF15 (6)</td><td>ORF46 (6)</td></tr><tr><td> Decoration</td><td>OB-fold</td><td>-</td><td>-</td><td>Beta-barrel<br>Fibre binding loop</td><td>-</td><td>Galactose-binding fold</td><td>Galectin fold<br>Spike binding loop</td><td>Galectin fold<br>Spike binging loop</td><td>Galectin fold</td></tr><tr><td>Fibres</td><td>Other domains</td><td>p140 (3)<br>p132 (12)<br>pb1 / LTF (3 x 3)</td><td>Stf</td><td>gp47 (3) / gp48 (3)</td><td>-</td><td>-</td><td>gp68 / FibU (6)<br>gp62 / FibL (6)</td><td>-</td><td>-</td></tr><tr><td rowspan="10">Baseplate module</td><td rowspan="2">hdl (TTP fold)</td><td>1-159</td><td>gpL (3)</td><td>1-154</td><td>gp50 / BppU (3)</td><td>1-128</td><td>ORF14 / Hub (3)</td><td>1-129</td><td>gp27 / BH1P (3)</td><td>2-126</td><td>2-88</td><td>1-109</td></tr><tr><td>175-201</td><td></td><td>171-232</td><td>147-158<br>239-255</td><td></td><td></td><td>147-169<br>244-261</td><td>146-157<br>237-254</td><td></td><td></td><td></td></tr><tr><td rowspan="2">Metal-binding<br>cluster</td><td></td><td>438-570</td><td>gpJ (3)</td><td>242-367</td><td></td><td>125-243</td><td></td><td>900-976</td><td></td><td></td><td>110-160<br>185-217</td></tr><tr><td>hdl</td><td>160-209<br>263-437</td><td>gpL (3)</td><td>Anchor<br>191-209<br>1-82<br>208-241</td><td>gp53 / BHP (3)</td><td>2-123</td><td>61-209</td><td></td><td>126-252</td><td>89-179</td><td></td><td></td></tr><tr><td rowspan="5">BHP</td><td>hdlII<br>insertion</td><td>pb3 / BHP (3)</td><td>586-658</td><td>393-458</td><td>gpJ (3)</td><td>2-123</td><td>ORF16 / Megatron (3)</td><td>997-1064</td><td>gp30 / BH2P (3)</td><td>2-125</td><td>gp59 / Tal (3)</td><td></td></tr><tr><td>hdlII</td><td>571-585<br>659-709</td><td></td><td>368-392<br>459-485<br>565-602</td><td></td><td>244-256<br>331-384</td><td></td><td>977-996<br>1065-1112</td><td></td><td>269-339</td><td>201-268</td><td>ORF16 / Tal (3)</td><td>233-319</td></tr><tr><td>hdlIV (TTP fold)</td><td>711-742</td><td></td><td>591-621</td><td></td><td>385-414<br>2-11<br>112-118</td><td></td><td>1113-1139</td><td></td><td>253-268<br>340-394</td><td>180-200<br>269-330</td><td></td><td>320-375</td></tr><tr><td>Linker</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>395-423<br>2-17<br>112-119</td><td>361-388</td><td></td><td></td></tr><tr><td>Plug / closing loops and termini</td><td>210-262</td><td>gpL (3)</td><td>162-175<br>115-129<br>142-154</td><td>gp50 / BppU (3)</td><td>262-273</td><td>2-43<br>96-121<br>189-204</td><td>gp27 / BH1P (3)</td><td>2-17<br>112-119</td><td>263-272</td><td>331-360</td><td></td><td>162-178<br>247-258</td></tr><tr><td rowspan="2">RBD / Other<br>domains</td><td rowspan="2">-</td><td>gpJ (3)</td><td>FNIII: 83-207<br>OB: 486-564</td><td>gp50 / BppU (3)</td><td>OB: 159-238</td><td>ORF14 / Hub (3)</td><td>OB: 160-243</td><td>gp27 / BH1P (3)</td><td>OB: 158-236</td><td></td><td></td><td>na</td></tr><tr><td>gpL (3)</td><td>Anchor<br>218-220</td><td>gp50 / BppU (3)</td><td></td><td>ORF16 / Megatron (3)</td><td>Hydrolase<br>OB: 210-899</td><td>gp28 / TAP (3)</td><td>50-62</td><td></td><td></td><td></td></tr><tr><td>RBP</td><td>Spikes</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>gp61 (3 x6)</td><td>ORF18 (3 x6)</td><td>ORF48/BppU (6)<br>ORF49 (3 x18)</td></tr><tr><td rowspan="3">Extension module</td><td rowspan="2">Structural domains<br>and RBP</td><td>pb3 (3)</td><td>2 FNIII</td><td>2 FNIII</td><td>gp53 (3)</td><td>2 FNIII</td><td>ORF16 / Megatron (3)</td><td>Cter = FNIII</td><td>gp30 / BH2P (3)</td><td>FNIII<br>3 lg-like</td><td>gp59 (3)</td><td>Cter = Hydrolase</td><td>ORF47 (3)</td><td>Cter = Hydrolase</td></tr><tr><td>pb4 (3)</td><td>3 FNIII<br>8-spike</td><td>gpJ (3)</td><td>gp54 (3)</td><td>Hydrolase</td><td>ORF17 (3)</td><td>Cter = Lectin</td><td>gp31 (x)</td><td>Tail<br>fibre</td><td></td><td></td><td></td></tr><tr><td>pb5 (1)</td><td>RBD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>References</td><td></td><td></td><td>[3-4, 22, 33, 48]</td><td></td><td>[5, 6]</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>[46, 49]</td><td>[50-51]</td></tr></tbody></table>

AHS, α-helical shaft; BHP, baseplate hub protein; BppU, baseplate upper protein; Cter, C-terminal domain; hydrolase, catalytic hydrolase domain; Dit, distal tail protein; FNIII, fibronectin type III; hd, hub domain; Ig, immunoglobulin-like fold; LTF, lateral tail fibres; OB-fold, oligosaccharide/oligonucleotide fold; RBD/RBP, receptor-binding domain/protein; Sym., symmetry; Tal, tail-associated lysin; TMP, tape measure protein; TTP, tail tube protein; -, no such domain observed; na, no data available. Underlined sequences are predicted to form transmembrane helices. Putative domain assignments are in italic.

Bold: names of phages, proteins and predicted transmembrane sequences.

[39,40]. This has recently been the case for siphophages T5 [3,7] and λ [16] tails. *In vitro*, interaction between tails and receptors leads to the opening of the tail tip, expulsion of the TMP and DNA release. On the molecular level, no changes were observed in the structures of the terminator, the TTP and the Dit, but dramatic conformational changes occur at the tail tip level, where the BHP opens the tail tube and the central fibre bends on the side to bring the tube close to the membrane.

## Approaching the membrane

Upon binding of T5 to its receptor FhuA, it has been proposed that RBP disorder to order transition induces subtle constraints on the β-spike [3,33]. In λ, binding of the three RBD to its receptor LamB induces a relative swivel of the three domains [16], also constraining the β-spike. In both cases, these constraints are amplified along the β-spike (Figure 3a), resulting in pulling the linker between the β-spike and the FNIII in T5, and

between the β-spike and the AHS in λ. In both cases, this disengages the tight interaction between the FNIII or the AHS, respectively, which results in fibre bending at the β-spike-FNIII hinge and FNIII domain reorganisation around the β-spike in both phages (Figure 3c). This fibre bending brings the tail tube closer to the OM. Even though both fibres are very different in length (~41 nm in T5 vs. ~30 nm in λ, Figure 1a), bending occurs in their respective middle, resulting in a very similar tube-OM distance (Figure 3b).

## Tail tube opening

In both T5 and λ phages, the central fibre bending pulls on the linker that connects the extension module to the baseplate module. This disrupts the interaction network stabilising neighbouring BHP subunits, allowing rigid body outwards shift of hdIII, hdII and hdII insertion, thus disrupting interactions with the plug, all together opening the tube. Large conformational changes then
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

**The conserved core of siphophage baseplates.** a. Ribbon structure of the BHPs of siphophages. One of the three subunits of the BHP is shown, seen from the inside of the tail tube. Colour code: hdl, blue; hdII, green; hdIII insertion, cyan; hdIII, orange; hdIV, yellow; Fe-S binding cluster, purple; linker, black; extension module, red; plug and closing loops and termini, brown; additional decoration domains, beige. BHPs missing domains, as part of the extension module, are embodied by red circles, with a dotted line for unknown but predicted structures. PDB IDs: T5, 7zhj; λ, 8iyk; JBD30, 8rk3; R4C, 8gtc; c,8vjh; 80a, 6v8i; p2, 2wzp. b. The topology representation of T5 and λ respective BHP (one and three polypeptides, respectively) highlights a conserved domain organisation. C-D. BHPs of the different siphophages were aligned on that of T5. c. The baseplate alignment of Gram-negative-infecting siphophages shows superposition of the BHP core (yellow), decoration domains (white), the linker (dark blue) and the extension module (pink). d. TMP-conserved position when aligning the baseplates on the respective BHPs. One monomer of the resolved TMP C-terminal is highlighted within the molecular surface of T5 baseplate. Notably, the R4C megatron protein iris helix aligns with that of other TMPs. Colour and PDB codes: grey, T5 (7zhj); black, λ (Biyk); pink, JBD30 (8rk3); white, R4C (8gtc); orange, χ (8vjh); dark blue, 80α (6v8i). Figure edited with ChimeraX [52]. BHP, baseplate hub protein; hd, hub domain; TMP, tape measure protein.

occur: in T5, *i*) the plug unfolds as a β-hairpin whose tip is composed of three hydrophobic residues that dip into the OM outer leaflet (Figure 3c), flanked by positive residues that would interact with the OM-lipopolysaccharide (LPS) phosphate groups. *ii*) The large TMP* polypeptide C-terminus inserts into the cleft between two BHP subunits. It then plunges into the OM with hydrophobic helices that form a trans-membrane channel (Figure 3c) [3]. In λ, *i*) the BHP<sub>gpJ</sub>-Nterminus swings towards the OM with also positively charged residues that would interact with the LPS phosphate groups. *ii*) The BHP<sub>gpI</sub> plug unfolds and pivots towards the OM to allow the tube opening and the insertion of the H1/H2 hydrophobic helices into the OM to form a transmembrane channel, BHP<sub>gpI</sub> C-terminus being tightly bound to the hdII insertion domain of BHP<sub>gpJ</sub> (Figure 3c) [16]. In both cases, the protein forming the OM channel is firmly anchored to the tail

tube. In addition, the tube is anchored to the OM through the strong RBP receptor-binding and the BHP "legs" that dip into the LPS outer leaflet. In the case of T5, a channel is visible in the nanodisc [3], whereas it is not in the case for λ [16]. This probably stems from the fact that FhuA being a monomer, its membrane cross-section is smaller than that of the trimeric LamB, leaving a patch of lipids available in the nanodisc for BHP and TMP channel insertion.

In both T5 and λ, a peptidoglycan activity has been proposed in TMP<sub>pb2Cter</sub> and in gpK, respectively [41,42]. These proteins would travel through the OM channel and be liberated into the periplasm. Because phage DNA is protected from periplasmic nucleases, it is proposed that the TMP forms a channel across the periplasm and the inner-membrane [17,43] but these hypotheses need to be confirmed.
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

**Tail tip interaction with the receptor.** **a.** Alignment of T5 (left) and λ (right) central fibre β-spike before (red) and after (blue) their interaction with their receptor as boxed in the inset schemes. The alignment was performed on the spike lower half. Side and top views of the spike upper and lower sections as indicated. **b.** Isosurface view of the tail tip C1 map of T5 (left, EMD-14799) and λ composite map (right, EMD-38242, 38244 and 38245) after interaction with their respective receptor reconstituted into a nanodisc. Centre: superimposition of both tail tip maps aligned on their BHP. In both phages, bending of the central fibre occurs in the centre of the fibre, bringing the tail tube to the membrane. **c.** Ribbon representation of the tail tips of T5 and λ after interaction with their respective receptor (purple). Dit: dark orange; BHP hdl: blue, hdlI: lime, hdII: orange, hdIV: yellow, linker: black, plug: brown; T5-TMP<sup>+</sup> (large polypeptide) and λ-gplCter: magenta, λ-tail tip decoration domains: white; extension module (FNIII, β-spike and RBD): shades of red. Note that the spike-binding domain of RBP<sub>pb5</sub> is not resolved in FhuA-RBP<sub>pb5</sub>, leaving a gap in the composite structure. Inset: blow up of the distal extremity of the open tube, in dashed line the approximate position of the OM In the T5 tail tip panel, the side-chains of Leu<sub>333</sub>-Gly-Phe-Leu<sub>336</sub> that are proposed to plunge into the OM outer leaflet hydrophobic core are shown in sticks and balls. Figure edited with ChimeraX [52]. BHP, baseplate hub protein; Dit, distal tail protein; FNIII, fibronectin type III; hd, hub domain; Ig, immunoglobulin-like fold; OM, outer membrane; RBD/RBP, receptor-binding domain/protein; TMP, tape measure protein.

**Conclusion**

All tail tip structures of phages infecting Gram-nega-
tive bacteria show a baseplate module, sharing a
conserved linker to an extension module (Figure 2a and

c). This remarkable structural conservation points to a
similar mechanism of infection trigger and tail tube
opening: when proximity to the host membrane is
ensured by sensing a membrane element (OM protein,
the LPS lipid A moiety...) by a domain/subunit of the
extension module, conformational changes in the latter
would pull on the linker, prying the tail tube tip open
and exposing membrane inserting elements, thus trig-
gering infection.

In this context, for Gram-negative bacteria-infecting
phages, we can categorise RBPs into two groups:
RBPs linked to the baseplate module, determining host
specificity, assisting adsorption and the correct phage
orientation with respect to the OM, and RBPs linked to
the extension module, that could be renamed “mem-
brane-sensing module”, which would recognise a mem-
brane element and trigger tube opening and cell wall
perforation, committing the phage to infection. For
example, pilus binding by JBD30, through the Dit-
attached lateral tail fibres, allows specific host
recognition but does not trigger DNA ejection. This
only occurs after pilus retractation, and membrane
interaction of the foot domain of the extension module
tripod RBP [14]. This latter would sense the mem-
brane vicinity and only then would tail opening occur.
In the case of T5 and λ, the RBP/RBDs have both roles
of host specificity and infection trigger, with, however,
the lateral tail fibres increasing the adsorption rates and
thus modulating host range [44,45]. In the case of
Gram-positive bacteria-infecting phages, a similar
mechanism of trigger after membrane sensing can be
proposed for 80α and TP901-1, in which the BHP bears
polysaccharide-binding domains in their C-terminus,
directly linked to the closure elements of the protein.
p2 trigger for infection is, however, completely
different [46]. Both the baseplate and the extension/
membrane-sensing modules also often bear enzymatic
activities, both in Gram-negative and Gram-positive
bacteria-infecting phages, which would selectively
digest the thick peptidoglycan/LPS/capsule layer,
bringing the tail tube close to the membrane. In the
case of phages with a central fibre, as for T5 and λ, the
central fibre, assisted by the lateral fibres, would be
thin enough to reach the membrane through the O-
antigen forest without need for polysaccharide diges-
tion, but would necessitate the fibre bending to bring
the tail tube close to the OM.

We are entering very exciting times as we can expect
more siphophage tail structures, including, hopefully,
structures after interaction with the receptor·s. This
will lead to describing other infection mechanisms,
most probably discovering new ones, also setting the
basis for phage engineering. Indeed, structure predic-
tion software, although extremely powerful, are yet
unable to correctly predict the complex structure of
these very large molecular assemblies [47], yet alone
predict the large conformational changes induced by
receptor binding. Efforts should also be made to
identify and characterise the receptors and the
necessary and sufficient elements to trigger DNA

ejection, as it will help solve precise infection mecha-
nisms and phage behaviours *in vivo*. Attention should
also be raised on understanding how variations of
the siphophage tails affect virus behaviour in more
complex media.

**Declaration of competing interest**

The authors declare no competing interest.

**Acknowledgements**

We thank Guy Schoehn for useful discussion and support. We acknowledge the European Synchrotron Radiation Facility for provision of beam time on CM01. This work acknowledges funding from the Grenoble InstructERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), and from the grants ANR-16-CE11-0027-01, ANR-20-CE11-0023 and ANR-22-CE92-0046-01. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

**Data availability**

No data was used for the research described in
the article.

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