Kyrie P Grasekamp

Basile Beaud

Najwa Taib

Bianca Audrain

Benjamin Bardiaux

Yannick Rossez

Nadia Izadi-Pruneyre

Maylis Lejeune

Xavier Trivelli

Zina Chouit

Benjamin Bardiaux #

Yann Guerardel

Jean-Marc Ghigo

Simonetta Gribaldo
email: simonetta.gribaldo@pasteur.fr

Christophe Beloin
email: christophe.beloin@pasteur.fr

Bridges instead of boats? The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope core of phospholipid trafficking

Keywords: phospholipid trafficking, outer membrane biogenesis, Mla system, lipid asymmetry, evolution

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Introduction

The diderm bacterial envelope is a complex structure consisting of two membranes: a cytoplasmic -or inner-membrane (IM) composed primarily of glycerophospholipids (GPLs), and an outer membrane (OM) which exhibits lipid asymmetry, comprising GPLs in its inner leaflet and primarily lipopolysaccharide (LPS) molecules in its outer leaflet. This asymmetry manifests as a robust exclusion barrier against a range of antibiotics and host components, such as vancomycin and bile salts 1,2 . Although GPLs are one of the most ubiquitous amphipathic components of the diderm envelope, their transport systems remain poorly understood, and most current information stems from a handful of model bacterial species. Recent studies implicate large AsmA-like proteins, such as TamB and YhdP, in the mediation of anterograde GPL trafficking [3][START_REF] Douglass | Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria[END_REF][START_REF] Grimm | The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli[END_REF] , whilst MCE proteins -namely MlaD within the maintenance of lipid asymmetry (Mla) machinery -are proposed to facilitate retrograde GPL trafficking [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF][START_REF] Malinverni | An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane[END_REF] . In E. coli, the Mla system is composed of six proteins (Fig 1): MlaBDEF form the IM complex [START_REF] Thong | Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry[END_REF][START_REF] Tang | Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB[END_REF] , MlaC is the periplasmic chaperone [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF][START_REF] Huang | Molecular dynamic study of MlaC protein in Gram-negative bacteria: conformational flexibility, solvent effect and protein-phospholipid binding: Molecular Dynamic Study of MlaC Protein[END_REF] , and MlaA is the OM lipoprotein that associates with porin trimers [START_REF] Chong | Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in E scherichia coli: An outer membrane complex important for lipid asymmetry[END_REF] . Despite extensive research of the Mla system within model diderms for over a decade, several mechanistic details -including substrate specificity and even directionality, until recently -remain controversial [START_REF] Tang | Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB[END_REF][START_REF] Powers | The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport[END_REF][START_REF] Kamischke | The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane[END_REF][START_REF] Hughes | Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system[END_REF] .

Here, we investigate GPL trafficking in Veillonella parvula, a genetically tractable member of the Firmicutes (Negativicutes) which presents a diderm envelope with LPS [START_REF] Antunes | Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes[END_REF][START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] . V. parvula is phylogenetically distant from classical models such as E. coli as it belongs to the Terrabacteria, one of the two large clades into which Bacteria are divided (containing phyla such as Actinobacteria, Cyanobacteria and Candidate Phyla Radiation (CPR)), and whose cell envelopes are largely understudied [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF][START_REF] Megrian | One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide[END_REF] . Remarkably, the Firmicutes is the only phylum so far identified to contain a mixture of both monoderm and diderm clades, offering an ideal platform on which to study both the monoderm-diderm transition [START_REF] Taib | Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition[END_REF] and the diversity of OM biogenesis systems [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] . We previously demonstrated that this diderm architecture is an ancestral feature, present in both the ancestor of the Firmicutes and the Last Bacterial Common Ancestor (LBCA) [START_REF] Antunes | Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes[END_REF][START_REF] Megrian | One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide[END_REF][START_REF] Taib | Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition[END_REF][START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] , an inference also supported by a recent study [START_REF] Coleman | A rooted phylogeny resolves early bacterial evolution[END_REF] . Studying diderm Firmicutes can therefore shed light on the evolution of ancestral systems in the biogenesis and maintenance of the bacterial OM. Interestingly, the V. parvula genome contains an operon embedded in a large OM biogenesis and maintenance cluster [START_REF] Antunes | Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes[END_REF][START_REF] Taib | Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition[END_REF] with only three homologues of the Mla system: the IM proteins MlaDEF, directly followed by a homologue of the OM efflux protein TolC [START_REF] Bavro | Assembly and channel opening in a bacterial drug efflux machine[END_REF] , and TamB (Fig 1). This arrangement is conserved in all Negativicutes (Extended Fig 1). However, no homologues of the periplasmic chaperone MlaC or the OM lipoprotein MlaA were identifiable at the sequence level, consistent with the fact that no OM lipoproteins or homologues of the Lol system have so far been identified in V. parvula [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF][START_REF] Poppleton | Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome[END_REF] . This raises the question of how these V. parvula Mla homologues are involved in retrograde lipid trafficking, and how they can accomplish this function in the absence of MlaABC.

perfringens [START_REF] Jackson | Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operon Responsible for Plasmalogen Production in Clostridium perfringens[END_REF] , encoded within a single gene (Supplementary Fig S1A). Using TLC, matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI-QIT-TOF) and nuclear magnetic resonance (NMR) spectroscopy on lipid extracts from the WT, we then revealed that the major lipid species in the envelope of V. parvula are PE (~68%) and PG (~20%), along with two other uncharacterised lipids representing a further ~9% and ~3% of the envelope (Supplementary Fig S1 ,     Supplementary Table S1). NMR analyses also revealed that ~15-35% of each lipid species present in the envelope of V. parvula contains a vinyl ether bond, which could result from the activity of the identified plasmalogen biosynthesis homologue (Supplementary Fig S2). No cardiolipin was observed via MALDI-QIT-TOF, consistent with the absence of homologues of genes involved in the synthesis of this lipid in the V. parvula genome.

Both whole-cell and membrane lipid extracts from WT and ∆mla strains were used to perform TLC, however no differences in GPL composition were observed, as previously described in E. coli [START_REF] Malinverni | An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane[END_REF] (Supplementary Fig S3A). We posited that this lack of difference may be due to the observed hypervesiculation of ∆mla strains, compensating for OM lipid imbalance by shedding excess envelope material. We therefore purified OMVs produced by both the WT and ∆mlaD strains, performed lipid extraction and TLC, and revealed different classes of lipid species by staining with iodine vapor, Schiff reagent and phosphomolybdic acid (PMA). Across all iodine vapour-stained plates, OMVs produced by ∆mlaD strains display a ~40% enrichment for PE, whilst also containing a marked reduction in another lipid species that is abundant in WT OMVs (Fig 2C, Supplementary Fig S3B). These results suggest that ∆mla mutants accumulate PE in their OM, which is subsequently shed from the envelope via excessive vesicle formation. Taken together, this analysis strongly suggests that the minimal MlaEFD system of V. parvula is involved in retrograde GPL trafficking.

Suppressor analysis implicates Mla and TamB in coordination of GPL homeostasis in V. parvula

To further strengthen the proposed function of the Mla system in V. parvula, we investigated the genes able to suppress detergent hypersensitivity of ∆mla strains. By performing random transposon mutagenesis in the ∆mlaD background, we identified ten chromosomal insertion mutants able to grow in SK media supplemented with 0.004% SDS and 312. 5 Considering the recent implications of TamB and other large AsmA-like proteins in anterograde GPL trafficking 3,[START_REF] Douglass | Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria[END_REF] , we were interested to identify this gene as a suppressor of ∆mlaD detergent sensitivity. Indeed, we had previously identified a spontaneous suppressor of ∆mlaD with a SNP encoding a STOP codon in tamB, though the phenotype was unstable and reverted (Fig 3B). We therefore generated clean, stable deletion mutants to further probe the relationship between these two genes. As with the suppressor screens, loss of tamB partially rescues SDS/EDTA sensitivity in ∆mlaD and reduces vancomycin resistance (Fig 3C). Interestingly, the tamB mutant presents a permeability phenotype which appears to be the opposite of ∆mla mutants, with no change in SDS/EDTA sensitivity and an increased sensitivity to vancomycin, as previously described in E. coli 3

(Fig 3C, Supplementary Fig S4)

. Much like ∆mlaD, however, ∆tamB also displays a hypervesiculation phenotype, with a ~10-fold increase in vesicle production as compared to the WT strain (Fig 3D). Strikingly, loss of tamB in the ∆mlaD background significantly reduces hypervesiculation; the ∆tamB∆mlaD mutant produces only ~5-fold more vesicles than the WT (Fig 3D). This rate of reduction in OMV production is comparable to the phenotype of the fully-complemented ∆mlaEFD::mlaEFD strain, suggesting that deletion of tamB is as effective at rescuing this envelope defect as reintroducing the mla genes. The contrasting OM permeability phenotypes of ∆mlaD and ∆tamB, together with the striking complementation of the double ∆mlaD∆tamB strain, hints to the antagonistic functions of these two genes in envelope biogenesis and maintenance. Overall, these results suggest that MlaEFD and TamB may represent the two major systems maintaining GPL homeostasis in the OM of V. parvula -likely retrograde and anterograde, respectively.

Structural modelling suggests a transenvelope Mla system in V. parvula

Though phenotypic and suppressor analyses elucidated the functional role of MlaEFD, we sought to understand how three IM proteins could be structurally arranged within the envelope of V. parvula to facilitate GPL trafficking in the absence of MlaABC. Whilst the mlaE and mlaF homologues closely resemble those of model diderms such as the Proteobacteria, mlaD in V. parvula is over double the size of the one of E. coli (419 amino acids vs 183 amino acids respectively) (Fig 4A). Both versions of this gene contain a transmembrane domain (TMD) that anchors the protein in the IM, and a conserved MCE domain that binds lipids [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF] . However, MlaD in V. parvula extends with a long predicted α-helical region spanning ~130 residues, and a predicted C-terminal β-barrel (Fig 4A).

AlphaFold2 29 (AF2) was used to predict the structure of this unusually long MlaD protein, but was unable to confidently model the elongated α-helical region, and indeed the positioning of this domain relative to the N-and C-termini of the protein (Extended Fig 3). However, high confidence predictions were achieved for the TMD and MCE domain, and for the C-terminal β-barrel. This C-terminal domain displays characteristic features of a membrane-embedded β-barrel, composed of ten alternating antiparallel β-strands with a hydrophobic and neutral surface for the β-sheets, and with 'girdles' of negatively charged and aromatic residues which flank the top and bottom of the structure (Extended

Fig 4).

Considering all characterised MCE proteins to date form hexamers, we merged overlapping predicted structures of all domains of MlaDVp with hexameric stoichiometry, generating a model for the fulllength protein (Fig 4B). Even when generated without templates, a 6:2:2 stoichiometry of the MlaDEF complex is confidently predicted for V. parvula, with high similarity to the resolved structure of the MlaDEF complex from E. coli (Extended Fig 3). Interestingly, two major models are generated for the hexameric α-helical region of MlaDVp: one with a closed tunnel structure, and one with an open pore or groove with little interaction between the first and last chains of the hexamer (Extended Fig 1). We suspect that the "open groove" configuration may represent an artifact of the prediction, owing to the way AlphaFold-Multimer concatenates sequences of all chains prior to structure inference. Instead, the closed tunnel model (Extended Fig 5) more closely reflects the resolved crystal structure of PqiB, another MCE-domain protein from E. coli, which forms a closed α-helical tunnel on top of three hexameric MCE rings [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF] , and the recently resolved heterohexamer of Mce1A-1F from Mycobacterium tuberculosis [START_REF] Chen | Structure of an endogenous mycobacterial MCE lipid transporter[END_REF] . Like these resolved structures, the predicted tunnel formed by the α-helices generates a hydrophobic interior of ~13 Å (Fig 4C ), supporting the possibility of a role in hydrophobic substrate transport. Further, both the N-and C-termini of the full-length model generate highly hydrophobic bands where both regions are predicted to embed within the IM and OM, respectively. The rest of the structure, which would reside in the aqueous periplasm, displays a hydrophilic exterior (Fig 4C). It is interesting to note the prediction of a small external cluster of hydrophobic residues at the lowest confidence region of the tunnel -which coincides with the region that forms a groove in the higher confidence model.

5, Extended Table

From these high-confidence structural predictions of a membrane-embedded C-terminal β-barrel and an IM MCE-domain hexamer, we hypothesised that MlaD from V. parvula may have domains anchored in both the IM and OM, forming a transenvelope bridge. This would remove the necessity for a periplasmic chaperone boat and OM lipoprotein, as MlaD would be capable of connecting the two membranes directly to facilitate transport. To verify this hypothesis experimentally, we localised full-length MlaD and its separate domains by performing membrane fractionation by sedimentation on sucrose density gradients. We used total membrane pellets (containing both IM and OM vesicles) obtained from WT and ∆mlaD strains complemented with HA-tagged versions of the TMD-MCE domain and the β-barrel. Localisation of these domains and of the full-length protein was examined via immunoblotting with our developed MlaD antisera, and the separate domains with a commercial anti-HA antibody. Whilst the full-length MlaD was distributed across almost all membrane fractions, the TMD-MCE domain construct was most abundant in fractions F4-5, similar to the localisation profile of the IM control SecA, and the β-barrel was located almost exclusively in the final few OM fractions, F9-11, mirroring the localisation of TolC (Fig 4D). These results support the hypothesis that MlaD can span the periplasm, with domains anchored in both the IM and OM.

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Phylogenomic analysis reveals an ancestral core of the Mla system

The possibility of a minimal, transenvelope MlaEFD system was intriguing, and we sought to understand if this type of pathway exists in other bacterial lineages by exploring the distribution of all Mla genes. We therefore carried out an exhaustive search for homologues of the six components (MlaABCDEF) in a local databank containing 1083 genomes, representing all current phyla. To identify MlaA, MlaC, MlaD, and MlaE homologues, we used the Pfam domains PF04333, PF05494, PF02470, and PF02405, respectively. As MlaB and MlaF belong to the generic STAS-domain and ATPase families, respectively, we used MacSyFinder2 [START_REF] Hao | Novel prosthecate bacteria from the candidate phylum Acetothermia[END_REF] to identify them when found in synteny with at least one of the other Mla components in the genome. The distribution of all six components was then mapped onto a reference phylogeny of Bacteria [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] 2).

In agreement with the fundamental role of the Mla system in maintaining OM asymmetry, Mla homologues are largely found in diderm bacteria (Fig 5). Some diderm phyla in the Terrabacteria lack any identifiable homologues, possibly due to their atypical envelope architectures: the Thermotogae and their neighbor Candidate phyla Bipolaricaulota and Fraserbacteria have an OM detached at the cell poles [START_REF] Brock | Fine Structure of Thermus aquaticus , an Extreme Thermophile[END_REF] ; Dictyoglomi and Deinococcus-Thermus both form rotund bodies where cell aggregates share a single OM [START_REF] Katayama | Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure[END_REF][START_REF] Hayat | Inclusion of dyad-repeat pattern improves topology prediction of transmembrane β-barrel proteins[END_REF] ; some members of Deinococcus-Thermus have lost LPS; and cryo-electron tomography of a member of Atribacteria (Atribacter laminatus) has recently revealed an atypical cell envelope architecture with an intracytoplasmic lipidic bilayer surrounding the nucleoid [START_REF] Konovalova | Outer membrane lipoprotein biogenesis: Lol is not the end[END_REF] . Within the three diderm lineages belonging to the Firmicutes [START_REF] Taib | Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition[END_REF] , Mla components are found in the Negativicutes 2).

Conversely, and consistently with its presence in M. tuberculosis, some monoderm phyla contain MCE-domain proteins; we identified such homologues in some members of Actinobacteria, and also within the Candidate Dormibacteraeota, usually in association with a Mce4_CUP1 domain (Extended Table 2). Further, our analysis suggests the MCE homologues present in these monoderm phyla were horizontally acquired from the Proteobacteria (Extended Figs 678).

More striking is the overall distribution of MlaDEF vs MlaABC; whilst MlaDEF are widely distributed across almost all diderm phyla, with the exception of those mentioned above, the MlaABC components have a much narrower distribution, present mostly within Proteobacteria, and totally absent in diderm Terrabacteria (Fig 5

, Extended Fig 6, Extended Table 2).

We then focused specifically on MlaD to investigate whether other bacteria possess the longer version, similar to V. parvula. Surprisingly, we found that the short MlaD sequences -similar to that of E. coli -are restricted to taxa within Proteobacteria, whereas 85% of MlaD homologues are of the longer version (corresponding to an average of 374 residues) (Extended Figs 678). To understand if these longer MlaD versions have a similar predicted structure to that of V. parvula, we screened them for the presence of β-sheet structures using BOCTOPUS2 [START_REF] Hayat | Inclusion of dyad-repeat pattern improves topology prediction of transmembrane β-barrel proteins[END_REF] and AlphaFold2 [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] . We could indeed infer the presence of a C-terminal β-barrel in 103 sequences, with a conserved fold and physicochemical properties, some containing an additional helical extension (Extended Fig 4F ,4H). These long versions of MlaD with a β-barrel are widely distributed in the diderm phyla belonging to the Terrabacteria division, whilst in the Gracilicutes they are found mainly in some Proteobacteria and 2). Interestingly, many of the long MlaD sequences with no identifiable complete β-barrel domain contain a disordered C-terminal region with some βstructure, often short β-hairpins, which may represent the remnants or precursors of a β-barrel

(Extended Fig 9).

Finally, to infer the origin and evolutionary history of the Mla system, we concatenated MlaD and MlaE sequences into a character supermatrix (538 leaves and 479 amino acid positions) and a maximum likelihood tree was inferred. Only markers found in a conserved cluster arrangement (MlaDEF and MlaDE) were used, due to the uncertainty of MlaF annotations. The resulting tree (Extended Fig 7) is consistent with the reference phylogeny of Bacteria. It shows, in fact, a clear separation between the Terrabacteria and the Gracilicutes (Ultra-Fast Bootstrap=95%), with monophyly of phyla within Terrabacteria -indicating vertical inheritance. Contrastingly, the evolutionary history of MlaDE within the Gracilicutes appears more complex, involving several duplications and horizontal transfer events (Extended Fig 7).

Together, our phylogenomic analyses strongly indicate that the majority of diderms may employ a transenvelope three-component Mla system for GPL homeostasis composed of MlaDEF, with the long version of MlaD, like the one present in V. parvula. The six-component Mla system characterised in Proteobacteria, containing MlaABC, may therefore represent an exception, rather than the rule. Moreover, we show that this core three-component Mla system is ancestral and was present in the diderm ancestor of all Bacteria. This system was then maintained in diderm Terrabacteria and many Gracilicutes, while it underwent progressive complexification in Proteobacteria with the acquisition of additional Mla components, forming the well-characterised Mla system that we recognise in E. coli today.

Discussion

In this work, we investigated a novel Mla system in the non-model diderm Firmicute Veillonella parvula, consisting only of MlaEFD, with no homologues of the OM lipoprotein MlaA or the periplasmic chaperone MlaC. This minimal architecture is consistent with our recent in silico studies suggesting that OM biogenesis systems present in V. parvula ressemble streamlined versions of those typically seen in Proteobacterial models. For example, the V. parvula genome encodes a homologue of BamA, but none of the associated OM lipoproteins, BamB-E; a gene encoding TamB is present, but no TamA [START_REF] Antunes | Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the Firmicutes[END_REF][START_REF] Taib | Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition[END_REF] . These bioinformatic findings spark a fundamental question: are these 'missing' components replaced by non-homologous proteins? Or do these systems function without them?

Our results show that MlaEFD likely perform a similar role in V. parvula as has been described in model diderms; deletion of these genes results in a loss of OM lipid asymmetry, indicated by a detergent hypersensitivity and hypervesiculation phenotype. Based on the enrichment of phosphatidylethanolamine (PE) in lipid extracts from ∆mla OMVs, we suggest the regular role of MlaEFD in V. parvula is to perform retrograde PE trafficking. We also determined that ~15% of each lipid species present in these bacteria are in their plasmalogen form, therefore it is likely that the system shuttles plasmenylethanolamine (PlsPE) as well as typical diacyl PE.

Structural modelling and subcellular localisation helped us to understand how just three mla genes may perform the role of GPL trafficking in V. parvula; MlaEFD likely form a minimal, periplasm-spanning complex, removing the requirement for the 'missing' MlaABC components by directly connecting the IM and OM with a transenvelope bridge. However, future work is required to understand the stoichiometry of this protein complex, and to further explore the structure of MCE proteins across diverse bacterial lineages. Interestingly, MCE proteins in E. coli [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF] , A. baumannii [START_REF] Kamischke | The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane[END_REF] and -recently -M. tuberculosis [START_REF] Chen | Structure of an endogenous mycobacterial MCE lipid transporter[END_REF] have been shown to form both hetero-and homohexamers, and for two of these structures (PqiB and Mce1A-1F), the hexameric nature of the α-helical region results in a closed tunnel that spans the periplasm, with a hydrophobic interior. These studies suggest the tunnel structures could facilitate the transport of a hydrophobic substrate and, from homology, conservation and phenotypic characterisation, the proposed substrate is a glycerophospholipid (or in the case of M. tuberculosis, cholesterol). However, it is difficult to understand how such a structure could support the hydrophilic head of GPLs. As phospholipid trafficking systems remain elusive even in the extensively characterised model diderms, little comparison is currently available to understand if this MlaDVp tunnel is a realistic transporter of GPLs.

So far, the only structurally characterised protein that performs retrograde GPL trafficking is MlaC, the periplasmic chaperone that can shield the acyl chains of a phospholipid whilst the head portion remains solvent-exposed [START_REF] Ekiert | Architectures of Lipid Transport Systems for the Bacterial Outer Membrane[END_REF][START_REF] Huang | Molecular dynamic study of MlaC protein in Gram-negative bacteria: conformational flexibility, solvent effect and protein-phospholipid binding: Molecular Dynamic Study of MlaC Protein[END_REF] . This chaperone-based transport is a common theme across diderm OM biogenesis important for the putative lipid importer function across bacteria. However, the recently resolved structure of M. tuberculosis Mce1A-1F -which do not contain complete C-terminal β-barrels -shows that each monomer contributes a single β-strand, forming a six-stranded β-barrel as a heterohexamer.

Perhaps this structural organisation is repeated in other long MlaD sequences without complete barrel structures, suggesting a functional convergence across diverse bacterial lineages. The function of these barrels remains to be determined, and may differ across species; perhaps this domain is required for stability or anchoring in some bacteria, and for substrate passage in others.

Regardless of the role of the β-barrel domain, or whether the extended MlaD in V. parvula is capable of transporting GPLs via a winding fairground slide or a direct tunnel, the implications are the same: this mechanism is one of higher throughput than a chaperone system. Indeed, this is an important element to consider in the argument about the directionality of the Mla system. Whilst a wealth of studies support the function of Mla in retrograde GPL trafficking [START_REF] Malinverni | An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane[END_REF][START_REF] Tang | Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB[END_REF][START_REF] Powers | The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport[END_REF][START_REF] Low | ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry[END_REF] , and several studies argue for the anterograde direction [START_REF] Kamischke | The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane[END_REF][START_REF] Hughes | Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system[END_REF][START_REF] Ercan | Characterization of Interactions and Phospholipid Transfer between Substrate Binding Proteins of the OmpC-Mla System[END_REF][START_REF] Mann | Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii[END_REF] , efficiency is a key factor to consider. In the case of anterograde GPL trafficking to facilitate cell growth, GPL flow would have to be enormously high throughput, considering the sheer volume of GPLs required to build a bilayer. In these cases, large periplasmspanning AsmA-like proteins, which can transport several GPLs at once, would likely facilitate the high flow rate required. On the other hand, a chaperone-based system which can only offer a 1:1 ratio of protein:GPL could not accommodate this high flow rate, and likely stands as a fine-tuning mechanism -which fits with the proposed function of the Mla system, i.e. to remove surface-exposed GPLs from the OM to restore lipid asymmetry. This idea is mirrored by the Lpt system that has evolved to transport LPS into the outer leaflet of the OM, which requires a trans-envelope bridge -and not singular protein chaperones. We do not yet understand where the long and likely transenvelope MlaD fits into these scenarios; as MCE proteins are primarily implicated in importer functions, would long MlaD provide high-throughput retrograde GPL trafficking? Or could this process be bi-directional with the same transporter? Perhaps sites of hemifusion, along with accessory proteins, are necessary to control and facilitate the functioning of these trans-envelope structures, as has been suggested for the functioning of YhdP in an mlaA* background 5 . Intriguingly, as the long MlaD version is widely distributed across Bacteria, whatever role this protein performs is likely common across a wide diversity of lineages, waiting to be uncovered.

Furthering our understanding of GPL homeostasis in V. parvula is the discovery that TamB is a major suppressor of the ∆mla phenotype. Given the recent implications of AsmA-like proteins in anterograde GPL trafficking [3][START_REF] Douglass | Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria[END_REF][START_REF] Grimm | The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli[END_REF] , it is very interesting to discover that this protein likely performs a similar role in V. parvula. These findings, in combination with the recent literature, strongly advocate for contrasting but similar roles of TamB and MlaEFD in maintaining the OM in V. parvula, performing anterograde and retrograde GPL trafficking respectively. However, as the double ∆mlaD∆tamB mutant is viable, we propose that there are as yet undiscovered systems that perform lipid trafficking in V. parvula.

To conclude, our results reinforce the notion that the paradigms set by extensive studies within the Proteobacteria can be challenged by using non-model and phylogenetically distant bacteria. Research within diderm models such as E. coli, A. baumannii and N. meningitidis is crucial for our understanding of the functioning and virulence of many pathogens, enabling us to expand research into effective treatment options and aid identification of novel targets. However, learnings from fundamental microbiology -including those from commensals such as V. parvula -are often transferable, and may help us fill in the gaps regarding the structure, function and diversity of OM biogenesis and maintenance systems across the Bacterial tree of life.

MATERIAL AND METHODS

Bacterial strains and growth conditions

Veillonella parvula SKV38 was grown in SK medium (10 g/L tryptone [Difco], 10 g/L yeast extract [Difco], 0.4 g/L disodium phosphate, 2 g/L sodium chloride, and 10 ml/L 60% [wt/vol] sodium DLlactate; described in [START_REF] Knapp | Natural Competence Is Common among Clinical Isolates of Veillonella parvula and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome[END_REF] . Cultures were incubated at 37°C in anaerobic conditions, either in anaerobic bags (GENbag anaero; bioMérieux no. 45534) or in a C400M Ruskinn anaerobic-microaerophilic station. Escherichia coli was grown in lysogeny broth (LB) (Corning) medium under aerobic conditions at 37°C. Where required, V. parvula cultures were supplemented with 20 mg/L chloramphenicol (Cm), 200 mg/L erythromycin (Ery), or 2.5 mg/L tetracycline (Tc), and E. coli cultures were supplemented with 100 mg/L ampicillin (Amp). 100 μg/L anhydrotetracycline (aTc) was added to induce the Ptet promoter and 1mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce the T7 promoter.

All chemicals were purchased from Sigma-Aldrich unless stated otherwise. All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Primers and strains used in this chapter are listed in Supplementary Tables S2-3.

Generation & Screening of Transposon mutagenesis library

A random transposon mutagenesis library was generated in V. parvula SKV38 WT, or the ∆mlaD mutant, using pRPF215, a plasmid previously used in Clostridium difficile that contains an aTcinducible transposase and mariner-based transposon (Addgene 106377) [START_REF] Dembek | High-throughput analysis of gene essentiality and sporulation in Clostridium difficile[END_REF] . pRPF215 was transformed into V. parvula SKV38 by natural transformation and selected on SK agar supplemented with Cm (20μg/mL). Several independent overnight cultures of V. parvula SKV38-pRPF215 were diluted to OD600 = 0.1 in SK medium supplemented with aTc (100ng/μL) and incubated for 5 h to induce the transposase. Following induction, cultures were diluted and plated onto SK supplemented with erythromycin (200ug/μL) and aTc (100ng/μL) for selection, then incubated in anaerobic conditions for 48h. Resultant colonies were used to inoculate Greiner Bio-one polystyrene flat-bottom 96-well plates (655101) in SK supplemented with either Ery and aTc or Cm to confirm both the presence of the transposon and the loss of pRPF215. After 24h incubation, aliquots were taken from each well to inoculate fresh 96-well plates filled with SK and SK supplemented with detergents and antibiotics for the screening process. Original transposon mutagenesis plates were stored in 15% glycerol at -80°C, whilst the screening plates were incubated for 24h then assessed for growth and OM permeability defects. Mutants of interest were tested for stability of phenotype, then harvested for genomic DNA using the Wizard® Genomic DNA Purification Kit (Promega). Genomic DNA was sent for wholegenome sequencing at the Mutualized Platform for Microbiology at Institut Pasteur.

Efficiency of plating / serial dilution assays

Sensibility to different detergents was performed on SK agar plates supplemented with detergents and antibiotics at different concentrations. Serial dilutions of strains (10 0 -10 -5 ) were inoculated with a starting OD600nm = 0.5. Plates were incubated in anaerobic conditions for 48h and cfu were calculated.

Chromosomal Mutagenesis by Allelic Exchange

To generate deletion strains of V. parvula SKV38, site-directed mutagenesis was performed as previously described [START_REF] Knapp | Natural Competence Is Common among Clinical Isolates of Veillonella parvula and Is Useful for Genetic Manipulation of This Key Member of the Oral Microbiome[END_REF] . Briefly, 1kb regions upstream and downstream of the target sequence were PCR amplified using Phusion Flash high-fidelity PCR master mix (Thermo Scientific, F548). For the selection process, three main resistance cassettes were PCR amplified with overlapping primers for the upstream and downstream regions of the target gene: the V. atypica tetracycline resistance cassette (tetM in pBSJL2), the C. difficile chloramphenicol resistance cassette (catP in pRPF185; Addgene 106367 [START_REF] Fagan | Clostridium difficile has two parallel and essential Sec secretion systems[END_REF] and the Saccharopolyspora erythraea erythromycin resistance cassette (ermE). PCR products were ligated to generate deletion cassettes via Gibson cloning or used as templates in a second PCR reaction to generate linear dsDNA with the resistance cassette flanked by the upstream and downstream sequences. These deletion constructs were transformed into V. parvula by natural transformation (detailed below), and its genomic integration was selected by plating on tetracycline, chloramphenicol or erythromycin-supplemented medium. Positive candidates were further confirmed by PCR and sequencing.

Natural Transformation

Recipient strains were grown on SK agar for 48h in anaerobic conditions at 37°C. Biomass was resuspended in 1 ml SK medium adjusted to OD600 = 0.4 -0.8. 10 μL aliquots were spotted onto SK agar, then 0.5 -1 μg plasmid or 75 -200 ng/μL linear double-stranded DNA (dsDNA) PCR product was added, using distilled water as a negative control. Following 48h incubation, the biomass was resuspended fresh SK medium, plated onto SK agar supplemented with the relevant antibiotic, and incubated for a further 48h. Colonies were streaked onto fresh selective plates, and correct integration of the construct was confirmed by PCR and sequencing.

Growth kinetics

Overnight cultures were diluted to 0.05 OD600 in 200 μL SK that had previously been incubated in anaerobic conditions overnight to remove dissolved oxygen in Greiner flat-bottom 96-well plates. To maintain anaerobic conditions, a plastic adhesive film (adhesive sealing sheet, Thermo Scientific, AB0558) was used to seal the plate whilst inside the anaerobic station. The sealed plates were then incubated in a TECAN Infinite M200 Pro spectrophotometer for 24h at 37°C. OD600 was measured every 30m after 900 seconds orbital shaking of 2 mm amplitude.

LPS silver staining (AgNO3)

Sample Preparation

Overnight cultures were concentrated to OD600 = 10 in Tricine Sample Buffer (BioRad 1610739: 200 mM Tris-HCl, pH 6.8, 40% glycerol, 2% SDS, 0.04% Coomassie Blue) and incubated at 100°C for 10min. After cooling to room temperature, Proteinase K was added to a final concentration of 1mg/mL and incubated at 37°C for 1h. 10 μL of samples were loaded onto tris-tricine gels (BioRad 4563063: 16.5% Mini-PROTEAN® Tris-Tricine Gel) and migrated in TTS buffer (BioRad 161-0744: 100 mM Tris-HCl pH 8.3, 100 mM Tricine, 0.1% SDS) for 2h at 50mA.

Staining

To visualise LPS profiles after gel electrophoresis separation, all development solutions were prepared immediately before use. Gels were fixed in ethanol 30%, acetic acid 10% for 1h, then washed 3 times in dH2O. Gels were incubated for 10m with metaperiodate 0.7%, again washed 3 times in dH2O, and incubated in thiosulfate 0.02% for 1m. Following a further 3 washes in dH2O, gels were incubated in AgNO3 25mM for 10-15m, washed in dH2O for 15s, then incubated in development solution (K2CO3 35mg/ml, formaldehyde 0.03% (w/v), thiosulfate 0.00125%) until bands appeared. To stop the reaction, gels were finally incubated in 4% Tris Base, 2% acetic acid for 30min, then imaged.

Spontaneous suppressor selection

Spontaneous suppressor mutations were selected by passaging overnight cultures of ∆mlaEFD and ∆mlaD, either in liquid media or on agar, supplemented with SDS 0.004% and EDTA 312.5 μM, until growth occurred. Colonies were restreaked onto the same SDS / EDTA concentration, or directly grown in a liquid culture overnight, then genomic DNA was harvested and sent for whole genome sequencing to identify the corresponding SNPs that enabled growth of the mutants in SDS / EDTA.

Lipid extraction and Thin Layer Chromatography (TLC)

Sample Preparation (Lipid Extraction)

To extract lipids, typical Bligh-Dyer extraction methods were used: briefly, overnight cultures were pelleted and concentrated to OD600 = 15 in 1mL of distilled water, then incubated on ice for 5-10min.

Using glass pipettes, a monophasic mixture of 4mL methanol and 2mL chloroform were added to the resuspended cells and vortexed vigorously for 5min. Following a further 10min incubation on ice, 4ml chloroform and 2ml of distilled water were added and samples were vortexed before centrifugation at 1500 x g for 5min. This centrifugation step allowed separation of the two phases: the upper water phase, containing solutes, ions, sugars, DNA and RNA, and the lower chloroform phase, containing the lipids.

The upper phase was discarded then 1mL sodium chloride solution (0.5M) was added and the samples were vortexed. Following a second centrifugation at 1500 x g for 5min, the lower phase was carefully extracted using glass micropipettes and dried in a vacuum centrifuge or under a flow of nitrogen gas (N2). If the initial purity was not satisfactory, a secondary sodium chloride step (and centrifugation) could be repeated. Dried lipid extracts were resuspended in 30μL chloroform.

Thin Layer Chromatography (TLC) and Staining

To visualise lipids, TLC plates (Silica gel 60, non-fluorescent, 0.25mm thick, Merck Millipore) were cut to a width that allowed ~1cm between each lipid sample. Plates were pre-run in acetone before use and dried at 100°C for 30min. 10µL of each sample was loaded with 5µL glass microcapillary tubes (Sigma Aldrich) 1cm from the bottom of the TLC plate. The plate was then placed in a solvent system optimised for the separation of phospholipids by head group polarity: 65:25:4 (v/v/v) or 80:15:2.5 chloroform/methanol/water. For separation of lipids from OMVs, we used the ratio of 80:20:2.5. When the solvent front reached ~2cm from the top of the plate, the plate was removed from the solvent system and dried at 100°C for 30m or air-dried for 10min. To reveal phospholipid species, the dried plate was incubated in a glass tank saturated with iodine vapour from iodine crystals until lipid spots were clearly visible (~5min). After imaging this stained plate, the iodine was allowed to decolourise and the same plate was re-stained with phosphomolybdic acid (PMA, 10%), followed by vigorous heating to develop spots, or Schiff reagent (Sigma-Aldrich). Amines were detected by spraying TLC plates with 0.2g Ninhydrin (Sigma-Aldrich) dissolved in 100ml ethanol, followed by vigorous heating to develop spots.

For the relative quantification of each lipid class, ImageJ was used.

Lipid identification

Nuclear Magnetic Resonance (NMR)

After labile proton to deuteron exchange with CD3OD, phospholipids were dried under vacuum and dissolved in a mixture of CDCl3/CD3OD (2:1, v/v). The samples were then introduced into a 3mm NMR tube. Solution NMR recordings were conducted at 10 or 20 °C on Bruker AVANCE NEO 400 and 900 spectrometers equipped with a 5mm TBI and 5mm cryo-TCI probe, respectively. Standard experiments

were run: 1D-1 H, 2D-1 H-COSY, 2D-1 H-TOCSY, 2D-1 H-ROESY, 2D-1 H-13 C-HSQC-DEPT, 2D-1 H-

Mass spectrometry (MS)

MALDI-QIT-TOF Shimadzu AXIMA Resonance mass spectrometer (Shimadzu Europe, Manchester, UK) in the positive mode was used to identify the different lipids. All lipids were dissolved in CHCl3/CH3OH (1:1, v/v) and mixed with the same volume (10µL) of 2,4,6-trihydroxyacetophenone (THAP) dissolved in methanol.

Outer Membrane Vesicle (OMV) Extraction and Purification

Overnight cultures were used to generate 1 litre cultures of the relevant V. parvula strains (WT SKV38 and ∆mla mutants). Bacterial cells were pelleted by centrifugation at 5000 x g for 10min, and supernatants were passed through 0.45µm and 0.22µm pore-size filters to remove any leftover cells or debris (Sarstedt). The filtered supernatant was then concentrated using Centricon Plus-70 centrifugal filters (100kDa cutoff, Millipore) centrifuged at 3,500 x g 4°C for 30min, 50mL at a time. ~3mL of concentrated OMV solution was purified from 1 litre of supernatant. This OMV solution was then pelleted by ultracentrifugation at 100,000 x g for 3h at 4°C (Beckman Coulter Optima L-80 XP Ultracentrifuge, Type 50.2 Ti rotor). The mass of the OMV pellets was recorded and used for subsequent lipid extraction and analysis via Thin Layer Chromatography (TLC). Purified OMVs were analysed via NanoFCM confirming an average size of ~60 nm, matching the average size of vesicles in whole cell cultures observed via both NanoFCM and cryo-electron tomography (Supplementary Fig

S5).

OMV quantification

Overnight cultures were adjusted for OD600 and diluted 1/25 and 1/50 in filtered PBS solution. The size distribution and particle concentration within the samples were analysed by nano-flow cytometry (nFCM) (NanoFCM, Inc., Xiamen, China). First, the instrument was calibrated for particle concentration using 200 nm PE and AF488 fluorophore-conjugated polystyrene beads, and for size distribution using Silica Nanosphere Cocktail (NanoFCM, Inc., S16M-Exo and S17M-MV). The flow rate and side scattering intensity were then calculated based on the calibration curve, and used to infer the size and concentration of the large and small events present in each sample using the NanoFCM software (NanoFCM Profession V2.0). All samples were diluted to ensure the particle count fell within the optimal range of 2000-12,000/min, and all particles that passed by the detector during time intervals of 60s were recorded for each test.

Membrane fractionation by sucrose gradient sedimentation

1.5L overnight cultures were pelleted at 5,000 x g for 15min and resuspended in ~10mL HEPES buffer.

Resuspended pellets were supplemented with 100µL benzonase and a small amount of lysozyme prior to lysis by two passages through a high-pressure French press (French Press G-M, Glen Mills) homogenizer at 20,000 psi. Following lysis, cell debris was pelleted at 15,000 x g for 90min. Resulting supernatant was spun in an ultracentrifuge (Beckman Coulter Optima L-80 XP Ultracentrifuge, Type 50.2 Ti rotor) at 35,000 x g for 90min to obtain the membrane pellet, containing both IM and OM vesicles. Membrane pellets were resuspended in 750µl of HEPES buffer using a Dounce homogeniser, then 500µL was loaded onto a discontinuous sucrose gradient comprising a bottom 2mL layer of 51%, a 3ml layer of 37%, a 4mL layer of 31%, and a 2.5mL top layer of 23% (w/w) sucrose. Sucrose gradient columns were centrifuged at 35,000 x g for 40h (Beckman Coulter Optima L-80 XP Ultracentrifuge, Type SW41 Ti rotor). At the end of the sucrose gradient sedimentation, 1mL by 1mL was extracted from the top of the gradient column to the bottom, resulting in 1ml membrane fractions.

Structural modelling

For the prediction of protein domains, SMART (Simple Modular Architecture Research Tool) [START_REF] Letunic | SMART: recent updates, new developments and status in 2020[END_REF] and I-TASSER [START_REF] Zhang | Fully automated protein structure prediction in CASP8[END_REF] were used. AlphaFold2 29 was used to generate structural predictions of the Mla proteins in V. parvula. To specifically analyse the presence of β-barrels within our target sequences, the BOCTOPUS2 database was used [START_REF] Hayat | Inclusion of dyad-repeat pattern improves topology prediction of transmembrane β-barrel proteins[END_REF] . As another transmembrane site-determining web-based tool, TMHMM 2.0 (Transmembrane Hidden Markov Model) was also used to analyse the transmembrane sites of proteins [START_REF] Krogh | Predicting transmembrane protein topology with a hidden markov model: application to complete genomes11Edited by F. Cohen[END_REF] .

All structure predictions with AlphaFold2 were performed through the ColabFold 53 implementation.

Output models of monomeric full-length MlaD (5 models) were ranked according to the pLDDT score.

MlaEFD1-130 predictions (10 models) were obtained without structural templates and sorted by predicted TM-scores (pTM) since it predates the release of AlphaFold-multimer [START_REF] Evans | Protein complex prediction with AlphaFold-Multimer[END_REF] .MCE, α-helical and β-barrel domains in Chimera [START_REF] Pettersen | UCSF Chimera?A visualization system for exploratory research and analysis[END_REF] and later refined with Modeller 56 to correct the topology of the rotated hinges.

MlaEFD1-130 predictions (10 models) were obtained without structural templates and sorted by predicted TM-scores (pTM) since it predates the release of AlphaFold-multimer 54 . To impose the 2:2:6 stoichiometry of the complex, 2 copies of the MlaE and MlaF sequences were concatenated together with 6 copies of the MlaD1-130 sequence. Model of the hexameric full-length MlaD complex was obtained by stitching together predictions for the MlaD1-130, MlaD36-263 and MlaD239-419 domains, via structural superimposition of the overlapping regions. Predictions for the MlaD36-263 and MlaD239-419 domains were first obtained using the AlphaFold2-multimer v2.3 parameters with templates found in the PDB70 database [START_REF] Steinegger | HH-suite3 for fast remote homology detection and deep protein annotation[END_REF] and ranked by multimer score [START_REF] Evans | Protein complex prediction with AlphaFold-Multimer[END_REF] .

Full-length monomeric models of long MlaD sequences were obtained with AlphaFold2 as described above. Secondary structure class of all positions in the best model (highest plDDT) for each sequence were obtained using DSSP [START_REF] Kabsch | Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features[END_REF] and PROSS [START_REF] Srinivasan | A physical basis for protein secondary structure[END_REF] . To automatically assess the presence of residues in β-strand conformation after the MCE domain, the start and end positions of the MCE domain in each sequence was obtained using HMMSEARCH 60 with the "MlaD protein" pfam profile (PF02470).

Phylogenetic Analysis

We assembled a databank of 1,083 genomes representing all bacterial phyla present at the National Center for Biotechnology (NCBI). We selected three species per order for each phylum. The number of genomes per phylum therefore reflects their taxonomical diversity (Extended Data Table 2). We chose preferably genomes from reference species and the most complete assemblies. We then queried this databank for the presence of MlaA, MlaB, MlaC, MlaD, MlaE, MlaF, and TamB, using the pfam domains PF04333, PF05494, PF02470, PF02405 and PF04357 respectively. The alignments of protein families NF033618 (MlaB) and PRK11831 (MlaF) were downloaded from NCBI (Conserved Domain Database) and used to build HMM profiles using HMMBUILD from the HMMER3.3.2 package [START_REF] Johnson | Hidden Markov model speed heuristic and iterative HMM search procedure[END_REF] MlaA, MlaC, MlaD, MlaE and TamB homologs were searched using HMMSEARCH from the HMMER3.3.2 package. As MlaB and MlaF belong to large protein families, we used MacSyFinder2 31 to identify their occurrences when they are in genomic synteny with at least one of the other components (mlaACDE) of the Mla system, with no more than five genes separating them. This also allowed the identification of the occurrences of mlaACDE when they were in genomic cluster. All the retrieved hits were curated using functional annotations, domains organization, alignments, and phylogeny.

We identified 1119 MCE-domain containing sequences (Extended Data Table 2). Among these, homologues of PqiB and LetB (containing more than one MCE domain) were identified only within Proteobacteria and closely related phyla.

For each protein, curated sequences were aligned with MAFFT v7.407 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] with the L-INS-I option and trimmed using BMGE-1.12 62 with the BLOSUM30 substitution matrix. Maximum likelihood trees were generated for each protein using IQ-TREE.2.0.6 [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] with the best evolutionary model assessed with ModelFinder 64 according to the Bayesian Information Criterion, and ultrafast bootstrap supports computed on 1000 replicates of the original dataset. Finally, the presence or absence of each protein was mapped onto a reference tree of Bacteria taken from [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] . All tree figures were generated using custom made scripts and iTOL [START_REF] Letunic | Thermoleophilum album~29539_Talbum@S EH10484.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL54395.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL60232.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL58372.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL59947.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae[END_REF] .

STATISTICS AND REPRODUCIBILITY

Data are presented as the mean ± standard deviation (SD) and with individual data points from at least three independent biological experiments. Statistical analyses were performed using Prism 9.5.0 (GraphPad Software Inc.). The distribution of data was analysed by Shapiro-Wilk test and their variance using Bartlett test. Since some of the data generated did not follow a normal distribution or had two high variance we performed non-parametric two-tailed Mann-Whitney test. P-values <0.05 were defined as the level of statistical significance.

DATA AVAILABILITY STATEMENT

The authors declare that all data supporting the findings of this study are either available within the paper and its supplementary information files or, otherwise, are available from the corresponding author upon request. produced by the WT, ∆mlaD, ∆mlaEFD and complemented strains were quantified via NanoFCM (see methods) and used to generate a ratio of hypervesiculation. The WT produces a similar number of vesicles to cells, resulting in a ~1:1 ratio, whilst the ∆mla deletion strains produce ~17-fold more vesicles than the WT. This phenotype can be significantly rescued, reducing vesicle production to ~5fold in the ∆mlaEFD::mlaEFD strain. 5 biological replicates and 3 technical replicates were tested per strain; n = 5; significance calculated by Mann Whitney U, p = 0.0079; two-tailed p-value. C) Enrichment of phosphatidylethanolamine (PE) in OMVs produced by ∆mlaD. Thin layer chromatography (TLC) of lipid extracts from OMVs produced by both the WT and ∆mlaD strains show a ~40% (+/-16%) enrichment for PE in the vesicles produced by the ∆mla mutants when stained with iodine vapour (labelled +++, quantified by ImageJ over 3 biological replicates). We also notice a decrease in the abundance of another as yet unidentified lipid species, highlighted in the black box.

MAIN TEXT FIGURES + LEGENDS

Schiff reagent staining shows a ~130% increase in the abundance of plasmenyl PE in ∆mlaD OMV lipid extracts. 5A4, 5B5, 27C1 and 90E10. B) Suppressor mutations identified in tamB. From both spontaneous suppressor analysis, and random Tn insertion suppression analysis, we identified mutations in a homologue of tamB. The two identical Tn insertions were at K925, whilst the SNP in the spontaneous suppressor was mapped to Q1329*, generating a STOP codon (CAA à TAA). C) OM permeability of WT, ∆tamB and ∆mla mutants. Clean deletion mutants were generated for ∆tamB, ∆mlaD and ∆mlaD∆tamB, then overnight cultures of these strains and the WT were serially diluted onto SDS / EDTA to assess OM permeability. ∆tamB does not display a hypersensitivity to SDS 0.004% / EDTA 312.5µM, whilst ∆mlaD is almost unable to grow at this concentration. Deletion of tamB in this ∆mlaD background partially rescues the detergent hypersensitivity of ∆mlaD. D) Outer Membrane Vesicle (OMV) production of WT, ∆tamB and ∆mla mutants. OMV production of WT, ∆tamB, ∆mlaD and ∆mlaD∆tamB strains were analysed via NanoFCM quantification. At least 5 biological replicates and 2-3 technical replicates were tested per strain. Whilst ∆mlaD and ∆tamB display hypervesiculation, the double mutant (∆mlaD∆tamB) shows a significant reduction in OMV production as compared to ∆mlaD; n = 5; significance calculated by Mann Whitney U, p = 0.0079; two-tailed p-value. This reduction in hypervesiculation is non-significant between ∆tamB and ∆mlaD∆tamB (p = 0.0952). WT and ∆mlaD OMV production data previously shown in The distribution of all six Mla components and of LolA as a proxy for the Lol system was mapped onto a reference phylogeny of Bacteria taken from [START_REF] Witwinowski | An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition[END_REF] (for a more detailed version of the tree, see Extended Fig 6). In the inner ring of the tree, diderms are represented by light blue and yellow, whilst the monoderms are colored in grey. On the outer ring of the tree, each colored block corresponds to the presence or absence of the six Mla components, A-F, and of LolA. Two purple blocks are used to represent MlaD; the first block represents presence / absence of the gene itself, and the second block represents the presence / absence of the β-barrel domain. From this tree, we can see MlaEFD are widely distributed across the bacteria kingdom and were likely present in the LBCA. In contrast, MlaABC are sparsely distributed and restricted mostly to the Proteobacteria, suggesting these components evolved later (and notably the lipoprotein MlaA with the emergence of the Lol system).

We also see that, in general, MlaD versions with a β-barrel tend to co-occur with the absence of MlaA and MlaC. (Yellow / Grey = Terrabacteria; Blue = Gracilicutes). Tree generated using custom made scripts and iTOL [START_REF] Letunic | Thermoleophilum album~29539_Talbum@S EH10484.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL54395.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL60232.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL58372.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae; Conexibacter; NA; Conexibacter sp. Seoho-28~2133960_Csp@ PTL59947.1| Bacteria; Actinobacteria; Thermoleophilia; Solirubrobacterales; Conexibacteraceae[END_REF] . For detailed species names, see Extended Fig 6.

Extended Data

Extended Figure 1: Conserved synteny of mla genes with tamB and tolC in the Negativicutes, and presence of mla genes in Limnochordia Within the Negativicutes, the three mla homologues, mlaEFD, are directly followed by a homologue of tolC and a homologue of tamB. This synteny is not conserved within the other diderm Firmicutes; no mla homologues were identified in the Halanaerobiales, whilst mla genes in the Limnochordia are spare and not in synteny with tolC and tamB homologues.

Extended Figure 2: Phenotypes unchanged by deletion of mla genes. A) Comparison of growth kinetics of WT and ∆mla mutants. All strains were assessed for growth kinetics via OD600 measurements via TECAN spectrophotometer (see methods). ∆mlaD, ∆mlaF and ∆mlaEFD were found to display similar growth rates to the WT, with no defects. At least 3 biological replicates and 3 technical replicates were performed per strain. B) Comparison of LPS profiles. LPS was extracted from WT and all ∆mla cultures, in triplicate, normalised by OD600, then observed with silver staining. All strains display similar LPS profiles, in both banding pattern and quantity. C) Comparison of autoaggregation capabilities. OD600 was recorded for WT and ∆mla strains once an hour for a period of seven hours. All strains display a similar decrease in OD600 over time, suggesting a similar capability of autoaggregation. At least 3 biological replicates and 3 technical replicates were performed per strain. D) Comparison of biofilm formation. Biofilm formation was assessed via crystal violet staining of biofilms formed over 24h in 96-well plates. ∆mla strains display a small but nonsignificant decrease in biofilm formation capability, as assessed by a decrease in fluorescence signal at A575nm. Due to variability in this technique, 10 biological replicates with 3 technical replicates each were tested per strain. Extended Figure 7: Phylogeny concatenation of MlaE with the three types of MlaD: short, long and long with β-barrel Sequences of MlaE and MlaD were concatenated into a character supermatrix (538 sequences and 479 amino acid positions) and a maximum likelihood tree was inferred. The length of MlaD sequence is indicated by the length of the purple bars, and the presence / absence of the β-barrel is depicted with a small purple at the end of the sequence. MlaE sequences are represented in pink. We clearly see from this analysis that the majority of MlaD sequences are long, and that the short MlaD sequences are restricted mostly to the Proteobacteria. We also see a clear separation between the Terrabacteria (yellow) and the Gracilicutes (blue), with monophyly of phyla within the Terrabacteria, indicating vertical inheritance of these Mla genes. Highlighted in the red box is the branch at which loss of this elongated form of MlaD seems to have occurred.

Extended

Extended Figure 8: Distribution of MCE-containing sequences across Bacteria

Maximum likelihood tree built from an alignment of 1119 sequences and 194 amino acid positions. The scale bar corresponds to the average number of substitutions per site. MlaD sequences are marked in green for Actinobacteria and C. Dormibacteraeota, yellow for other Terrabacteria and blue for Gracilicutes. We highlighted the short version of MlaD in purple, and PqiB and LetB sequences in red. The presence of a β-barrel is highlighted by a green bar at the outer ring of the tree.

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  µM EDTA. Efficiency of plating revealed that only four of these mutants could significantly rescue the ∆mlaD phenotype (Fig 3A), of which two were of particular interest: sequencing revealed two identical Tn insertions in the downstream homologue of tamB (Fig 3B).

  (Fig 5, Extended Fig 6, Extended Table

  and a few Limnochordia (Extended Fig 1), whilst they are completely absent from the Halanaerobiales (Extended Fig 6, Extended Table

  closely related phyla (Extended Fig 6). The β-barrel is completely absent in the MlaD from the PVC (Planctomycetes, Verrucomicrobia, Chlamydia) and FCB (Fibrobacteres, Chlorobi, Bacteroidetes) groups (Extended Fig 6, Extended Table

  [START_REF] Kamischke | The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane[END_REF] C-HSQC-TOCSY. After acquisition and phase correction, chemical shifts calibration on CHD2OD signals were performed for 1 H (3.31 ppm) and 13 C (49.29 ppm). The data were analysed using the TopSpin software (Bruker).

Figure 1 :

 1 Figure 1: Comparison of Mla operons in V. parvula and E. coli.

Figure 1 :

 1 Figure 1: Phenotypic characterisation of ∆mla mutants reveals detergent hypersensitivity and hypervesiculation.

Figure 2 :

 2 Figure 2: TamB is a suppressor of the ∆mlaD OM permeability and hypervesiculation phenotype.

  Fig 2 above.

Figure 3 :

 3 Figure 3: Structural modelling and experimental localisation of a transenvelope MlaD in V. parvula.

Figure 5 :

 5 Figure 5: Taxonomic distribution of MlaABCDEF and LolA across Tree of Bacteria.

Figure 3 :Extended Figure 5 :

 35 AlphaFold2 modelling of MlaD in V. parvula A) Predicted structure of full-length MlaD. When the whole sequence is processed by AlphaFold2, the TMD, MCE and β-barrel domain are of very high confidence, and the α-helical domain is of lower confidence. The confidence rating is indicated by Predicted Local Distance Different Test (pLDDT) colouring. B) Predicted Local Distance Difference Test (pLDDT) per position of full-length MlaD. Graphical depiction of the pLDDT per position of the full-length models shows low confidence for the α-helical region, and high confidence for the TMD, MCE and β-barrel domains. C) Predicted Aligned Error (PAE) of full-length MlaD. Graphical depiction of the PAE shows the confidence in the relative positioning of the domains. The relative positioning of main chain atoms within the TMD and MCE domains and within the connecting α-helical region are low error, suggesting the predicted local conformation of these individual domains to be likely. However, the high predicted alignment error of atoms across these domain regions indicates that the global conformation of full-length MlaD is likely inaccurate. D) Modelling of MlaEFD complex in V. parvula with a 2:2:6 stoichiometry. MlaEF modelled as dimers in complex with hexameric MlaD (residues 1-130) closely resemble the resolved structure of the MlaEFD complex in E. coli. E) pLDDT per position of MlaEFD in V. parvula shows high confidence with the exception of 2 pairs of TM helices. Extended Figure 4: The C-terminal domain of MlaD288-419 folds as a membrane β-barrel. A) Hydrophobicity potential of the MlaD C-terminal β-barrel surface. The bottom view is rotated 180°. Dotted lines represent the possible position of GPL polar heads when inserted in a lipid bilayer. B) Electrostatics potential of the MlaD C-terminal β-barrel surface. The bottom view is rotated 180°. C) Dimensions of the β-barrel (right) and radius profile (calculated using the program HOLE) along the Z-axis (membrane normal). The height of the barrel matches the thickness of a lipid bilayer. D) Position of side-chains of R310, R383 et R385 at the constriction point of the β-barrel. E) Rings of surface accessible negatively charged and aromatics residues at each side of the β-barrel. F) Weblogo of MlaD C-terminal β-barrel sequences coloured by hydrophobicity (Hydrophilic in blue, neutral in green and hydrophobic in black). G) Superimposition of the V. parvula MlaD C-terminal β-barrel model (red) onto the S. marcescens OmpF structure (light cyan). Its small diameter, compared to large porins such as OmpF, and very narrow inner radius (~1 Å) would likely prevent the passage of any small molecule. H) Superimposed ensembles of MlaD C-terminal β-barrel models for long MlaD sequences predicted with AlphaFold2. Helices are coloured in red and β-strands in yellow. Insertions in the barrel and C-terminal helical extensions found in some sequences are labelled. AlphaFold2 models for the alpha-helical domain of MlaD36-263 in hexameric configuration. A) Predicted Aligned error (PAE) for the 5 ranked models obtained with AlphaFold-Multimer v2.2. The regions corresponding to each chain in the hexamer (from A to G) are labeled. B) PAE between the first (A) and last (G) chain for the 5 ranked models obtained with AlphaFold-Multimer v2.2. The regions corresponding the MCE and helical domains are labeled. C) Models obtained AlphaFold-Multimer v2.2. colored by predicted local distance difference test (pLDDT). D) Predicted Aligned error (PAE) for the 5 ranked models obtained with AlphaFold-Multimer v2.3. The regions corresponding to each chain in the hexamer (from A to G) are labeled. E) PAE between the first (A) and last (G) chain for the 5 ranked models obtained with AlphaFold-Multimer v2.3. The regions corresponding the MCE and helical domains are labeled. F) Models obtained AlphaFold-Multimer v2.3 colored by predicted local distance difference test (pLDDT). Extended Figure 6: Taxonomic distribution of Mla components (and LolA) with species names This figure is adapted from main text Fig 5 to include detailed species names. All other aspects of the figure are the same. Presence of absence of Mla component indicated in red (MlaA), green (MlaB), blue (MlaC), purple (MlaD), pink (MlaE), turquoise (MlaF) and LolA is indicated in yellow. (Yellow / Grey = Terrabacteria; Blue = Gracilicutes). Tree generated using custom made scripts and iTOL 1 .

  ; Cytophagal es; Flammeovir gaceae; Marivirga; Marivirga tractuosa; Marivirga tractuosa DSM 4126~6438 67_Mtractuo sa@ADR20 102.1| Bacteria; Bacteroidet es; Cytophagia ; Cytophagal es; Bernardetia ceae; Bernardetia ; Bernardetia litoralis; Bernardetia litoralis DSM 6794~8800 71_Blitoralis @AFM0302 4.1| Bacteria; Bacteroidet es; Saprospiria ; Saprospiral es; NA; NA; NA; Saprospiral es bacterium~2 026790_Sb acterium@M AT55036.1| Bacteria; Bacteroidet es; Saprospiria ; Saprospiral es; Saprospirac eae; Saprospira; Saprospira grandis; Saprospira grandis str. Lewin~9842 62_Sgrandi s@AFC247 88.1| Bacteria; Bacteroidet es; Saprospiria ; Saprospiral es; Lewinellace ae; NA; NA; Lewinellace ae bacterium SD302~201 4804_Lbac terium@PH I21176.1| Bacteria; Bacteroidet es; Chitinophag ia; Chitinophag ales; Chitinophag aceae; Niabella; NA; Niabella ginsenosidi vorans~117 6587_Ngin senosidivor ans@ANH7 9845.ta; NA; NA; NA; Candidatus Cyclonatro num; NA; Candidatus Cyclonatro num proteinivoru m~145736 5_CCyclon atronum@A XI99323.1| Bacteria; Balneolaeo ta; Balneolia; NA; NA; NA; NA; Balneolia bacterium~2 094027_Bb acterium@ tes Order II. Incertae sedis; Rhodother maceae; NA; NA; Rhodother maceae bacterium RA~17793 82_Rbacte rium@ARA 92711.1| Bacteria; Bacteroide tes; NA; Bacteroide tes Order II. Incertae sedis; NA; NA; NA; Bacteroide tes bacterium UBA7368~1 950766_Bb acterium@ DKOG010 00029.1_4 | Bacteria; Bacteroide tes; NA; Bacteroide tes Order II. Incertae sedis; Rhodother maceae; Salinibacte r; Salinibacte r ruber; Salinibacte r ruber DSM 13855~309 807_Srube raceae; Salinivenu s; NA; Salinivenu s lutea~8904 26_Slutea @NCRC01 000005.division KSB1; NA; NA; NA; NA; NA; candidate division KSB1 bacterium division KSB1; NA; NA; NA; NA; NA; candidate division KSB1 bacterium onadetes; Gemmatim onadetes; Gemmatim onadales; Gemmatim onadacea e; Gemmatim onas; NA; Gemmatim onas phototroph ica~13792 70_Gphot otrophica@ AMW0632 6.

  ; Legionellaceae; Legionella; Legionella pneumophila; Legionella pneumophila subsp. pneumophila str. division KSB1; NA; NA; NA; NA; NA; candidate division KSB1 bacterium division KSB1; NA; NA; NA; NA; NA; candidate division KSB1 bacterium division KSB1; NA; NA; NA; NA; NA; candidate division KSB1 bacterium division KSB1; NA; NA; NA; NA; NA; candidate division KSB3

1 Extended Figure 9 :

 19 Presence of disordered β-structures in long MlaD sequences with no βbarrel A) Presence of β-structures in long MlaD sequences with no β-barrel. The start of the sequence represents the presence of the MCE domain, to align all long MlaD sequences. The total length of each sequence is represented by the grey shaded area. Later, in some of these long sequences, we see the presence of β-hairpins / sheets, but no real folded β-barrel domain. For comparison, we also show the ~100 sequences of long MlaD that do possess a predicted folded β-barrel. Predicted Local Distance Difference Test (pLDDT) values, corresponding to the confidence of these predictions, are coloured and labelled in the key above. B) AlphaFold modelling of long MlaD sequences with no β-barrel. As seen from these two predicted structures, these long MlaD sequences do not possess a βbarrel, but do possess unstructured, disordered regions at their C-termini that contain fragments of βhairpins / sheets.

Table S2 :

 S2 Primers used in this study.

	Supplementary Primer name DNA sequence
	tamB_3F	CTTCACTCGCCTGCATTTTACTAATGATACTCCCATACACATTATG
	tamB_3R	GTTGGTTTAGTGGCGAGCTCCG
	tamB_5F	GTATAGCCAGCTTGTACCTCAGC
	tamB_5R	ATTGTGTCCTGTGGATCCACGAGTATGGCTAGAATGTGTAAAC
	mlaD_3F_er	ATTATTTAACGGGAGGAAATAATGACTAACACATTTACTCTATATGAGTATGGA
	y	GGT
	mlaD_3R	AGTAACCATCTGGATCAC
	mlaD_5F	CGTGTATGATTATGGTGC
	mlaD_5R_er	GAGAATATTTTATATTTTTGTTCATTTATATATCTCCCATCGCAGATTCGTTAATC
	y	G
	ery_F_mod	ATGAACAAAAATATAAAATATTCTCAAAACTTTTTAACGAGTG
	ery_R_mod	TTATTTCCTCCCGTTAAATAATAGATAACTATTAAAAATAGACAATACTTG
	catP_F	GGCCTTTTGCTCACATGTTC
	catP_R	CCTGAAGTTAACTATTTATCAATTCCTGC
	tetM_F	AGTAAAATGCAGGCGAGTGAAG
	tetM_R	GTGGATCCACAGGACACAAT
	mlaE_5F	GCAGCTACAGCCTTATAC
	mlaE_5R	TCACTCGCCTGCATTTTACTCAGTGTCTCCCTTTCCC
	mlaD_pRPF 185OH_F	AGCGTTAACAGATCTGAGCTGTACAACAATTTATTCATGGTGAAGCAGAGC
	mlaD_pRPF	TCTCCTTTACTGCAGGAGCTTTAGAATACTTGTTTAATACCGAAATAGGTACCTC
	185OH_R	CA
	mlaE_pRPF 185OH_F	CGTTAACAGATCTGAGCTACTGTAAGGGAAAGGGAGACACTGTTG
	mlaD_F_pR PF185_2	CTCTAAGAAAGAAGGAATTCACATGAAGTGGACGACGGAGG
	MCE_R_lin
	k_pRPF185	TACCGCTGCCGCTACCACTATTGATAGACTGTAGCATCTTCTCAGTG
	_2
	MCE_R_pR PF185_2	CCAGGAGAGTTGTTGATCACTATTGATAGACTGTAGCATCTTCTCAGTG
	Barrel_F_O
	mpSP_
	pRPF185_2

ACKNOWLEDGEMENTS

We gratefully thank Noa Guzzi, and members of the UGB and EBMC units, for their assistance in generating the transposon mutant libraries used in this study. We also thank Robert Smith for his help

We also thank Philippe Delepelaire for generously providing us with anti-SecA and anti-TolC serum, and Jean-Michel Betton for his help in developing the membrane fractionation protocol. This work was supported by funding from the French National Research Agency (ANR) (Fir-OM ANR-16-CE12-0010) and (OM-LipAsy-CE44-008), by the French government's Investissement d'Avenir Program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID) and by the Fondation pour la Recherche Médicale (grant DEQ20180339185). KG was supported by the Pasteur Paris University program (PPU). B-Beaud was supported by a MENESR (Ministère

COMPETING INTEREST STATEMENT

All the authors declare that they have no competing interests. The first number is the average of all i,i+1 pairs of chains except between the 1 st and the last chain (i being the index of the chain in the hexamer). The second number (in bold) is between the 1 st and the last chain in the hexamer.

Extended Table 1: Statistics of AF2-Multimer models generated for MlaD36-263 hexamers

# Number of residue pairs with a Predicted Aligned Error (PAE) < 8 Å for the helical regions (MlaD132-263) between neighboring chains. The first number is the average of all i,i+1 pairs of chains except between the 1 st and the last chain (i being the index of the chain in the hexamer). The second number (in bold) is between the 1 st and the last chain in the hexamer.

Extended Table 2: Distribution and accession numbers of Mla components homologs among 1083 genomes representing the bacterial diversity.

The presence of MlaB and MlaF is only indicated when they are in cluster with at least one of the other components of the Mla system.

Supplementary Figure S4: OM permeability phenotypes of ∆mlaD, ∆tamB and ∆mlaD∆tamB strains

A) Overnight cultures of WT, ∆mlaD, ∆tamB, ∆mlaD∆tamB and ∆mlaD::mlaD were plated onto SDS / EDTA and vancomycin to assess OM permeability. At this concentration of SDS (0.003%) and EDTA (312.5µM), the rescue of detergent hypersensitivity of ∆mlaD by deletion of tamB is striking, and similar to the rescue obtained from reintroducing the expression of mlaD itself. ∆tamB strains are also highly sensitive to vancomycin. B) Cultures of WT, ∆mlaD, ∆tamB and 4 independent biological replicates of the double ∆mlaD∆tamB mutant were plated onto SDS 0.004% / EDTA 312.5µM and vancomycin 250µg/ml. The slight increase in vancomycin sensitivity of the ∆tamB can be observed as compared to the WT, which is in direct contrast to the high resistance of ∆mlaD to vancomycin.

Supplementary Figure S5: Average size of outer membrane vesicles (OMVs) is consistent across whole cell cultures and purified OMV samples.

A) Cryo-electron tomography images of ∆mlaD with outer membrane vesicles (OMVs). Cultures of WT and ∆mlaD strains were observed via cryo-electron tomography (cryo-ET). In all tomograms of ∆mlaD, outer membrane vesicles (OMVs) were observed either attached to the cell or externally (highlighted by white arrows). These vesicles were on average ~60nm in diameter, matching the average size observed of these vesicles (present both in whole-cell cultures and in purified samples) via NanoFCM. Scale bar = 100nm. B) NanoFCM report showing size distribution of purified OMV sample. OMVs were purified from large-scale supernatants of WT and ∆mlaD cultures. Prior to concentration of these vesicles via ultracentrifugation, a small sample was extracted and processed via NanoFCM, confirming the average size of OMVs is ~60nm, matching the average size of OMVs present in full cell cultures of WT and ∆mlaD strains, and also the average size observed via cryo-ET.

Supplementary Table S3: Strains and plasmids used in this study.