Introduction

The bacterial cell envelope is a highly complex compartment and protective barrier against the external environment, ensuring survival in response to chemical threats while maintaining individual cellular identity and communication of signals within communities. In Gram-negative species, including clinical pathogens such as Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and Helicobacter pylori, the envelopes are characterised by an organisation of two lipid bi-layer membranes and an interstitial partition, referred to as the periplasm, that encompasses a peptidoglycan sacculus conferring mechanical robustness (Guest and Silhavy, 2023; Gumbart et al., 2021). The envelope is home to a diversity of dedicated machineries that selectively move molecules across the barrier, facilitated by energy transduction. Harmful compounds such as antibiotics and bactericidal agents as well as effector proteins are displaced through this barrier by energy-dependent machines, and representative and well-characterised examples are the tripartite assemblies AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli, which are powered respectively by proton motive force and ATP binding and hydrolysis (Du et al., 2018). The AcrA-AcrB-TolC assembly drives efflux of chemically diverse compounds, and MacA-MacB-TolC can transport macrolides, peptide toxins and host antibacterial peptides (Honeycutt et al., 2020; Zhang et al., 2025). Structures of these and analogous assemblies have provided insight into how the machines operate to recognise and move complex transport substrates in a preferred direction against concentration gradients (Du et al., 2018, 2018; Fitzpatrick et al., 2017; Glavier et al., 2020; Tsutsumi et al., 2019).

The outer membrane component of the efflux assemblies, TolC, is a homotrimeric protein with a self-closing β-barrel topology that inserts into the outer membrane and a long helical structural domain that interacts with periplasmic partners such as AcrA or MacA. TolC homologs are found throughout diderm bacteria, highlighting the functional importance of this superfamily (Stubenrauch et al., 2022). The long helical portion of TolC can be subdivided into an α-helical barrel domain, which appears to be a stable structural unit (Calladine et al., 2001; Huang et al., 2014), and conventional coiled-coil segments that dilate from a sealed resting state to an open-state when interacting with a periplasmic partner (Wang et al., 2017). At the junction of the two helical regions, the polypeptide meanders to form a circumferential ring known as the equatorial domain that is proposed to interact with the peptidoglycan layer of the periplasm (Shi et al., 2019).

Here, in our analysis of the MacA-MacB-TolC tripartite assembly, we observed unexpected cryo-EM density spanning between the equatorial domain of TolC and the transmembrane boundary. Using a structural topology pattern recognition query against predicted folds of E. coli proteins, we identified the protein as a lipoprotein of unknown function, YbjP (UniProt ID P75818). The N-terminus of YbjP trails over the surface of TolC to reach the outer membrane where its lipid moiety is embedded. Our structural comparisons, together with functional and proteomic results from YbjP deletion strains, do not support an allosteric or signalling role for the lipoprotein in TolC-mediated efflux systems but suggest that YbjP is required for expression of transporters including TnaB tryptophan permease and YojI ABC transporter that exports toxins such as microcin J25 through TolC (Baquero et al., 2024; Delgado et al., 2005).

Results

Cryo-EM structure of TolC bound to a lipoprotein

Earlier cryo-EM work on the MacA-MacB-TolC assembly used samples reconstituted in detergents (Fitzpatrick et al., 2017). We explored the preparation of MacA-MacB-TolC in peptidiscs as a better mimic of the membrane environment (Carlson et al., 2018). From these novel preparations, we were able to solve the structure by cryo-EM with high-quality density for all three components of the assembly (Figure 1A). In one of our preparations, additional density was observed near the equatorial domain of TolC, extending towards the outer membrane β-barrel domain (Figure 1B). We refined this structure to a final resolution of 2.48 Å (Supplementary Figure S1 and Table S1) and fitted the additional density with secondary structural elements (Figure 1C). The incomplete candidate fold was then searched against the database of E. coli protein structures predicted by AlphaFold2 (Jumper et al., 2021) using the DALI topology recognition program (Holm, 2022).The result matches with the lipoprotein YbjP (P75818) which could be fitted into the cryo-EM map. The refined model is presented in Figure 1D. The lipoprotein globular domain is formed by a series of four consecutive α-helices (α1-α4), followed by four antiparallel β-strands (β1-β4) and a C-terminal α-helix (α5) (Figure 1E). Structural alignment of the predicted YbjP and cryo-EM refined models reveals a strikingly high degree of similarity (RMSD of 0.53 Å over 118 residues), confirming the accuracy of the prediction (Figure 1D, inset). A superposition of the MacAB-TolC-YbjP complex with our previously reported MacAB-TolC structure (Fitzpatrick et al., 2017) revealed that TolC and the tripartite pump are largely unaffected upon YbjP binding with an RMSD of 1.03 Å over 1258 residues for TolC trimer (Figure 2A). Nonetheless, the side chains of a few residues in TolC, which mainly correspond to positively charged amino acids (R18, R24, K214, R227, R234), reorient to interact with the YbjP lipoprotein partner (Figure 2B).

Figure 1.

Figure 2.

Interactions between TolC and lipoprotein YbjP

YbjP straddles two adjacent TolC protomers, and the interface is stabilised by multiple hydrogen bonding and ionic interactions involving the linker and globular domain of YbjP and two TolC subunits (Figure 2C). The intermolecular contacts engage almost 20 residues in both TolC and YbjP. The lipoprotein flexible linker runs along TolC N-terminal helix from the equatorial to the transmembrane domains and is stabilised by several hydrogen bonds involving N32, R35, R24, Y98 and L17 from one TolC monomer (Figure 2C, panel 1). This cluster is consolidated by hydrogen bonding interactions between S107, S109, T110 and N113 from YbjP loop connecting beta-strands β1 and β2 and R234, Q306 and Y307 from the proximal adjacent TolC monomer, maintaining this flexible loop perpendicular to the lipoprotein linker. YbjP D115 and N118 also make contacts with R18 and E314 from TolC.

Interestingly, the YbjP globular domain is strongly associated via a cluster of ionic interactions (Figure 2C, panel 2), mediated by a trio of positive, negative, positive residues (R122, E135, R151) which each contacts a residue of opposite charge in TolC (D231, R227, E317). Finally, numerous hydrogen bonds are formed between YbjP globular domain and helices from both TolC monomers (Figure 2C, panel 3) engaging G155, V157 and A159 backbones, and K70, N118, E135 and R151 side chains from the lipoprotein. Residue conservation of YbjP was evaluated visually with the server CONSURF, revealing that the conserved residues are located at the interface with TolC (Figure 2D).

Like other lipoproteins, YbjP is predicted to be processed by cleavage of the signal sequence at residue C19 (also referred as to the +1 Cys of the matured lipoprotein), then lipidated as N-palmitoyl and S-diacylglycerol cysteine. These processing and modification steps are likely to occur at the inner membrane (Guest and Silhavy, 2023). Although the density is not well defined for the lipid moiety due to its flexibility, the map does reveal a portion of the hydrocarbon embedded into the inner leaflet of the outer membrane. This is consistent with mass spectrometry studies that have identified YbjP as an outer membrane protein (Molloy et al., 2000). Interestingly, there is no density for the lipoprotein R3 acyl chain suggesting that the cysteine α-amino group has not been modified while E. coli lipoproteins are presumed to be tri-acylated to traffic via the Lol system (Grabowicz and May, 2026; Noland et al., 2017). Further characterisation is needed to establish if YbjP is naturally di- or tri-acylated.

Validation of the TolC-YbjP interaction by in vitro pull-down and in vivo photo-crosslinking

To validate the TolC-YbjP interactions, we used an in vitro affinity pull-down assay with purified proteins. For this, we produced a soluble mutant of YbjP, hereafter referred to as YbjP_s, removing the lipobox conserved cysteine that carries the acyl chains, and the 8 following residues corresponding to a flexible linker joining the acylated cysteine to the protein globular domain. The protein (D28-R171) with a N-terminal His-tag was purified from the cytoplasm and immobilised on nickel resin. As shown in Figure 3A, detergent-purified TolC remained attached to the resin only in the presence of YbjP_s, confirming the association between the two proteins and demonstrating that the lipoprotein lipid moiety is not required for the interaction to occur. We could not detect an interaction between YbjP_s and Pal (peptidoglycan associated protein) lipoprotein or between Pal and TolC using the pull-down assay.

Figure 3.

Interactions between TolC and YbjP_s were also measured by isothermal titration calorimetry. (Figure 3B and Supplementary Table S3). The binding event is exothermic, consistent with electrostatic interactions at the protein-protein interface. The dissociation constant is in the micromolar range (K_D ∼ 4 µM). Although this value represents a weak to moderate association, the affinity is likely to be substantially higher in the presence of full-length YbjP which includes the 8-residue linker connecting the lipid modification to the globular domain. This region is absent here but participates in favourable interactions with TolC and is likely to increase the affinity (Figure 2A, panel 1). Additionally, the lipid modification would localise YbjP to the membrane, reducing its configurational entropy and thereby increasing TolC binding.

To validate that YbjP and TolC interact within the cell, we engineered C-terminally His-tagged full-length YbjP (YbjP_FL) to contain the photo-crosslinkable amino acid para-benzophenone, pBPA (Chin et al., 2002; Young et al., 2010) at positions guided by the YbjP-TolC structure. We replaced residues at the protein-protein interface (T110, N113) or at a distance (N43, N90 and H104) as negative controls. To validate the experiment, we also constructed amber mutants of His-tagged AcrA, an inner membrane protein known to interact with TolC, and substituted Q136 and Y137, two residues proximal to TolC in the AcrABZ-TolC efflux pump structure (Wang et al., 2017). Cells were grown in a medium supplemented with pBPA, induced and then irradiated for 15 min with ultraviolet light. After cell lysis, His-tagged proteins were retrieved from the samples by affinity chromatography. Purified fractions were analyzed by SDS-PAGE and immunoblotting (Figure 3C). We observed covalent photo-adducts that migrate at higher molecular weight compatible with AcrA-TolC or YbjP-TolC adducts only for the pBPA-supplemented cells and only after illumination with UV light for the residues that are proximal to the TolC surface, but not those that are distal. We confirmed the presence of both YbjP and TolC in the N113 YbjP crosslinked product by Western blot (Supplementary Figure S2). These results are consistent with a model for YbjP lipoprotein interactions with TolC in vivo.

TolC-YbjP interaction in the AcrA-AcrB-AcrZ-TolC assembly

To examine whether YbjP can interact with other TolC-mediated assemblies, we supplemented the AcrABZ-TolC pump reconstituted in peptidisc with YbjP_s and resolved the cryo-EM structure of the complex at 3.17 Å resolution (Supplemental Figure S3 and Table S1). Structural comparison of the two assemblies reveals that YbjP binds at the same location on TolC (Figure 4A, B and Supplementary Figure S3) and makes the same interactions (Figure 4C). The AcrABZ-TolC-YbjP assembly was also reconstituted into peptidoglycan (Supplementary Figure S4A, B), and the position of the peptidoglycan layer between AcrA and TolC corresponds with the localization previously inferred from low-resolution cryo-ET data (Shi et al., 2019) (Supplementary Figure S4C). The reconstructed maps indicate that the YbjP may not interact with the peptidoglycan layer, but this cannot be certain due to the limited resolution of the cryo-EM map. In vitro characterizations indicated that YbjP can interact with peptidoglycan in pull-down assays, but this is likely to be non-specific as alanine substitutions in the domain proximal to the peptidoglycan (R80, D81, R85 and E86) had little impact on the efficiency of the pull-downs. Photo-crosslinking in substitutions of the photoreactive amino acid para-benzophenone (pBPA) at positions D77, Q166, E169 and R171 did not give evidence for peptidoglycan-linked species. The data suggest that YbjP may not form avid interactions with the peptidoglycan.

Figure 4.

Evolutionary profile of YbjP

To explore the possible function of YbjP, we investigated its evolutionary profile. In the PFAM database, YbjP is annotated to contain one DUF3828 domain. The most well-characterized DUF3828-containing protein is Tai3, a periplasmic type VI cognate immunity protein of the T6SS amidase effector Tae3 (Dong et al., 2013; Russell et al., 2014). Another periplasmic protein YqhG contains one full and one partial DUF3828 domain and is involved in regulating type I fimbriae expression in E. coli by an unknown mechanism (Bessaiah et al., 2019). Structural alignment of YbjP with the Tai3 crystal structure and YqhG AlphaFold2 model indicates that these proteins share a common fold (Figure 5A).

Figure 5.

To investigate whether YbjP is functionally homologous to Tai3 or YqhG, we reconstructed a phylogenetic history of the DUF3828-containing proteins, which could be classified into three major families based on taxonomy, gene synteny, presence of a signal peptide and a conserved QDX motif, involved in Tae3 inhibition (Supplementary Table S4). The result indicates that the function of YbjP is likely distinct from that of Tai3 or YqhG (Figure 5B). We noted that Tai3 is found in a broader range of bacterial groups (Gammaproteobacteria, Betaproteobacteria, and Bacteroidota/Chlorobiota) compared to YbjP. Similarly, YbjP is restricted to some members of the Enterobacterales Order within the class of Gammaproteobacteria, unlike TolC which is more widespread (Supplementary Figure S5). Notably, the TolC-YbjP interface is more conserved in Enterobacterales with YbjP than without (Figure 5C), suggesting a scenario where YbjP evolved recently from an ancestral Tai3-like protein to become a TolC interactor.

YbjP is not required to compensate for lack of TolC lipidation

Outer membrane efflux proteins structurally homologous to TolC often carry a N-terminal acylation. This is the case for the drug exit duct OprM in Pseudomonas and the outer membrane efflux proteins in E. coli MdtP, MdtQ, and CusC, but with the notable exception of TolC. The reason for the presence or absence of acylation remains unclear, but it has been proposed that acylation has roles in anchorage to the membrane and in membrane insertion. We first postulated that YbjP could compensate for the absence of lipid modification in TolC. Indeed, YbjP linker and N-acetylation mimic the linker and acetylation arrangement seen in OprM and CusC (Figure 6A). Similarly, AlphaFold3 predicted the interaction of YbjP with TolC but not with the other TolC structural homologues that are lipidated in E. coli (Figure 6B). Consistent with this, we found that E. coli TolC is distinct from its lipidated structural homologues (Figure 6C), suggesting the possibility that YbjP evolved to compensate for the absence of lipidation. However, further bioinformatic analysis reveals that while most organisms containing the ybjP gene possess non-lipidated TolC, non-lipidated TolC is also present in most organisms lacking ybjP (Figure 6D). This indicates that YbjP may not be necessary to compensate for the lack of lipidation of TolC.

Figure 6.

Exploring in vivo function of YbjP

To explore the physiological function of YbjP, we compared the proteomic profiles of ΔybjP, ΔtolC, ΔtolC ΔybjP and parental strains under exponential and stationary phase conditions (Figure 7). Only minor differences were detected in the proteome of the ΔybjP strain compared with the parental wild-type in both growth stages, while numerous significant changes were observed for the ΔtolC and ΔtolC ΔybjP strains. Levels of TolC were not impacted in the ΔybjP strain, indicating that YbjP is not required for TolC biogenesis. Previous RNA-seq analysis highlighted an upregulation of ybjP in response to membrane-disrupting chemicals, including vanillin (Pattrick et al., 2019) and EDTA (Janssens et al., 2024). However, we do not observe an impact on growth under standard laboratory conditions, as the minimal inhibitory concentrations for growth inhibition were unchanged in the ΔybjP compared to the parental strains in the presence of vanillin and EDTA; nor where changes observed with benzalkonium chloride and different classes of antibiotics, under either aerobic or microaerobic conditions (Supplementary Table S5).

Figure 7.

Considering that YbjP expression is regulated by the stress response sigma factor rpoS (Lacour and Landini, 2004), its physiological role is likely context-dependent and not readily appear under standard laboratory conditions. Indeed, the few proteins whose levels were influenced in the ΔybjP strain are associated with specific contexts, including stress and changes in lifestyle (Figure 7). We observed the downregulation of Antigen 43 (flu), which is an autotransporter porin with an extensive extracellular domain that is involved in auto-aggregation and which promotes the shift from a planktonic state to sessile state. While previous work noted that YbjP overexpression influences motility (Tenorio et al., 2003), we did not observe any effect of the ybjP deleted strain on LB motility agar plates (Supplementary Table S6).

In addition, changes in specific transporters were noticed. The YojI ABC transporter, proposed to be involved in the efflux of the microcin J25 toxin in conjunction with TolC, was decreased in relative abundance in the ΔybjP strain in stationary phase (Figure 7). However, we did not observe any change in MIC values for the ΔybjP strain in the presence of microcin J25 during exponential phase, while the ΔtolC and ΔtolC ΔybjP strains did show increased sensitivity of >4,000-fold (Supplementary Table S5). Conversely, the TnaB low-affinity tryptophan permease level increased. We did observe a small increase in growth for the ΔybjP strain compared to the parental strain in minimal media in the presence of tryptophan. Interestingly, the ΔtolC and ΔtolC ΔybjP strains did show marked decreased growth in the absence of tryptophan (Supplementary Figure S6). The outer membrane pore OmpW, which plays a minor role in the efflux of quaternary cationic compounds together with the EmrE transporter (Beketskaia et al., 2014), was downregulated. Taken together, the changes in the proteome implicate nuanced, multiple roles of YbjP, but the phenotypic impact of the null strain is likely to be seen under environmental stress conditions.

Discussion

Our structural and functional results indicate that the lipoprotein YbjP is located to the outer membrane, where it can interact with the membrane protein TolC. The mode of interaction between TolC and YbjP resembles closely the intramolecular interactions of the lipidated N-terminus in TolC paralogs, such as OprM from Salmonella. The interaction is likely functionally important within some Enterobacterales. It is proposed that the TolC archetype may bear sequence or environmental features that have limited its distribution, or it is a more recently evolved form in the TolC-like protein family (Stubenrauch et al., 2022). It is possible that TolC and YbjP co-evolved in this lineage.

The biogenesis of TolC presents intriguing mechanistic puzzles. The insertion stage of outer membrane proteins involves the β-barrel assembly machine (BAM) complex (Voulhoux et al., 2003; Wu et al., 2005), but the process is not well characterised for a non-canonical porin-like protein with an extended hydrophilic portion, such as TolC, nor are the earlier steps of translocation across the periplasm. The chaperone translocation and assembly machinery (TAM) facilitates assembly of TolC into the outer membrane in a potentially redundant pathway (Stubenrauch et al., 2022). Reflecting on the potential requirements suggests key aspects to consider for understanding the process. First, the protomers must be moved across the periplasm from the translocon where the nascent chains emerge. Second, this insertion would be coordinated to deliver three protomers to fold into the full trimeric architecture. Lastly, the folding does not require exogenous energy and is likely driven by hydrophobic interactions with the highly crowded and asymmetric lipid bilayer (Horne et al., 2020).

Mechanistic puzzles also arise when considering how lipoproteins such as OprM and YbjP arrive at the outer membrane and are inserted. Lipoproteins are processed and lipid groups are attached to a conserved cysteine residue that immediately follows the signal sequence in the inner membrane (Guest and Silhavy, 2023). Acylated thiols are found in components of other bacterial efflux assemblies. The structure of CusC indicates the presence of an acylated thiol on the N-terminal cysteine, and acylation is also found in CmeC, but nothing has been found to be added to the N-terminal cysteine of the MtrE structure. Post-translational lipidation is particularly essential for the secretion and localization of some membrane proteins, a process involving different acyl transferases (Aicart-Ramos et al., 2011; Kovacs-Simon et al., 2011; Linder and Deschenes, 2007; Nakayama et al., 2012). Why proteins that are embedded within cellular membranes via a large hydrophobic structural domain need modifications such as the N-terminal lipidation appears puzzling. Akama et al. (2004) suggested that these proteins must first be anchored to the membrane by an N-terminal lipid so that the insertion of their large hydrophobic domain may be triggered. Structural evidence indicates that this later step is critical for the correct folding of outer membrane factors (OMFs) and highlight the possible involvement of membrane-interacting components in the OMF opening process and consequently the efflux-pump function (Lei et al., 2014).

Translocation of lipoprotein to the outer membrane in E. coli requires five essential proteins (LolA-E) (Guest and Silhavy, 2023). The mechanism involves lipoprotein extraction from the inner membrane by the ABC transporter LolCDE, then transfer to the periplasmic carrier LolA for delivery to the outer membrane acceptor LolB, which facilitates insertion into the outer membrane. It is envisaged that this pathway is involved in the movement of OprM and its lipidated outer membrane homologs. As E. coli TolC is not lipidated, it is possible that YbjP acts as a chaperone that intercepts the TolC and aids its translocation across the periplasm via the Lol system. However, this proposed pathway is likely redundant or not essential, as TolC is still found in the outer membrane in strains null for YbjP. The BAM complex impacts on TolC assembly in E. coli (Malinverni et al., 2006; Werner and Misra, 2005), but surprisingly it is not involved in assembly of the TolC-like proteins OprM in P. aeruginosa (Hoang et al., 2011). The pathway for insertion is likely to be redundant and divergent for the TolC family (Stubenrauch et al., 2022). Notably, a broad screen of cell envelope protein complexes in E. coli did not identify physical or genetic interactions between YbjP and TolC (Babu et al., 2018), but such interactions were found between YbjP and the partners of TolC in the tripartite assembly, namely the RND transporter AcrB and the periplasmic AcrA. It may be that the interactions between TolC and YbjP in vivo are weak and transient, as might occur for example during TolC insertion into the outer membrane.

Our results do not support an allosteric or signalling role for YbjP but suggest that the lipoprotein impacts on expression of transporters tryptophan permease TnaB and ABC family member YojI, which transports toxins such as microcin J25 through TolC (Baquero et al., 2024). It is also possible that YbjP may facilitate the biogenesis of TolC during times of rapid adaptation or its distribution in the membrane. To our knowledge, YbjP is the first protein identified to interact with the lateral surface of TolC. Interestingly, the outer membrane protein SlyB has been shown to encapsulate TolC-like proteins during stress conditions and cryo-EM 2D classification images predict the binding of SlyB at the TolC-YbjP interface (Janssens et al., 2024). Structural predictions from AlphaFold3 further support that SlyB binds TolC at the YbjP location (Supplementary Figure S7). Given that both YbjP and TolC are upregulated in response to EDTA treatment (Janssens et al., 2024), YbjP and SlyB might compete for binding to TolC under conditions of outer membrane stress. Possibly YbjP might support clustering and partitioning of transport machineries during cell division or during stress (Zhang et al., 2025), which in turn can influence the efficiency of adaptation to environmental conditions.

Materials and methods

Preparation of disulphide-engineered MacAB-TolC and reconstitution into peptidisc

E. coli BL21 (DE3) strain was transformed with plasmids pET20b-hexaHis-MacA_D271C-MacB_G465C expressing the MacA_D271C and MacB_G465C mutants, allowing a disulphide link, with a C-terminal hexaHis-tag on MacB and pRSFDuet-TolC-FLAG expressing full-length TolC with a C-terminal FLAG-tag (Fitzpatrick et al., 2017). Transformed cells were spread onto LB agar plates containing 50 μg mL^-1 carbenicillin and 50 μg mL^-1 kanamycin and were incubated at 37°C overnight. A single colony was used to inoculate 50 mL of LB medium containing 50 μg mL^-1 carbenicillin and 50 μg mL^-1 kanamycin. The preculture was grown at 37°C, with an orbital shaker set at 200 rpm for 4 h. When the absorbance at 600 nm reached between 0.5 and 0.7, the preculture was used to inoculate 6 L of 2YT medium containing 50 μg mL^-1 carbenicillin and 50 μg mL^-1 kanamycin in 6x2 L baffled flasks. Bacteria were grown at 37°C, 200 rpm until OD_600nm reached 0.5 and were subsequently induced with 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The culture was further incubated at 20°C, 200 rpm overnight. Cells were collected by centrifugation (5,000 g for 30 min at 4°C) and pellets were resuspended in a buffer containing 20 mM Tris-HCl pH 8.0 and 400 mM NaCl (Buffer A). One tablet of EDTA-free protease inhibitor per 50 mL, 5 μg mL^-1 lysozyme and 5 U mL^-1 DNase I were added to the cell suspension and incubated 1 h at 4°C. The mixture was then passed five times through a water-cooled high-pressure homogeniser (Emulsiflex), keeping the pressure below 14,500 psi throughout. The resulting lysate was centrifuged (9,000 g, 30 min, 4°C) to remove cell debris and then ultracentrifuged (175,000 g, 4 h, 4°C) to pellet membranes.

Membranes were solubilized in 50 mL of buffer 20 mM Tris pH 8.0, 400 mM NaCl with 1.5% (wt/v) β-dodecyl maltoside (β-DDM) and one tablet of EDTA-free protease inhibitor for 3 h at 4°C with gentle stirring. The solution was then purified by Ni-NTA chromatography after addition of 10 mM imidazole. The elution peak was further purified with ANTI-FLAG M2 affinity resin (0.5 mL, Sigma Aldrich), pre-equilibrated with buffer A supplemented with 0.05% wt/v β-DDM. The elution fraction was added to the resin and incubated at 4°C for 1 h with gentle agitation. The sample was then washed with buffer A containing 0.05% β-DDM and then 0.03% (wt/v) decyl maltose neopentyl glycol (DMNG). Peptidisc (Carlson et al., 2018) at 1 mg mL^-1 in 400 mM Tris-HCl and additional E. coli lipids (100 μL at 20 mg mL^-1) were added to the sample on the resin. After 16 h at 4°C, the resin was washed with buffer A containing no detergent and then eluted with 3xFLAG peptide (Sigma-Aldrich). Flow-through was collected at each step and analysed by SDS-PAGE. The presence of the full assembly was confirmed by mass photometry (Refeyn).

MacAB-TolC cryo-EM sample preparation and data collection

For single-particle cryo-EM structure solution, the samples of MacAB-TolC complex reconstituted in peptidisc (Carlson et al., 2018) were thawed and diluted to 0.5 mg mL^-1 in a buffer composed of 20 mM Tris pH 8.0, 100 mM KCl, 2 mM TCEP and 1 mM NaN₃. A 3-μL aliquot of dilute protein was applied onto a Cu 300-mesh EM grid layered with R1.2/1.3 holey carbon support film (Quantifoil Micro Tools, GmbH), which had been glow-discharged immediately prior to use. The sample deposited on the EM grid was blotted of excess solution and vitrified in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific, Inc.) robotic cryo-EM plunger.

The grids were imaged using Titan Krios G3 transmission electron microscope (Thermo Fisher Scientific Inc.) operating at accelerating voltage of 300 kV and liquid nitrogen temperature. The images were recorded in EFTEM mode using AFIS on K3 direct electron detector (Gatan, Inc.) and data acquisition software EPU (Thermo Fisher Scientific, Inc.). Exposures were for 1 s and were collected as 40-frame movies using total electron fluency (dose) of 49.75 e⁻/Ų and defocus values between -2.4 and -1.0 µm. The movies were collected in super-resolution mode with nominal pixel size 0.415 Å, but were binned 2× following exposure, yielding effective pixel size of 0.83 Å/px.

MacAB-TolC cryo-EM data processing

The cryo-EM data were processed using cryoSPARC 4.5 software (Structura Biotechnology, Inc.). Patch motion correction was used to correct for beam-induced motion and Patch CTF used to calculate the CTF. Circular Gaussian blob picking with a min/max diameter of 120/380 Å was used to extract an initial set of pixels in 878-px boxes Fourier-cropped to 600-px (effective pixel size 1.21 Å/px). After 2D classification these were used as input to a template-picking model and particles re-extracted at 600-px and Fourier-cropped to 400-px (effective pixel size 1.245 Å/px). This set of particles was pruned again with another round of 2D classification and then subject to ab-initio reconstruction. The best volume and particles from 2D classification was used for heterogeneous refinement along with 5 ‘junk’ volumes. The best output was then subjected to non-uniform refinement in C₁ symmetry with optimize per-particle defocus, optimize per-group CTF parameters, and fit spherical aberration options used to further improve the refinement. This yielded a 2.74 Å map with 233,835 particles. Finally, local CTF refinement, global CTF refinement, and another round of non-uniform refinement in C₁ was performed followed by reference-based motion correction. The unbinned output particles (233,775 particles at 600-px box size, 0.83 Å/px) were subjected to a final round of non-uniform refinement in C₃ symmetry yielding a final map with a GSFSC reported resolution of 2.48 Å. To facilitate model-building, Resolve from Phenix package was also run on this map. Data collection and processing parameters are shown in Supplementary Table S1.

MacAB-TolC-YbjP model building and refinement

The part of the experimental map containing an unidentified protein density adjacent to TolC trimer was visualised and traced as a poly-alanine chain using Coot 0.9 (Emsley et al., 2010). The resulting structure model was submitted to the DALI server (Holm, 2022) to query the E. coli species subset of the AlphaFold Database (Hierarchical AF-DB Search). The search showed strong structural homology with the uncharacterised lipoprotein YbjP (AF-P75818-F1). The AlphaFold model was fitted into the experimental map and manually adjusted in Coot 0.9, allowing the identification of the density as YbjP. Following minor structure adjustments, the model was refined using ISOLDE 1.6 plugin (Croll, 2018) for ChimeraX-1.6.1 (Pettersen et al., 2021). Molecular refinement was completed in Phenix-1.20 (Liebschner et al., 2019). Refinement statistics are listed in Supplementary Table S1.

Plasmid construction and purification of soluble YbjP

Soluble YbjP bearing a thrombin-cleavable N-terminal His-tag or a TEV-cleavable C-terminal His-tag were constructed by amplifying the protein globular domain (residues 28-171) from E. coli MG1655 genomic DNA. The PCR product was digested NdeI-BamHI or NdeI-XhoI prior insertion into pET28a or pET24a respectively, digested with the same enzymes. E. coli BL21 (DE3) cells were then transformed with pET28-YbjP_28-171 or pET24-YbjP_28-171 plasmid encoding YbjP_s. Bacteria were grown at 37°C in 2YT media supplemented with kanamycin and protein expression induced with addition at mid- to late-log phase of 0.1 mM IPTG. After 16 h at 18°C, cells were harvested by centrifugation (4,000 g, 10 min), and pellets resuspended in 50 mM Tris pH 7.5, 200 mM NaCl and 10% glycerol supplemented with lysozyme and DNase I. Cells were then lysed by passage through a cell disruptor (Constant Systems) at 30,200 psi. Unbroken cells and debris were removed by ultracentrifugation (107,000 g, 1 h, 4°C). The supernatant was supplemented with 20 mM imidazole and loaded onto a 5 mL HisTrap FF column (GE Healthcare), which was washed with 15 column volumes of the same buffer before elution with 250 mM imidazole. Protein fractions were pooled, dialysed twice against buffer containing 50 mM Tris pH 7.5 and 200 mM NaCl and concentrated using a Vivaspin filter with a 10 kDa MWCO before flash freezing and storage at -80°C.

Preparation of AcrABZ-TolC in peptidisc reconstituted with YbjP

Co-expression of the recombinant AcrABZ-TolC-3xFLAG and cellular membrane preparation was as previously described with some modifications (Du et al., 2014).The AcrABZ-TolC-3xFLAG components were expressed into E. coli strain C43 (ΔacrAB and ΔtolC). Cell pellets were resuspended in lysis buffer (150 mM NaCl and 50 mM Tris pH 7.5) and membranes pelleted as previously described (Du et al., 2014) and solubilized with a buffer containing 400 mM NaCl, 50 mM Tris pH 7.5 and 1.5% (wt/v) β-DDM by stirring overnight at 4°C. The soluble extract was incubated with Ni-NTA beads (Qiagen) for nickel affinity purification (2 mL for 4.5 g membrane extract) of the His-tagged AcrABZ_6xHis-TolC_3xFLAG. The beads were loaded onto a gravity flow-column and the column washed with 4 column volumes (CVs) of a buffer composed of 200 mM NaCl, 50 mM Tris pH 7.5 and 0.02% (wt/v) β-DDM. The AcrABZ_6xHis-TolC_3xFLAG protein complex was eluted with the same buffer containing 0.5 M imidazole. Eluted fractions were analysed on a 4-12% SDS-PAGE gel and fractions enriched for the pump were then pooled for a second purification step using anti-FLAG M2 affinity beads (Sigma). The fractions were incubated with 0.5 mL anti-FLAG resin for 3 h at 4°C with gentle stirring and the beads were then washed with 2 CVs of buffer (200 mM NaCl, 50 mM Tris pH 7.5 and 0.02% β-DDM) and 3 CVs of buffer 200 mM NaCl, 50 mM Tris pH 7.5 to gradually decrease the detergent of the solubilized pump to below the critical micelle concentration. For reconstitution of the AcrABZ-TolC sample, the NSPr peptidisc was used based on the protocol by Carlson et al. (2018) with some modifications. Next, 2 mg mL^-1 of peptidisc (Peptidisc Biotech) in 20 mM Tris-HCl pH 8.0 peptide solution was loaded to the column and incubated for 20 min at room temperature with gentle shaking. The flow-through was collected by gravity and 2 mg mL^-1 peptide solution containing 40 μL of E. coli lipids (6 mg mL^-1) was loaded to the column and incubated with the beads. Finally, 0.5 mg mL^-1 of 3xFLAG peptide in buffer 50 mM Tris pH 7.5, 200 mM NaCl was added to the column and incubated with the beads for 15 min. The fractions were analysed on a 4-12% gradient SDS-PAGE gel and concentrated to 5-6 mg mL^-1 using a Vivaspin filter with a 100 kDa MWCO. Prior grid freezing, samples of AcrABZ-TolC in peptidisc were mixed with 10-fold excess of C-terminally His-tagged soluble YbjP.

AcrABZ-TolC-YbjP cryo-EM sample preparation, data collection and processing

Sample of AcrABZ-TolC in peptidisc and YbjP was applied on a Cu 300-mesh EM grid layered with R1.2/1.3 holey carbon support film (Quantifoil), which had been glow-discharged immediately prior to use. The sample deposited on the EM grid was blotted of excess solution and vitrified in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific, Inc.) robotic cryo-EM plunger. Images were collected on a Titan Krios microscope operating at 300 kV. Electron fluency was 56 e⁻/Ų and defocus values between -2.5 and -1.0 µm. Data were processed with cryoSPARC as detailed in Supplementary Figure S3. After patch motion correction and CTF correction, particles were selected using Blob picker and 8,603 particles were extracted prior 2D classification and best classes were used for template picking. Particles were then subjected to two rounds of 2D classification. The selected 257,681 particles were used to build three ab initio models. The tripartite pump was further refined by heterogenous refinement and non-uniform refinement in C₃ symmetry, yielding a final map at 3.17 Å resolution.

AcrABZ-TolC-YbjP preparation with mature peptidoglycan and cryo-EM processing

The AcrABZ-TolC-YbjP prepared in peptidisc were mixed with roughly 1 mg mL^-1 of peptidoglycan slurry. Samples of mature and early forms of peptidoglycan (PG) were kindly provided by Professor Waldemar Vollmer (University of Newcastle and now at University of Queensland, Brisbane, Australia), prepared from strains BW25113 ΔGLDT and C5703-1. The mixture was incubated on ice for 1 hour and then used to prepare grids on glow-discharged Ultrafoil R 1.2/1.3 using a FEI Vitrobot (IV) at settings of 95% humidity, 4°C, 3 seconds blot time, -5 force. Cryo-EM data processing and 3D-reconstructions were carried out using cryoSPARC (Punjani et al., 2017). Multiple rounds of 3D refinement were conducted including heterogeneous refinement to remove non-pump particles, along with homogeneous refinement and non-uniform refinement to improve the resolution yielding a final map of AcrABZ-TolC-PG-YbjP at 3.97 Å. The data collection and processing parameters are summarised in Supplementary Table S2.

Isothermal titration calorimetry (ITC)

Experiments were performed with a ITC200 calorimeter (MicroCal, Malvern Panalytical) at 25°C. After overnight dialysis in 20 mM HEPES pH 7.5, 200 mM NaCl, 0.03% (wt/v) DDM, N-terminally His-tagged soluble YbjP at 230 µM was injected into 40 µM TolC in the same buffer. The titration program consisted of 25 injections of 1.5 µL with an initial injection of 0.4 µL every 300 s, with cell stirring rate of 750 rpm. For each condition, a control titration was performed by injecting YbjP into a cell containing buffer only. The control signal was then subtracted from the experimental titration, and the binding affinity, stoichiometry and thermodynamic parameters were obtained by nonlinear least-squares fitting of the background corrected experimental data using a single-site binding model from the MicroCal PEAQ-ITC analysis software (Malvern). ITC parameters are listed in Supplementary Table S3.

IMAC (immobilised metal affinity chromatography) binding assay of TolC and YbjP

Detergent-purified FLAG-tagged TolC was mixed to N-terminally His-tagged YbjP_s for 5 min in 50 mM Tris pH 7.5, 200 mM NaCl and 0.03% (wt/v) β-DDM. Each protein was at 15 µM final concentration in a final volume of 125 µL. A control was performed with only the TolC protein, or only the YbjP, or by replacing YbjP with another periplasmic protein, soluble LolB, produced as described in Kaplan et al. (2018). Proteins were added to 100 µL of Ni-resin (50% slurry, profinity Biorad) in a microbatch spin column and after 5 minutes the flow-through was recovered. The resin was washed four times with 250 µL of buffer and bound proteins were eluted with the same volume of buffer containing 250 mM imidazole. Elution fractions were analysed on a gradient (4-12%) SDS-PAGE gel. As a reference the TolC flow-through fraction was also loaded.

In vivo photo-crosslinking

Plasmid constructions

Full-length YbjP was amplified from E. coli MG1655 genomic DNA. A Ser-Gly linker followed by a 6xHis-tag was added on the reverse primer. The PCR product was digested by NdeI-Xho and inserted into pET24a digested by the same enzymes. AcrA was cloned as described for YbjP. Amber codons were introduced at desired positions by Quikchange site-directed mutagenesis.

Culture and photo-crosslinking

E. coli C43 cells were co-transformed with pEVOL encoding an amber suppressor tyrosyl tRNA and the engineered tyrosyl tRNA synthetase (Chatterjee et al., 2014) and with YbjP_s or AcrA amber mutant for incorporation of pBPA into the TAG amber codon at desired positions. Cultures of 20 mL LB media, supplemented with 20 and 50 µg mL^-1 of chloramphenicol and kanamycin respectively, were inoculated with 1/100 of overnight cultures and grown at 30°C. When an optical density of 0.3 was reached, 1 mM of freshly prepared pBPA (Bachem) in NaOH was added in each culture with additional HCl to balance pH. When indicated, a control without addition of pBPA was carried out. After 30 min at 30°C, cells were induced with 0.02% (wt/v) L-arabinose and 0.5 mM IPTG and grown for 2 h at 30°C. Cells corresponding to 4 mL of culture were collected by centrifugation (4,000 g, 4°C, 4 minutes) washed with 1 mL of PBS and resuspended in 700 µL PBS. Aliquots of 150 µL of each culture were transferred in a 96-well plate and exposed under UV (365 nm) for 15 min at room temperature using a UVGL-58 handheld UV lamp.

His-tag enrichment

The next day, samples were lysed by 3 cycles of freezing in liquid nitrogen and thawing in 37°C water bath. Lysozyme at 0.5 mg mL^-1, DNase at 50 µg mL^-1 and 1% (wt/v) DDM were added. After 30 min at room temperature, samples (300 µL) were transferred into spin columns containing 100 µL of Ni-NTA resin (50% suspension, profinity Biorad) previously equilibrated with PBS (phosphate buffered saline). Proteins were allowed to bind for 5 min at room temperature. The resin was then washed 3 times with 400 µL of PBS supplemented with 0.03% DDM and bound proteins eluted with 150 µL of PBS containing 0.03% DDM and 250 µM imidazole.

Western-blot

Samples (20 µL) were analysed by SDS-PAGE and immunoblotting with mouse anti-His primary antibodies (Penta-His, Qiagen) and goat anti-mouse secondary antibodies (IRDye 800 CW, Licor) prior to imaging with an Odyssey Licor system. For the YbjP and TolC double detection (Supplementary Figure S2), proteins were simultaneously detected with mouse anti-His and rabbit anti-TolC primary antibodies and with goat anti-mouse secondary antibodies (IRDye 800 CW, Licor) and donkey anti-rabbit secondary antibodies (IRDye 680 RD, Licor).

Strains and culture conditions

E. coli BW25113 wild-type and ΔtolC were obtained from the Keio collection (Baba et al., 2006). BW25113 ΔtolC ΔybjP was generated by λ-Red recombination from the ΔtolC strain. Due to consistent deletion of the yecT- flhD region of the Keio collection BW25113 ΔybjP strain following FLP expression, ΔybjP was generated by λ-Red recombination from the wild-type strain. The kanamycin-resistance cassettes were excised from these strains using pCP20 and the genomes were sequenced for confirmation.

Cells were cultured routinely in Mueller Hinton (MH) broth at 37 °C, with shaking at 200 rpm. Where specified, cells were cultured in Luria-Bertani (LB) broth (0.5 g L^-1 NaCl) or M9 minimal media supplemented with 0.4% glycerol. Cells containing a gentamicin-resistance version of the pKD46 plasmid were cultured in LB broth containing 10 µg mL^-1 gentamicin, except ΔtolC, which was cultured with 5 µg mL^-1 gentamicin. LB agar plates containing 50 µg mL^-1 kanamycin or 50 µg mL^-1 carbenicillin were used for routine transformant selection. Genome-integrated kanamycin-resistance cassettes were selected for on LB agar containing 30 µg mL^-1 kanamycin, except for ΔybjP and ΔtolC ΔybjP, which were plated on LB agar containing 20 µg mL^-1 and 10 µg mL^-1 kanamycin, respectively.

λ-Red recombination

λ-Red recombination (Datsenko and Wanner, 2000) was carried out using a modified version of the protocol described in Peng et al. (2022). Briefly, cultures of cells containing a gentamicin-resistance version of the pKD46 plasmid were prepared in LB broth containing gentamicin and incubated overnight at 30°C. The overnight cultures were diluted 1:200 into fresh LB broth containing gentamicin and incubated at 30°C until the OD_600nm reached 0.4, at which point L-arabinose was added to a final concentration of 0.3% (w/v) and the cultures were incubated at 37 °C for 1 h. The cultures were transferred to ice, then washed by four cycles of centrifugation and resuspension in ice-cold distilled water. After the final centrifugation, the distilled water was discarded, and the cells were resuspended in the residual liquid volume. The OD_600nm of the resuspended cells were measured and appropriate volumes of ice-cold distilled water were added to bring the cells to a final density of OD_600nm 50.

Amplicons of kanamycin-resistance cassettes with 50 bp homology to the gene of interest were designed as described in Baba et al. (2006), amplified using Q5 polymerase (NEB), and gel purified (NEB). 100 ng of each purified amplicon was added to 70 μL of the pKD46 induced electrocompetent cells and transformed by electroporation. Cells were recovered in SOC media (NEB) at 37°C for 1 h, then plated on selective agar plates. The kanamycin-resistance cassettes were excised from the strains using the plasmid pCP20, as described in Baba et al. (2006).

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Briefly, overnight cultures in MH broth were diluted 1:1000 in fresh MH broth and incubated at 37°C until reaching an OD_600nm of 0.2-0.5. The cultures were then diluted to an OD_600nm of 0.001 in fresh MH broth and 50 μL were added to wells of flat-bottom 96-well microtiter plates already containing 50 μL of 2-fold serially diluted compounds in MH broth, to a final volume of 100 μL. The plates were sealed with a gas-permeable seal and incubated at 37°C for 18 h. Growth was determined by visual inspection and OD_600nm measurement using a SpectraMax iD3 microplate reader.

For antimicrobial susceptibility testing under microaerobic conditions, prior to incubation, the 96-well microtiter plates were placed in a 2.5 L anaerobic jar and Millipore Anaerocult™ C was used following the manufacturer’s instructions to generate an environment containing 5-6% O₂ and 8-10% CO₂.

Bacterial growth curves

Bacterial growth curves were carried out by diluting overnight cultures to an OD_600nm of 0.02 in fresh media containing the relevant concentrations of supplemented solutions. 200 μL of the diluted cultures were added to wells of a flat-bottom 96-well microtiter plate and the plate was sealed with a gas-permeable seal. OD_600nm was read every 10 min for 24 h at 37°C with shaking using a SpectraMax iD3 microplate reader.

Swimming motility assay

LB motility agar plates were prepared by adding 25 mL of LB broth containing 0.3% agar in 94 mm diameter sterile petri dishes. Overnight cultures of the relevant strains were prepared in LB broth. A volume of 2 μL of overnight culture was injected into the centre of each motility agar plate, followed by incubation at 37°C for 24 h. The diameters of the colonies at the widest point were measured using a ruler.

Proteomics sample preparation and analysis

Cultures were prepared in triplicate under aerobic conditions in MH media for the four strains ΔybjP, ΔtolC, ΔtolC ΔybjP and the BW25113 parent, all prepared by the recombineering approach described above. Cultures were grown to exponential phase (0.6-0.8 OD_{600 nm}, 15 mL) and to stationary phase (4.8-5 OD_{600 nm}, 5 mL) and pelleted by centrifugation at 4,000 g, 5 min and frozen. The pellets were resuspended in 1 mL lysis buffer for set 1 (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 1.4 mg mL^-1 hen egg white lysozyme, 1% (v/v) Triton X-100). The suspension was sonicated with a microprobe tip (Sonics vibracell) on ice for 60 sec, with 4 sec pulses, 6 sec wait time, 50 % amplitude. To the sonicate lysate, 2 units of DNase I were added to digest DNA, followed by incubation on ice for 10 min. Trichloroacetic acid (Sigma) was then added to 5 % (wt/v), then incubated on ice and collected by centrifugation in an Eppendorf centrifuge at (13,000 rpm, 5°C, 10 min). The pellet was washed with 5% TCA in water, then spun (set 1) or with 50% ethanol in 20 mM Tris-Cl pH 7.5 (set 2), and the collected pellet provided to the University of Cambridge Centre for Proteomics. Samples were digested with trypsin and analysed by quantitative label free LC-MS/MS. Signals from total unique spectral counts were normalised for each replicate and averaged.

Raw mass spectrometry data was processed with DIA-NN v2.1.1 (Demichev et al., 2020) using the reference E. coli proteome from UniProt (acc. no. UP000000625) along with contaminant protein sequences provided by the Cambridge Centre for Proteomics (Dawson & Smith, 2024). Precursor and fragment mass tolerances were automatically optimized within the DIA-NN search. Trypsin was set as the enzyme of choice and a maximum of one missed cleavage was allowed. Carbamidomethylation of cysteine was set as a fixed modification, one variable modification was allowed from either N-terminus acetylation or oxidation of methionine. An FDR cutoff of 1% was used at the precursor level.

Quantitative analysis was performed from the abundances at the peptide level using qfeatures and limma (Gatto and Vanderaa, 2025), adapting the code from (Hutchings et al., 2025). Briefly: peptides matching contaminant proteins, peptides with q-values higher than 0.01, ambiguously mapping peptides and peptides with a proportion of missing values greater than 0.5 across all samples were removed from the analysis. Missing values were imputed using minDet imputation for missing values not at random, and knn for missing values at random. Peptide quantitation information was aggregated at the protein level using qfeatures with the function colMedians, quantitation information was log₂ transformed and normalised. Protein-wise linear models were built with limma which were subsequently adjusted with the eBayes function. Differentially expressed proteins were considered if they had log₂ fold changes (LFC) greater than 1 or smaller than -1, statistically significant proteins were considered if their Bonferroni-Hochberg adjusted p-value was smaller than 0.05.

Phylogenetic analysis of DUF3828 domain

3327 proteins containing DUF3828 domain (PF12883) were acquired from InterPro (v103), and the domain architectures represented by E. coli YbjP and YqhG were selected. Taxonomy information was retrieved from UniProt on Dec 22, 2024, and the signal peptide was predicted by SignalP 6.0 (Teufel et al., 2022). After removing proteins with unidentified taxonomy, representative sequences were selected by manual inspection of E. coli and Salmonella and CD-HIT with a 95% identity threshold (Fu et al., 2012), yielding 356 sequences. The multiple sequence alignment was generated by hmmalign using the PF12883 HMM profile and processed with TrimAl on automated mode (Capella-Gutiérrez et al., 2009). A phylogenetic tree was constructed from the resulting multiple sequence alignment using IQ-Tree2 on a default parameter (Nguyen et al., 2015). The tree was rooted using the minimal ancestor deviation method (Tria et al., 2017), yielding the same result. Annotation was performed with TreeViewer (Bianchini and Sánchez-Baracaldo, 2024) and webFlaGs (Saha et al., 2021) was used to analyze the gene synteny of DUF3828-containing proteins.

Bioinformatic analysis of outer membrane efflux protein

Outer membrane efflux proteins (IPR003423) and TolC proteins (IPR010130) were acquired from InterPro (v103) and the taxonomic information was retrieved from UniProt on Dec 22 2024. The signal peptide was predicted by SignalP6.0. Outer membrane efflux proteins of Pseudomonadota were selected and clustered with CLANS (Frickey and Lupas, 2004).

For the phylogenetic analysis of TolC, the method described for DUF3828 analysis was used with some modifications. Sequences of Pseudomonadota were collected, and the sequence showing the highest sequence similarity to E. coli TolC in each organism was selected. Redundant sequences were removed by CD-HIT with a 90% identity threshold. In addition, a phylogenetic tree was constructed using FastTree on -mlacc 2 -slowni parameter (Price et al., 2010).

Supplementary Information

Supplementary Figure S1.

Supplementary Figure S2.

Supplementary Figure S3.

Supplementary Figure S4.

Supplementary Figure S5.

Supplementary Figure S6.

Supplementary Figure S7.

Table S1.

Table S2.

Table S3.

Table S4.

Table S5.

Table S6.

Data availability

The MacAB-TolC-YbjP and AcrABZ-TolC-YbjP atomic models and electron microscopy maps have been deposited in the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) under the accession codes 9QGY, EMDB-53150, and 9TG4, EMD-55890 respectively. Proteomics data have been deposited with the PRIDE database with identification code 1-20251119-140222-7962809

Acknowledgements

JG was supported by an Amgen scholarship. Grids were prepared and cryo-EM data collected at the BIOCEM facility, Department of Biochemistry, University of Cambridge. We thank Dimitri Y. Chirgadze, Steven Hardwick, and Lee Cooper for assistance with data collection and processing at the Cryo-EM Facility. We thank Yulia Yuzenkova for providing samples of microcin J25. We thank Somenath Bakshi, Liz Sockett and Waldemar Voller for helpful discussions, and Waldemar for providing mature and early forms of peptidoglycan.

Additional information

Funding

This work is supported by an ERC Advanced Award (742210) and Wellcome Trust Investigator Awards (222451/Z/21/Z and 200873/Z/16/Z). AZ is a recipient of a Transition To Independence (TTI) fellowship from the School of Biological Sciences at the University of Cambridge, supported by funding from the Rosetrees Trust (JS16/TTI2021\1) and the Isaac Newton Trust (21.22(a)iii) and the School of Biological Sciences at the University of Cambridge.

Funding

Wellcome Trust (WT)

https://doi.org/10.35802/222451

• Ben F Luisi

• Yvette Ntsogo

• Andrzej Harris

• Elise Kaplan

• Ben Jin

• Emmanouela Petsolari

• Jan Gradon

• Dingquan Yu

EC | European Research Council (ERC)

https://doi.org/10.3030/742210

• Yvette Ntsogo

• Ben F Luisi

• Jim Horne

• Elise Kaplan

cambridgeuniversity | School of the Biological Sciences, University of Cambridge (School of the Biological Sciences) (Transition to Independence)

• Ashraf Zarkan

Rosetrees Trust (Rosetrees) (JS16/TT12021\1)

• Ashraf Zarkan

Isaac Newton Trust (21.22(a)iii)

• Ashraf Zarkan