# An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes

Augustinas Silale, Yiling Zhu, Jerzy Witwinowski, Robert Smith, Kahlan Newman, Satya Bhamidimarri, Arnaud Baslé, Syma Khalid, Christophe Beloin, Simonetta Gribaldo, et al.

► To cite this version:

Augustinas Silale, Yiling Zhu, Jerzy Witwinowski, Robert Smith, Kahlan Newman, et al. An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes. 2023. pasteur-04252443

HAL Id: pasteur-04252443

https://pasteur.hal.science/pasteur-04252443

Preprint submitted on 20 Oct 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Dual function of OmpM as outer membrane tether and
nutrient uptake channel in diderm Firmicutes

Augustinas Silale¹, Yiling Zhu¹, Jerzy Witwinowski², Robert E. Smith², Kahlan E.

Newman³, Satya P. Bhamidimarri¹, Arnaud Baslé¹, Syma Khalid⁴, Christophe

Beloin⁵*, Simonetta Gribaldo²*, Bert van den Berg¹*

¹ Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Framlington
Place, NE2 4HH, Newcastle upon Tyne, UK

² Institut Pasteur, Université de Paris Cité, UMR CNRS 6047, Unit Evolutionary Biology of
the Microbial Cell, Paris, France

³ School of Chemistry, University of Southampton, Southampton, SO17 1BJ, UK

⁴ Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK

⁵ Institut Pasteur, Université de Paris Cité, UMR CNRS 6047, Genetics of Biofilms
Laboratory, Paris, France

* Correspondence to: bert.van-den-berg@newcastle.ac.uk;

simonetta.gribaldo@pasteur.fr

christophe.beloin@pasteur.fr
28 Abstract

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group have recently been shown to lack well-characterised OM attachment systems, but instead have OmpM, which could represent an ancestral tethering system in Bacteria. Here, we have determined the structure of the most abundant OmpM protein from *Veillonella parvula* (diderm Firmicutes) by single particle cryo-electron microscopy. We also characterised the channel properties of the transmembrane β-barrel of OmpM and investigated the structure and PG-binding properties of its periplasmic stalk region. Our results show that OM tethering and nutrient acquisition are genetically linked in *V. parvula*, and probably other diderm Terrabacteria. This dual function of OmpM may have played a role in the loss of the OM in ancestral bacteria and the emergence of monoderm bacterial lineages.

42

43
44 **Introduction**

The defining feature of diderm, or Gram-negative, bacteria is the presence of an outer
membrane (OM)¹,². This complex cell envelope component is an asymmetric lipid
bilayer, usually containing lipopolysaccharide (LPS) in the outer leaflet and
phospholipids in the inner leaflet. The first formal definition of an OM was subsequent
to an electron microscopy analysis of the cell envelope of the diderm Firmicute
*Veillonella parvula* (Negativicutes class)³. However, most of our knowledge about the
OM derives from studies of Proteobacteria, especially *Escherichia coli*. While the OM
provides mechanical stabilisation to the cell⁴, diderm bacteria must tether the OM to
peptidoglycan (PG) for additional mechanical stability and to maintain cell morphology.
*E. coli* has three OM tethering systems: Braun's lipoprotein (Lpp) that covalently links
the PG with the OM⁵,⁶, the lipoprotein Pal and the OM protein OmpA which associate
with PG non-covalently⁷⁻¹¹. Mutants of these systems exhibit cell envelope defects
and increased susceptibility to cell envelope stressors, such as detergents⁷,¹²⁻¹⁵.

58

Recent phylogenetic analysis of the distribution of OM attachment systems throughout
the Bacteria Tree of Life has highlighted a striking bimodal distribution across the two
major clades in which Bacteria are divided, the Terrabacteria (including both classical
monoderms such as Bacilli and Clostridia, and diderms such as Cyanobacteria and
Deinococcus-Thermus) and the Gracilicutes (including only diderms such as
Proteobacteria and Bacteroidetes)¹⁶. Braun's lipoprotein was shown to be only present
in a subset of Proteobacteria, and Pal restricted to the Gracilicutes, together with the
Lol machinery for lipoprotein export to the OM. OmpA showed a patchy distribution,
being absent in many diderms. In contrast, the OmpM protein is a fourth OM tethering
system that appears to be widespread in diderm Terrabacteria but is completely absent in the Gracilicutes¹⁶.

70

OmpM consists of an N-terminal periplasmic S-layer homology (SLH) domain connected via a linker region to a C-terminal OM β-barrel¹⁷⁻²⁰. The SLH domain has been mostly studied for its role in the building of S-layers in monoderm bacteria²¹. The primary role of the SLH domain is binding to the cell wall. Similarly to the SLH domain found in monoderm S-layer proteins²²⁻²⁴, the SLH domain of OmpM proteins of some diderm Terrabacteria phyla such as Thermus and Cyanobacteria has been shown *in vitro* to bind pyruvylated secondary cell wall polymers (SCWPs)²⁵⁻²⁷, whereas in members of the Negativicutes (including *V. parvula*) it anchors the OM to PG modified with polyamines (e.g. cadaverine and putrescine) on the D-glutamate of the peptide stem¹⁸,²⁸,²⁹.

81

Deletion of three out of the four OmpM paralogues in *V. parvula* resulted in a dramatic phenotype where the OM detaches, the periplasmic space is greatly enlarged and multiple cells share a single OM¹⁶. Complementation by the most abundant *V. parvula* OmpM paralogue, OmpM1, reverted this phenotype back to wild type, demonstrating *in vivo* that OmpM is responsible for tethering the OM to PG¹⁶. This dramatic phenotype after deletion of OmpM-like proteins has been reported also in two other representatives of diderm Terrabacteria, *Thermus aquaticus*³⁰ and *Deinoccoccus radiodurans*³¹,³².

90

These results led to the proposal that OmpM proteins represent an ancestral OM tethering system in Terrabacteria and possibly the last bacterial common ancestor,
which united anchoring and nutrient acquisition functions¹⁶. The presence of this
unique OM tether in Terrabacteria might therefore have been involved in the multiple
OM-loss events inferred to have occurred specifically in this clade, perhaps via OmpM
mutations weakening (and eventually abolishing) the SLH-PG interactions¹⁶.

Due to its LPS component, the OM presents a permeability barrier for hydrophilic and
hydrophobic small molecules, both detrimental (e.g. antibiotics) and essential (e.g.
nutrients)³³,³⁴. Controlled permeability of the OM is established by OM proteins, which
either mediate energized transport of specific nutrients in the case of TonB-dependent
transporters, or allow size-limited diffusion in the case of porins (<~600 Da for rigid
spherical molecules³³,³⁴, although larger flexible molecules can also go through³⁵).
Porins are well-characterised in many Proteobacteria, but much less information is
available on small-molecule permeation in diderm Terrabacteria. Earlier work on the
OmpM-like protein Mep45 from the Negativicute *Selenomonas ruminantium*
suggested that this protein can transport nutrients via its β-barrel domain¹⁹, but
another study in *Synechocystis* (Cyanobacteria) claimed that its OmpM orthologues
form channels too small for most simple nutrients²⁰. In *V. parvula*, the role of OmpM
in nutrient acquisition has not been studied. In addition to the four OmpM paralogues,
there are two other putative porin genes in the *V. parvula* genome: an OmpA-like
homologue (*FNLLGLLA_00518*) and a porin (*FNLLGLLA_00833*)³⁶. Proteomic
analysis of the *V. parvula* OM showed that OmpM1, OmpM2 and FNLLGLLA_00833
are, respectively, the first, third, and sixth most abundant OM proteins³⁶, suggesting
that these proteins could play major roles in determining OM permeability.

116
117 A recent analysis has proposed that the barrels of most OmpM-like proteins have 16-
118 30 β-strands³⁷, and could therefore form pores that are large enough for small
119 molecule diffusion across the OM, a hypothesis supported by electrophysiology
120 experiments with *Deinococcus radiodurans* SlpA protein³⁸ and liposome swelling
121 studies on Mep45 from *S. ruminantium*¹⁹. The recently determined structures of SlpA
122 revealed that it forms a large trimeric complex composed of 30-stranded β-barrels, i.e.
123 much larger than those of the general *E. coli* porins OmpC and OmpF³⁷,³⁹. However,
124 since *D. radiodurans* has an unusually complex cell envelope with an S-layer³¹,⁴⁰,⁴¹, it
125 is unclear how generalisable these findings are to OmpM proteins in other diderm
126 Terrabacteria, especially in Negativicutes which tether the OM to polyaminated PG
127 rather than pyruvylated SCWP.

128

129 Here we use single particle cryo-EM, X-ray crystallography, molecular dynamics
130 simulations, bioinformatic analyses and functional assays to show that OmpM1 from
131 *V. parvula* (*VpOmpM1*) is a general porin with similar structural and functional
132 properties to *E. coli* OmpF, with an additional mobile periplasmic region that can bind
133 *V. parvula* PG and take on different folds. We also show that the four OmpM
134 paralogues are likely the only porins in *V. parvula*, implying that nutrient acquisition
135 and OM attachment are genetically linked in diderm Firmicutes, and likely all diderm
136 Terrabacteria, via OmpM. Our results support an ancestral dual function of OmpM as
137 both an OM tether and nutrient uptake porin which may have supported life in early
138 bacteria.

139

140
141

# Results

142

## VpOmpM1 is a trimeric porin with an extended periplasmic region

143

We determined the structure of VpOmpM1 expressed in *E. coli* by single particle cryo-EM (Figure 1a-d and Supplementary Table 1). Reconstructions up to 3.2 Å without enforced symmetry revealed a trimeric arrangement of the protomers. The C-terminal 16-stranded β-barrels that reside in the OM form a classical three-fold symmetrical porin assembly reminiscent of the well-characterised *E. coli* porins OmpF (EcOmpF) and OmpC<sup>42,43</sup>. However, the β-barrel lumens are constricted by inward-folded extracellular loops 3 and 7 in VpOmpM1 (Figure 1b), rather than solely by loop 3 as in the *E. coli* porins. The inter-protomer contact area, mediated by hydrophobic sidechains on the outside of the β-barrel, completely excludes lipid/detergent and solvent. The β-barrels exhibit other features common to known porin structures, including an aromatic girdle at the membrane-solvent interfaces and strong density surrounding the β-barrels that likely corresponds to the lipid moieties of LPS and phospholipid or detergent (Figure 1b, d and Supplementary Fig. 1). An unusual feature observed in the cryo-EM reconstructions was a weaker, elongated density that corresponds to the N-terminal SLH domain at the distal part, connected to the β-barrel via a triple coiled coil (Figure 1c, d). We term this periplasmic region of VpOmpM1 the ‘stalk’. Only the region of the stalk proximal to the OM was resolved well enough for model building, as the cryo-EM density deteriorates towards the SLH domain end of the stalk. We speculated that the poor density is the result of movement of the stalk relative to the β-barrels. Measurements of the blurry SLH domain density observed in 2D class averages suggest that the distal part of the stalk spans 50-60 Å (Figure 1c). The VpOmpM1 trimer is pseudosymmetrical: while the β-barrels exhibit three-fold symmetry, the stalk is tilted relative to the membrane plane normal (Figure 1d). The

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165
pseudosymmetry was confirmed by enforcing C3 symmetry during data processing, which resulted in a better-resolved β-barrel region (2.8 Å) but uninterpretable density for the periplasmic stalk (Supplementary Fig. 2-4).

The *E. coli* cell wall does not contain polyaminated PG, the native ligand of VpOmpM1. Therefore, to exclude the possibility that in our structure of VpOmpM1 purified from *E. coli* the stalk region is misfolded due to the absence of polyaminated PG, we purified a His-tagged OmpM1 construct expressed in *V. parvula* (native VpOmpM1) and determined its structure by single particle cryo-EM to 3.3 Å resolution (Supplementary Table 1, Supplementary Fig. 2-4). The β-barrel structures are virtually identical (Cα-Cα root-mean-square deviation 0.239 Å), but there are differences in the relative orientation of the stalk (Figure 1e). In reconstructions without enforced symmetry, the α-helices forming the coiled-coil are not equivalent in either dataset, as they exit the OM plane in different orientations (Figure 1f). The reasons for these structural differences are not obvious. We speculate that intra- or inter-protomer hydrogen bonds at the stalk-barrel interface could be responsible for the subtle conformational differences. In the *E. coli* VpOmpM1 structure, the sidechain of R113 in one protomer interacts with the sidechain of N155 (part of the periplasmic end of the β2 strand) of another protomer. This interaction is observed only in two inter-protomer interfaces, but not the third one (Figure 1g). A similar interaction was observed in the native VpOmpM1 structure: at two inter-protomer interfaces, R113 interacts with the carbonyl oxygen of G115 and the sidechain of N116 (both part of the β-barrel end of the stalk) (Figure 1h). Interestingly, the R113-N155 interaction was not observed in the native VpOmpM1 structure, and the R113-G115/N116 interaction was not observed in the
190 VpOmpM1 structure from *E. coli*. It is possible that the two protein constructs differ
191 slightly in their lowest energy states which are observed in the averaged cryo-EM data.

192

193 We performed all-atom molecular dynamics (MD) simulations to investigate the
194 conformational space sampled by the stalk. We simulated the native VpOmpM1 model
195 to which an AlphaFold2<sup>45</sup> prediction of the unmodeled region of the stalk was grafted
196 and fit into the low resolution cryo-EM density (Figure 2a). The simulations revealed
197 substantial movement of the stalk in the periplasmic space (Supplementary Movie 1).

198 The SLH domain end of the stalk sampled a span of ~50 Å (Figure 2b) during the
199 simulations, in agreement with the cryo-EM data (Figure 1c). The coiled-coil does not
200 kink throughout the 1 µs simulation (Figure 2c), and the movement of the stalk is due
201 to flexibility at the interface between the stalk and the β-barrels. We observed that the
202 inter-protomer hydrogen bonds identified in our static cryo-EM structures, R113-N155
203 and R113-G115, are broken and re-formed throughout the simulation (Figure 2d). The
204 R113 interaction with the sidechain of N116 was not stable, and R113 instead
205 interacted with the backbone carbonyl oxygen of N116 (Figure 2d). Similar results
206 were obtained from a replicate simulation with the AlphaFold2 graft model as well as
207 the native VpOmpM1 cryo-EM model by itself (Supplementary Fig. 5). The stalk would
208 presumably be bound to the relatively immobile PG in vivo, which would significantly
209 impair its mobility relative to the OM. We speculate that the flexible interface between
210 the stalk and the β-barrels would result in the ability of the OM to move slightly relative
211 to the PG, imparting mechanical resistance to the cell envelope.

212

213 The stalk binds polyaminated peptidoglycan
214 The SLH domain of OmpM binds polyaminated PG *in vivo*, as demonstrated in the
215 Negativicute S. *ruminantium*¹⁸,²⁸. Recent evidence for VpOmpM1 binding to PG comes
216 from fluorescence microscopy and cryogenic electron tomography experiments that
217 showed OM detachment when three *ompM* paralogues were deleted¹⁶. We tested
218 binding of recombinant full-length and barrel-only VpOmpM1 to sacculi isolated from
219 *V. parvula* and *E. coli* (Figure 3a). Only full-length protein binds to *V. parvula* sacculi,
220 as expected. Minor amounts of full-length VpOmpM1 were found in the wash fraction,
221 suggesting that the non-covalent PG-SLH domain interaction is reasonably strong. No
222 binding was observed to sacculi from *E. coli*, confirming that PG stem peptide
223 polyamination is required for binding.
224

225 The cryo-EM density for VpOmpM1 SLH domains was not sufficiently resolved for
226 model building due to movement of the stalk, as demonstrated by MD simulations. We
227 therefore used bioinformatics and structural comparisons to investigate where
228 polyaminated PG could bind within the AlphaFold2 predicted model of VpOmpM1 SLH
229 domains. Monoderm bacteria attach cell surface proteins to SCWPs using single-
230 chain proteins that have triple tandem SLH domain repeats, which fold into a core
231 three α-helix bundle with additional helices and loops packing against this bundle²²,⁴⁶-
232 ⁴⁹. Available crystal structures of the SLH domains 1-3 of *Bacillus anthracis* surface
233 array protein (Sap) and *Paenibacillus alvei* S-layer protein SpaA are similar to the
234 predicted VpOmpM1 SLH domain⁴⁶,⁴⁷ (Figure 3b) with Cα-Cα r.m.s.d values of 1.0
235 and 1.3 Å, respectively. The main difference is that both Sap and SpaA have short
236 core helical bundles, but the VpOmpM1 central bundle extends into the long coiled-
237 coil that connects it to the β-barrels. Structures of SpaA bound to defined SCWP
238 ligands⁴⁷ show that the sugars bind in grooves between SLH repeats (Figure 3b) and
239 interact with conserved motifs therein: conserved tryptophan and glycine residues bind
240 the sugar moiety (W34 and G58 in VpOmpM1), and the arginine of the TRAE motif
241 forms a salt bridge with the ketal-pyruvate modification of the SCWP (residues 63-66,
242 TRYE, in VpOmpM1). It is not surprising that these motifs are conserved in most
243 OmpM-like proteins from diderm Terrabacteria because they are thought to bind
244 pyruvylated SCWP, but not in the Negativicutes (Figure 3c).
245

246 Negativicute OmpM proteins are thought to recognise the polyamine modification on
247 the α-carboxylate of the D-glutamate in the PG peptide stem. Crystal structures of
248 proteins bound to putrescine<sup>50-52</sup> and cadaverine<sup>53,54</sup> indicate that aromatic residues
249 are involved in binding these polyamines via stacking interactions with the aliphatic
250 chain of the polyamine. We next investigated if the SLH domains are different between
251 OmpM and non-OmpM proteins (Figure 3c). We found that the tyrosine residue Y36
252 is highly conserved in the OmpM SLH domains from the Negativicutes and slightly
253 less in the other two lineages of diderm Firmicutes (Limnochordia and
254 Halanaerobiales), but it is not conserved in the SLH domains of OmpM homologues
255 from other diderm Terrabacteria as well as non-OmpM SLH domains (Figure 3c and
256 d). Also, Y65 of the TRYE motif is more conserved in the OmpM SLH domain of
257 Negativicutes than in the other groups of sequences.
258

259 Mapping these residues onto the predicted VpOmpM1 SLH domain structure reveals
260 that the conserved tyrosines are located away from the grooves between the SLH
261 chains that contain the putative PG disaccharide binding site (Figure 3e). We suggest
262 that the peptide stem containing the polyamine moiety specifically in Negativicutes
263 OmpM could extend away from the disaccharide binding groove to interact with either
264

of the conserved tyrosines. Alternatively, or perhaps additionally, PG interaction could
induce conformational changes that bring the tyrosine side chains closer to the binding
groove.

265

266

267

268

The stalk domain of OmpM may exist in compact and extended conformations

269

We generated a soluble construct encompassing almost the entire VpOmpM1 stalk
region (residues 22-107) in E. coli and determined its crystal structure to 1.7 Å (Figure
270
271 4a, Supplementary Table 2, PDB 8BZ2) in an attempt to verify the AlphaFold2
272 prediction for this region. Surprisingly, the crystallized stalk does not fold into a coiled-
273 coil but forms a compact trimer composed mainly of α-helices (residues 23-105). The
274 protomer interfaces form grooves on one side of the trimer that could potentially
275 accommodate ligands (Figure 4b). However, mapping of the previously identified
276 putative PG-binding residues onto the stalk crystal structure shows they are spread
277 across the structure (Supplementary Fig. 6), suggesting that this conformation does
278 not bind PG. The residues that form the coiled-coil in the extended AlphaFold2
279 prediction instead form helical hairpins and extensive intra-protomer interactions
280 rather than interact with the other chains in the asymmetric unit (Figure 4c). We
281 obtained a very similar structure, albeit at a lower resolution, with a longer construct
282 that encompasses the entire stalk region (residues 22-118). A DALI⁵⁶ search of the
283 PDB using the SLH model (PDB 8BZ2) showed that it has low structural similarity to
284 other proteins in the database (Supplementary Figure 5). The highest ranking hit was
285 a blood group antigen binding adhesin (PDB 5F7L⁵⁷) with a 5.7 Z-score and 2.5 Å Cα-
286 Cα RMSD.

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287
The protomer fold of the stalk observed in the crystal structures aligns well with an alternative, compact conformation predicted by AlphaFold2 (Figure 4d and e, Supplementary Fig. 8). This was surprising as the confidence for this prediction was low, yet it agreed with the experimental structure. However, the trimerization interface in the compact stalk prediction is different: residues that are far away in the crystal structure (e.g., M75) interact in the predicted compact conformation (Figure 3e). We considered that this compact state could be an unstable high energy conformation (hence the low prediction confidence), and that the stalk construct is stabilized in a similar conformation in the crystallization condition. Notably, the soluble SLH domain did not bind to *V. parvula* or *E. coli* sacculi (Figure 4f), unlike the full-length VpOmpM1 (Figure 3a). This suggests that the structure of the isolated SLH domain is different than in the full-length protein, as observed by cryo-EM. Therefore, we do not believe that the conformation observed in the crystal structure is the result of crystal packing.

All-atom MD simulation of VpOmpM1 with the compact stalk showed the stalk helix of one protomer reaching towards the OM, partially occluding the β-barrel of another protomer and forming salt bridges with its periplasmic turns (Figure 4g and Supplementary Movie 2). Additionally, this conformation was stabilised by inter-protomer hydrogen bonds in the stalk region (Supplementary Fig. 9). The role of this interaction is unclear, but this simulation supports the dynamic nature of the stalk.

The two different stalk conformations of VpOmpM1 imply that either there is conformational switching under the appropriate conditions (e.g., cell envelope stress, presence of specific ligands), or that the protein commits to a particular conformation during biogenesis. In one replicate of the all-atom MD simulations of the native
313 VpOmpM1-AlphaFold2 graft model (Figure 2a) we observed unfolding of the very N-
314 terminus of the SLH domain of one chain, which then proceeded to interact with
315 residues of the coiled-coil (Supplementary Movie 3, Supplementary Fig. 10). This
316 suggests some flexibility for the SLH domain, and perhaps *in vivo* this flexibility is
317 important for the SLH domain to find its PG ligand. The coiled-coil, however, remained
318 stable during the simulation. Protein melting temperature analysis by dynamic
319 scanning calorimetry suggested that the melting temperature of the stalk in the full-
320 length VpOmpM1 construct is ~87°C (Supplementary Fig. 11), which means that the
321 coiled-coil interaction is very stable and it is unlikely to unfold under physiological
322 conditions. Therefore, the more favourable hypothesis is that the VpOmpM1 trimer is
323 committed to either the extended or compact stalk conformation during biogenesis.
324

325

OmpM paralogues are the only general diffusion porins in V. parvula

325

326 The β-barrel of VpOmpM1 consists of 16 anti-parallel β-strands connected by
327 extracellular loops and periplasmic turns similar to the E. coli porin OmpF (Figure
328 1b,c). Although sequence similarity of VpOmpM1 to EcOmpF is only 25%, the overall
329 structure is similar, but with some prominent differences in the extracellular loops
330 (Figure 5a and Supplementary Fig. 12). VpOmpM1 extracellular loops 3 and 7 fold into
331 the β-barrel to form the constriction. In EcOmpF and similar Proteobacterial general
332 porins the constriction is formed solely by loop 3 (Figure 5a). The eyelet (the narrowest
333 region of the pore) is formed by sidechains projecting from the β-barrel strands into
334 the lumen as well as sidechain and backbone atoms of the constricting loop residues
335 (Figure 5b). Analysis of the pore radius of experimental structure β-barrels by the
336 HOLE program⁵⁸ reveals a maximal constriction of ~3.7 Å (Figure 5c). However, in
337 EcOmpF the extent of the constriction (± 0.1 Å) along the axis perpendicular to the
338

membrane plane is ~2.4 Å, and the VpOmpM1 constriction extends for ~7 Å. All-atom

339

MD simulations show that the pore constriction in VpOmpM1 is constant due to an

340

extensive interaction network restricting movement of the eyelet loops 3 and 7

341

(Supplementary Figure 13). In agreement with a previous report⁵⁹, we observed that

342

this is not the case in EcOmpF, where fluctuations in loop 3 can completely close the

343

pore.

344

345

An electric field exists across the eyelet of general porins as a result of asymmetric

346

distribution of charged residues⁶⁰,⁶¹, and a similar charge asymmetry is seen in the

347

eyelets of VpOmpM1 and EcOmpF (Figure 5b). We calculated the average dipole

348

moment of water molecules inside the channel during the MD simulations and used

349

this as a proxy for comparing the strength of the transverse electric field inside the two

350

porins. The largest average dipole moment magnitudes are observed in the

351

constriction regions (Supplementary Figure 14), and the direction of the dipole points

352

from the acidic residues towards the basic residues of the eyelet. This analysis shows

353

that the EcOmpF constriction has a stronger localised transverse electric field, while

354

VpOmpM1 has a weaker and more diffuse transverse electric field.

355

356

We tested the ability of VpOmpM1 to transport small molecules across membranes in

357

liposome swelling assays (Figure 6a). Notably, VpOmpM1 could transport lactate,

358

which is essential for *V. parvula* growth, and putrescine, which is used for modification

359

of the PG peptide stem. We also observed transport of the amino acids arginine,

360

lysine, glutamate, aspartate, glycine, alanine, leucine, and methionine. VpOmpM1

361

transported monosaccharides, even though *V. parvula* is asaccharolytic, but not

362

disaccharides, which underlines the non-specific, size-limited nature of transport. We
carried out experiments with a VpOmpM1 construct that lacks the stalk to test if the stalk is involved in regulating transport, but this construct had similar transport properties to the full-length protein (Figure 6a). The only exception was impaired transport of arginine and alanine, and the reasons for this are unclear. VpOmpM1 had a similar substrate permeation profile to EcOmpF but showed consistently lower transport rates, which could be the consequence of a longer constriction region or weaker transverse electric field (Figure 5c and Supplementary Figure 14). One notable difference is that there was little transport of ampicillin, kanamycin and gentamicin by VpOmpM1 compared to EcOmpF (Figure 6a), but the relevance of this for antibiotic resistance *in vivo* is unclear.

We further characterised the channel properties of VpOmpM1 in bilayer electrophysiology experiments. Initially, we obtained conductance value distributions from recordings with multiple channel insertion events (Figure 6b and Supplementary Fig. 15a,b). The resulting distributions were very broad and centred at 4.87 ± 1.86 nS and 4.66 ± 1.88 nS for the full-length and barrel-only constructs of VpOmpM1, respectively. The broad conductance distributions are likely due to multiple subconductance states of the channel, as observed in recordings where the channels can randomly close to different extent (Figure 6c and Supplementary Fig. 15c,d). We were able to get more accurate conductance values from single channel recordings: 4.34 ± 0.46 nS (mean ± SD, n=8) for full-length VpOmpM1 and 4.37 ± 0.28 nS (mean ± SD, n=8) for barrel-only VpOmpM1. In single channel recordings at higher voltages, we occasionally observed classical trimeric porin behaviour where the three protomer pores close sequentially (Figure 6c and Supplementary Fig. 15c,d). Using our setup we obtained a conductance of 4.28 ± 0.19 (mean ± SD, n=9) for EcOmpF, which is
388

similar to previously reported values⁶²,⁶³ and to VpOmpM1 conductance. Current-voltage characteristics for both VpOmpM1 constructs and EcOmpF were again very similar (Figure 6d).

389

390

391

392

We conclude that VpOmpM1 is a general diffusion channel in *V. parvula*, with similar properties to EcOmpF. We expect that the VpOmpM2-4 paralogues also function as nutrient uptake channels based on their sequence similarity¹⁶. We think it is unlikely that the other two putative porins of *V. parvula*, FNLLGLLA_00518 and FNLLGLLA_00833, also transport nutrients because they are predicted to only have 10 strands in their β-barrels (Figure 7a,b). Previous work on 10-stranded β-barrels has shown that their lumen is occluded by amino acid residue sidechains and that they cannot perform major transport roles⁶⁵,⁶⁶. To verify this experimentally, we produced the β-barrel region of FNLLGLLA_00518 in sufficient quantities for liposome swelling experiments. It did not transport lactate and arabinose, and could only transport putrescine and glycine at much slower rates than VpOmpM1 and EcOmpF (Figure 7c), in agreement with reported transport properties of small OM β-barrels⁶⁷,⁶⁸. Therefore, FNLLGLLA_00518 is not a general porin. We were not able to test the transport activity of FNLLGLLA_00833 because the protein could not be expressed in *E. coli*, but we expect this 10 β-strand OM protein to have very low substrate permeation rates as well.

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409
410 Discussion

411 Together, our results show that OmpM has a dual function as an OM tethering system
412 and a nutrient uptake channel. Our structures, simulations and functional data provide
413 insight into how these key OM-related functions are linked in diderm Firmicutes and
414 probably many other diderm Terrabacteria that possess OmpM-like attachment
415 systems and lack the well-characterised tethers present in the Gracilicutes.

416

417 Interestingly, structural data and MD simulations show that the extended stalk of
418 VpOmpM1 is highly mobile. The functional consequences of this mobility are not clear,
419 and neither is it clear why it may be preferable to a rigid connection between the PG
420 and OM. One possibility is that the stalk samples the local environment to find
421 polyaminated-PG. Although one would expect the polyamine modification to be
422 present on most peptide stems, PG spatial organization is not well understood, and it
423 is unclear what proportion of the modified peptide stems is easily accessible to the
424 SLH domains of VpOmpM1. Another possibility is that the flexible interface between
425 the stalk and the β-barrels imparts favourable mechanical properties to the cell
426 envelope, as having a somewhat flexible PG-OM tether would allow the OM to deform
427 when external mechanical forces are applied.

428

429 The combination of structural, functional and bioinformatics data suggests the
430 possibility that the stalk exists in multiple conformations, despite the fact we did not
431 observe any particle populations with a compact stalk in our cryo-EM datasets.
432 Unravelling the roles of both potential states of the stalk is a challenging problem that
433 will likely have to be resolved using *in vivo* studies. Based on structural comparisons
434 with SCWP-binding SLH domains from monoderm bacteria and our PG binding assay
435

results, we can conclude that the extended stalk state observed in cryo-EM reconstructions of VpOmpM1 is the PG-binding state.

436

437

438

The stalked OmpM-like porins represent a new variation of SLH domains. The three copies of the SLH domain from separate protein chains come together to form three likely identical ligand-binding sites, whereas previously characterised triple SLH domains are encoded in a single chain and their ligand-binding grooves have different affinities for SCWPs due to differences in binding motif residues in each SLH repeat$^{47,49,69,70}$. The functional consequences of having equivalent or non-equivalent ligand binding sites are not clear in the context of protein anchoring to the PG/SCWP.

439

440

441

442

443

444

445

446

SlpA from *D. radiodurans* is the only other OmpM-like protein for which structures have been determined$^{37-39}$. SlpA, like VpOmpM1, plays an important role in maintaining cell envelope integrity$^{16,31}$. VpOmpM1 is similar to SlpA in its overall architecture: both proteins are trimers and have an N-terminal SLH domain connected via a coiled coil to a C-terminal β-barrel domain. However, the VpOmpM1 β-barrel trimer is approximately the size of a single SlpA β-barrel (Supplementary Fig. 16).

447

448

449

450

451

452

Consequently, the pore inside the SlpA β-barrel, although constricted by extracellular loop insertions, is much larger than in VpOmpM1 and is ~14 Å in diameter at the narrowest point. VpOmM1 clearly imposes a size filter for diffusion across the OM (Figures 5 and 6) and prevents leakage of large periplasmic contents from the cell.

453

454

455

456

SlpA has been shown to transport nutrients$^{38}$, but it is unclear how escape of periplasmic material via the massive SlpA β-barrel pore is prevented *in vivo*. Other components of the complex and unusual cell envelope characteristics of *D. radiodurans* could partially obstruct the SlpA pore$^{40,71}$. A superoxide dismutase

457

458

459
(DR_0644) was observed bound at the top of the stalk and the β-barrel interface in SlpA structures³⁷,³⁹, and there are no homologues of this protein coded in the V. parvula genome. The authors of one study noted that large (28-30 strand) β-barrels in OmpM-like proteins appear to be confined to the Deinococcus-Thermus phylum³⁷. Thus, the unusual β-barrel structure of SlpA and the superoxide dismutase partner protein might be adaptations of this phylum to resist environmental extremes, such as oxidative stress, radiation, vacuum conditions, and high temperatures⁷².

VpOmpM1, on the other hand, could be more representative of OmpM proteins in mesophilic diderm Terrabacteria. In fact, the β-barrel of Mep45, the OmpM homologue from the Negativicute S. ruminantium, has been shown to form a diffusion channel with similar properties to VpOmpM1, albeit with a larger estimated diameter (11.6 Å) than that observed for VpOmpM1 (7.4 Å)¹⁹. Cyanobacterial OmpM homologues have been shown to form small channels that facilitate transport of inorganic ions²⁰. Our VpOmpM1 structures suggest that OmpM homologues from these and perhaps most diderm Terrabacteria might be similar to proteobacterial porins, unlike D. radiodurans SlpA. Our results indeed show that nutrient acquisition and OM tethering are genetically linked via OmpM in V. parvula and likely in all Negativicutes and other diderm Terrabacteria.

In conclusion, these data support the recent hypothesis that OmpM is an ancestral system that may have been present in the last common bacterial ancestor, possibly consolidating two key OM-related functions, tethering and nutrient uptake¹⁶. Therefore, deletion of or mutations in *ompM* may have promoted loss of the OM due to the concurrent loss of these fundamental cell functions. Although we envisage that
485 OmpM plays similar roles in all diderm Terrabacteria, experimental characterisation of

486 OmpM orthologues from diverse members of this clade will be required to fully capture

487 the conservation and variation of this ancient OM tethering system.
488

# Methods

489

## Bacterial strains, culture conditions and strain manipulation

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

Bacterial strains used in this work are listed in Supplementary Table 3. *E. coli* strains were genetically manipulated using standard laboratory procedures⁷³. When needed, the following compounds were added to *E. coli* cultures at the following concentrations: ampicillin (liquid media) or ticarcillin (solid media) – 100 mg/l, chloramphenicol – 30 mg/l (liquid media) or 25 mg/l (solid media), kanamycin – 50 mg/ml, apramycin – 50 mg/l, diaminopimelic acid – 300 µM.

*V. parvula* was manipulated as described previously¹⁶,⁷⁴,⁷⁵. When needed, the following compounds were added to *V. parvula* cultures at the following concentrations: chloramphenicol – 25 mg/l, anhydrotetracycline – 250 µg/l. The anaerobic conditions were generated using the GenBag Anaer generator (Biomérieux), or the GP Campus anaerobic chamber (Jacomex). The anaerobic chamber was filled with a H₂/CO₂/N₂ (5%/5%/90%) mixture.

## Plasmids, primers, and DNA manipulation

505

506

507

508

509

510

511

512

All plasmids and primers used in this study are listed in Supplementary Tables 4 and 5, respectively. Cloning was performed using either NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) or standard restriction cloning methods. Chemically competent *E. coli* DH5α or TOP10 cells⁷⁶ were used for transformation of cloning products or plasmids. *V. parvula* genomic DNA was extracted according to a protocol previously described for *Streptomyces* gDNA extraction⁷⁷ from stationary phase cultures in SK medium⁷⁸. PCR reactions for cloning applications were carried out using Phusion HiFi Master Mix (Thermo Fisher Scientific) according to
538

the native ribosome binding site into pRPF185 using the JW172/JW206 primer pair and *V. parvula* SKV38 gDNA as a template, yielding vector pJW48. The vector was then transferred by conjugation into the *ΔompM1-3 V. parvula* mutant strain as described previously¹⁶.

539

540

541

542

543

## Protein expression and purification in *E. coli*

544

*E. coli* C43(DE3) Δ*cyoABCD* (Cyo complex deletion to improve purity) cells were transformed with a pB22 plasmid carrying either the full-length VpOmpM1 (pJW46), barrel-only VpOmpM1 (pJW45), barrel-only FNLLGLLA_00518 (pB22-00518_21-200) or full-length FNLLGLLA_00833 (pB22-00833). After overnight incubation at 37°C, transformants were picked from Lysogeny Broth (LB)-ampicillin plates and used to inoculate a starter LB-ampicillin culture incubated at 37°C with shaking for 2 h. Flasks with 1 L LB were inoculated with 8-12 ml of the starter culture and incubated at 37°C, 160 rpm until OD600~0.5-0.6. Protein expression was induced by supplementing the cultures with 0.1% arabinose, followed by a further 3-4 h incubation at 37°C with shaking. Cultures were harvested and cell pellets were stored at -20°C. Cell pellets were thawed, resuspended in cold 20 mM Tris-HCl pH 8.0, 300 mM NaCl (TBS) and supplemented with DNase I. Cells were lysed by passing the cell suspension once through a cell disruptor (Constant Systems) at 23 kpsi. The lysate was clarified by centrifugation at 30,000 x g, 4°C for 30 min. The membranes were isolated from the clarified lysate by ultracentrifugation at 140,000 x g, 4°C for 50 min. The membranes were solubilised in 2.5% Elugent (Millipore) in TBS for 1 h at 4°C. Insoluble material was pelleted by centrifugation at 44,000 x g, 4°C for 30 min. The solubilised fraction was passed through a ~4 ml chelating sepharose column charged with Ni²⁺ ions. The column was washed with 20 column volumes of TBS with 30 mM imidazole and 0.15%

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562
563 lauryldimethylamine oxide (LDAO), and bound protein was eluted with TBS
564 supplemented with 200 mM imidazole and 0.2% decyl maltoside (DM). The eluate was
565 concentrated using an Amicon Ultra filtration device (50 kDa cut-off membrane),
566 loaded on a HiLoad Superdex 200 16/60 column and eluted in 10 mM HEPES-NaOH
567 pH 7.5, 100 mM NaCl, 0.12% DM. Fractions were analysed by SDS-PAGE, pooled,
568 concentrated by filtration (100 kDa cut-off membrane) and flash-frozen in liquid
569 nitrogen. Protein samples were stored at -80°C.

570

571 *E. coli* BL21 (DE3) cells were transformed with the pET28b-SLH_22-107 plasmid for
572 production of the VpOmpM1 stalk region. Expression cultures were set up as above,
573 except kanamycin instead of ampicillin was used in all media, and protein expression
574 was induced by adding 0.4 mM isopropyl β-D-1-thiogalactopyranoside. Protein
575 purification was performed as above, omitting the ultracentrifugation and solubilisation
576 steps, and without detergents in buffers. A 10 kDa cut-off membrane was used for
577 concentrating protein.

578

579 *E. coli* BL21 (DE3) cells were transformed with the pBAD24-EcOmpF plasmid for
580 production of EcOmpF. Protein expression and purification was performed as for the
581 pB22 constructs up to the membrane isolation stage. Given that pBAD24-EcOmpF
582 doesn't encode a His-tag, inner membranes were selectively solubilised in 20 mM
583 HEPES-NaOH pH 7.5 and 0.5 % (w/v) sodium lauroyl sarcosinate for 30 min at room
584 temperature with stirring. The insoluble fraction containing the outer membranes was
585 recovered by ultracentrifugation for 30 min at 140,000 x g. The sarcosinate wash and
586 ultracentrifugation steps were repeated once. Outer membranes were solubilised in
587 20 mM HEPES-NaOH pH 7.5, 50 mM NaCl and 1.5% LDAO for 1 h at 4°C. The extract
588 was clarified by ultracentrifugation and loaded on a 1 ml ResourceQ anion exchange
589
588

column (Cytiva). The column was washed and eluted with a 0-500 mM NaCl gradient.

589

Fractions containing EcOmpF were concentrated and subjected to size exclusion

590

chromatography on a Superdex200 10/300 GL column (10 mM HEPES-NaOH pH 7.5,

591

100 mM NaCl, 0.05% LDAO). The protein was further purified on a MonoQ 5/50 GL

592

anion exchange column (Cytiva), and detergent was exchanged to 0.12% DM via a

593

final size exclusion chromatography run.

594

595

## Expression and purification of VpOmpM1 from V. parvula

596

15 l of *V. parvula* was grown overnight in anaerobic SK media containing 1.2% sodium

597

lactate and supplemented with 700 µg/l sodium resazurin as an oxygen indicator.

598

Briefly, media was mixed from sterile components, boiled to remove oxygen from the

599

solution (until resazurin appeared colourless) and flushed for 15 minutes with N₂ to

600

remove oxygen from the bottle head volume, before being sealed with a pierceable

601

lid. Anaerobic bottles were inoculated from overnight starter cultures at an OD of 0.04

602

and supplemented with 25 mg/l chloramphenicol and 250 µg/l anhydrotetracycline.

603

Cultures were grown at 37°C overnight with 180 rpm agitation. Cells were harvested

604

by centrifugation at 8,000 x g for 10 minutes. Pellets were pooled to represent 1.5 l of

605

original culture each and resuspended in 5 ml HEPES pH 7.4. 100 µl 3.5x10⁴ U/ml

606

benzonase and a small spatula of lysozyme was added and sample was incubated on

607

ice for 15 minutes. Cells were lysed by two rounds of French press and the debris was

608

pelleted by centrifugation for 90 minutes at 15,000 x g at 4°C. The supernatant was

609

carefully transferred to an ultracentrifuge tube containing 2 ml of a 50% sucrose

610

cushion and centrifuged for 4 hours at 35,000 x g. The volume at the sucrose

611

supernatant interface was extracted, resuspended in 20 ml of HEPES pH 7.4 and
612

pelleted at 35,000 x g for 4 hours. Pelleted membranes were frozen in liquid nitrogen and stored at -80°C.

613

614

615

V. *parvula* membrane solubilization and the protein purification procedure was the same as for VpOmpM1 expressed in *E. coli*, except that the flow-through from the chelating sepharose column was put through the column again due to poor binding of protein of interest to the resin. The final size exclusion chromatography step was done on a Superdex 200 10/300 Increase column.

616

617

618

619

620

621

## Cryo-EM structure determination

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

Purified VpOmpM1 from *E. coli* (11.6 mg/ml) or *V. parvula* (8 mg/ml) was applied to glow discharged Quantifoil R 1.2/1.3 copper 200 mesh holey carbon grids. The grids were immediately blotted and plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific) device operating at 4°C and 100% humidity. Data were collected on a FEI Glacios microscope operating at 200 kV using a Falcon 4 direct electron detector (ThermoFisher Scientific) at the University of York (Supplementary Table 1). A total of 4,284 and 6,505 movies with the *E. coli* and *V. parvula* samples, respectively, were recorded in electron event representation (EER) mode at 240,000 magnification, corresponding to a pixel size of 0.574 Å.

All image processing was done in cryoSPARC v3.3.2<sup>79,80</sup>. Data processing workflows are depicted in Supplementary Fig. 2. Movies were motion corrected using patch motion correction, and CTF parameters were estimated using patch CTF estimation. For VpOmpM1 expressed in *E. coli*, 2D classes generated from manually picked particles were used for template-based picking. 838,051 particles were extracted in
637 600 pixel boxes and Fourier cropped to 300 pixel boxes corresponding to a pixel size
638 of 1.148 Å. Three rounds of 2D classification were used to discard bad particles,
639 followed by generation of an ab initio 3D map using a stochastic gradient descent
640 algorithm with 3 classes and 145,518 particles. All three classes were very similar,
641 and a single class was used as a template in non-uniform refinement with the whole
642 particle stack either without symmetry (C1) or with C3 symmetry enforced, with per-
643 particle defocus and CTF group parameter (beam tilt and trefoil) refinement enabled.
644 The 145,518 particle stack was subjected to heterogeneous refinements against the
645 C1 map from non-uniform refinement and three decoy templates, or against the C3
646 map and the same three decoy templates. Particles were re-extracted in 600 pixel
647 boxes with a pixel size of 0.574 Å. The final particle stacks (96,280 particles for the
648 C1 map; 119,001 particles for the C3 map) were subjected to non-uniform refinement
649 either with C1 or C3 symmetry enforced. The native VpOmpM1 dataset was processed
650 similarly, except that two rounds of 2D classification were performed, four classes
651 were used to make the ab initio map, four decoy templates were used in
652 heterogeneous refinement, and the pixel size remained 1.148 Å throughout. Local
653 resolution estimates, gold-standard Fourier shell correlations, and particle angular
654 distribution plots for all maps are shown in Supplementary Fig. 3.

655

656 The final B-factor-sharpened maps from non-uniform refinement were used to build
657 the models. A de novo model was built into the C3 map using Bucaneer⁸¹ (part of
658 CCPEM package⁸²), followed by cycles of manual building in COOT⁸³ and real space
659 refinement in Phenix⁸⁴. The C3 model was then docked into the C1 map, manually
660 extended in COOT and real-space-refined. The model built into the C1 map of
661 VpOmpM1 expressed in *E. coli* was docked into the native VpOmpM1 C1 map,
662

manually adjusted in COOT and real-space-refined. Models were validated using
MolProbity<sup>85</sup>. Model refinement statistics are shown in Supplementary Table 1, and
representative map-to-model fits are shown in Supplementary Fig. 4.

663

664

665

666

## Crystal structure determination

667

The purified VpOmpM1 stalk construct was concentrated to ~20 mg/ml. Sitting drop
vapour diffusion crystallisation screens were set up using a Mosquito robot (SPT
Labtech). Crystals grew in 0.1 M citric acid pH 3.5, 2.0 M ammonium sulphate at 20°C.
Crystals were cryo-protected in mother liquor supplemented with ~20% PEG400 and
flash-cooled in liquid nitrogen. Diffraction data were collected at the synchrotron
beamline I03 at Diamond Light Source (UK) at a temperature of 100 K (Supplementary
Table 2). The dataset was processed with XIA2<sup>86</sup>, scaled with Aimless<sup>87</sup>, and the
space group was confirmed with Pointless<sup>88</sup>. Data quality was evaluated in Xtriage<sup>84</sup>.
Arcimboldo<sup>89</sup>, part of the CCP4i2 suite<sup>90</sup>, was used for ab initio phasing. The initial
model was extended by Buccaneer and subjected to cycles of manual building and
refinement in Phenix<sup>84</sup> (Supplementary Table 2).

668

669

670

671

672

673

674

675

676

677

678

679

## Isolation of sacculi

680

Peptidoglycan was isolated using the protocol of Wheeler et al.<sup>91</sup>. Briefly, cells were grown to exponential phase, harvested and boiled for one hour in 4% SDS with agitation. The resulting insoluble sacculi were isolated by ultracentrifugation at 150,000 x g for 2 h. The pellet was then washed by 8 rounds of resuspension in milliQ H<sub>2</sub>O and ultracentrifugation. Proteins were removed from the sample by incubating the cell pellet with 100 µg/ml trypsin in digestion buffer (50 mM Tris-HCl pH 7.0, 10 mM CaCl<sub>2</sub>) overnight at 37°C with agitation. To inactivate and remove the trypsin, sacculi

681

682

683

684

685

686
687

were again boiled in 4% SDS and washed to remove residual SDS. Sacculi were then

688

resuspended in MiliQ H₂O and lyophilized in preweighed Eppendorf tubes.

689

690

## Peptidoglycan binding assay

691

Dry *V. parvula* and *E. coli* sacculi were resuspended in binding assay buffer (10 mM

692

HEPES-NaOH pH 7.5, 150 mM NaCl) at 20 mg/ml. Sacculi suspensions were

693

sonicated for 10 min in an ultrasonic bath. Binding assays with full-length and barrel-

694

only VpOmpM1 were carried out in 200 µl aliquots, each containing binding assay

695

buffer with 1 mg/ml sacculus suspension, ~12 µg protein, and 0.12% DM. Binding

696

reactions were incubated at 20°C for 2 h. The sacculi were pelleted by centrifugation

697

in a benchtop microfuge (21,000 x g) for 15 min at 8°C. The supernatant was saved

698

(the soluble fraction), the pellet was resuspended in binding buffer, and the sacculi

699

were pelleted again. The second supernatant (the wash fraction) was saved, and the

700

pellet was resuspended in binding buffer (the pellet fraction). Samples of the soluble,

701

wash and pellet fractions were boiled in SDS loading buffer and analysed by SDS-

702

PAGE on 12% FastCast gels (BioRad). The SLH domain binding assay was carried

703

out with the following modifications. 36 µg of protein was used and the detergent was

704

omitted. The resuspended pellet fraction was split into two 100 µl aliquots, and one

705

aliquot was supplemented with 25 U mutanolysin (Sigma) and incubated at 37°C for

706

30 min. The mutanolysin digestion was included to ensure that no SLH domain that

707

could be bound to PG was prevented from entering the gel due to insolubility of the

708

sacculi. The SLH binding assay was analysed by SDS-PAGE using pre-cast Bis-Tris

709

4-12% gels (Invitrogen) in MES running buffer.

710

711

## Computational Modelling & Simulation
712 **VpOmpM1 model building.** Computational models of trimeric full-length VpOmpM1
713 were predicted using AlphaFold2⁴⁵. Two unique structures were predicted: one with
714 an extended coiled-coil stalk, and one with a compacted stalk region (Supplementary
715 Fig. 6). The AlphaFold2 predicted structure with the extended stalk displays no tilt of
716 the stalk relative to the z-axis, in contrast to the reconstructed cryo-EM density. To
717 generate a full-length VpOmpM1 structure with a tilted stalk consistent with the
718 experimental data, the extended stalk of the AlphaFold2 prediction was fit into the low
719 resolution cryo-EM density and grafted to the well-resolved experimental structure
720 using ChimeraX⁹².

721

722 **System generation.** Three different models of VpOmpM1 were used to build protein-
723 membrane systems: full-length VpOmpM1 with the grafted extended stalk; full-length
724 VpOmpM1 with the compact stalk (AlphaFold2 predicted structure); and truncated
725 VpOmpM1 from the reconstructed cryo-EM density (from residue L100 onwards). For
726 each protein model, the β-barrel domain of the VpOmpM1 trimer was embedded in a
727 model *Escherichia coli* outer membrane using the CHARMM-GUI Membrane Builder
728 module⁹³. The inner leaflet consisted of 90% 1-palmitoyl-2-oleoyl-sn-glycero-3-
729 phosphoethanolamine (POPE), 5% 1-palmitoyl-2-oleoyl-sn-glycero-3-
730 phosphoglycerol (POPG), and 5% 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-
731 phospho]glycerol (cardiolipin), and the outer leaflet consisted of 100% LPS (R1 core,
732 5 × O1 O-antigen units). This asymmetric bilayer system was solvated in 150 mM KCl,
733 with additional calcium ions to neutralise the LPS headgroups (~115 mM in each
734 system). The system composition is detailed in Supplementary Table 7.

735
**Atomistic Molecular Dynamics Simulations.** All simulations used the CHARMM36m forcefield⁹⁴ with the TIP3P water model⁹⁵. Simulations were carried out using the GROMACS simulation package (version 2021.2)⁹⁶. For all simulation steps, a cut-off of 1.2 nm was applied to Lennard-Jones interactions and short-range electrostatics using the potential shift Verlet scheme. Long-range electrostatics were treated using the particle mesh-Ewald (PME) method⁹⁷. Atoms were constrained using the LINCS algorithm to allow the use of a 2 fs timestep in NPT equilibration and production phases⁹⁸. The three systems were energy minimised in 5000 steps using the steepest descent method⁹⁹. The subsequent systems were equilibrated in six phases (two NVT and four NPT phases) in which the protein and lipid headgroups were subjected to position restraints with varying force constants (Supplementary Table 6). All equilibration phases used the Berendsen thermostat¹⁰⁰ to bring the system to 303.15 K (coupling constant of 1.0 ps). NPT equilibration phases used semi-isotropic Berendsen pressure coupling scheme¹⁰⁰ to equilibrate with a pressure bath of 1 bar (τp = 5.0 ps, compressibility of 4.5x10⁻⁵ bar⁻¹).

These equilibrated systems were then simulated. The extended stalk system was simulated in duplicate for 1 µs, and the compacted and truncated stalk systems were each simulated as a single replicate for 1 µs. These production simulations utilised the Nosé-Hoover thermostat¹⁰¹ (coupling constant of 1.0 ps) and the semi-isotropic Parrinello-Rahman barostat¹⁰² (τp = 5.0 ps, compressibility of 4.5x10⁻⁵ bar⁻¹). Trajectories were analysed using GROMACS tools and MDAnalysis utilities¹⁰³,¹⁰⁴. Water orientation was analysed using a locally-written python script (available with deposited data on Zenodo). Molecular graphics and supplementary animations were generated using VMD (version 1.9.4a51)¹⁰⁵ and Molywood (version 0.22)¹⁰⁶.
761

762

**OmpF Simulation.** To compare the dynamics of the constriction regions, EcOmpF was also simulated. The crystal structure of trimeric EcOmpF (PDB ID: 2OMF<sup>42</sup>) was embedded in a model *E. coli* outer membrane and solvated using CHARMM-GUI, then equilibrated as described for the VpOmpM1 systems above. The system composition is shown in Supplementary Table 8. This system was simulated once for 500 ns under the same conditions as the above VpOmpM1 simulations.

763

764

765

766

767

768

769

**Bilayer electrophysiology**

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

Electrophysiology measurements were carried out in a custom-made cuvette at the centre of which was suspended a 25 µm thick Teflon film (Goodfellow Cambridge Ltd) with a 80-100 µm aperture and 1.25 ml electrode chambers either side of the film. The Teflon film aperture was made lipophilic by painting with 1.5-3 µl 1% hexadecane in hexane solution on both sides, and the hexane was allowed to evaporate for at least 30 min. Electrophysiology buffer (10 mM HEPES-KOH pH 7.5, 1 M KCl) was added to both cuvette chambers, and 3 µl of 5 mg/ml 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) dissolved in *n*-pentane was added. The *n*-pentane was allowed to evaporate for at least 5 min. DPhPC bilayers were formed using the method of Montal and Mueller<sup>107</sup>. Pure, concentrated protein (7-15 mg/ml) was serially diluted in 1% Genapol X-100 (Sigma). 0.3-0.5 µl of diluted protein was added to the *cis* (ground side) chamber, and diluted with electrophysiology buffer as required to promote protein insertion into the bilayer. Protein insertion events were detected as sudden jumps in current in constant-voltage mode. All measurements were carried out using Ag/AgCl pellet electrodes attached to an Axopatch 200B amplifier headstage, and a Digidata 1440A digitiser. The cuvette and headstage were enclosed in a custom-
786

made Faraday shield during recording. Clampex software was used for recording.

787

Clampfit software was used to analyse data.

788

789

## Liposome swelling assays

790

Liposome swelling assays were carried out using the method of Nikaido and

791

Rosenberg¹⁰⁸ with modifications. Lipid solution (4 mg/ml phosphatidylcholine and 0.46

792

mg/ml dihexadecyl phosphate in chloroform) was dried under an air stream, and

793

completely dried under vacuum for at least 2 h. 80 µl of lipid solution was used per

794

condition, i.e. per protein to be reconstituted. The dried lipids were resuspended in

795

100 µl deionised water per condition. Equimolar amounts of protein were added to the

796

resuspended lipid so that the total protein quantity was 15-30 µg, followed by

797

immediate vortexing. All conditions were supplemented with DM containing buffer to

798

ensure that equal amounts of detergent were present in all samples. DM-containing

799

buffer was added to control liposomes. Solutions were sonicated in a water bath for 1-

800

1.5 min until translucent, and dried under vacuum overnight. The following day the

801

proteoliposomes were rehydrated in 200 µl 10 mM HEPES-NaOH pH 7.5 and 12 mM

802

stachyose per condition for 1-2 h at room temperature. Control liposomes were used

803

to determine the concentration of each substrate that is isosmotic to the intraliposomal

804

milieu (i.e. there is no swelling of the control liposomes), usually between 7.5 and 15

805

mM substrate in 10 mM HEPES-NaOH pH 7.5. Substrate concentrations used can be

806

found in Supplementary Table 9. The decrease in A₄₀₀ due to liposome swelling was

807

measured after adding 200 µl of the substrate solution to 15 µl of the proteoliposome

808

suspension. Readings were taken on a Perkin Elmer Lambda 35 spectrophotometer

809

in 1 s intervals for 30 s. A line was fit to the absorbance data corresponding to the

810

linear phase of swelling (2-15 s), and the slope of the line was recorded as the swelling
811 rate. Control liposome swelling rates were subtracted from all proteoliposome swelling

812 rates. All data were normalized to EcOmpF-containing liposome swelling in the

813 presence of arabinose.

814

815
816 Data availability

817 Electron microscopy maps have been deposited in the Electron Microscopy Data Bank
818 with the accession codes EMD-16328 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-
819 16328] (VpOmpM1 from E. coli in C1), EMD-16333
820 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-16333] (VpOmpM1 from E. coli in C3)
821 and EMD-16332 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-16332] (VpOmpM1
822 from V. parvula in C1). Atomic coordinates have been deposited in the Protein Data
823 Bank under accession codes 8BYM [https://doi.org/10.2210/pdb8BYM/pdb]
824 (VpOmpM1 from E. coli in C1), 8BYT [https://doi.org/10.2210/pdb8BYT/pdb]
825 (VpOmpM1 from E. coli in C3), 8BYS [https://doi.org/10.2210/pdb8BYS/pdb]
826 (VpOmpM1 from V. parvula in C1), and 8BZ2 [https://doi.org/10.2210/pdb8BZ2/pdb]
827 (VpOmpM1 stalk crystal structure). The following atomic coordinates used for
828 comparison purposes were downloaded from the Protein Data Bank:

829 3PYW [https://doi.org/10.2210/pdb3PYW/pdb], 6CWH
830 [https://doi.org/10.2210/pdb6CWH/pdb], 3POQ
831 [https://doi.org/10.2210/pdb3POQ/pdb], 5F7L [https://doi.org/10.2210/pdb5F7L/pdb],
832 2OMF [https://doi.org/10.2210/pdb2OMF/pdb], 2ZFG
833 [https://doi.org/10.2210/pdb2ZFG/pdb], 8AGD [https://doi.org/10.2210/pdb8AGDpdb].
834 Trajectories and run input files for the Molecular Dynamics simulations are available
835 on Zenodo (https://doi.org/10.5281/zenodo.8239075). Source data for
836 electrophysiology, liposome swelling assays, and HOLE and MD analyses are
837 available. Other data presented in this paper, constructs and strains are available on
838 reasonable request.

839

840 Code availability
841 The water orientation analysis script is available on Zenodo

842 (https://doi.org/10.5281/zenodo.8239075).

843

844
845 **References**

846 1. Silhavy, T. J., Ruiz, N. & Kahne, D. Advances in understanding bacterial outer-membrane biogenesis. *Nat. Rev. Microbiol.* 2006 *41* 4, 57–66 (2006).

848 2. Sun, J., Rutherford, S. T., Silhavy, T. J. & Huang, K. C. Physical properties of the bacterial outer membrane. *Nat. Rev. Microbiol.* 20, 236–248 (2022).

850 3. Bladen, H. A. & Mergenhagen, S. E. Ultrastructure of *Veillonella* and morphological correlation of an outer membrane with particles associated with endotoxic activity. *J. Bacteriol.* 88, 1482 (1964).

853 4. Rojas, E. R. et al. The outer membrane is an essential load-bearing element in Gram-negative bacteria. *Nature* 559, 617–621 (2018).

855 5. Braun, V. & Rehn, K. Chemical Characterization, Spatial Distribution and Function of a Lipoprotein (Murein-Lipoprotein) of the *E. coli* Cell Wall. *Eur. J. Biochem.* 10, 426–438 (1969).

858 6. Braun, V. Covalent lipoprotein from the outer membrane of *Escherichia coli*. *Biochim. Biophys. Acta - Rev. Biomembr.* 415, 335–377 (1975).

860 7. Cascales, E., Bernadac, A., Gavioli, M., Lazzaroni, J. C. & Lloubes, R. Pal lipoprotein of *Escherichia coli* plays a major role in outer membrane integrity. *J. Bacteriol.* 184, 754–759 (2002).

863 8. Bouveret, E., Bénédetti, H., Rigal, A., Loret, E. & Lazdunski, C. In vitro characterization of peptidoglycan-associated lipoprotein (PAL)- peptidoglycan and PAL-TolB interactions. *J. Bacteriol.* 181, 6306–6311 (1999).

866 9. Parsons, L. M., Lin, F. & Orban, J. Peptidoglycan recognition by Pal, an outer membrane lipoprotein. *Biochemistry* 45, 2122–2128 (2006).
868

10. Park, J. S. et al. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram-negative bacterial outer membrane. *FASEB J.* 26, 219-228 (2012).

869

870

871

11. Samsudin, F., Boags, A., Piggot, T. J. & Khalid, S. Braun's Lipoprotein Facilitates OmpA Interaction with the *Escherichia coli* Cell Wall. *Biophys. J.* 113, 1496-1504 (2017).

872

873

874

12. Sonntag, I., Schwarz, H., Hirota, Y. & Henning, U. Cell envelope and shape of *Escherichia coli*: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. *J. Bacteriol.* 136, 280 (1978).

875

876

877

13. Suzuki, H. et al. Murein-lipoprotein of *Escherichia coli*: a protein involved in the stabilization of bacterial cell envelope. *Mol. Gen. Genet.* 167, 1-9 (1978).

878

879

14. Bernadac, A., Gavioli, M., Lazzaroni, J. C., Raina, S. & Lloubès, R. *Escherichia coli tol-pal* mutants form outer membrane vesicles. *J. Bacteriol.* 180, 4872-4878 (1998).

880

881

882

15. Wang, Y. The Function of OmpA in *Escherichia coli*. *Biochem. Biophys. Res. Commun.* 292, 396-401 (2002).

883

884

16. Witwinowski, J. et al. An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition. *Nat. Microbiol.* 7, 411-422 (2022).

885

886

887

17. Kalmokoff, M. L. et al. Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe *Mitsuokella multacida*. *Anaerobe* 15, 74-81 (2009).

888

889

890

891

18. Kojima, S. et al. Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-

892
homologous domain of major outer membrane protein Mep45 in *Selenomonas ruminantium*. *J. Bacteriol.* 192, 5953–5961 (2010).

19. Kojima, S. et al. Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe *Selenomonas ruminantium* forms a non-specific diffusion pore via its C-terminal transmembrane domain. *Biosci. Biotechnol. Biochem.* 80, 1954 (2016).

20. Kowata, H., Tochigi, S., Takahashi, H. & Kojima, S. Outer Membrane Permeability of Cyanobacterium *Synechocystis* sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability. *J. Bacteriol.* 199, (2017).

21. Ravi, J. & Fioravanti, A. S-layers: The Proteinaceous Multifunctional Armors of Gram-Positive Pathogens. *Front. Microbiol.* 12, 663468 (2021).

22. Mesnage, S. et al. Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. *EMBO J.* 19, 4473–4484 (2000).

23. Kern, J., Ryan, C., Faull, K. & Schneewind, O. *Bacillus anthracis* Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires *csaB* and *tagO*. *J. Mol. Biol.* 401, 757–775 (2010).

24. Missiakas, D. & Schneewind, O. Assembly and Function of the *Bacillus anthracis* S-Layer. *Annu. Rev. Microbiol.* 71, 79–98 (2017).

25. Olabarría, C., Carrascosa, J. L., De Pedro, M. A. & Berenguer, J. A conserved motif in S-layer proteins is involved in peptidoglycan binding in *Thermus thermophilus*. *J. Bacteriol.* 178, 4765 (1996).

26. Cava, F., De Pedro, M. A., Schwarz, H., Henne, A. & Berenguer, J. Binding to pyruvylated compounds as an ancestral mechanism to anchor the outer envelope in primitive bacteria. *Mol. Microbiol.* 52, 677–690 (2004).
918

27. Kojima, S. & Okumura, Y. Outer membrane-deprived cyanobacteria liberate periplasmic and thylakoid luminal components that support the growth of heterotrophs. *bioRxiv* 2020.03.24.006684 (2020). doi:10.1101/2020.03.24.006684

919

920

921

28. Kamio, Y., Itoh, Y. & Terawaki, Y. Chemical structure of peptidoglycan in *Selenomonas ruminantium*: Cadaverine links covalently to the D-glutamic acid residue of peptidoglycan. *J. Bacteriol.* 146, 49–53 (1981).

922

923

924

29. Kamio, Y. & Nakamura, K. Putrescine and cadaverine are constituents of peptidoglycan in *Veillonella alcalescens* and *Veillonella parvula*. *J. Bacteriol.* 169, 2881–2884 (1987).

925

926

927

30. Fernandez-Herrero, L. A., Olabarria, G., Caston, J. R., Lasa, I. & Berenguer, J. Horizontal transference of S-layer genes within *Thermus thermophilus*. *J. Bacteriol.* 177, 5460 (1995).

928

929

930

31. Rothfuss, H., Lara, J. C., Schmid, A. K. & Lidstrom, M. E. Involvement of the S-layer proteins Hpi and SlpA in the maintenance of cell envelope integrity in *Deinococcus radiodurans* R1. *Microbiology* 152, 2779–2787 (2006).

931

932

933

32. von Kügelgen, A., van Dorst, S., Alva, V. & Bharat, T. A. M. A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria. *Proc. Natl. Acad. Sci. U. S. A.* 119, e2203156119 (2022).

934

935

936

33. Nikaido, H. & Vaara, M. Molecular basis of bacterial outer membrane permeability. *Microbiol. Rev.* 49, 1 (1985).

937

938

34. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. *Microbiol. Mol. Biol. Rev.* 67, 593–656 (2003).

939

940

35. Ruggiu, F. et al. Size Matters and How You Measure It: A Gram-Negative Antibacterial Example Exceeding Typical Molecular Weight Limits. *ACS Infect. Dis.* 5, 1688–1692 (2019).

941

942
943

36. Poppleton, D. I. et al. Outer Membrane Proteome of *Veillonella parvula*: A Diderm Firmicute of the Human Microbiome. *Front. Microbiol.* 8, (2017).

944

945

37. Farci, D. et al. The cryo-EM structure of the S-layer deinoxanthin-binding complex of *Deinococcus radiodurans* informs properties of its environmental interactions. *J. Biol. Chem.* 298, (2022).

946

947

948

38. Farci, D., Graça, A. T., Iesu, L., de Sanctis, D. & Piano, D. The SDBC is active in quenching oxidative conditions and bridges the cell envelope layers in *Deinococcus radiodurans*. *J. Biol. Chem.* 299, 102784 (2023).

949

950

951

39. Farci, D., Haniewicz, P. & Piano, D. The structured organization of *Deinococcus radiodurans' cell envelope. Proc. Natl. Acad. Sci. U. S. A.* 119, e2209111119 (2022).

952

953

40. von Kügelgen, A. et al. Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium *Deinococcus radiodurans*. *Proc. Natl. Acad. Sci. U. S. A.* 120, e2215808120 (2023).

954

955

956

41. Cowan, S. W. et al. Crystal structures explain functional properties of two *E. coli* porins. *Nature* 358, 727–733 (1992).

957

958

42. Baslé, A., Rummel, G., Storici, P., Rosenbusch, J. P. & Schirmer, T. Crystal structure of osmoporin OmpC from *E. coli* at 2.0 Å. *J. Mol. Biol.* 362, 933–942 (2006).

959

960

43. Xie, Y. et al. IBS 2.0: an upgraded illustrator for the visualization of biological sequences. *Nucleic Acids Res.* 50, W420–W426 (2022).

961

962

44. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. *Nature* 2021 596, 583–589 (2021).

963

964

45. Kern, J. et al. Structure of Surface Layer Homology (SLH) Domains from *Bacillus anthracis* Surface Array Protein. *J. Biol. Chem.* 286, 26042–26049 (2011).

965

966

46. Blackler, R. J. et al. Structural basis of cell wall anchoring by SLH domains in *Paenibacillus alvei*. *Nat. Commun.* 2018 91 9, 1–11 (2018).

967
968

47. Sychantha, D. et al. Molecular Basis for the Attachment of S-Layer Proteins to the Cell Wall of *Bacillus anthracis*. *Biochemistry* 57, 1949–1953 (2018).

969

970

48. Legg, M. S. G. et al. The S-layer homology domains of *Paenibacillus alvei* surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue. *J. Biol. Chem.* 298, 101745 (2022).

971

972

973

49. Vassylyev, D. G., Tomitori, H., Kashiwagi, K., Morikawa, K. & Igarashi, K. Crystal Structure and Mutational Analysis of the *Escherichia coli* Putrescine Receptor: STRUCTURAL BASIS FOR SUBSTRATE SPECIFICITY. *J. Biol. Chem.* 273, 17604–17609 (1998).

974

975

976

977

50. Wu, D. et al. Structural Basis of Substrate Binding Specificity Revealed by the Crystal Structures of Polyamine Receptors SpuD and SpuE from *Pseudomonas aeruginosa*. *J. Mol. Biol.* 416, 697–712 (2012).

978

979

980

51. Bale, S. et al. Structural basis for putrescine activation of human S-adenosylmethionine decarboxylase. *Biochemistry* 47, 13404–13417 (2008).

981

982

52. Sagong, H. Y. et al. Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from *Selenomonas ruminantium*. *PLoS One* 11, e0166667 (2016).

983

984

985

53. Kröger, P., Shanmugaratnam, S., Ferruz, N., Schweimer, K. & Höcker, B. A comprehensive binding study illustrates ligand recognition in the periplasmic binding protein PotF. *Structure* 29, 433-443.e4 (2021).

986

987

988

54. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. *Nucleic Acids Res.* 44, W344–W350 (2016).

989

990

991

55. Holm, L. Dali server: structural unification of protein families. *Nucleic Acids Res.* 50, W210–W215 (2022).

992
993

56. Moonens, K. et al. Structural Insights into Polymorphic ABO Glycan Binding by *Helicobacter pylori*. *Cell Host Microbe* 19, 55–66 (2016).

994

995

57. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. *HOLE: A program for the analysis of the pore dimensions of ion channel structural models*. *J. Mol. Graph*. 14, 354–360 (1996).

996

997

998

58. Vasan, A. K. et al. Role of internal loop dynamics in antibiotic permeability of outer membrane porins. *Proc. Natl. Acad. Sci. U. S. A.* 119, e2117009119 (2022).

999

1000

59. Gutiérrez, S. A., Bodrenko, I., Scorciapino, M. A. & Ceccarelli, M. Macroscopic electric field inside water-filled biological nanopores. *Phys. Chem. Chem. Phys.* 18, 8855–8864 (2016).

1001

1002

1003

60. Bajaj, H. et al. Bacterial Outer Membrane Porins as Electrostatic Nanosieves: Exploring Transport Rules of Small Polar Molecules. *ACS Nano* 11, 5465–5473 (2017).

1004

1005

1006

61. Nestorovich, E. M., Rostovtseva, T. K. & Bezrukov, S. M. Residue Ionization and Ion Transport through OmpF Channels. *Biophys. J.* 85, 3718–3729 (2003).

1007

1008

62. Schmitt, E. K., Vrouenraets, M. & Steinem, C. Channel activity of OmpF monitored in nano-BLMs. *Biophys. J.* 91, 2163–2171 (2006).

1009

1010

63. Efremov, R. G. & Sazanov, L. A. Structure of *Escherichia coli* OmpF porin from lipidic mesophase. *J. Struct. Biol.* 178, 311–318 (2012).

1011

1012

64. Vandeputte-Rutten, L. et al. Crystal structure of the outer membrane protease OmpT from *Escherichia coli* suggests a novel catalytic site. *EMBO J.* 20, 5033–5039 (2001).

1013

1014

1015

65. Eren, E., Murphy, M., Goguen, J. & van den Berg, B. An Active Site Water Network in the Plasminogen Activator Pla from *Yersinia pestis*. *Structure* 18, 809–818 (2010).

1016

1017
1018 66. Hong, H., Patel, D. R., Tamm, L. K. & Van Den Berg, B. The Outer Membrane Protein OmpW Forms an Eight-stranded β-Barrel with a Hydrophobic Channel. *J. Biol. Chem.* 281, 7568–7577 (2006).

1021 67. Hong, H., Szabo, G. & Tamm, L. K. Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening. *Nat. Chem. Biol.* 2006 211 2, 1023 627–635 (2006).

1024 68. Mader, C., Huber, C., Moll, D., Sleytr, U. B. & Sára, M. Interaction of the 1025 Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall 1026 Polymer of *Geobacillus stearothermophilus* PV72 Assessed by Real-Time Surface 1027 Plasmon Resonance Biosensor Technology. *J. Bacteriol.* 186, 1758–1768 (2004).

1028 69. Janesch, B., Messner, P. & Schäffer, C. Are the surface layer homology 1029 domains essential for cell surface display and glycosylation of the S-layer protein from 1030 *Paenibacillus alvei* CCM 2051T? *J. Bacteriol.* 195, 565–575 (2013).

1031 70. von Kügelgen, A. et al. Interdigitated immunoglobulin arrays form the 1032 hyperstable surface layer of the extremophilic bacterium *Deinococcus radiodurans*. 1033 *bioRxiv* 2022.09.15.508085 (2022). doi:10.1101/2022.09.15.508085

1034 71. Merino, N. et al. Living at the extremes: Extremophiles and the limits of life in a 1035 planetary context. *Front. Microbiol.* 10, 447668 (2019).

1036 72. Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Fourth 1037 Edition). (Cold Spring Harbor Laboratory Press, 2012).

1038 73. Knapp, S. et al. Natural Competence Is Common among Clinical Isolates of 1039 *Veillonella parvula* and Is Useful for Genetic Manipulation of This Key Member of the 1040 Oral Microbiome. *Front. Cell. Infect. Microbiol.* 7, (2017).

1041 74. Béchon, N. et al. Autotransporters Drive Biofilm Formation and 1042 Autoaggregation in the Diderm Firmicute *Veillonella parvula*. *J. Bacteriol.* 202, (2020).
75. Inoue, H., Nojima, H. & Okayama, H. High efficiency transformation of *Escherichia coli* with plasmids. *Gene* 96, 23–28 (1990).

76. Jacques, I. B. et al. Analysis of 51 cyclodipeptide synthases reveals the basis for substrate specificity. *Nat. Chem. Biol.* 11 721–727 (2015).

77. Knapp, S. et al. Natural Competence Is Common among Clinical Isolates of *Veillonella parvula* and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome. *Front. Cell. Infect. Microbiol.* 7, 139 (2017).

78. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. *Nat. Methods* 14 290–296 (2017).

79. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. *Nat. Methods* 17 1214–1221 (2020).

80. Hoh, S. W., Burnley, T. & Cowtan, K. Current approaches for automated model building into cryo-EM maps using Biscaneer with CCP-EM. *Acta Crystallogr. Sect. D, Struct. Biol.* 76, 531–541 (2020).

81. Wood, C. et al. Collaborative Computational Project for Electron cryo-EMicroscopy. *Acta Crystallogr. Sect. D Biol. Crystallogr.* 71, 123–126 (2015).

82. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. *Acta Crystallogr. D. Biol. Crystallogr.* 66, 486–501 (2010).

83. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. *Acta Crystallogr. Sect. D, Struct. Biol.* 75, 861–877 (2019).

84. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. *Acta Crystallogr. D. Biol. Crystallogr.* 66, 12–21 (2010).
1068 85. Winter, G. Xia2: An expert system for macromolecular crystallography data
1069 reduction. *J. Appl. Crystallogr.* 43, 186–190 (2010).

1070 86. Evans, P. R. & Murshudov, G. N. How good are my data and what is the
1071 resolution? *Acta Crystallogr. Sect. D Biol. Crystallogr.* 69, 1204–1214 (2013).

1072 87. Evans, P. Scaling and assessment of data quality. *Acta Crystallographica*
1073 *Section D: Biological Crystallography* 62, 72–82 (International Union of
1074 Crystallography, 2006).

1075 88. Rodríguez, D. D. et al. Crystallographic ab initio protein structure solution below
1076 atomic resolution. *Nat. Methods* 2009 69 6, 651–653 (2009).

1077 89. Potterton, L. et al. CCP4i2: the new graphical user interface to the CCP4
1078 program suite. *Acta Crystallogr. Sect. D, Struct. Biol.* 74, 68 (2018).

1079 90. Wheeler, R., Veyrier, F., Werts, C. & Boneca, I. G. Peptidoglycan and Nod
1080 Receptor. *Glycosci. Biol. Med.* 1–10 (2014).

1081 91. Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers,
1082 educators, and developers. *Protein Sci.* 30, 70–82 (2021).

1083 92. Lee, J. et al. CHARMM-GUI Membrane Builder for Complex Biological
1084 Membrane Simulations with Glycolipids and Lipoglycans. *J. Chem. Theory Comput.*
1085 15, 775–786 (2019).

1086 93. Huang, J. & Mackerell, A. D. CHARMM36 all-atom additive protein force field:
1087 Validation based on comparison to NMR data. *J. Comput. Chem.* 34, 2135–2145
1088 (2013).

1089 94. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M.
1090 L. Comparison of simple potential functions for simulating liquid water. *J. Chem. Phys.*
1091 79, 926 (1998).
1092 95. Van Der Spoel, D. et al. GROMACS: Fast, flexible, and free. *Journal of Computational Chemistry* 26, 1701–1718 (2005).

1093 96. Essmann, U. et al. A smooth particle mesh Ewald method. *J. Chem. Phys.* 103, 8577–8593 (1995).

1096 97. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: A Linear Constraint Solver for molecular simulations. *J. Comput. Chem.* 18, 1463–1472 (1997).

1098 98. Goldstein, A. A. On Steepest Descent. *J. Soc. Ind. Appl. Math. Ser. A Control* 3, 147–151 (1965).

1100 99. Berendsen, H. J. C., Postma, J. P. M., Van Gunsteren, W. F., Dinola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. *J. Chem. Phys.* 81, 3684–3690 (1984).

1103 100. Nosé, S. A unified formulation of the constant temperature molecular dynamics methods. *J. Chem. Phys.* 81, 511–519 (1984).

1105 101. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method. *J. Appl. Phys.* 52, 7182 (1998).

1107 102. Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: A toolkit for the analysis of molecular dynamics simulations. *J. Comput. Chem.* 32, 2319–2327 (2011).

1110 103. Gowers, R. et al. MDAnalysis: A Python Package for the Rapid Analysis of Molecular Dynamics Simulations. *Proceedings of the 15th Python in Science Conference* 98–105 (SciPy, 2016). doi:10.25080/Majora-629e541a-00e

1113 104. Humphrey, W., Dalke, A. & Schulten, K. VMD: Visual molecular dynamics. *J. Mol. Graph.* 14, 33–38 (1996).
1115 105. Wieczór, M., Hospital, A., Bayarri, G., Czub, J. & Orozco, M. Molywood: streamlining the design and rendering of molecular movies. *Bioinformatics* 36, 4660–4661 (2020).

1118 106. Montal, M. & Mueller, P. Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties. *Proc. Natl. Acad. Sci. U. S. A.* 69, 3561 (1972).

1121 107. Nikaido, H. & Rosenberg, E. Y. Porin channels in *Escherichia coli*: studies with liposomes reconstituted from purified proteins. *J. Bacteriol.* 153, 241–252 (1983).

1123

1124
1125 Acknowledgements

1126 We thank the University of York (UK) electron cryo-microscopy facility for access to
1127 instrumentation and support. We acknowledge the Diamond Light Source for I03
1128 beamline access (proposal mx-24948) and support. We thank David Staunton
1129 (University of Oxford) for carrying out the dynamic scanning calorimetry experiments.

1130 This work was supported by a Wellcome Trust Investigator award (214222/Z/18/Z) to
1131 B.v.d.B., providing salary support to A.S. and Y.Z. C.B., S.G., J.W., and R.E.S. were
1132 supported by funding from the French National Research Agency (ANR) (no. Fir-OM
1133 ANR-16-CE12-0010), the Institut Pasteur Programmes Transversaux de Recherche
1134 (no. PTR 39-16), and the French government Investissement d'Avenir Program,
1135 Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant
1136 no. ANR-10-LABX-62-IBEID). The authors acknowledge the use of the IRIDIS High
1137 Performance Computing Facility, and associated support services at the University of
1138 Southampton, in the completion of this work. K.E.N. was supported by a Ph.D.
1139 Studentship from the Engineering and Physical Sciences Research Council (Project
1140 Number: 2446840), and S.K. by an EPSRC established Career Fellowship (EPSRC
1141 grant no. EP/V030779/1).

1142

1143 Author contributions

1144 A.S. made constructs, purified proteins, determined cryo-EM and crystal structures,
1145 carried out functional assays, and wrote the manuscript with input from all authors.
1146 Y.Z. purified proteins and carried out functional assays. J.W. made constructs and
1147 strains and prepared the dataset for ConSurf analysis. R.E.S. grew V. *parvula*,
1148 prepared membrane pellets, and isolated sacculi. K.E.N. performed simulations,
1149 supervised by S.K. S.P.B. collected preliminary electrophysiology data. A.B. collected
1150 X-ray diffraction data and managed the Newcastle Structural Biology Laboratory.

1151 B.v.d.B. made constructs, purified and crystallised proteins. S.G., C.B. and B.v.d.B.

1152 conceived and supervised the project.

1153

1154 Competing Interests

1155 The authors declare no competing interests.

1156
1157 **Figure Legends**

1158

1159 **Figure 1.** Cryo-EM structures of VpOmpM1 expressed in *E. coli* and *V. parvula*.
1160 **a** Schematic depicting VpOmpM1 (UniProt A0A100YN03) domain arrangement and boundaries. SP, signal peptide; SLH, S-layer homology; CC, coiled coil. Generated using IBS 2.0⁴⁴. **b** Structure of recombinant VpOmpM1. The cryo-EM density is shown on the left and the model is shown on the right as viewed towards the OM from outside the cell. **c** Representative 2D class averages. White arrowheads point to the diffuse SLH domain density. The edge of each square is 344.4 Å long. **d** Side view of the cryo-EM density at high (left) and low (right) contour level. The grey bar represents the outer membrane (OM). The diffuse grey density around the membrane region in the high contour view likely corresponds to lipid or detergent. **e** Superposition of structures from recombinant VpOmpM1 (in colour) and native VpOmpM1 purified from *V. parvula* (white). **f** Superposition of coiled-coils of protomers from the recombinant (yellow, pink, blue) and native (grey, white, black) VpOmpM1 structures. The structural alignment was performed on the β-barrel region only (not shown). Left – side view, right – view from the periplasm towards the OM. **g, h** Inter-protomer contacts at the β-barrel-stalk interface in the recombinant (g) and native (h) structures. The protomer (A, B, C) for each sidechain is indicated in superscript.

1165

1166
1167
1168
1169
1170
1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
1186
1187
1188
1189
1190
1191
1192
1193
1194
1195
1196
1197
1198
1199
1200
1201
1202
1203
1204
1205
1206
**Figure 2.** All-atom molecular dynamics simulations with native VpOmpM1. **a** The AlphaFold2 prediction for the stalk (grey) was grafted onto the N-terminus of the first residue (L100) modelled into the experimental density of native VpOmpM1 (in colour). R64 (bottom of SLH domain) and R113 are shown as space filling models. **b** Bird's-eye-view of the β-barrel region from outside the OM (left) and a plot of the stalk end projection (centre of mass of the R64 Cα atoms) over the 1 µs simulation (right). The observation point in the left and right panels is equivalent. **c** Frequency distribution of stalk lengths (R64-R113 Cα-Cα) observed throughout the simulation. **d** Distances between the R113 sidechain and interacting sidechain atoms throughout the simulation. The dotted line denotes an inter-atomic distance of 3 Å. The equivalent plots for a replicate simulation and a simulation without the AlphaFold2 graft are presented in Supplementary Fig. 5.

1207

1208
1209
1210
1211
1212
1213
1214
1215
1216
1217
1218
1219
1220
1221
1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
1235
1236
1237
1238
1239
1240
1241
1242
1243
1244
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
1255
1256
1257
1258
1259
1260
**Figure 3.** Analysis of the putative peptidoglycan binding site within the SLH domain. **a** Binding assay of recombinant full-length and barrel-only VpOmpM1 to sacculi (PG) isolated from *V. parvula* and *E. coli*. After incubation of protein with PG, the insoluble PG was pelleted by centrifugation, washed by resuspending in buffer, and pelleted again. The supernatant from the binding reaction (S), the wash (W) and the resuspended PG pellet (P) were boiled and analysed by SDS-PAGE. Three independent binding experiments were performed and yielded similar results. **b** Close up view of the VpOmpM1 SLH domains predicted by AlphaFold2, the crystal structure of SLH domains 1-3 from *Bacillus anthracis* Sap (surface array protein) (PDB 3PYW)⁴⁶, and the crystal structure of SLH domains 1-3 from *Paenibacillus alvei* SpaA in complex with the monosaccharide 4,6-pyr-β-D-ManNAcOMe (space-filling representation) (PDB 6CWH)⁴⁷. Views were generated from a superposition. **c** Multiple sequence alignment results from different subsets of SLH domain-containing proteins mapped onto the sequence of VpOmpM1 SLH domain and coloured by conservation using ConSurf ⁵⁵ (colour key). Row 1 - OmpM homologues from Negativicutes; row 2 – representative OmpM homologues from all diderm Firmicutes; row 3 – representative OmpM homologues from diderm Terrabacteria, excluding
1207 Firmicutes; row 4 – SLH domains from non-OmpM from diderm Firmicutes. The arrows
1208 point to Y36 and Y65, which could be important for binding polyaminated PG. d
1209 ConSurf results mapped onto the predicted SLH domains of VpOmpM1 (same
1210 sequence subsets and colour key as b). Arrows point to the grooves between SLH
1211 protomers. Y36 is shown in space-filling representation. The datasets used for
1212 ConSurf analysis correspond to the datasets used in a previous study¹⁶ that were
1213 subsampled using custom scripts. e Conserved residues shown as stick models on
1214 the predicted SLH structure, coloured by chain, except for Y36 and Y65 which are in
1215 purple. SpaA bound to monosaccharide (PDB 6CWH) was superposed onto the
1216 VpOmpM1 SLH structure; the monosaccharide is in grey, the SpaA protein model is
1217 not shown.
1218

1219 **Figure 4. Crystallography, AlphaFold2, and binding assay show evidence of alternate stalk conformations.** a Crystal structure of VpOmpM1 stalk trimer at 1.7 Å. 1220 Residues 23-105 were resolved. One chain is in rainbow: N-terminus is blue, C- 1221 terminus is red. b Electrostatic surface presentation of the stalk crystal structure. (Left) 1222 Putative view from the OM and (right) from the periplasm. c Comparison of the stalk 1223 crystal structure (left) and AlphaFold2 predicted extended conformation (middle). Only 1224 residues 23-105 are displayed for both. (Right) Residues 64-105 of the stalk crystal 1225 structure that form the extended coiled-coil in the AlphaFold2 prediction are in grey. d 1226 Alternative, compact stalk conformation predicted by AlphaFold2. e Close up view of 1227 the compact stalk prediction with a single chain from the crystal structure (black) 1228 superposed (Cα-Cα r.m.s.d. 0.78 Å) (left). (Middle) Top view down the section marked 1229 by the dashed line. (Right) Stalk crystal structure. The sidechain of M75 is shown in 1230 green space-filling representation in each panel. f Binding assay of recombinant 1231 VpOmpM1 SLH domain to sacculi (PG) isolated from *V. parvula* and *E. coli*. After 1232 incubation of protein with PG, the insoluble PG was pelleted by centrifugation, washed 1233 by resuspending in buffer, and pelleted again. The PG was then resuspended and split 1234 into two aliquots, one of which was incubated with mutanolysin (Mut). The supernatant 1235 from the binding reaction (S), the wash (W) and the resuspended PG pellet without 1236 (P) and with Mut treatment (P Mut) were boiled and analysed by SDS-PAGE. Three 1237 independent binding experiments were performed and yielded similar results. g All- 1238 atom MD simulation of AlphaFold2 predicted compact stalk model over 1 µs. The SLH 1239 domain of one of the protomers (pink) reaches towards the OM and interacts with the 1240 periplasmic turns of the β-barrel of another protomer (blue). The salt bridges shown 1241 on the right had occupancies of 5.8%, 3.44% and 1.89% (top to bottom) throughout 1242 the simulation. 1243

1244 **Figure 5. VpOmpM1 and EcOmpF eyelet comparison.** a β-barrel from the C3 cryo-EM reconstruction of VpOmpM1 (top) and an EcOmpF protomer (PDB 3POQ)⁶⁴ (bottom). The views are generated from a superposition. VpOmpM1 loops 3 and 7 are in green and orange, respectively; EcOmpF loop 3 is in black. Residues forming the eyelet are shown in stick representation. b Close-up view of the eyelet regions of VpOmpM1 (top) and EcOmpF (bottom). Colours as in a. Positively and negatively charged residues lining the eyelet are annotated with a blue plus or red minus sign, respectively. c HOLE⁵⁸ profiles calculated from aligned experimental structures of VpOmpM1 β-barrel and EcOmpF. The z-coordinate is perpendicular to the membrane plane and its origin is at the narrowest part of the VpOmpM1 eyelet.
1245
1246
1247
1248
1249
1250
1251
1252
1253
1254
1255
**Figure 6.** VpOmpM1 channel properties. **a** Liposome swelling in the presence of indicated substrate. Liposomes with embedded full-length VpOmpM1, VpOmpM1 barrel-only or EcOmpF were tested. Swelling rates were normalised to the rate of swelling of EcOmpF-containing liposomes in the presence of arabinose. Each condition was measured in technical triplicates (except EcOmpF with fructose, where n=2) from the same proteoliposome preparation. Error bars show mean ± SD. **b** Bilayer electrophysiology conductance distribution plots obtained from recordings with multiple insertion events (see Supplementary Fig. 15 for representative traces). Full-length VpOmpM1 distribution is centred on 4.87 ± 1.86 nS (SD, n=117); VpOmpM1 barrel-only distribution is centred on 4.66 ± 1.88 nS (SD, n=269). **c** Representative bilayer electrophysiology recordings of full-length VpOmpM1 (top) showing multiple sub-conductance states and of the barrel-only construct (bottom) showing classical trimeric porin sequential channel closure at high voltage. **d** Current-voltage characteristics from single channel recordings of full-length VpOmpM1 (n=4), barrel-only VpOmpM1 (n=4), and EcOmpF (n=3). Individual data points are shown as open black circles and means as coloured dots. The error bars show the SD.

1272

**Figure 7.** *V. parvula* OM proteins other than OmpM paralogues are unlikely to be general diffusion channels. **a** AlphaFold2⁴⁵ models of the FNLLGLLA_00518 (OmpA-like) and FNLLGLLA_00833 proteins. The colours represent the per residue confidence of the prediction (pLDDT). **b** Molecular surface models of *V. parvula* OM proteins and EcOmpF as viewed from outside the cell. **c** Comparative liposome swelling assays with FNLLGLLA_00518 β-barrel, VpOmpM1 β-barrel, and EcOmpF. Each condition was measured in technical triplicates from the same proteoliposome preparation. Bars show mean ± SD.

1274

1275

1276

1277

1278

1279

1280

1281

1282
<figure><img src="image_2.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>h</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_38.png" /></figure>
<figure><img src="image_39.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_41.png" /></figure>
<figure><img src="image_42.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_45.png" /></figure>
<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>
<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /><figcaption>**Supplementary Figure 1.** VpOmpM1 aromatic girdle and putative lipid density. **a** Aromatic residues forming the aromatic girdle shown in space-filling representation on the cartoon of C1 VpOmpM1 reconstruction. Brown, phenylalanine; red, tyrosine; grey, tryptophan. **b** Cryo-EM density of the C3 VpOmpM1 reconstruction. Protein density coloured by chain; grey, putative lipid or detergent density.</figcaption></figure>
<figure><img src="image_52.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_53.png" /></figure>

**Supplementary Figure 2. Cryo-EM data processing.** Cryo-EM data processing workflows for VpOmpM1 expressed in *E. coli* (a) and purified from *V. parvula* (b), showing representative motion-corrected movies. 2D class averages, and intermediate and final cryo-EM maps. A total of 4,284 and 6,505 movies were collected of grids with VpOmpM1 expressed in *E. coli* and *V. parvula*, respectively.
<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /><figcaption>**Supplementary Figure 3.** Cryo-EM map local resolution estimates, FSC curves, and angular distribution plots. **a, d** VpOmpM1 purified from *E. coli*, C1 symmetry. **b, e** VpOmpM1 purified from *E. coli*, C3 symmetry. **c, f** VpOmpM1 purified from *V. parvula*, C1 symmetry.</figcaption></figure>
<figure><img src="image_58.png" /><figcaption>b VpOmpM1 from *V. parvula*, C1 symmetry</figcaption></figure>

<figure><img src="image_59.png" /><figcaption>C VpOmpM1 from *E. coli*, C3 symmetry</figcaption></figure>

<figure><img src="image_60.png" /></figure>

**Supplementary Figure 4.** Cryo-EM representative map-to-model fits. Densities and models for the stalk coiled-coil helix for each protomer and the eyelet loops 3 and 7 from a single protomer are shown for VpOmpM1 purified from *E. coli* (a) and from *V. parvula* (b) in C1 symmetry. Only the eyelet model fit for VpOmpM1 purified from *E. coli* with applied C3 symmetry is shown (c), as it was not possible to build a model into the stalk density in the symmetrized map. Protein models are shown in stick representation. Cryo-EM density is represented by a mesh.
<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /><figcaption>**Supplementary Figure 5. All-atom molecular dynamics simulation replicates.** a stalk length and end projection plots for the two replicates of all-atom simulations with the native VpOmpM1 and AlphaFold2 graft model (full-length). The bird's-eye view coordinates correspond to the plot coordinates in the stalk projection plot. b hydrogen bond distances throughout the simulation. The 'truncated stalk' simulation was performed with the native VpOmpM1 model without the AlphaFold2 graft. Data for replicate 1 shown in this figure are the same as the data shown in Figure 2.</figcaption></figure>
<figure><img src="image_64.png" /><figcaption>**Supplementary Figure 6.** Putative PG-binding motifs in the SLH crystal structure. Residues that are part of motifs conserved in SLH domains are shown in stick representation. Motif residues are not located in intra-protomer grooves as seen in crystal structures of SLH domains from monoderm Firmicutes and in the extended conformation of the VpOmpM1 stalk predicted by AlphaFold2 (Figure 3).</figcaption></figure>
<table><thead><tr><th>Rank</th><th>Chain</th><th>Z</th><th>rmsd</th><th>lali</th><th>nres</th><th>%id</th><th>Description</th></tr></thead><tbody><tr><td>1</td><td>5f7l-C</td><td>5.7</td><td>2.5</td><td>57</td><td>375</td><td>11</td><td>Blood group antigen binding adhesin BabA</td></tr><tr><td>2</td><td>6bml-B</td><td>5.2</td><td>3.3</td><td>67</td><td>294</td><td>12</td><td>Human palmitoyltransferase DHHC20</td></tr><tr><td>3</td><td>6fws-B</td><td>5.2</td><td>3.3</td><td>68</td><td>683</td><td>4</td><td>Helicase DinG</td></tr><tr><td>4</td><td>6gmm-A</td><td>5.2</td><td>2.9</td><td>61</td><td>433</td><td>11</td><td>Adhesin LabA</td></tr><tr><td>5</td><td>6fws-A</td><td>5.0</td><td>2.7</td><td>65</td><td>686</td><td>8</td><td>Helicase DinG</td></tr><tr><td>6</td><td>7khm-A</td><td>4.8</td><td>3.3</td><td>68</td><td>290</td><td>12</td><td>Human palmitoyltransferase DHHC20</td></tr><tr><td>7</td><td>5f7w-A</td><td>4.8</td><td>2.7</td><td>60</td><td>419</td><td>10</td><td>Blood group antigen binding adhesin BabA</td></tr><tr><td>8</td><td>5f7y-A</td><td>4.8</td><td>3.2</td><td>62</td><td>417</td><td>10</td><td>Blood group antigen binding adhesin BabA</td></tr><tr><td>9</td><td>5has-A</td><td>4.7</td><td>3.2</td><td>64</td><td>381</td><td>5</td><td>DCB-HUS domain of Sec7</td></tr><tr><td>10</td><td>5f8q-A</td><td>4.7</td><td>2.4</td><td>56</td><td>419</td><td>11</td><td>Blood group antigen binding adhesin BabA</td></tr></tbody></table>

**Supplementary Figure 7. SLH domain crystal structure DALI search results.** DALI¹ analysis shows that the VpOmpM1 SLH domain crystal structure (PDB 8BZ2) has low similarity to other proteins from the PDB.
<figure><img src="image_65.png" /><figcaption>**Supplementary Figure 8.** VpOmpM1 trimer AlphaFold² predictions. Cartoons coloured by per residue confidence score (colour key): pLDDT>90 – very high; 90>pLDDT>70 confident; 70>pLDDT>50 low; pLDDT<50 – very low.</figcaption></figure>
<figure><img src="image_66.png" /><figcaption>**Supplementary Figure 9.** VpOmpM1 compact stalk simulation. **a** The stalk α-helices in each protomer are kinked at residues 97-102 (arrows). This kink is the hinge that allows movement of the stalk towards the OM, as seen for the pink protomer in the simulation. **b** The conformation of the pink protomer is stabilised by salt bridge formation with the stalk of the yellow protomer. Salt bridge occupancy throughout the simulation (top to bottom): 14.33%, 3.21% and 1.27%.</figcaption></figure>
a

<figure><img src="image_67.png" /></figure>

b

<figure><img src="image_68.png" /><figcaption>**Supplementary Figure 10.** Interaction of SLH domain with coiled-coil during extended stalk MD simulations. **a** During one all-atom simulation of VpOmpM1 with the grafted stalk, the SLH domain of one protomer (pink) unfolded and formed contacts with the coiled-coil region of another protomer (yellow). The SLH domains of the yellow and cyan protomers are not shown for clarity. **b** Close-up views of the SLH-coiled coil interactions. The percentage occupancy of each interaction throughout the 1 µs simulation is shown under each panel.</figcaption></figure>
<figure><img src="image_69.png" /></figure>

<table><caption>**Supplementary Figure 11. Protein melting temperature analysis.** a Dynamic scanning calorimetry thermograms for full-length VpOmpM1, VpOmpM1 barrel domain, and EcOmpF. Measurements were performed on a Malvern VP Capillary DSC instrument, using 18.7 µM protein in 10 mM HEPES-NaOH pH 7.0, 100 mM NaCl and 0.12% DM in each experiment. b Data were fitted and the melting temperatures (T<sub>m</sub>) were extracted via the instrument manufacturer's software.</caption><thead><tr><th rowspan="2">Sample</th><th colspan="2">Melting temperature</th></tr><tr><th>T<sub>m1</sub></th><th>T<sub>m2</sub></th></tr></thead><tbody><tr><td>VpOmpM1</td><td>87.46°C</td><td>95.74°C</td></tr><tr><td>VpOmpM1 barrel</td><td>92.27°C</td><td>101.06°C</td></tr><tr><td>EcOmpF</td><td>89.93°C</td><td>N/A</td></tr></tbody></table>
<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /><figcaption>**Supplementary Figure 12.** Comparison of VpOmpM1 and EcOmpF. **a** Alignment of VpOmpM1 β-barrel and EcOmpF amino acid sequences. The alignment was generated by sequence-independent pairwise structure alignment of VpOmpM1 and EcOmpF (PDB 2ZFG) using TM-align³ (RMSD 3.84 Å). Residues forming the barrel β-strands are annotated above and below the alignment for VpOmpM1 and EcOmpF, respectively. The sequences share 8% identity and 25% similarity. **b** Comparison of the VpOmpM1 β-barrel from C3 reconstruction (rainbow, N-terminus blue, C-terminus red) and EcOmpF (2ZFG; grey) structures. Views generated from a superposition. **c** Comparison of the extracellular loops (L1-8) of the two β-barrels as viewed from inside the β-barrel.</figcaption></figure>
<figure><img src="image_72.png" /><figcaption>**Supplementary Figure 13. Fluctuations in the channel regions of VpOmpM1 and EcOmpF during MD simulations.**

**a-f** Analysis of residue fluctuations in VpOmpM1 and EcOmpF during MD simulations. HOLE⁴ analyses of VpOmpM1 (**a**) and EcOmpF (**d**) calculated averaged from all-atom MD simulation frames: 1 frame every 10 ns over the final 100 ns from 3 simulations, averaged over the 3 protomers (VpOmpM1, n=90 traces); 1 frame every 10 ns over the final 300 ns of a single all-atom simulation, averaged over the three protomers (EcOmpF, n=90 traces). Lines show the average values ± S.D. depicted by the shaded area. **b, e** Per-residue root-mean-square fluctuation (RMSF) plots for VpOmpM1 (**b**) and EcOmpF (**e**) during MD simulations. Same sampling as in **a** and **d**. Lines show the average values ± S.D. depicted by the shaded area. Residues forming the constriction loops are highlighted: VpOmpM1 L3, green; VpOmpM1 L7, orange; EcOmpF L3, grey. **c, f** Per-residue RMSF values converted to B-factors and displayed on VpOmpM1 (**c**) and EcOmpF (**f**) structures on the same scale. Cartoon thickness and colour (blue to red) are proportional to B-factor value. **g** Examples of interactions observed during simulations that likely restrict movement of the eyelet-forming loops of VpOmpM1: between the L3 and L7 loops (left), between L3 and L4 (middle), and between L7 and the internal barrel wall (right).</figcaption></figure>
<figure><img src="image_73.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_74.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>**Supplementary Figure 14.** Transverse electric field properties in VpOmpM1 and EcOmpF channels. **a** Total water dipole moment by z-coordinate, normalised to the number of water molecules in each z-slice. **b**, **c** Arrows indicate the direction of the dipole moment averaged across the waters in a particular z-slice over the final 100 ns of equilibrium simulations for VpOmpM1 (b) and EcOmpF (c). The length of the arrow is proportional to the magnitude of the dipole moment. VpOmpM1 loop 3 is green and loop 7 is orange; EcOmpF loop 3 is black. Basic loop residues are in blue and acidic residues are in red.</figcaption></figure>
<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

<figure><img src="image_80.png" /></figure>

<figure><img src="image_81.png" /><figcaption>**Supplementary Figure 15.** Representative electrophysiology recordings. **a, b** Representative ion-current traces of full-length VpOmpM1 (**a**) and the barrel-only construct (**b**) showing multiple channel insertion events observed as 'steps' or sudden increases in current. **c, d** Ion-current traces for full-length VpOmpM1 (**c**) and the barrel-only construct (**d**) showing channel gating activity at high potential (left), with the corresponding all-current point histograms shown on the right. A 2.5 kHz low-pass eight-pole Bessel filter was applied to all traces. The applied voltage is indicated on each trace.</figcaption></figure>
<figure><img src="image_82.png" /><figcaption>**Supplementary Figure 16. Comparison of VpOmpM1 and DrSlpA structures.** a To-scale surface representations of VpOmpM1 and *Deinococcus radiodurans* SlpA (DrSlpA) (PDB 8AGD⁵) from outside the cell (top) and from the OM plane (bottom). b HOLE⁴ profiles of VpOmpM1 and DrSlpA. Note that the profiles show the path of a ball-shaped probe via the narrowest part of the pore and, therefore, this analysis grossly underestimates the size of the SlpA pore, since the constriction is not roughly circular as in VpOmpM1.</figcaption></figure>
<table><caption>**Supplementary Table 1.** Cryo-EM data collection, processing and refinement statistics.</caption><thead><tr><th></th><th>VpOmpM1 from <i>E. coli</i></th><th>VpOmpM1 from <i>V. parvula</i></th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td><td></td></tr><tr><td>Electron microscope</td><td>FEI Glacios</td><td>FEI Glacios</td></tr><tr><td>Voltage (kV)</td><td>200</td><td>200</td></tr><tr><td>Spherical aberration (µm)</td><td>2.7</td><td>2.7</td></tr><tr><td>Camera</td><td>Falcon 4 (counting)</td><td>Falcon 4 (counting)</td></tr><tr><td>Energy filter</td><td>none</td><td>none</td></tr><tr><td>Magnification</td><td>240,000</td><td>240,000</td></tr><tr><td>Pixel size (Å)</td><td>0.574</td><td>0.574</td></tr><tr><td>Total dose (e/Å<sup>2</sup>)</td><td>50.1</td><td>50</td></tr><tr><td>Defocus minimum maximum (µm)</td><td>-1.0 to -2.0</td><td>-0.8 to -2.0</td></tr><tr><td>Number of movies collected</td><td>4,284</td><td>6,505</td></tr><tr><td><strong>Image Processing</strong></td><td></td><td></td></tr><tr><td>Imposed symmetry</td><td>C1</td><td>C3</td></tr><tr><td>Initial number of particles</td><td>838,051</td><td>838,051</td></tr><tr><td>Final number of particles</td><td>96,280</td><td>119,001</td></tr><tr><td>Global resolution (FSC = 0.143)</td><td>3.15</td><td>2.78</td></tr><tr><td>Map sharpening B-factor (Å<sup>2</sup>)</td><td>-70.2</td><td>-85.5</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td></tr><tr><td>Model composition</td><td></td><td></td></tr><tr><td>Non-hydrogen atoms</td><td>7,386</td><td>7,077</td></tr><tr><td>Protein residues</td><td>963</td><td>924</td></tr><tr><td>R.m.s. deviations</td><td></td><td></td></tr><tr><td>Bonds lengths (Å)</td><td>0.003</td><td>0.002</td></tr><tr><td>Bond angles (°)</td><td>0.493</td><td>0.420</td></tr><tr><td>Validation</td><td></td><td></td></tr><tr><td>MolProbity score</td><td>1.56</td><td>1.39</td></tr><tr><td>Clash score</td><td>4.52</td><td>3.57</td></tr><tr><td>Rotamer outliers (%)</td><td>0</td><td>0</td></tr><tr><td>Ramachandran plot</td><td></td><td></td></tr><tr><td>Favoured (%)</td><td>95.30</td><td>96.41</td></tr><tr><td>Outliers (%)</td><td>0</td><td>0</td></tr><tr><td>PDB</td><td>8BYM</td><td>8BYT</td></tr><tr><td>EMDB</td><td>EMD-16328</td><td>EMD-16333</td></tr></tbody></table>
<table><caption>**Supplementary Table 2.** Crystallography data collection, processing and refinement parameters.</caption><thead><tr><th></th><th>VparOmpM1 stalk*</th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td></tr><tr><td>DLS beamline</td><td>I03</td></tr><tr><td>Wavelength</td><td>0.89842</td></tr><tr><td>Space Group</td><td>H3</td></tr><tr><td>Unit cell parameters</td><td></td></tr><tr><td>a, b, c (Å)</td><td>118.71, 118.71, 47.24</td></tr><tr><td>α, β, γ (°)</td><td>90, 90, 120</td></tr><tr><td>Molecules in AU</td><td>3</td></tr><tr><td>Resolution range (Å)</td><td>34.78-1.57 (1.57-1.6)</td></tr><tr><td>I/σI</td><td>17.1 (0.7)</td></tr><tr><td>Completeness (%)</td><td>99.5 (92.9)</td></tr><tr><td>Multiplicity</td><td>9.0 (4.7)</td></tr><tr><td>Rpim (%)</td><td>1.7 (88.4)</td></tr><tr><td>CC<sub>1/2</sub> (%)</td><td>100 (23)</td></tr><tr><td><strong>Phasing</strong></td><td></td></tr><tr><td>Ab initio</td><td>Arcimboldo<sup>6</sup></td></tr><tr><td><strong>Refinement</strong></td><td></td></tr><tr><td>Resolution (Å)</td><td>34.78-1.7</td></tr><tr><td>Rwork/Rfree (%)</td><td>21.9 /24.8</td></tr><tr><td>Reflections</td><td></td></tr><tr><td>Non-hydrogen atoms</td><td>2,015</td></tr><tr><td>Protein only</td><td>1,918</td></tr><tr><td>Average B-factor (Å<sup>2</sup>)</td><td>39.68</td></tr><tr><td>Rmsd</td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.011</td></tr><tr><td>Bond angles (°)</td><td>1.21</td></tr><tr><td>MolProbity clashscore</td><td>3.75</td></tr><tr><td>Ramachandran plot</td><td></td></tr><tr><td>Favoured (%)</td><td>99.59</td></tr><tr><td>Outliers (%)</td><td>0</td></tr><tr><td>PDB</td><td>8BZ2</td></tr></tbody></table>

<footnote>*Statistics for the highest-resolution shell are shown in parentheses.</footnote>
<table><caption>Supplementary Table 3. Bacterial strains used in this study.</caption><thead><tr><td>Strain</td><td>Genotype</td><td>Reference or Source</td></tr></thead><tbody><tr><td><i>E. coli</i> DH5α</td><td>F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80d lacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK<sup>-</sup> mK<sup>+</sup>), λ<sup>-</sup></td><td>Promega</td></tr><tr><td><i>E. coli</i> TOP10</td><td>F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-</td><td>Invitrogen</td></tr><tr><td><i>E. coli</i> BL21(DE3)</td><td>F- ompT lon hsdSB (rB- mB-) gal dcm (DE3)</td><td>Invitrogen</td></tr><tr><td><i>E. coli</i> C43(DE3) ΔcyoABCD</td><td>F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) lacUV5** ΔcyoABCD</td><td>7 (modified)</td></tr><tr><td><i>E. coli</i> MFDpir</td><td>MG1655 RP4-2-Tc ::[Δmu1 ::aac(3)IV-ΔaphA-Δnic35-Δmu2 ::zeo] ΔdapA :erm-pir) ΔrecA</td><td>8</td></tr><tr><td><i>V. parvula</i> SKV38</td><td>Wild type strain</td><td>9</td></tr><tr><td><i>V. parvula</i> SKV38 ΔompM1-3</td><td>ΔFNLLGLLA_01389-7::tetM</td><td>10</td></tr></tbody></table>
<table><caption>Supplementary Table 4. Plasmids used in this study.</caption><thead><tr><td>Plasmid</td><td>Description</td><td>Reference or Source</td></tr></thead><tbody><tr><td>pB22</td><td>pBAD22 vector with <i>E. coli</i> TamA signal sequence, N-terminal His<sub>7</sub>-tag</td><td>11,12</td></tr><tr><td>pJW45</td><td>pB22 with beta barrel of <i>ompM1</i> cloned between Xhol and Xbal restriction sites</td><td>This study</td></tr><tr><td>pJW46</td><td>pB22 with <i>ompM1</i> (without signal peptide) CDS cloned between Xhol and Xbal restriction sites</td><td>This study</td></tr><tr><td>pJW48</td><td>pRPF185 with His-tagged <i>ompM1</i> (native RBS) cloned into the <i>SacI</i> restriction site</td><td>This study</td></tr><tr><td>pRPF185</td><td><i>Escherichia/Clostridium</i> shuttle conjugative expression vector with <i>tet</i> promoter</td><td>13</td></tr><tr><td>pBAD24</td><td>Vector for expressing proteins with their native signal sequence in <i>E. coli</i></td><td>11</td></tr><tr><td>pBAD24-EcOmpF</td><td>pBAD24 vector with the full-length <i>E. coli</i> OmpF coding sequence cloned between EcoRI and Xbal restriction sites</td><td>This study</td></tr><tr><td>pET28b</td><td><i>E. coli</i> expression vector</td><td>Novagen</td></tr><tr><td>pET28b-SLH_22-107</td><td>pET28b with <i>ompM1</i> SLH domain and stalk residues 22-107 coding region cloned between Ncol and Xhol sites (C-terminal His<sub>6</sub>-tag)</td><td>This study</td></tr><tr><td>pB22-00518_21-200</td><td>pB22 with beta barrel (residues 21-200) of <i>FNLLGLLA_00528</i> cloned between Xhol and Xbal restriction sites</td><td>This study</td></tr><tr><td>pB22-00833</td><td>pB22 with beta barrel of <i>FNLLGLLA_00833</i> cloned between Xhol and Xbal restriction sites</td><td>This study</td></tr></tbody></table>
<table><caption>Supplementary Table 5. Primers* used in this study.</caption><thead><tr><td>Primer</td><td>Sequence</td><td>Description</td></tr></thead><tbody><tr><td>JW172</td><td>agcgttaacagatctgagcttaagaaagaaggaattcattatgaa<br/>aaaac</td><td>Forward primer for amplifying full length ompM1 with its native RBS with homology arm to the pRPF185 digested with Sacl</td></tr><tr><td>JW201</td><td>ccatcaccatcaccatcacctcgagcgcttggaagaccgtgtag</td><td>Forward primer for amplifying OmpM1 beta barrel with homology arm to the pB22 digested with Xhol</td></tr><tr><td>JW202</td><td>ctgaagcggtgtgaatactctagattagaatttgtaatttaattctgc<br/>acg</td><td>Reverse primer for amplifying ompM1 with homology arm to the pB22 digested with Xbal</td></tr><tr><td>JW203</td><td>ccatcaccatcaccatcacctcgaggctgcaaattccattctcc</td><td>Forward primer for amplifying ompM1 without signal peptide with homology arm to the pB22 digested with Xhol</td></tr><tr><td>JW206</td><td>tctcctttactgcaggagctttaatgatgatgatgatgatgaccac<br/>cgaatttgtaatttaattctgcacggtag</td><td>Reverse primer for amplifying ompM1, adding C-terminal 6His tag (indicated in bold) with homology arm to the pRPF185 digested with Sacl.</td></tr><tr><td>00833_F</td><td>atactcgaggcattttgctgcagctcc</td><td>Forward primer for amplifying FNLLGLLA_00833 without signal peptide with Xhol site</td></tr><tr><td>00833_R</td><td>atatctagattagaaggagtagcctacacc</td><td>Reverse primer for amplifying FNLLGLLA_00833 without signal peptide with Xbal site</td></tr><tr><td>00518_F</td><td>atactcgagactcctcaaaactcaattcaataaag</td><td>Forward primer for amplifying FNLLGLLA_00518 barrel with Xhol site</td></tr><tr><td>00518_R</td><td>atatctagattaaccaccgaaacggtaagataatc</td><td>Reverse primer for amplifying FNLLGLLA_00518 barrel with Xbal site</td></tr><tr><td>stalk_F</td><td>ataccatggctgcaaattccattctccg</td><td>Forward primer for amplifying ompM1 stalk region with Ncol site</td></tr><tr><td>stalk_R</td><td>atactcgagctttacattacctacacggtc</td><td>Reverse primer for amplifying ompM1 stalk region with Xhol site</td></tr></tbody></table>

<footnote>* The part of the primer hybridizing to the template is indicated by an underscore.</footnote>
<table><caption>**Supplementary Table 6.** Position restraints, timesteps (dt), and durations of the equilibration phases in the all-atom MD simulations.</caption><thead><tr><th rowspan="2">Equilibration phase</th><th colspan="4">Position restraint / kJ mol<sup>-1</sup> nm<sup>-2</sup></th><th rowspan="2">dt/ fs</th><th rowspan="2">Duration / ns</th></tr><tr><th>Protein Backbone</th><th>Protein Sidechains</th><th>Lipid Headgroups</th><th>Dihedrals</th></tr></thead><tbody><tr><td>NVT1</td><td>4000</td><td>2000</td><td>1000</td><td>1000</td><td>1</td><td>0.125</td></tr><tr><td>NVT2</td><td>2000</td><td>1000</td><td>400</td><td>400</td><td>1</td><td>0.125</td></tr><tr><td>NPT1</td><td>1000</td><td>500</td><td>400</td><td>200</td><td>2</td><td>0.5</td></tr><tr><td>NPT2</td><td>500</td><td>200</td><td>200</td><td>200</td><td>2</td><td>0.5</td></tr><tr><td>NPT3</td><td>200</td><td>50</td><td>40</td><td>100</td><td>2</td><td>0.5</td></tr><tr><td>NPT4</td><td>50</td><td>-</td><td>-</td><td>-</td><td>2</td><td>0.5</td></tr></tbody></table>
<table><caption>Supplementary Table 7. VpOmpM1 simulation composition.</caption><thead><tr><th rowspan="2">System</th><th colspan="10">Contents</th><th rowspan="2">Total atoms</th><th rowspan="2">Box dimensions, nm</th></tr><tr><th>OmpM protomers</th><th>POPE</th><th>POPG</th><th>Cardiolipin</th><th>LPS</th><th>Potassium</th><th>Chloride</th><th>Calcium</th><th>TIP3P Water</th></tr></thead><tbody><tr><td>Truncated stalk (residue 100 onwards)</td><td>3</td><td>180</td><td>10</td><td>10</td><td>62</td><td>258</td><td>178</td><td>279</td><td>69,146</td><td>317,563</td><td>12.74 x 12.74 x 21.56</td></tr><tr><td>Full length extended stalk</td><td>3</td><td>162</td><td>9</td><td>9</td><td>55</td><td>324</td><td>241</td><td>247</td><td>91,443</td><td>377,626</td><td>12.22 x 12.22 x 27.40</td></tr><tr><td>Full length compacted stalk</td><td>3</td><td>162</td><td>9</td><td>9</td><td>56</td><td>268</td><td>185</td><td>252</td><td>71,804</td><td>319,677</td><td>12.25 x 12.25 x 23.18</td></tr></tbody></table>
<table><caption>Supplementary Table 8. EcOmpF simulation composition.</caption><thead><tr><th rowspan="2">System</th><th colspan="10">Contents</th><th rowspan="2">Total atoms</th><th rowspan="2">Box dimensions, nm</th></tr><tr><th>OmpF protomers</th><th>POPE</th><th>POPG</th><th>Cardiolipin</th><th>LPS</th><th>Potassium</th><th>Chloride</th><th>Calcium</th><th>TIP3P Water</th></tr></thead><tbody><tr><td>EcOmpF</td><td>3</td><td>180</td><td>10</td><td>10</td><td>63</td><td>273</td><td>143</td><td>283</td><td>56,781</td><td>282,355</td><td>12.71 x 12.71 x 19.61</td></tr></tbody></table>
<table><caption>**Supplementary Table 9.** Substrate concentrations used in liposome swelling assays.</caption><thead><tr><th>Substrate</th><th>Concentration (mM)</th></tr></thead><tbody><tr><td colspan="2"><strong>Figure 6 – VpOmpM1, VpOmpM1 barrel and EcOmpF</strong></td></tr><tr><td>Lactate</td><td>8</td></tr><tr><td>Acetate</td><td>8</td></tr><tr><td>Putrescine</td><td>8</td></tr><tr><td>Arginine</td><td>8</td></tr><tr><td>Lysine</td><td>8</td></tr><tr><td>Glutamate</td><td>10</td></tr><tr><td>Aspartate</td><td>8</td></tr><tr><td>Glycine</td><td>15</td></tr><tr><td>Alanine</td><td>15</td></tr><tr><td>Leucine</td><td>12</td></tr><tr><td>Methionine</td><td>15</td></tr><tr><td>Arabinose</td><td>12</td></tr><tr><td>Glucose</td><td>12</td></tr><tr><td>Fructose</td><td>12</td></tr><tr><td>Lactose</td><td>15</td></tr><tr><td>Maltose</td><td>15</td></tr><tr><td>Sucrose</td><td>15</td></tr><tr><td>Kanamycin</td><td>8</td></tr><tr><td>Ampicillin</td><td>8</td></tr><tr><td>Gentamicin</td><td>8</td></tr><tr><td colspan="2"><strong>Figure 7 – FNLLGLLA_00518 barrel, VpOmpM1 barrel and EcOmpF</strong></td></tr><tr><td>Lactate</td><td>10</td></tr><tr><td>Putrescine</td><td>8</td></tr><tr><td>Glycine</td><td>15</td></tr><tr><td>Arabinose</td><td>10</td></tr></tbody></table>
Supplementary references

1. Holm, L. Dali server: structural unification of protein families. *Nucleic Acids Res.* **50**, W210–W215 (2022).

2. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. *Nat. 2021* **596**7873 **596**, 583–589 (2021).

3. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. *Nucleic Acids Res.* **33**, 2302–2309 (2005).

4. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. HOLE: A program for the analysis of the pore dimensions of ion channel structural models. *J. Mol. Graph.* **14**, 354–360 (1996).

5. Farci, D., Graça, A. T., Iesu, L., de Sanctis, D. & Piano, D. The SDBC is active in quenching oxidative conditions and bridges the cell envelope layers in Deinococcus radiodurans. *J. Biol. Chem.* **299**, 102784 (2023).

6. Rodríguez, D. D. et al. Crystallographic ab initio protein structure solution below atomic resolution. *Nat. Methods* **2009** *69*, 651–653 (2009).

7. Miroux, B. & Walker, J. E. Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. *J. Mol. Biol.* **260**, 289–298 (1996).

8. Ferrières, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. *J. Bacteriol.* **192**, 6418–6427 (2010).

9. Knapp, S. et al. Natural Competence Is Common among Clinical Isolates of Veillonella parvula and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome. *Front. Cell. Infect. Microbiol.* **7**, (2017).

10. Witwinowski, J. et al. An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition. *Nat. Microbiol.* **7**, 411–422 (2022).

11. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J. Bacteriol.* **177**, 4121–4130 (1995).

12. Van den Berg, B. et al. X-ray structure of a protein-conducting channel. *Nature* **427**, 36–44 (2004).

13. Fagan, R. P. & Fairweather, N. F. Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems. *J. Biol. Chem.* **286**, 27483–27493 (2011).