BamA self-insertion drives inner-to-outer membrane remodelling in diderm Firmicutes

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: major_revision

Abstract
Exclusive to Firmicutes, endospore formation is a complex process that results in the release of a mature spore after the mother cell lyses. The spore is protected by multiple layers, including inner and outer spore membranes (IsM and OsM), both originating from the mother cell’s inner membrane (IM). While well understood in monoderm bacteria like Bacillus subtilis, less is known about diderm sporulators such as Acetonema longum, which retain and remodel their OsM into a functional outer membrane (OM) during germination. Here, we demonstrate that outer membrane proteins (OMPs), including the essential BAM complex component BamA and the LPS translocon protein LptD, are present in vegetative and germinating cells but absent from mature spores, indicating that OM biogenesis occurs without pre-existing OMPs. Growth-stage proteomics and expression profiling identify two previously uncharacterized proteins, SonA and SonB, co-expressed with BamA and in a conserved operon among diderm Firmicutes. SonA is a β-barrel OMP with three POTRA domains, while SonB is a predicted OM lipoprotein. In vitro, BamA and SonA spontaneously fold and insert into liposomes, supporting a model where BamA and/or SonA initiate self-insertion into the OsM, driving its stepwise transformation into a fully functional OM.

Introduction
Members of the phylum Firmicutes rely on endospore formation (sporulation) as a complex stress response strategy when faced with starvation and unfavorable environmental conditions1. Endospores (spores) are metabolically inactive cell forms with intrinsic resistance to heat, ultraviolet radiation, desiccation and pressure2. These properties allow for the preservation of genetic material for millions of years, rendering spores the oldest life form on Earth3. Notable examples of sporulating bacteria include members of the classes Bacilli and Clostridia, with Bacillus subtilis and Clostridioides difficile as some of the most extensively characterized representatives1. These model spore-formers and their close relatives within the phylum Firmicutes are also classified as monoderm based on the composition of their cell envelope, which includes a single cytoplasmic membrane (CM) and a thick layer of peptidoglycan (PG). Thus, our current understanding of sporulation-associated processes has been based on studies performed primarily in monoderm species.

The discovery of diderm Firmicutes challenged the universality of past findings4. Deeply rooted within the Firmicutes, members of the class Negativicutes possess typical diderm cell envelopes comprising an inner membrane (IM), a thin layer of PG, and a true outer membrane (OM) that contains lipopolysaccharides (LPS) and integral β-barrel outer membrane proteins (OMPs)5. Notably, in addition to having an OM, some Negativicutes, such as Acetonema longum, can sporulate6,7. Characterization of sporulation in monoderm and diderm Firmicutes using cryo-electron tomography (cryo-ET) helped identify major structural similarities and differences7–9. Overall, in both cell envelope types, sporulation begins with the development of an asymmetric septum and the formation of two compartments with different sizes and fates9. The larger compartment, known as the mother cell, engulfs the smaller compartment, or prespore, in a phagocytosis-like process9. Multiple protective layers develop around the prespore before it is released as a mature spore upon lysis of the mother cell. The mechanisms of septum formation and cell envelope remodeling during germination, however, differ between monoderm and diderm species7,8. In monoderms, the vegetative and sporulative septa form by invagination of the CM and PG, while diderms invaginate their IM, PG, and OM during vegetative septum formation, and only the IM and PG during sporulation (Fig. 1a,b). These structural differences imposed by the sporulative septa result in both monoderm and diderm species producing mature spores surrounded by two IM-like membranes: an inner and outer spore membrane (IsM and OsM) (Fig. 1c,d)9. Upon germination, monoderm species discard their OsM, whereas diderm species retain and remodel it into a true OM (Fig. 1e). This remodeling represents a biological phenomenon where an IM-like membrane is converted into a functional OM, highlighting the remarkable dynamic nature of bacterial membranes7.

The OM is an ancient structural feature that is conserved across most bacterial phyla and defined by the presence of hallmark components with distinct functional roles5,10. Importantly, it acts as a permeability barrier, with integral OMPs restricting the diffusion of small hydrophilic molecules, while LPS in the outer leaflet repels hydrophobic molecules11. Bacteria require dedicated systems to maintain the OM. For example, integral OMPs are inserted by the β-barrel Assembly Machinery complex (BAM), while the LPS transport complex (LPT) incorporates LPS into the OM11. BamA and LptD are the core OM components of the BAM and LPT complexes, respectively. BamA forms a 16-stranded β-barrel with several N-terminal periplasmic polypeptide transport-associated (POTRA) domains, which recognize substrates and interact with the accessory lipoproteins BamBCDE10,12. The current model for OMP insertion by the BAM complex involves direct interaction between the hydrogen bonds of BamA and its substrate and the assembly of a hybrid β-barrel intermediate via a mechanism termed β-augmentation13. During LPS transport, the periplasmic domain of LptD accepts the LPS monomers from the LPT transenvelope bridge and passes them to its C-terminal 26-stranded β-barrel, which facilitates their insertion in association with LptE11. BamA and LptD are essential proteins that are strictly conserved among all organisms with a typical OM10. They are also functionally linked, as BamA mediates the folding and insertion of LptD into the OM11.

Not all bacteria possess the same BAM and LPT accessory proteins, with differences reported at both phylum and species levels14. For example, the BAM complex in E. coli includes the four lipoproteins BamB-E, of which only BamD is considered essential, while other species show variation in the presence and functional importance of accessory components12,15,16. Some organisms, such as members of the Cyanobacteria, lack homologs of conserved BAM lipoproteins altogether, suggesting that BamA may either function independently or in association with currently unidentified proteins17. Similarly, some diderm bacteria (for example, members of the phyla Thermotogae, Deinococcus–Thermus, and Cyanobacteria) lack the canonical LPS in their OM and have been shown to possess incomplete LPT systems or highly diverged homologs10. In addition to maintaining the protein and lipid composition, diderm bacteria have developed tethering systems to attach the OM to the PG and preserve the structural integrity of the cell envelope. In E. coli, this function is performed by the Brown’s lipoprotein, or Lpp, and the OmpA-like proteins10. In contrast, diderm Firmicutes rely on the homotrimeric protein OmpM, composed of an OM β-barrel and a periplasmic coiled-coil domain that connects an S-layer Homology (SLH) domain to PG18.

In this work, we elucidate the mechanism of inner-to-outer membrane remodeling during germination. Previous studies using cryo-ET showed that the IsM and OsM were continuous with the IM of the mother cell9. Density profiles of the membranes at different growth stages further revealed that IsM and OsM were similar to the IM and not the OM, suggesting shared structure and composition9. Thus, we hypothesize that BamA and other OM hallmarks are absent from mature A. longum spores, and that the OsM was structurally and functionally remodeled from an IM-like into a true OM during germination. Homology searches reveal that, despite the overall typical diderm architecture, A. longum encodes only for the TamB component of the Translocation and Assembly Module (TAM) complex, entirely lacks the Lipoprotein outer membrane localization (Lol) pathway, and possesses non-canonical components of the BAM, LPT and tethering systems. By tracking the expression and localization of proteins involved in OM biogenesis at different growth stages, we demonstrate that BamA and other OMPs are absent from mature spores yet highly abundant in vegetative and germinating cells. Synteny analysis identified the presence of two previously uncharacterized, conserved proteins within the BamA operon: a β-barrel OMP F7NM22 (referred to as SonA) and a putative OM lipoprotein F7NM24 (referred to as SonB). Folding assays reveal that BamA and SonA can self-incorporate into liposomes under physiological conditions. Together, these observations support a model in which BamA self-insertion initiates inner-to-outer membrane remodeling, a process that may be supported by the proteins SonA and SonB.

Results
Computational prediction and detection of OMPs
Secondary and tertiary structure predictions were used to identify hydrophobic β-barrels or β-hairpin-containing protomers of integral OMPs in the A. longum genome. Using sequence homology to known OMPs and predictions of subcellular localization, we identified 46 putative β-barrel OMPs (Table 1). This list included homologs of the conserved proteins BamA, LptD and PilQ, four homologs of the PG anchor OmpM1-4, five TolC family proteins, nine TonB-dependent receptors, and 12 proteins of unknown function. The number of predicted β-barrel OMPs was comparable to previously published proteome surveys in diderm bacteria, estimating 40-100 integral OMPs19–23. HMM and PSI-BLAST searches were conducted to identify and annotate sequences that exhibited significant similarity with conserved β-barrels OMPs and other proteins involved in cell envelope maintenance, such as components of the pathways for OMP biogenesis, LPS biosynthesis and transport (Supplementary Table 1).

Transcriptomic and proteomic analyses of vegetative and sporulating cells, as well as dormant and germinating spores, were conducted to verify protein expression during specific growth stages (Fig. 2). Of the 46 predicted β-barrel OMPs, 25 were expressed, with 23 detected during vegetative growth and 12 during germination (Fig. 2a, Supplementary Table 2). These included BamA and LptD, along with OmpM1 and OmpM2, the OM phospholipid transporter MlaD, the lipid A 3-O-deacylase PagL, two putative porins, TolC proteins, TonB receptors, and three Type Vb autotransporters (Fig. 2a). While only approximately 50% of the predicted OMPs were identified with LC-MS/MS, our transcriptomic data indicated that the number of target peptide spectra generally aligned with the levels of their respective transcripts, suggesting that, under our experimental growth conditions, undetected proteins were likely expressed at levels below the detection limits or not expressed at all (Supplementary Table 3).

OMP biogenesis pathway and other β-barrel OMPs
A detailed analysis of the detected proteins revealed several distinct features in the OMP biogenesis pathways of A. longum. Structural prediction of BamA indicated that the protein consisted of a 16-stranded β-barrel followed by three periplasmic POTRA domains (Fig. 3a, Supplementary Fig. 1a). Notably, homology searches did not detect the canonical BamB-E lipoproteins, despite the known essential role of BamD reported in other diderms15. Homologs of the IM holotranslocon SecYEG-SecDF-YajC-YidC, which is associated with BamA in E. coli, were identified in the genome and also detected through proteomics (Supplementary Table 1)15,24. A. longum encoded four homologs of OmpM (OmpM1-4), featuring the characteristic β-barrel, coiled-coil, and SLH domains (Fig. 3a). Among these homologs, sequence similarity was primarily observed within the SLH domains rather than the transmembrane domains (Table 1, Supplementary Fig. 2). OmpM1 exhibited the highest level of transcription and consistently ranked among the most enriched OMPs (Supplementary Table 2, Supplementary Table 3). The expression of OmpM1 and OmpM2 was linked to the sporulation cycle, with OmpM1 produced constitutively and OmpM2 only observed during germination (Fig. 2a,c, Supplementary Table 2). No peptides were detected from OmpM3 and OmpM4 (Supplementary Table 2). No homologs of TamA were identified in the genome of A. longum, whereas TamB was detected in vegetative cells and germinating spores (Supplementary Table 2). MlaD was detected in vegetative and sporulating cells but not in germinating spores (Supplementary Table 2).

The most highly expressed protein-encoding gene was for the β-barrel OMP F7NLZ1-2, initially annotated as two distinct proteins, F7NLZ1 and F7NLZ2 (Supplementary Table 3). Sanger sequencing of the whole gene F7NLZ1-2 (GenBank accession PX233182) and tertiary structure prediction suggested that the two proteins comprised halves of a single porin (Table 1, Supplementary Fig. 1b, Supplementary Fig. 3). Structural predictions identified four additional proteins, F7NFH8, F7NF30, F7NFT1, and F7NML1, as Type Vb autotransporters, while five other OMPs, F7NDN8, F7NF31, F7NFH7, F7NHH4, and F7NI93, were predicted as ten-stranded β-barrels with several β-strands extending into the extracellular environment, similar to the eight-stranded attachment protein OmpX (Table 1, Supplementary Fig. 1b)25,26. Overall, these proteins lacked any further conserved domains that could shed light on their function.

Synthesis of LPS and lipid transport pathways
The OM is defined by lipid asymmetry, with LPS and phospholipids (PLs) serving as the primary components of the outer and inner leaflets, respectively (Fig. 2a)11. The synthesis of LPS occurs through the conserved IM-associated enzymes of the Raetz pathway, while the LPT system, which spans the IM, PG, and OM, transports LPS molecules to the outer leaflet of the OM11. Our analyses indicated that the primary components of the LPS biosynthesis and transport pathways in A. longum were largely conserved, with some deviations from previously characterized systems (Fig. 2a, Fig. 3a, Supplementary Table 1). For example, in the Raetz pathway of A. longum, the intermediate lipid X was predicted to be generated by the pyrophosphatase LpxI, rather than the non-homologous LpxH or LpxG found in E. coli and Chlamydia, respectively (Fig. 2a, Supplementary Table 1)27–29. LpxI, LpxH, and LpxG perform the same overall reaction but target different phosphates, indicating that their distinct catalytic strategies reflect organism-specific adaptations in the early steps of lipid A biosynthesis29. We also identified four homologs of the lipid A lauroyltransferase LpxL (F7NG76, F7NM01, F7NM16, F7NFR4), but not the myristoyltransferase LpxM. Since fatty acids with chains of both 12 (lauroyl) and 14 (myristoyl) carbons were previously detected in the LPS fraction of A. longum7, we conducted a reciprocal BLAST, which showed that enzymes annotated as LpxL in A. longum were homologous to LpxM in E. coli, suggesting that LpxL could attach both lauroyl and myristoyl groups to lipid A in A. longum (Supplementary Table 4). As part of the LPS transport pathway, we identified the MsbA permease that directs lipid A across the IM, along with most of the LPT complex proteins: the IM components LptB2FG, the periplasmic proteins LptA and LptC, and the OM translocon LptD (Supplementary Table 1). A homolog of the OM lipoprotein plug LptE, typically present in model diderms11, was not identified in A. longum with either homology searches or proteomics. Interestingly, proteomics results indicated that the LPT permease in A. longum was composed of a homodimer of a single protein with sequence homology to both LptF and LptG (LptF/G, F7NLZ6) rather than the canonical LptFG heterodimer found in E. coli (Supplementary Table 1, Supplementary Table 2)5. Additionally, two putative A. longum OMPs, F7NLY8 and F7NII2, were predicted to be structurally similar to the canonical LptD, featuring a C-terminal β-barrel and an N-terminal β-jellyroll region (Fig. 3a, Supplementary Fig. 1). While the C-terminal β-barrel of the canonical LptD consists of 26 β-strands, in F7NLY8 this domain was predicted to contain 16 β-strands with over 90% confidence, and in F7NII2 the domain contained 26 β-strands with less than 70% confidence (Table 1, Fig. 3a, Supplementary Fig. 1)11. Both F7NLY8 and F7NII2 were encoded close to other genes associated with LPS biosynthesis and transport (Supplementary Fig. 4). However, only F7NLY8 yielded a significant match to LptD through HMM searches, exhibited differential gene expression throughout the sporulation cycle, and was consistently detected with proteomics (Supplementary Table 1, Supplementary Table 2, Supplementary Table 3). Based on these observations, we conclude that F7NLY8 is the functional LptD homolog in A. longum. F7NII2 may be a distant homolog of LptD that serves a different function, such as lipoprotein transport, as reported in other bacteria30,31.

Our searches predicted the presence of a TamB homolog but not TamA, and transcriptomics and proteomics detected the expression of TamB in vegetative cells and germinating spores (Supplementary Table 1). These findings led us to hypothesize that, rather than performing a protein transport role as part of the TAM complex, TamB may be implicated in the anterograde transport of OM phospholipids, which has been suggested for its ortholog in another Negativicute32. Interestingly, tertiary structure predictions revealed that A. longum possessed another AsmA-like family protein, F7NHB8, whose expression was detected by both transcriptomics and proteomics in vegetative cells and germinating spores (Supplementary Fig. 1b, Supplementary Table 2, Supplementary Table 3)26. We hypothesize that while TamB may be involved in OM phospholipid transport, F7NHB8 may perform an overlapping function or associate with BamA as a potential component of a hybrid OMP transport system, as proposed in the spirochete Borrelia burgdorferi33.

Investigation of the components of the Mla system showed that A. longum lacked homologs of the periplasmic proteins MlaA and MlaC, as well as the ABC transporter binding protein MlaB, which are found in E. coli34 (Supplementary Table 1). Of the remaining Mla components (MlaDEF), MlaD was predicted to possess an N-terminal transmembrane α-helix anchoring the protein to the IM, a Mammalian Cell Entry domain that spans the periplasm, and a C-terminal β-barrel embedded in the OM (Table 1, Supplementary Fig. 1b)35. Transcriptomics captured the expression of all three proteins, while proteomics detected peptide fragments of MlaD only, possibly due to low counts of MlaE and MlaF (Supplementary Table 2, Supplementary Table 3). Our data thus suggested that the A. longum Mla system consists of only three proteins, MlaDEF, where MlaE and MlaF form an IM permease, and MlaD spans the entire cell envelope to facilitate phospholipid transport to the OM, similar to other Negativicutes32. In summary, A. longum and other diderm Firmicutes display the following unique characteristics in their OM biogenesis pathways: the Lol system is completely absent, and the BAM, LPT, TAM, and Mla systems tend to be simpler, with truncated versions. Future characterization of these complexes will offer insights into the minimal set of proteins that maintain the same functions in diderm species.

Differential gene and protein expression during sporulation
To identify pathways involved in OM remodeling, cells at various stages of the sporulation cycle were analyzed using transcriptomics and proteomics (Fig. 2b,c). A comparison between the vegetative and sporulating A. longum showed that genes associated with OM biogenesis were not differentially expressed, indicating they did not play significant roles during this transition phase (Supplementary Table 3). However, we observed an enrichment of transcripts related to LPS synthesis and transport during sporulation, indicative of overproduction of LPS (Fig. 2b,c, Supplementary Table 3). Other enzymes involved in OM lipid homeostasis, such as fabZ dehydratase, a member of the fatty acid synthesis (FAS II) pathway, were not upregulated (Supplementary Table 3)36. The genes encoding TamB, the second AsmA-like protein F7NHB8, and the Mla system also showed no significant change in expression (Supplementary Table 3)32. Our data thus indicated that sporulation is potentially characterized by a surplus of LPS in the cell, while the core OM biogenesis machinery remained constitutively expressed. These observations led us to speculate that LPS overproduction could have a function in destabilizing the OM and promoting lysis of the mother cell. We, therefore, propose that, along with the degradation of PG by autolysins, the disruption of OM lipid homeostasis could be the primary mechanism for spore release in diderm Firmicutes.

The precise biochemical composition of the IsM and OsM has not been characterized due to the lack of established isolation and separation protocols1. However, proteomic analyses of whole A. longum spores, including IsM and OsM, revealed that OMPs were absent during dormancy (Fig. 2a, Supplementary Table 2). Specifically, the absence of BamA and LptD indicated that mature spores, and notably the OsM of A. longum, lacked essential OMPs. The absence of the LPT periplasmic bridge proteins, LptC and LptA, further suggested that the spores also lacked LPS (Supplementary Table 2), although lipidomic analyses are required to definitively determine the presence or absence of LPS and its precursors. Our data thus demonstrated that the OsM, initially formed by the invagination of the IM of the mother cell, retained an IM-like protein and lipid composition throughout engulfment. All enzymes involved in the LPS synthesis pathway (LpxA-LpxH/I, LpxL/M, and Kdt) and the IM-localizing components of the LPT machinery (LptB and LptF/G) were present during dormancy, highlighting that mature spores were primed for LPS biosynthesis (Fig. 2a). While peptides from the β-barrel F7NLZ1-2 were also detected at this stage, their counts declined with more stringent spore purification, indicating contamination from mother cell debris (Supplementary Table 2).

The enrichment of OM-associated proteins was assessed by cross-referencing the proteomes of mature and germinating spores with the gene expression data. Out of the 46 predicted β-barrel OMPs, 12 were detected during germination, including BamA, LptD, OmpM1 and OmpM2, a TolC homolog, along with six β-barrels of unknown function (Fig. 2a). The detection of LptD and the periplasmic bridge protein LptA through both transcriptomics and proteomics highlighted LPS transport during germination, further supporting active OsM remodeling at this stage (Fig. 2, Supplementary Table 2, Supplementary Table 3). Interestingly, OmpM2 and the putative porin F7NDG7 were absent from vegetative cell membranes, sporulating cells, and dormant spores, potentially implicating these proteins in germination-specific cell envelope remodeling (Supplementary Table 2). Peptide and transcript levels of OmpM2 were also higher than those of OmpM1 during germination, identifying OmpM2 as the primary anchor during germination (Supplementary Table 2, Supplementary Table 3). Other transcripts enriched during germination and known to be involved in OM biogenesis included the skp1 chaperone, degP protease, and the general secretion system secYEG (Fig. 2c, Supplementary Table 3). Both TamB and the AsmA-like protein F7NHB8 also demonstrated transcript and peptide enrichment during germination, suggesting their functional involvement in OM biogenesis (Supplementary Table 2, Supplementary Table 3). Together, our results indicate that membrane remodeling is driven by the synthesis and insertion of OMPs and LPS at the onset of germination.

Immunodetection of BamA and OmpM1 in vegetative cells, mature spores, and germinating spores
To determine when and where key OMPs were localized throughout the sporulation cycle, we used super-resolution immunofluorescence, cryo-immuno-electron microscopy (cryo-immuno-EM), and Western blot analysis (Fig. 4). Since A. longum is non-genetically tractable, we generated polyclonal antibodies against the β-barrel domain of BamA and OmpM1, the two main proteins detected in the vegetative OM (Supplementary Table 2). Tracking the expression of these proteins with super-resolution immunofluorescence showed widespread distribution of BamA, which appeared as puncta on the surface of both vegetative and germinating cells (Fig. 4a,b). Quantitative spatial analysis showed that BamA formed ~8±2 puncta per μm² in vegetative cells, with an average width of 300 nm (n=51; Supplementary Table 5), which is comparable to the ~13 precincts per μm2 estimated for E. coli37. Given the resolution limit of our super-resolution fluorescence microscopy approach (~120 nm in x and y), we could not determine the exact number of BamA molecules within each punctum. However, considering that individual BamA molecules are ~5 nm in diameter, each punctum could potentially contain several molecules.

Immuno-EM of chemically decoated spores and spore cross-sections further confirmed the absence of BamA from mature spores (Supplementary Fig. 5a). Despite the literature reporting that decoating may result in stripping of the OsM38,39, two intact membranes were visible in chemically-decoated A. longum spores (Supplementary Fig. 5a). Neither the surfaces of dormant spores nor their cross-sections exhibited differential colloidal gold accumulation compared to the control sample, consistent with the absence of BamA from mature spores (Supplementary Fig. 5). In contrast, colloidal gold clustering was evident during germination in the 2D projection images using cryo-immuno-EM (Fig. 4b). Western blot analysis of vegetative cells and dormant spores using anti-BamA serum detected BamA in vegetative cells and germinating spores, but not in whole or chemically de-coated dormant spores (Fig. 4c). OmpM1 showed similar detection patterns across the sporulation stages with Western blot and fluorescence microscopy, displaying fluorescent puncta on the surface of vegetative cells and at the tip of germinating spores (Supplementary Fig. 6, Supplementary Fig. 5b). Western blot analysis detected some non-specific binding with the anti-OmpM1 serum, likely due to cross-reactivity with abundant host proteins of similar molecular weight (Supplementary Fig. 6). In contrast, the anti-BamA serum showed minimal background, especially in lysates from germinating spores, supporting the specificity of the labeling observed with both immunofluorescence and immunoEM (Fig. 4). Taken together, these results demonstrate that BamA and OmpM1 are present in vegetative and germinating cells but absent from mature spores.

OM-associated proteins are conserved among diderm Firmicutes
To identify the proteins involved in the inner-to-outer membrane remodeling during germination, we assessed the presence and synteny of OM-associated genes. In particular, bamA, skp, and the genes for LPS synthesis and transport (such as lpx and lpt), were found organized in conserved genomic clusters, characteristic of typical Gram-negative bacteria, including diderm Firmicutes4. Analysis with Operon-mapper predicted nine operons in the largest OM cluster of A. longum (Supplementary Table 6)40. This list included the mla operon, along with five operons containing the genes for LPS biosynthesis (lpx, kds/kdt, rfa, waa, wba) and transport (msbA, lpt) (Supplementary Fig. 4). Additionally, data from the Comprehensive Directory of Bacterial Promoters (CDBProm) identified a shared promoter between bamA and three other genes encoding for SonB (F7NM24), SonA (F7NM22), and the skp1 chaperone, involved in the folding and assembly of OMPs41 (Fig. 5a). Structure predictions further indicated that SonA comprised a ten-stranded β-barrel with three N-terminal POTRA domains exhibiting the characteristic β-α-α-β-β topology26,42, reminiscent of a smaller version of BamA (Table 1, Fig. 3a, Supplementary Fig. 1). The presence of POTRA domains specifically suggested a role in protein transport or possible interactions with TamB. The tertiary structure of SonB, on the other hand, was predicted to include a transmembrane helix (Fig. 3a, Supplementary Fig. 1). Finally, proteomics and transcriptomics data confirmed that BamA, SonA, and SonB were co-expressed and upregulated during germination (Supplementary Table 2, Supplementary Table 3).

To assess gene synteny, we examined the distribution of key OM-associated proteins across the 36 Negativicute species with fully sequenced genomes and confirmed sporulation ability. BamA, LptD, and TamB were universally conserved, while homologs to BamD were completely absent (Fig. 5b, Supplementary Table 7, Supplementary Table 8). SonA and SonB were also conserved among all members, except for V. parvula and Acidaminococcus fermentans. Furthermore, sonB, bamA, sonA, and skp1 were syntenic across all genomes, suggesting they form a conserved genetic cluster that is co-regulated and functionally linked to OM biogenesis (Fig. 5, Supplementary Table 8). Notably, the presence or absence of SonA and SonB homologs did not correlate with the ability of these organisms to sporulate, indicating that their functions are unrelated to sporulation.

No accessory proteins detected in association with BamA
To identify potential interacting partners of BamA in A. longum, we conducted co-immunoprecipitation (co-IP) with chemical cross-linking using anti-BamA serum (day 28), followed by immunoblotting and LC-MS/MS analysis of the recovered complexes (Fig. 6a, Supplementary Table 11). Comparison to the control sample prepared using pre-immunization serum failed to detect major BamA interaction partners. Only two proteins co-eluted with BamA (17 peptides) at >95% confidence: the putative generic porin F7NLZ1-2 (10 peptides) and a peptidase S55 domain-containing protein F7NM31 (3 peptides) (Supplementary Table 11). As an alternative approach, we also analyzed whole cell preparations before and after reversible cross-linking using Western blot with anti-BamA antibodies. Protein complexes with BamA were expected to show an additional high molecular weight band in cells that were cross-linked by formaldehyde, while non-cross-linked and heat-treated samples were expected to show only one band corresponding to BamA (~63 kDa). No difference was observed between any of the samples, all of which displayed only a single ~63 kDa band (Fig. 6b). Despite combining cross-linking and co-IP, we were unable to identify proteins associating with BamA. These results suggest that BamA may function without accessory proteins, that interactions with other proteins are transient, or that they require specific expression conditions not replicated in our assays.

BamA and SonA self-insert into liposomes
Since A. longum and other Negativicutes do not encode alternative pathways for OMP assembly, such as TAM and Lol, we hypothesized that unassisted folding of BamA and/or SonA could influence OsM remodeling5. Previous in vitro studies have demonstrated that β-barrel OMPs tend to fold into liposomes spontaneously43. However, this process was limited to β-barrels with ten or fewer β-strands and took several hours to complete, unlike the minutes observed for BAM-mediated insertion44. While 16-stranded β-barrels like BamA can also self-assemble into liposomes in vitro, this process was reported to be less thermodynamically favorable and to require temperatures above 40°C45. Therefore, we decided to investigate whether BamA and SonA were capable of self-inserting into liposomes.

Trypsin and Proteinase K are common proteases used in protein folding assays, each with unique characteristics. Therefore, the suitability of each protease for different proteins must be determined experimentally. We conducted in vitro assays to test the folding of denatured proteins into liposomes, followed by diagnostic digests with trypsin or Proteinase K (Fig. 7, Supplementary Fig. 7). Since lipid-embedded residues are protected from protease cleavage, this method allowed us to distinguish between proteins in solution and those folded into membranes46. Western blot analysis of BamA proteoliposomes digested with trypsin showed a distinct band at ~50 kDa, and several prominent bands ~40 kDa (Fig. 7a). Based on the predicted structure and identified trypsin cleavage sites (Supplementary Table 10), we estimated that BamA without POTRA1 and POTRA1-3 domains is ~54 kDa and 38 kDa, respectively, aligning with the observed bands26. A faint band at ~63 kDa, representing intact BamA, was also observed. This was likely caused by incomplete digestion due to the excess BamA used to enhance protein visualization on gels without adjusting the trypsin amount. The ~63 kDa band increased in intensity with liposomes over time, which we interpret as BamA inserting into lipid vesicles, thereby blocking trypsin access even to the POTRA domains. When analyzed on semi-native PAGE gels at 4°C, the folded and denatured BamA displayed a ~10 kDa shift in electrophoretic mobility (Fig. 7b). These findings further support our conclusion that BamA adopts the correct spatial configuration within liposomes. Trypsin-treated SonA proteoliposomes examined on silver-stained SDS-PAGE gels showed an intact protein band (~45 kDa) along with several bands between 30-45 kDa, corresponding to the expected size of the β-barrel portion with two partially digested POTRA domains (Fig. 7c). Importantly, most of SonA was fully digested in the absence of liposomes, indicating that it was soluble and accessible to trypsin. These findings show that both A. longum BamA and SonA can self-insert into liposomes at physiological conditions.

Treatment of BamA proteoliposomes with Proteinase K resulted in complete BamA digestion, which aligns with Proteinase K having broad specificity (Supplementary Fig. 7a). Treatment of SonA with Proteinase K, on the other hand, produced a single prominent band at ~30 kDa (Supplementary Fig. 7b). Although this band was similar in size to the enzyme itself, its intensity increased after lipid pre-incubation, indicating that SonA was partially protected from Proteinase K cleavage.

Methods
Bacterial strains and cultures
Acetonema longum APO-1 DSM 6540 cells were routinely grown at 30°C in an anaerobic chamber (BactronEZ, Sheldon Manufacturing, Inc.) at an atmosphere of 5% H2, 10% CO2 and 85% N2. Liquid 4× YACO broth (ATCC Medium 2479) supplemented with yeast extract at 1.5 g/L, or AC-20 medium with 2% (w/v) agar as a solidifying agent, was used for cultivation50. Live A. longum, including dormant endospores, were handled under anoxic conditions. Liquid media and reagents were pre-flushed with N2 to remove oxygen. E. coli DH5α and BL21 (DE3) were cultivated in Luria-Bertani (LB) broth and agar under aerobic conditions unless otherwise specified.

Endospore purification
Dormant spores were extracted from stationary phase A. longum cultures using previously established protocols with slight modifications51. In brief, 800 mL of >2-week-old culture were harvested by centrifugation and washed in 200 mL buffer containing 1 M KCl and 0.5 M NaCl, then lysed by incubation at 37°C with shaking in 200 mL lysis solution containing Tris-HCl at pH 7.2, supplemented with 80 mg/L lysozyme and 1 mM EDTA. Lysed cells were centrifuged and washed in 200 mL 1 M NaCl, then 200 mL H2O, then 40 mL 0.05% (w/v) SDS, then 40 mL H2O. All centrifugation up to this stage was done at 5000×g for 10 min at 4°C. The TEP buffer washing step was also omitted. Each spore preparation was further resuspended in 400 µl PBS supplemented with 0.1% (v/v) Tween 20 and loaded on a 1 mL 20%-50% (w/v) urografin gradient52. Pellets remaining after centrifugation for >30 min at 14,500 rpm and 4°C in a tabletop centrifuge were transferred onto a repeat density gradient and washed three times in resuspension buffer prior to storage at −20°C.

Endospore germination
Endospore pellets from ~4 L starting culture were subject to heat shock at 60°C for 15 min and briefly chilled on ice prior to inoculation into 10 mL modified 4× YACO broth supplemented with 1.5 g/L yeast extract53. Cultures were incubated for 16 h at 30°C in Hungate tubes, placed horizontally to maximize spore dispersion. Cells were harvested anaerobically at ~5500×g, and pellets were washed once in PBS prior to downstream processing.

Computational predictions of OMPs
Acetonema longum APO-1 DSM 6540 proteome (Uniprot) was analyzed for putative integral β-barrels using BOMP54, as well as PRED-TMBB255. Proteins with an integral β-barrel score >0.5 were then assessed for predicted subcellular localization with PSORTb v3.056, CELLO57, SOSUIGramN58, and SignalP 6.059. Secondary structure predictions were performed using Quick2D60, and tertiary structures were generated using AlphaFold and AlphaPickle61,62. All searches were run with default parameters. The final list of candidates was cross-referenced with A. longum homologs of known OMPs5,21.

Sanger sequencing of the f7nlz1-2 gene
The putative OMPs F7NLZ1 and F7NLZ2, identified with computational methods outlined above, displayed low confidence levels in their respective 3D structural predictions and appeared to be encoded by two overlapping genes. The gene encoding the OMP F7NLZ1-2 was confirmed using Sanger sequencing by the UBC Sequencing and Bioinformatics Consortium using F7NLZ2_up1 (5’ to 3’: gtcgacatgaaaaagactcttgtattagctctgg) as the forward primer and F7NLZ1_down1 (5’ to 3’: ggatcctagaagaacagttcgagctgg) as the reverse primer.

Membrane protein extraction from vegetative cells
Sucrose gradient membrane isolation. Pellets from exponentially growing A. longum cultures (100 mL starting volume per replicate), were resuspended and sonicated in 50 mM Tris-HCl, pH 7.5, supplemented with 1 mM PMSF, then spun down at 4700 rpm to pellet unlysed bacteria and debris. The supernatant was centrifuged at 100,000×g at 4°C to obtain whole membranes. Membrane pellets were solubilized in 20% (w/v) sucrose and loaded on a 5 mL density gradient of 50% and 70% (w/v) sucrose, then centrifuged at 100,000×g at 4°C overnight to separate the IM and the OM. The membrane bands were collected, pelleted at 100,000×g and washed three times in sterile ice-cold H2O. The OM fractions were dissolved in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.3, supplemented with PMSF, and the IM fractions were dissolved in the same buffer, additionally containing 0.3% (v/v) N-lauroylsarcosine, prior to acetone precipitation. Briefly, solubilized membranes were combined with cold acetone at a sample-to-acetone ratio of 1:4, vortexed vigorously, incubated at −20°C for 60 min, centrifuged at 15,000×g at 4°C, and the pellets were air-dried for 30 min at room temperature after decanting the supernatant.

Sodium carbonate integral protein enrichment
Sonicated lysates from 100 mL cultures and after the 4,700 rpm spin described above, were combined with ice-cold 0.1 M Na2CO3 at the ratio of 1:10. This mixture was incubated at 4°C with slow agitation for 1 h, then centrifuged at 115,000×g for >1 h at 4°C to pellet integral proteins, followed by two washes in 0.1 M Tris-HCl, pH 7.3.

Sample preparation for LC-MS/MS
Pellets were resuspended in 1× Laemmli SDS-PAGE sample dye before boiling at 95°C for 10 min and running on a 10%, 1 mm SDS-PAGE gel at 100 V for ~25 min. Gel bands containing the entire sample were stained with Coomassie Brilliant Blue G250 and submitted to the UBC Proteomics Core Facility for downstream processing. Gel slices were then destained with 50 mM NH4HCO3:100% ethanol (6:4), dehydrated in ethanol and treated with 10 mM dithiothreitol (DTT) at 56°C for 45 min. Alkylation by 55 mM iodoacetamide (IAA) was carried out in the dark for 30 min at room temperature. Following dehydration as described above, gel pieces were treated with 50 mM NH4HCO3, pH 8, and dehydrated again before overnight digestion with 0.4−1.5 µg trypsin at 37°C. To stop digestion, 1% (v/v) trifluoroacetic acid (TFA) was added, and proteins were extracted with 50% (v/v) acetonitrile and 0.1% (v/v) TFA, then 100% acetonitrile. Digested peptides were concentrated using vacuum centrifugation and cleaned up with C18 StageTip purification. Briefly, the resolubilized and acidified samples were passed through the column, washed twice with 1% (v/v) TFA, eluted in 40% (v/v) acetonitrile and 0.1% (v/v) TFA, dried and reconstituted in 2% (v/v) acetonitrile and 0.5% (v/v) formic acid.

Data acquisition
LC-MS/MS was used to detect the protein composition of membrane fractions in vegetative cells. Samples were analyzed at the Proteomics and Metabolomics Core Facility (University of British Columbia) with a Quadrupole Time-of-Flight Impact II mass spectrometer (Bruker Daltonics) coupled to an EasyLC 1200 HPLC system (Thermo Fisher Scientific) using the CaptiveSpray nanospray ionization source (Bruker Daltonics). The mass spectrometer was operated with OTOF control (version 4.1, Bruker Daltonics), and LC and MS were operated with HyStar v4.1.21.2 (Bruker Daltonics). The ESI source was operated at 1.9 kV, 0.25 Bar pressure with methanol in the nanoBooster, 3 L/min drying gas and 150°C drying temperature. The Aurora Series Gen2 (CSI) analytical column (25 cm × 75 µm, 1.6 µm FSCC18) with Aurora nanoZero and CSI fitting (IonOpticks, Parkville, Victoria, Australia) was used at 50°C. Samples of 500 ng, reconstituted in solvent A (0.1% (v/v) formic acid, 2% (v/v) acetonitrile), were run over a 90 min stepwise gradient from 5% to 90% solvent B (0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 0.4 µl/min at 7°C. The mass spectrometer was set to acquire data in the data-dependent auto-MS/MS mode with inactive focus fragmenting the top 20 ions by abundance (individually, at 18 Hz) after each full-range scan of m/z between 200–2000 (at 5 Hz). The MS/MS isolation window was 2-3 Th, and the collision energy was between 23 and 65 eV. Strict active exclusion was applied. Singly charged ions were excluded, and parent ions were excluded for 0.3 min unless a subsequent intensity increase of >5-fold was observed. The maximum error of mass measurement was set to 10 ppm.

Data processing and analysis
The Byonic search algorithm (ProteinMetrics Inc.) was applied against the Acetonema longum DSM 6540 Uniprot and RefSeq databases, and common contaminant proteins. Parameters were set to specify trypsin digestion with a maximum 1 missed cleavage, mass accuracies of 20 ppm for precursors and 40 ppm for production masses, and a false discovery rate (FDR) threshold of 1%. For further analysis, only the hits with an FDR score of <1% or an intensity reading above the highest-ranking decoy (reversed peptide sequences) were selected, whichever applied first. The presence of a protein in the entire integral protein fraction was then defined as the detection of at least three peptides in at least two out of three biological replicates in the carbonate-enriched samples and/or sucrose gradient-isolated OM fraction.

Proteomic analysis of dormant and germinating endospores
Sample preparation. Dormant or germinating endospore pellets were solubilized in 60 µl 1× Laemmli SDS-PAGE dye, boiled at 95°C for 10 min, subjected to bead-beating at 5 m/s for 2×30 s with 0.1 mm Zirconia/Silica beads using FastPrep-24 (MP Biomedicals), then boiled again at 95°C for 10 min. The suspension was then spun at 13,000 rpm for 1 min, and the supernatant was designated as the SDS-soluble fraction. Pellet remaining after lysis was resuspended in 30 µl 6.7 M urea in PBS, spun at 13,000 rpm for 1 min, and the supernatant was designated as the Urea-soluble fraction. Each fraction (15 µl) was loaded on 10%, 1 mm Laemmli SDS-PAGE gels and submitted as ingel bands, stained with Coomassie Brilliant Blue G250, to the Southern Alberta Mass Spectrometry Facility for further processing. The bands were sliced and washed with 50 mM NH4HCO3/acetonitrile (1:1), then 100% acetonitrile, before air-drying. Following reduction with 10 mM DTT, the samples were alkylated with 50 mM IAA for 30 min in the dark at room temperature. Gel slices were then washed with NH4HCO3/acetonitrile and dehydrated as described above, prior to rehydration and digestion with 0.02 µg/µl trypsin in 40 mM NH4HCO3 in 10% (v/v) acetonitrile for 2 h on ice. After removal of excess trypsin, digestion was continued for 16 h at 37°C. Peptides were extracted using a 60:30:10 (v/v) mixture of acetonitrile/water/10% (v/v) TFA, lyophilized and rehydrated in 1% (v/v) formic acid.

Data acquisition
LC-MS/MS runs were performed at the Southern Alberta Mass Spectrometry Facility (University of Calgary) on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), operated with Xcalibur (version 4.4.16.14) and coupled to an Easy-nLC 1200 system (Thermo Fisher Scientific). The peptides were loaded onto a 75 µm × 2 cm Acclaim PepMap 100 C18 trap (Thermo Fisher Scientific) in solvent A (0.1% (v/v) formic acid) at a flow rate of 2 µl/min and eluted at 0.3 µl/min in a 5 to 40% gradient of solvent A and solvent B (0.1% (v/v) formic acid in 80% (v/v) acetonitrile). Separation was performed through a 75 µm × 50 cm Acclaim PepMap RSLC C18 column (Thermo Fisher Scientific). Electrospray was applied at 2.1 kV into the ion transfer tube (300°C) of the Orbitrap operating in positive mode, top speed with 3 s cycle time. Auto Gain Control and maximum injection time were set to 4×105 and 50 ms, respectively. Initial full scan was performed at 120,000 FWHM, m/z of 375–1575 and a charge range of +2 to +7. Precursors with a peptidic profile and an intensity threshold of ≥5000 were isolated using the quadrupole and fragmented in the ion routing multipole with HCD (30% collision energy). The fragmented MS2 ions were analyzed in the ion trap at a rapid scan rate, AGC of 1×104 and injection time of 35 ms, with dynamic exclusion enabled for 30 s.

Data processing and analysis
The Lumos raw data files (*.raw) comprising monoisotopic precursors with a charge state between +2 and +7 were converted into the Mascot Generic Format (*.mgf) with RawConverter (v1.1.0.18; The Scripps Research Institute) operating in a data dependent mode. The mgf files were then run against the Acetonema longum DSM 6540 Uniprot database using the Mascot algorithm (Matrix Sciences; version 2.7), specifying trypsin digestion with a maximum of 1 missed cleavage, peptide charge of ≥2, cysteine carbamidomethylation as the fixed modification, methionine oxidation as the variable modification, and mass error tolerance of 10 ppm (0.6 Da for fragmented ions). Using the spectral counts of peptides identified at a 95% confidence level, the normalized spectral abundance factor (NSAF) scores were calculated for label-free quantitation with Excel software (Version 16.16.27 for Mac)63. The spectral counts of the SDS- and urea-soluble fractions were combined for each replicate prior to NSAF calculation, and a pseudocount of 0.5 peptides was assigned in case of no detection. Significance of the fold changes between the mean NSAF scores in dormant and germinating spore proteomes was analyzed with an unpaired, two-tailed t-test using Excel software. Significant enrichment was defined as the fold change of ≥1.5 and ≤−1.5, with a p-value of <0.05. For profiling analysis, the presence of a protein was defined as the detection of at least three spectra at a 95% confidence level in at least two out of three biological replicates.

Transcriptomic analysis of vegetative, sporulating and germinating cells
RNA extraction. Pellets of vegetative cell cultures in early exponential (2 d subculture in broth) and stationary growth (5 d subculture in broth), as well as germinating endospores (16 h post-inoculation), were resuspended in RNAprotect Bacteria reagent (QIAGEN) prior to RNA isolation with the RNeasy Mini kit (QIAGEN) with enzymatic lysis and on-column DNase digestion. RNA was eluted in ~25 µl nuclease-free H2O and 1 µl SUPERase In (Invitrogen), and stored at −80°C.

mRNA library preparation and RNA sequencing
Altogether, the 19 total RNA isolates were processed and sequenced in two separate batches (Supplementary Table 3). Sample quality was assessed with Bioanalyzer on RNA 6000 Nano chip (Agilent Technologies), and library concentrations were measured with a Qubit Quant-iT kit (Fisher Scientific). For samples ET01-ET10, after rRNA depletion with the Ribo-Zero kit for bacteria (Illumina), clean-up was performed with Agencourt AMPure RNA Clean XP (Agencourt Bioscience), and the cDNA libraries were prepared using the KAPA RNA HyperPrep kit (Roche). For samples ET11-ET19, NEBNext rRNA Depletion Bacteria (New England BioLabs) and NEBNext Ultra II Directional RNA Library Prep (New England BioLabs) kits were used. Libraries were then sequenced on an Illumina NextSeq system with a read length of 150 bp.

RNAseq data processing and differential gene expression (DGE) analysis
The raw fastq files from all samples were processed simultaneously. Briefly, adaptors were trimmed using Cutadapt (v2.3)64, and the resulting reads were aligned to the Acetonema longum APO-1 DSM 6540 genome (GCA_000219125.2) using STAR (v2.7.10b)65. Gene counts were obtained with FeatureCounts (v2.0.1)66, and differential gene expression analysis was conducted using DESeq2 (v.1.34.0) in R (v3.6.1)67.

Generation of polyclonal antibodies to BamA and OmpM1
Plasmids for protein overexpression. A. longum protein BamA (F7NM23) was recombinantly expressed in E. coli with the following modifications: the native secretion (Sec) signal sequence was replaced with PelB signal sequence (for optimized secretion in E. coli), a 6-histidine (His6) tag was introduced after the PelB sequence to facilitate protein purification, and lastly, the three POTRA domains were removed, leaving only the β-barrel portion (residues 251-599) of BamA. Recombinant A. longum protein OmpM1 (F7NLZ0) was designed similarly, and the construct included the PelB signal sequence, followed by a His10 tag and only the β-barrel portion of the native protein (residues 120-436). Each protein was cloned into the multiple cloning site of the pET28a+ backbone (Addgene plasmid 2565) (Supplementary Table 9). Plasmids designated pET28a-pelB-6xHis-BamA and pET28a-pelB-10xHis-OmpM, respectively, were then electroporated into E. coli DH5α for routine propagation and BL21 (DE3) for protein overexpression under 50 µg/mL kanamycin selection.

Protein expression and purification
BL21 (DE3) strains harboring the BamA or OmpM1 target plasmids were subcultured in Terrific Broth (TB) media until exponential growth was achieved (OD600 ~0.6)68. Protein expression was induced with 1 mM IPTG for 2-4 h at 30°C with shaking at 200 rpm. Cells were harvested by centrifugation at 4700 rpm in an Allegra X-30R centrifuge (Beckman Coulter, Inc.), SX4400 rotor (Beckman Coulter, Inc.). Cells were then resuspended in lysis buffer composed of 50 mM Na-Pi, 300 mM NaCl, 1 mM PMSF, pH 7.5, prior to homogenizing with EmulsiFlex-C5 (Avestin). After removal of unlysed bacteria and debris through centrifugation at 4700 rpm for 10 min, membrane fractions were collected at 100,000×g at 4°C. Membranes were solubilized in lysis buffer supplemented with 1.5% (v/v) LDAO detergent and 10 mM imidazole for 2 h at 37°C. Undissolved material was then removed by centrifugation at 13,000 rpm (Ti50.2 rotor; Beckman Coulter, Inc.) for 15 min. Recombinant BamA was purified from the solubilized membrane fraction using affinity chromatography with a Ni-nitrilotriacetic acid (Ni-NTA) column (HisTrap FF crude 5 ml, Cytiva) using the ÄKTA Pure 25 system (Cytiva). Protein was purified under native conditions in Buffer A (50 mM Na-Pi, 300 mM NaCl, pH 7.5, 0.06% (v/v) LDAO) and eluted with Buffer A supplemented with 350 mM imidazole. Imidazole was then removed by repeated washing through a 30 kDa spin filter (Amicon), and the sample was concentrated to ~1 ml. Recombinant OmpM1 was purified from inclusion bodies using a Ni-NTA column under denaturing conditions. Briefly, the pellet remaining after membrane dissolution was solubilized in 0.1 M Na2HPO4, 0.01 M Tris, 8 M urea, pH 8, for 30–45 min at room temperature. Suspension was then centrifuged at 13,000 rpm (Ti50.2 rotor) for 15 min, and the supernatant was loaded onto the Ni-NTA column. Protein was eluted in the same buffer at pH 4. The eluate in ~400 µl volume was then dialyzed using regenerated cellulose tubing with a 6–8 kDa molecular weight cut-off (Fisherbrand) into 0.1 M Na2HPO4, 0.01 M Tris, 0.1% (w/v) SDS, pH 7.5, with gradual decrease in urea concentration at room temperature. Final dialysis into TBS + 0.05% (v/v) LDAO was carried out overnight at 16°C.

Antibody production
Purified recombinant BamA and OmpM1, at a concentration of ~1 mg/ml in 0.5–1 mL total volume, were separately injected into New Zealand White rabbits (day 0, pre-injection), followed by an identical booster injection at 14 d. Blood samples (5 mL each) were collected prior to antigen introduction at day 0, as well as at 14, 28 and 48 d after first injection. Blood was kept on ice prior to coagulation at 37°C and 4°C for 30 min each, and serum was collected by centrifugation for 10 min at 4000 rpm, 4°C, in an Allegra X-30R centrifuge with an SX4400 rotor (Beckman Coulter Inc.). Serum aliquots were stored at −80°C long-term or −20°C short-term. The pre- and post-immunization sera were assessed for binding specificity to inform the experimental design for Western blotting and immunomicroscopy (Supplementary Fig. 8). Animal work was performed under the Animal Care and Use Protocol (A21-0167) that was reviewed and approved by the UBC Animal Care Committee (University of British Columbia).

Western blot analysis
All protein samples were loaded onto a Laemmli SDS-PAGE gel (10%, 1 mm, unless otherwise specified) and run at 140 V for ~70 min. The proteins were transferred onto a nitrocellulose membrane in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3, by semi-dry transfer according to the Standard SD protocol on a Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was then blocked in 5% (w/v) milk in TBST, and rabbit serum diluted 1/50 or 1/150 in blocking buffer for detection of OmpM1 and BamA, respectively, was used as the primary antibody. Goat-anti rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used as the secondary antibody. Targets were visualized with a colorimetric method using 4-chloro-1-naphthol and H2O2.

Co-immunoprecipitation using anti-BamA serum
At least 800 mL of 3 d A. longum subcultures were harvested and washed with PBS prior to in vivo protein crosslinking with 1% (v/v) formaldehyde in PBS for 8 min at room temperature with slow agitation. The reaction was quenched by addition of 1 M glycine, pH 5, to a final concentration of 0.1 M and incubated for 15 min at room temperature with slow agitation. Cells were then washed once in PBS and resuspended in lysis buffer (25 mM Na-Pi, 150 mM NaCl, 1 mM PMSF, pH 7.5) before homogenization with EmulsiFlex-C5 (Avestin). Following a spin at 4500 rpm for 10 min at 4°C to pellet unlysed bacteria, the membrane fraction was isolated by ultracentrifugation at 100,000×g for 1.5 h at 4°C. Total membrane pellet was then solubilized in 1 mL lysis buffer supplemented with 1% (v/v) DDM at 37°C for 45 min with constant vigorous pipetting. The solution was then spun at 13,000 rpm for 2 min, and the supernatant was diluted 1/2 with lysis buffer to decrease the final concentration of DDM to 0.5% (v/v). This suspension was divided into 2 reactions, each mixed with rabbit serum diluted 1/100: 28 d anti-BamA or pre-injection (day 0) control. The reactions were incubated at 16°C, 60 rpm for 2 h prior to mixing with 15 mg Dynabeads Protein A (Invitrogen) for an overnight incubation under the same conditions. Subsequent steps were performed according to the Dynabeads Protein A manual, with denaturing elution carried out in 2× Laemmli sample buffer. Eluates were run ~2 cm into Laemmli SDS-PAGE gels, excised as whole-sample bands and analyzed using LC-MS/MS according to the protocol used in proteomic profiling of spores, as described above, except the peptide spectra were remapped to both the A. longum and rabbit proteomes to account for the presence of sera proteins in the samples.

Immunofluorescence and immuno-electron microscopy (immuno-EM)
Sample preparation. Harvested A. longum cells or endospores were washed with PBS and subsequently fixed under anaerobic conditions in PBS + 4% (v/v) formaldehyde for 30 min at room temperature. Fixed cells were then treated with blocking solution containing 2% (w/v) BSA in TBST for 1-2 h at room temperature, followed by three washes with TBST. Day 28 anti-BamA or anti-OmpM1 rabbit sera mixed with blocking solution at 1/150 and 1/50 dilutions, respectively, were used as primary antibodies. Equivalent amounts of pre-injection rabbit sera (day 0) were used for negative controls. Goat-anti rabbit IgG antibodies conjugated to Alexa Fluor 488™-10 nm colloidal gold (A-31566, Invitrogen), diluted 1/50 in blocking buffer, were used as the secondary antibody for both super-resolution immunofluorescence and immuno-EM. Antibody incubations were performed for 1–2 h at room temperature and followed by three consecutive washes with TBST. Lastly, cells were resuspended in TBS and immediately imaged with microscopy, frozen on EM grids for cryo-immuno-EM imaging, or stored at 4°C in the dark until further use.

Fluorescence light microscopy
Cells tagged with Alexa Fluor 488™ (and gold)-conjugated antibodies were imaged using the phase contrast and EGFP channel (488 nm excitation, 509 nm emission) settings on Zeiss Axio Examiner with an α-Plan-Apochromat 100×/1.46 oil immersion Ph3 objective lens (Zeiss). Images were obtained and subsequently processed with ZEN 2.6 Blue software.

Super-resolution fluorescence microscopy
Cells tagged with Alexa Fluor 488™ (and gold)-conjugated antibodies were imaged using the T-PMT (488 nm laser, detection at 400 nm) and EGFP (488 nm laser, detection at 490–614 nm) channel settings on Zeiss Axio Imager.Z2 equipped with an Airyscan 2 super-resolution detector and an α-Plan-Apochromat 100×/1.40 Oil DIC M27 objective (Zeiss). Images were obtained and subsequently processed with ZEN 3.4 Blue software.

Analysis of BamA puncta distribution on the surface of vegetative cells
Estimation of the distribution of fluorescent puncta from super-resolution images was performed using the MicrobeJ plugin (version 5.13p (27) beta) in ImageJ (version 1.54p)70,71. Region of interest (ROI) for each cell was defined manually as a curved line along the axis in the T-PMT channel of the processed images and imported into MicrobeJ using the filament setting with default parameters. Maxima were defined in the corresponding fluorescent channel with the tolerance cut-off of 10, and the outside association cut-off of 0.05 in each ROI to ensure that all puncta that could be attributed to each cell by eye would be captured while simultaneously avoiding noise. Images of cells tagged with pre-immunization serum were used as the negative control. The area of each cell was estimated by multiplying the measured cell length by the width (300 nm), and the number of maxima per μM2 was calculated using the number of total maxima per ROI divided by the estimated cell area. The maxima per area values between the test and control groups were compared using an unpaired, two-tailed t-test using Excel software (Supplementary Table 5).

Preparation of endospore cross-sections
Purified dormant endospores were embedded in resin as described previously7. Briefly, spore suspensions were treated with 2.5% gluter-aldehyde in Buffer A (0.1 M sodium cacodylate buffer, pH 7.2), washed with Buffer A three times, then fixed with 1% osmium tetraoxide in Buffer A. Plastic resin was applied by dissolution in ethanol, then cured at 60°C for 2 d. 70-nm thick sections were prepared with an ultramicrotome and applied to glow-discharged Formvar/Carbon 200 Mesh, Ni grids (Ted Pella, Inc.).

Preparation of decoated endospores
Dormant endospores were decoated as described previously72. Briefly, pure spores were resuspended in buffer containing 0.1 M DTT, 0.1 M NaCl, 0.1 M NaOH and 0.5% (w/v) SDS, pH 10, and incubated at 65°C for 30 min with regular vortexing, then washed three times with PBS. All incubation steps and washes were done anaerobically with anoxic solutions.

Transmission electron microscopy (TEM)
Cells for immuno-EM were applied to glow-discharged Formvar/Carbon 200 mesh, Ni grids (Ted Pella, Inc.) and stained with 2% (w/v) ammonium molybdate, pH 7, for 30 s, then washed three times with water sterilized through a 0.2 µm filter. Endospore cross-sections (on grids) were immunolabelled by applying 2% (w/v) BSA in TBST for blocking, then day 0 or day 28 anti-BamA sera diluted 1/150, or anti-OmpM1 sera diluted 1/50 in blocking buffer, washed three times with TBST, then incubated with Goat-anti rabbit IgG Alexa Fluor™ 488-10 nm colloidal gold (Invitrogen) diluted 1/50 in blocking buffer, then washed three times with TBST and three times with filter-sterilized water before staining with ammonium molybdate as described above. All incubations were carried out on parafilm at room temperature for 90 min. Grids were imaged using Talos L120C (Thermo Fisher Scientific) with a Ceta camera (Thermo Fisher Scientific) at 120 kV acceleration voltage. Colloidal gold amounts between day 0 and day 28 antisera samples were assessed with an unpaired, two-tailed t-test using Excel software.

Cryo-immuno-EM sample preparation and data collection
Cells for cryo-immuno-EM were prepared as described above and kept hydrated until freezing on glow-discharged Quantifoil Holey Carbon R2/2 200 mesh Cu grids (Quantifoil Micro Tools GmbH). Grids were plunge-frozen into liquid ethane-propane mixture cooled at liquid nitrogen temperatures with a Mark IV Vitrobot maintained at room temperature and 100% humidity. 2D projection images were collected using 10–30 s exposure on a Titan Krios G2 300 kV TEM equipped with a Selectris energy filter (Thermo Fischer Scientific). 

Phylogeny and homology searches
To generate a phylogeny of Firmicutes members, an alignment of 120 conserved bacterial single-copy marker genes was extracted from publicly available genomes using the Genome Taxonomy Database (GTDB) Toolkit v1.4.073,74. A maximum-likelihood phylogeny of 228 representative Firmicutes was constructed using IQ-TREE v.2.1.4 with 1000 ultrafast bootstraps and the substitution model LG+G475, with representative Cyanobacteria genomes as an outgroup. A reduced subset of the tree was visualized using iTOL v476. To identify the OMP, LPS and PL pathway proteins, the proteomes of members of Firmicutes were searched using a curated list of Hidden Markov Model profiles (HMMs) obtained from the KEGG, pFAM, and TIGRFAM databases77–79. HMM searches were performed using HMMER with default hmmsearch settings and standard thresholds (http://hmmer.org/). Proteins that returned ambiguous hits from the HMM searches in A. longum were cross-checked by Position-Specific Iterative (PSI)-BLAST using characterized homologs as queries and run under default parameters. To identify the homologs of SonA and SonB, a PSI-BLAST search using amino acids sequences from A. longum was performed against a set of sporulating and non-sporulating diderm Firmicutes, using an e-value of 1×10−30 and 25% identity cut-off parameters. Sporulation ability was established from literature reports combined with deepG-based genomic predictions available in the BacDive database (https://bacdive.dsmz.de/)80. Synteny of homologous genes obtained from the PSI-BLAST search was assessed manually. Computational predictions of operons were performed using the Operon-mapper tool (https://biocomputo.ibt.unam.mx/operon_mapper/)40, and the CDBProm database (https://aw.iimas.unam.mx/cdbprom/)41.

In vitro protein folding assays
Plasmids for protein overexpression. Recombinant A. longum proteins BamA (F7NM23, residues 30-599) and SonA (F7NM22, residues 23-426), missing their native Sec signal sequences, were cloned into the pET28a+ backbone to generate the plasmids pET28a-no-SS-BamA and pET28a-no-SS-SonA, respectively (Supplementary Table 9). The plasmids were introduced into E. coli DH5α and BL21 (DE3) as described above.

Protein expression and isolation
BL21 (DE3) cells with the BamA or SonA plasmid were subcultured in LB broth until exponential phase (OD600 ~0.6), then protein expression was induced with 0.5–1 mM IPTG for 4 h at 37°C with shaking at 200 rpm. After harvesting by centrifugation and washing in ice-cold PBS, bacteria were resuspended in lysis buffer and homogenized as described above. After unlysed cells and debris were pelleted at ~5000×g for 10 min, the supernatant was centrifuged at 15,000 rpm (Ti50.2 rotor) for 15 min. The pellet was first washed in the membrane solubilization buffer with LDAO, pre-warmed to 37°C, then over three times in ice-cold TBS, pH 8, to remove detergent. The isolated inclusion body fraction was solubilized in TBS, pH 8, supplemented with 8 M urea, for 30 min at room temperature and centrifuged again at 15,000 rpm for 15 min. After analysis by SDS-PAGE, the sample was additionally purified by passage through a 100 and/or 30 kDa spin filter (Amicon) as needed, and final protein concentration was determined using the Bradford assay.

Liposome preparation
Films of E. coli polar lipids (Avanti Research) dried under N2 gas were resuspended in TBS, pH 8, to a final concentration of 4 mg/mL and solubilized overnight at 15°C with gentle agitation. The suspension was then sonicated for 6 min with 15 s on and off intervals at 40% intensity using the Model 50 Sonic Dismembrator (Thermo Fisher Scientific), centrifuged at 11,000×g for 10 min at 4°C, and the supernatant was aliquoted and stored at 4°C short-term or −80°C long-term. In case of storage at 4°C, the suspension was further passed through a 27G syringe needle at least five times immediately before use.

Assessment of protein folding into phospholipid liposomes
In vitro reactions were setup by combining the denatured substrate in TBS, pH 8, with 8 M urea (5 µM stock to 0.5 µM final for BamA, and 1 mg/mL stock to 0.1 mg/mL final for SonA) and phospholipid liposomes (4 mg/mL stock to 0.4 mg/mL final) in TBS, pH 8, on ice. Using the molecular weight of 720 g/mol for E. coli phospholipids and the substrate’s respective molecular weight values, we generated lipid-to-protein ratios of 1000:1 for BamA and 263:1 for SonA unless specified otherwise81. The reactions were placed at 30°C with slight agitation, and aliquots were taken immediately before (t=0) and at regular intervals after incubation started. Aliquots were then either immediately boiled in 1× Laemmli dye, or incubated at 37°C with proteinase K (50:1 substrate to enzyme ratio by weight) or trypsin (20:1 substrate to enzyme ratio by weight, unless specified otherwise) for 5 h and 30 min for the BamA and SonA reactions, respectively, before boiling in 1× Laemmli dye. Samples were run on identical Laemmli SDS-PAGE gels for subsequent analyses by silver staining or Western blot using anti-BamA serum as the primary antibody, as described above. Band sizes in trypsin-digested samples were compared against the prediction of trypsin cut sites generated with PeptideCutter (https://web.expasy.org/peptide_cutter/)82.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by Polina Beskrovnaya and colleagues investigates the molecular mechanisms involved in the biogenesis of the\nouter membrane (OM) during spore germination in Acetonema longum, a diderm spore-forming bacterium from the\nFirmicutes phylum. A. longum is notable for its ability to form spores with two membranes: an inner spore membrane (IsM)\nand an outer spore membrane (OsM). The research focuses on how the OsM transitions into a true OM during germination.\nThe authors utilize structural biology, biochemistry, and genomics to explore the role of the β-barrel assembly machinery\n(BAM) complex, particularly BamA, in this transformation. In mature spores, key outer membrane proteins (OMPs), including\nBamA, are absent, suggesting that the true OM only forms once germination begins. In vitro experiments demonstrate that\nBamA can spontaneously insert into lipid bilayers under physiological conditions, indicating its potential role in de novo OM\nbiogenesis during germination. Additionally, proteomic and phylogenomic analyses identify two conserved proteins—SonA,\na β-barrel OMP, and SonB, a putative OM lipoprotein—that are likely auxiliary factors supporting BamA in the membrane\nremodeling process. The study proposes a model in which BamA, along with SonA and SonB, facilitates the formation of the\ntrue OM during spore germination. This remodeling also includes the insertion of other OMPs, such as LptD, which is crucial\nfor maintaining the proper lipid composition of the OM. These findings emphasize the dynamic nature of bacterial membrane\nremodeling and the central role of the BamA complex in this process. The study offers new insights into the evolutionary and\nfunctional aspects of OM biogenesis in diderm spore-forming bacteria, with a robust methodology supporting its conclusions.\nSpecific points:\n1, The article primarily focuses on the functional characteristics of BamA and its critical role in membrane remodeling and de\nnovo outer membrane biogenesis. However, the authors also delve into the biosynthesis and transport of\nlipopolysaccharides (LPS) and phospholipids in the results section. How does this discussion relate to the broader context\nof the study? Specifically, how do these lipid transport processes intersect with BamA’s function in outer membrane\nassembly?\n2, While the study suggests that SonA and SonB assist BamA in its function, this remains a speculative assertion. To\nstrengthen the study's conclusions, the authors could incorporate experimental approaches such as co-immunoprecipitation\nto validate the interactions between BamA, SonA, and SonB during the membrane remodeling process.\n3, The proteomic analysis identifies several outer membrane proteins (OMPs), but the detection of only about 50% of the\npredicted proteins indicates that some low-abundance proteins might have been overlooked. To further support the authors’\nconclusions, could more advanced mass spectrometry techniques be applied, or could membrane-associated proteins be\nselectively enriched to capture a wider range of proteins involved in membrane biogenesis?\n4, In the experiments demonstrating the insertion of BamA and SonA into liposomes (Figure 6), the authors used trypsin for\nBamA and both trypsin and proteinase K for SonA. What was the rationale behind using two different proteases for SonA?\nAdditionally, why did the inserted form of SonA disappear entirely following treatment with proteinase K?\n5, Since both BamA and SonA are β-barrel proteins, it would be beneficial to include a control group where samples are\neither heated or left unheated before undergoing SDS-PAGE. This would ensure that the proteins adopt the correct spatial\nconfiguration, providing further confidence in the folding and insertion data obtained from the experiments.", "reviewer_2": "The assembly of the diderm cell envelope has, so far, been studied primarily in a few model “Gram-negative” species from\nthe Proteobacteria. In contrast, very little is known about outer membrane biogenesis in diderm Firmicutes, an ancient group\nof bacterial species. This study focuses on diderm spore-formers such as Acetonema longum and investigates how the\norganism reconstructs a “normal” vegetative outer membrane following spore germination.\nThe authors demonstrate that the spores possess two membranes; however, the outer spore membrane (OsM) differs\nsignificantly from the vegetative outer membrane because it is derived from the cytoplasmic membrane of the mother cell.\nAlthough the system is not genetically tractable, the authors perform gene expression and proteomic profiling, suggesting\nthat the OsM lacks typical outer membrane proteins. These include essential components for outer membrane biogenesis,\nsuch as BamA (required for β-barrel protein assembly) and LptD (required for LPS transport).\nThe authors propose that during spore germination, BamA in Acetonema longum may be capable of self-inserting into the\nphospholipid bilayer of the OsM due to its unique structural properties, thereby initiating the conversion of the OsM into a\ntypical outer membrane. Additionally, they suggest that BamA may interact with two unique partner proteins, based on gene\nsynteny analysis.\nOverall, this is an interesting and thought-provoking study that provides a valuable starting point for investigating outer\nmembrane biogenesis in non-model organisms such as diderm Firmicutes. However, I have several important concerns, as\nsome of the data appear to be overinterpreted.\nMajor Comments:\n1. Misleading Use of “De Novo Outer Membrane Biogenesis”:\nThe phrase “De Novo Outer Membrane Biogenesis” is misleading. For most readers, this implies de novo synthesis of the\nouter membrane from scratch, not remodeling of a pre-existing membrane (OsM). While I understand that the OsM is\nfundamentally different from the vegetative outer membrane , being derived from the mother cell's cytoplasmic membrane\nand likely consisting of a phospholipid bilayer with distinct protein content, the process described in the manuscript is better\ncharacterized as remodeling rather than de novo synthesis. Furthermore, there are several known bacterial species in which\nLPS is non-essential under certain conditions, demonstrating that the outer membrane can transition to or from a\nphospholipid bilayer. Therefore, the transformation of the OsM into a functional outer membrane does not constitute true de\nnovo synthesis.\n2. Insufficient Evidence for the Absence of LPS in OsM:\nThe authors infer that the OsM lacks LPS based on the apparent absence of LptD and periplasmic Lpt components (lines\n284-286). However, this alone is not sufficient evidence. Interestingly, the authors also show that LPS biosynthesis genes are\nupregulated during sporulation. Since LPS biosynthesis occurs on the cytoplasmic leaflet of the mother cell's inner\nmembrane — which becomes the outer leaflet of the OsM — this raises the possibility that at least some LPS may be\npresent in the OsM. Direct assays are needed to confirm the presence or absence of LPS and support the conclusion.\n3. Protein Abundance and Detection Limitations:\nThe proteomic data show a significant reduction in outer membrane proteins (OMPs), including\nBamA. However, it is unclear whether these proteins are completely absent. In E. coli, essential OMPs such as BamA and BamD can be reduced by over\n100-fold and remain undetectable by mass spectrometry, yet cells remain viable under slow-growing conditions. What is the\ndetection limit in the current mass spectrometry experiments? Can low levels of these proteins truly be ruled out? The\nimmunofluorescence experiments are also inconclusive, as residual spore coat layers may obscure detection of proteins on\nthe OsM. Positive controls are needed to validate these results.\nAdditionally, based on the stages of endospore formation (Fig. 1), what prevents the transport and self-insertion of BamA,\nand therefore later OMPs and LPS, from the prespore into the mother cell-derived membrane during engulfment? From the\nprespore side, the mother cell membrane looks the same as the inner leaflet of the outer membrane.\n4. In Vitro Folding and Interpretation of BamA Assays:\nThe comparison between A. longum BamA and E. coli BamA relies on previously published data. A side-by-side in vitro\nfolding comparison would strengthen this point. The protease digestion assay used here is not a standard approach for\nstudying BamA folding. If protease sensitivity were only detecting membrane insertion, one would expect digestion of\nexposed domains (e.g., POTRA domains), with only the membrane-embedded β-barrel remaining intact. However, multiple\nundigested bands were observed (with no identification provided), suggesting unexpected protease resistance of\nsubdomains and complicating interpretation. Additionally, since these assays were conducted using E. coli phospholipids\nrather than native lipids, the physiological relevance is uncertain. It is also worth noting that E. coli can be viable with BamA\nalone in a strain lacking all Bam lipoproteins (with a specific bamA mutation), presumably because it self-assembles in vivo,\nraising questions about the uniqueness of A. longum BamA's behavior.\n5. SonA and SonB as Putative BamA Partners:\nThe identification of SonA and SonB as potential BamA partners is based on synteny. Since the authors generated anti-\nBamA antibodies, they could perform co-immunoprecipitation or pull-down experiments to test for physical interactions.\nIn Proteobacteria, synteny between bamA, LPS biosynthesis demonstrated functional linkage, rather than physical protein\npartnerships.\nOther Comments:\n1. The Introduction is overly long and reads more like a review article on outer membrane biogenesis. I suggest removing\ndetails that are not essential for understanding the manuscript’s focus.\n2. Lines 93–94: There is strong evidence suggesting non-overlapping functions.\n3. Line 122: This statement is misleading. HxcQ does not self-transport; it contains a lipoprotein signal sequence and is\ntransported via the Lol pathway.", "reviewer_3": "The emergence of the outer membrane (OM) is a poorly understood and important topic relevant to the emergence of diderm\nbacteria. Here Breskronaya et al cleverly use a sporulating Gram negative (A. longum) to study how the OsM transitions to a\nbona fide OM. They convincingly show that expression of OM biogenesis machinery is timed to germination and\nsubcellularly locate the appearance of BamA and SonA OMPs to the emerging OM during germination. With a few additional\ncontrols and experiments (Major points outlined below, related to concerns on the in vitro refolding assay) I would support\npublication of the manuscript.\nMajor:\n1) In vitro folding assay (Fig 6):\na): 6A: Where are the undigested controls?\nb) A proportion of the unfolded BamA and SonA is resistant to proteolysis. How do the authors know that retained bands are\nresistant to the protease due to folding and membrane insertion or instead merely due to aggregation forming precipitates\nthat are resistant to proteolysis? The experiments should be repeated by running heated and unheated samples. Folded\nbarrels are resistant the SDS denaturation and exhibit different electrophoretic migration than the unfolded form when the gel\nis run at 4C. Since the authors already have this assay established, repeating the this assay with the above conditions\nshould be achievable in a short time frame.\n2) The puncta of BamA is similar to the “assembly precincts” observed by the Lithgow group by super resolution microscopy.\nI think that this observation should be discussed with respect to this in the Discussion.\nGunasinghe SD, Shiota T, Stubenrauch CJ, Schulze KE, Webb CT, Fulcher AJ, Dunstan RA, Hay ID, Naderer T, Whelan\nDR, Bell TDM, Elgass KD, Strugnell RA, Lithgow T. The WD40 Protein BamB Mediates Coupling of BAM Complexes into\nAssembly Precincts in the Bacterial Outer Membrane. Cell Rep. 2018 May 29;23(9):2782-2794. doi:\n10.1016/j.celrep.2018.04.093. PMID: 29847806.\nMinor:\n3) How were the anti-BamA and anti-OmpM1 antisera validated? Although I understand that this species is genetically\nintractable, it is little worrying that the potential for off-target binding cannot be excluded due to the lack of a negative control.\nIt would be good to show a WB against whole protein extracts of the various life stages. Any intensely detected off-target\nbands should be identified to determine their potential surface exposure (and therefore whether there is the potential that the\ndetected molecules by microscopy are a mix of BamA and other surface antigens).\n4) Alphafold structure predictions where mentioned several times for various proteins by where not shown (or were tiny as\nfor SonB, SonB, and BamA.\nLiterature review:\nline 80: Revise sentence. In E. coli the accessory subunits are BamB-E. These vary considerably between species (as is\nthen said later in the paragraph which contradicts with this sentence).\nline 87-89: The citation on this sentence is to a review published 8 years prior to the primary articles in which the data\nestablishing the existence of the described mechanistic steps were published. Suggest updating references here and above.\nlines 102-104: Revise. I am unaware of any review suggesting that TAM transports lipoproteins. Neither of the reviews cited\nto this statement mention this idea. Indeed, one of the two reviews cited do not mention TAM. There are no primary articles\nthat I am aware of that provide any evidence of such a mechanism.\nlines 106-108: A hypothesis is that assembly of substrates by TamA is similar to the mechanism used by BamA…this has\nnever been tested (at least no published articles). The sentence is constructed as a statement of truth and therefore needs to be revised.\nDiscussion:\nline 496-498: An experiment to show that LPS is inserted into the OsM de novo was not conducted. Revise sentence."}, "author_response": "Response to Reviewers\nWe would like to thank the editorial team and reviewers once again for their thoughtful\ncomments and revisions, all of which have greatly improved the manuscript. All comments have\nbeen addressed.\nSummary of major changes:\n1. New Figure 6: Detec&on of BamA interac&ve partners with co-immunoprecipita&on and\ncross-linking.\n2. New Figure 7, new Supplementary Figure 7, new Supplementary Table 10: Protein\nfolding assays of BamA and SonA using trypsin and Proteinase K, mapping of trypsin cleavage\nsites\n3. New Figure 7b: Migration of folded and denatured BamA at room temperature and 4 °C\n4. New Figure 4, new Supplementary Figure 6: Detection of BamA and OmpM1 in vegetative\ncells, mature spores and germinating cells.\n5. New Supplementary Figure 8: Analysis of anti-BamA and anti-OmpM1 antisera specificity.\n6. New Supplementary Table 5: Analysis BamA puncta distribution in vegetative A. longum\ncells.\n7. Text: concise, more targeted introduction and discussion\n7. Text: replaced “de novo OM biogenesis” with “inner-to-outer membrane remodelling”\nBelow is a detailed summary of all changes.\nREVIEWER COMMENTS\nReviewer #1: The article primarily focuses on the functional characteristics of BamA and its\ncritical role in membrane remodeling and de novo outer membrane biogenesis. However, the\nauthors also delve into the biosynthesis and transport of lipopolysaccharides (LPS) and\nphospholipids in the results section. How does this discussion relate to the broader context\nof the study? Specifically, how do these lipid transport processes intersect with BamA’s function in\nouter membrane assembly?\nAuthors: The outer membrane (OM) is composed of both lipids and proteins. Therefore, its\nproper assembly requires the coordinated action of the BAM complex for protein assembly and\nthe LPT system for LPS transport. Importantly, BamA functionally connects outer membrane\nprotein (OMP) assembly to LPS transport by facilitating the incorporation of LptD, the key OMP\ncomponent of the LPT system, into the OM. Notably, our data show for the first time that protein\nincorporation precedes lipid remodelling, as BamA is necessary for the proper insertion of LptD.\nduring germination and, as such, is pertinent to the discussion of the paper. We have revised\nthe introduction to highlight the connection between BamA and LPS in the broader context of\nthe study.\nReviewer #1: While the study suggests that SonA and SonB assist BamA in its function, this\nremains a speculative assertion. To strengthen the study's conclusions, the authors could\nincorporate experimental approaches such as co-immunoprecipitation to validate the\ninteractions between BamA, SonA, and SonB during the membrane remodeling process.\nAuthors: To identify potential structural partners to BamA, we performed co-\nimmunoprecipitation (co-IP) and cross-linking experiments. Neither of these approaches\nidentified stable partners. These results have been added to the main text and presented as a\nnew Figure 6. Based on these results, synteny, operon structure and co-expression profiles, we\npropose BamA, SonA and SonB as functional partners that work together during OM\nbiogenesis.\nNew Figure 6. Detec%on of BamA interac%ve partners with co-immunoprecipita%on and\ncross-linking. a Silver stain and LC-MS/MS analysis of control (pre-immuniza&on serum) and\nan&-BamA (day 28 serum) samples iden&fied only two proteins at >95% confidence that co-\neluted with BamA: a puta&ve generic porin F7NLZ1-2, and a pep&dase S55 domain-containing\nprotein F7NM31. b Western blot analysis of whole-cell lysates, with or without chemical cross-\nlinking and subjected to heat or non-heat treatment, revealed no addi&onal bands or molecular\nweight shiWs indica&ve of BamA interac&on partners.\nReviewer #1: The proteomic analysis identifies several outer membrane proteins (OMPs), but\nthe detection of only about 50% of the predicted proteins indicates that some low-abundance\nproteins might have been overlooked. To further support the authors’ conclusions, could more\nadvanced mass spectrometry techniques be applied, or could membrane-associated proteins\nbe selectively enriched to capture a wider range of proteins involved in membrane biogenesis?\nAuthors: We predicted 46 β-barrel OMPs in the genome of A. longum, and 25 were identified\nthrough proteomics using well-established isolation protocols and a high-sensitivity Orbitrap\nmass spectrometer. These findings align with previous studies of the Negativicute Veillonella\nparvula, a close relative of A. longum, where 17 β-barrel OMPs were detected using MS\n2\n(Poppleton et al., Front Microbiol, 2017). Some proteins may be poorly digested by trypsin,\nnot\nexpressed under our growth conditions, or expressed at low levels (the instrument's detection\nlimit for low-abundant proteins is approximately 1-2 peptides). For the scope of this paper, the\nessential BamA and LptD are highly abundant and readily detected via proteomics and\ntranscriptomics. To verify that our protein detection matched protein expression levels, we\ncompared our proteomics results with transcriptomics data. We confirmed that protein counts\ncorrespond to transcript levels (Supplementary Table 3).\nReviewer #1: In the experiments demonstrating the insertion of BamA and SonA into liposomes\n(Figure 6), the authors used trypsin for BamA and both trypsin and proteinase K for SonA. What\nwas the rationale behind using two different proteases for SonA? Additionally, why did the\ninserted form of SonA disappear entirely following treatment with proteinase K?\nAuthors: Trypsin and Proteinase K are commonly used proteases in protein folding assays. We\ntested both on BamA and SonA and found that trypsin was more effective for both proteins,\nproducing clearer band patterns. The Methods and Results sections have been updated to\ninclude more detailed descriptions of the assays. Trypsin digests and relevant controls are now\npart of the main figures (new Figure 7), while Proteinase K digests and additional controls have\nbeen moved to the Supplementary Materials, new Supplementary Figure 7. Notably, our results\nshow that both BamA and SonA can self-insert into liposomes at 30 °C. SonA in liposomes\ndigested with Proteinase K appeared at ~ 30 kDa, overlapping with the size of Proteinase K\n(~29 kDa). Therefore, distinguishing the two proteins on a gel was challenging, except that the\nband with SonA appeared much denser (Supplementary Figure 7b).\nWestern blot analysis of trypsin digests of BamA with and without liposomes shows that\nliposomes protect the β-barrel of BamA over &me (0-6h). The observed bands include the\nexpected intact BamA (~63 kDa), the β-barrel part with two intact POTRA domains (~54 kDa),\nthe par&ally digested second POTRA domain (~40-45 kDa), and no POTRA domains (~38 kDa).\nWithout lipids, most BamA is digested by trypsin, whereas without trypsin, BamA remains\nstable over 6 h. b Denatured BamA was refolded into E. coli liposomes at 37 °C and 42 °C under\ndifferent lipid-to-protein ra&os. Samples were prepared in non-denaturing Laemmli dye\n(without heat treatment or reducing agent) and run on SDS-PAGE gels at 4 °C. A clear shiW of\n~10 kDa in migra&on was observed between folded (F) and denatured (D) BamA. c Silver stain\nanalysis of SonA folding into liposomes aWer 21 h, before (-) and aWer trypsin treatment. In the\npresence of lipids, a bright band at ~45 kDa represents intact SonA, sugges&ng protec&on from\ntrypsin diges&on. Addi&onal bands at ~30-40 kDa correspond to the expected size of the β-\nbarrel por&on with two par&ally digested POTRA domains. In the absence of lipids, most of\nSonA is digested (middle). Trypsin alone migrates at its expected size of ~24 kDa (right). TR,\ntrypsin.\nReviewer #1: Since both BamA and SonA are β-barrel proteins, it would be beneficial to include\na control group where samples are either heated or left unheated before undergoing SDS-\nPAGE. This would ensure that the proteins adopt the correct spatial configuration, providing\nfurther confidence in the folding and insertion data obtained from the experiments.\nAuthors: Previous studies using BamA from E. coli have shown that, when run at room\ntemperature, the folded and denatured states appeared indistinguishable on an SDS-PAGE gel.\nHowever, a difference in the electrophoretic mobility of BamA from E. coli was observed when\nthe folded and denatured states were analyzed on SDS-free gels at 4 °C (Patel and\nKleinschmidt, Biochemistry, 2013).\nWe conducted similar experiments with BamA from A. longum. As a control for correct spatial\nconfiguration, BamA from A. longum was expressed in E. coli and purified under non-denaturing\nconditions from the total membrane fraction. We ran heated and unheated samples with and\nwithout SDS to distinguish between folded and denatured states of BamA (New Figure 7b, see\nabove in response to Reviewer 1). At room temperature, under all tested conditions, the folded\nand denatured states appeared indistinguishable on an SDS-PAGE gel, consistent with\nprevious reports for BamA from E. coli. When analyzed on semi-native PAGE gels at 4 °C, the\nfolded and denatured BamA from A. longum showed a ~10 kDa shift in electrophoretic mobility.\nThese findings support our conclusion that the folded BamA from A. longum adopts the correct\nspatial configuration.\nReviewer #2: Misleading Use of “De Novo Outer Membrane Biogenesis”:\nThe phrase “De Novo Outer Membrane Biogenesis” is misleading. For most readers, this\nimplies de novo synthesis of the outer membrane from scratch, not remodeling of a pre-existing\nmembrane (OsM). While I understand that the OsM is fundamentally different from the\nvegetative outer membrane, being derived from the mother cell’s cytoplasmic membrane and\nlikely consisting of a phospholipid bilayer with distinct protein content, the process described in\nthe manuscript is better characterized as remodeling rather than de novo synthesis.\nFurthermore, there are several known bacterial species in which LPS is non-essential under\n4\ncertain conditions, demonstrating that the outer membrane can transition to or from a\nphospholipid bilayer. Therefore, the transformation of the OsM into a functional outer membrane\ndoes not constitute true de novo synthesis.\nAuthors: To avoid misinterpretation, the use of “de novo outer membrane biogenesis” in the\ntext has been replaced by “inner-to-outer membrane remodeling.”\nReviewer #2: Insufficient Evidence for the Absence of LPS in OsM:\nThe authors infer that the OsM lacks LPS based on the apparent absence of LptD and\nperiplasmic Lpt components (lines 284-286). However, this alone is not sufficient evidence.\nInterestingly, the authors also show that LPS biosynthesis genes are upregulated during\nsporulation. Since LPS biosynthesis occurs on the cytoplasmic leaflet of the mother cell's inner\nmembrane — which becomes the outer leaflet of the OsM — this raises the possibility that at\nleast some LPS may be present in the OsM. Direct assays are needed to confirm the presence\nor absence of LPS and support the conclusion.\nAuthors: We have several observations in strong support of LPS being absent from the OsM:\n1) Our proteomics analysis showed that LptD, along with the periplasmic bridge proteins LptA\nand LptC, are absent from mature spores. Since these proteins are essential for the proper\ninsertion of LPS, we conclude that the OsM also lacks LPS.\n2) While LPS production is initiated at the inner\nleaflet of the IM, additional proteins help\nensure that the precursors are correctly\noriented for subsequent insertion into the OM.\nIn short, synthesis begins with lipid A, which is\nflipped to the periplasmic side of the IM by\nMsbA. Once lipid A is loaded with Kdo and O-\nantigen, it is inserted into the OM by the LPT\ncomplex. The localization of the nucleotide-\nbinding domains of MsbA ensures\nunidirectional transport of precursors in the\npresence of ATP, which would be abundant in\nthe mother cell, as demonstrated by in vitro\nreconstitution experiments (Eckford & Sharom, Biochem J, 2010). Additionally, structural\nstudies indicate that the gate feature of LptF prevents reversion of lipid A transport (Owens et\nal, Nature, 2019). Thus, if the mother cell was targeting LPS to the engulfing membranes\nduring sporulation, in the absence of the LPT complex, the precursors would indeed end up\nin the OsM but would face into the periplasmic space between the two spore membranes with\nno way to reverse orientation.\n3) Previous studies using immunofluorescence showed that LPS was absent from mature A.\nlongum spores but present on the outgrowing cell immediately upon germination (Tocheva et\nal, Cell, 2011). The same study also compared the density profiles of the IM, IsM, OsM, and\nOM, showing that the IsM and OsM were similar to IM and not the OM, which is consistent\nwith the absence of OMPs and LPS from these membranes.\nThe proteomic data show a significant reduction in outer membrane proteins (OMPs), including\nBamA. However, it is unclear whether these proteins are completely absent. In E. coli, essential\nOMPs such as BamA and BamD can be reduced by over 100-fold and remain undetectable by\nmass spectrometry, yet cells remain viable under slow-growing conditions. What is the detection\nlimit in the current mass spectrometry experiments? Can low levels of these proteins truly be\nruled out? The immunofluorescence experiments are also inconclusive, as residual spore coat\nlayers may obscure detection of proteins on the OsM. Positive controls are needed to validate\nthese results.\nAuthors: Our proteomics data identified approximately 200-250 peptides in germinating spores\nand around 110-140 peptides in vegetative cells that matched BamA with more than 95%\nconfidence. This suggests that BamA is a highly abundant protein. The Orbitrap mass\nspectrometer used in our research is known for its very low detection limits, ranging from 10-15\nto 10-18 moles. In theory, even if BamA levels decreased by 100-fold, the Orbitrap LC-MS/MS\nshould still detect it. Beyond mass spectrometry, we examined A. longum lysates from\nvegetative, dormant, and germinating samples using anti-BamA antibodies (New Figure 4c).\nOur findings show that BamA is present in vegetative and germinating cells but not in dormant\nspores. As a control, we used chemically de-coated dormant spores, which we confirmed lacked\ntheir spore coat through TEM. Collectively, these results align with our LC-MS/MS,\nimmunofluorescence, and immuno-EM experiments and support our conclusion that dormant\nspores do not contain BamA.\nNew Figure 4c. Western blot analysis of vegeta&ve cells and dormant spores using an&-BamA\nserum detected BamA in vegeta&ve cells and germina&ng spores, but not in whole or chemically\nde-coated dormant spores. IM, inner membrane (orange); OM, outer membrane (blue).\nReviewer #2: Additionally, based on the stages of endospore formation (Fig. 1), what prevents\nthe transport and self-insertion of BamA, and therefore later OMPs and LPS, from the prespore\ninto the mother cell-derived membrane during engulfment? From the\nprespore side, the mother cell membrane looks the same as the inner leaflet of the outer membrane.\nAuthors: The reviewer is raising an important mechanistic question about how membrane\nasymmetry and protein sorting are maintained during engulfment. Specifically, can the prespore\nsynthesize BamA and LPS and localize them to the OsM?\nOur conclusion that BamA and LPS are not synthesized or inserted into the OsM by the\nprespore is based on several key considerations: 1) BamA and LptD are highly abundant β-\nbarrel proteins. Neither of these proteins was detected in mature spores using mass\n6\nspectrometry, immunofluorescence, immuno-EM, or Western blot analysis. If the prespore were\nto produce BamA and LPS and target them to the engulfing membranes, it would imply that the\nOsM transformed into a true OM during engulfment instead of during germination. In this case,\nBamA, LptD, and other essential proteins should have been identified through mass\nspectrometry and immunodetection. 2) Immediately after asymmetric septum formation, the\nmother cell deposits the amphipathic helix SpoVM and the scaffold protein SpoIVA onto the\nOsM (Price & Losick, J Bacteriol, 1999). The presence of these proteins triggers the deposition\nof numerous Cot proteins that form the spore coat. The presence of LPS on the mother side of\nthe OsM would hinder proper coat formation. 3) Lastly, during sporulation, the spore becomes\nmetabolically inactive, while the mother cell supports the spore's maturation. For example,\nstudies in B. subtilis have shown that lipid and cortex synthesis occur on the mother side of the\nOsM (Pedrido et al., Mol Microbiol, 2013; Vasudevan et al., Mol Microbiol, 2007).\nThese studies suggest that BamA and LPS are synthesized by the mother cell rather than the\nprespore.\nReviewer #2: In Vitro Folding and Interpretation of BamA Assays:\nThe comparison between A. longum BamA and E. coli BamA relies on previously published\ndata. A side-by-side in vitro folding comparison would strengthen this point. The protease\ndigestion assay used here is not a standard approach for studying BamA folding. If protease\nsensitivity were only detecting membrane insertion, one would expect digestion of\nexposed domains (e.g., POTRA domains), with only the membrane-embedded β-barrel remaining intact.\nHowever, multiple undigested bands were observed (with no identification provided), suggesting\nunexpected protease resistance of subdomains and complicating interpretation.\nAuthors: Protease digestion and folding in native and denaturing PAGE gels are the two\nprimary methods used to study β-barrel folding in vitro, and we employed both approaches to\nexamine the ability of BamA to self-insert in liposomes. As mentioned earlier in response to\nReviewer 1, our experiments demonstrate that folded and denatured BamA migrate similarly on\ngels at room temperature (New Figure 7b). This finding led us to continue exploring protein\nfolding through protease digestion assays. Western blot analysis of BamA proteoliposomes\ndigested with trypsin showed distinct bands at ~ 63 kDa, 50 kDa, and 40-45 kDa (Figure 7a).\nThe 63 kDa band corresponds to undigested BamA (since we added an excess of BamA to\nensure adequate visualization on the gel), while the 50 and 40-45 kDa bands are consistent with\nBamA missing one or two POTRA domains (~10 kDa each). We have mapped the POTRA\ndomain trypsin cleavage sites in the new Supplementary Table 10.\nReviewer #2: Additionally, since these assays were conducted using E. coli phospholipids\nrather than native lipids, the physiological relevance is uncertain.\nAuthors: We used commercially available E. coli phospholipids for two main reasons: 1)\nlipidomic analyses indicate that IM lipids are highly conserved among diderm bacteria, with\n~70–80% comprising phosphatidylethanolamine (PE) (Sohlenkamp & Geiger, FEMS Microbiol\nRev, 2016); and 2) isolating native IM lipids from A. longum risks contamination from OM lipids,\nwhich could interfere with our in vitro experiments.\nlacking all Bam lipoproteins (with a specific bamA mutation), presumably because it self-\nassembles in vivo, raising questions about the uniqueness of A. longum BamA's behavior.\nAuthors: A previous study has indeed shown that BamAE470K can bypass the requirement for\naccessory BAM lipoproteins in vivo (Wang et al., J Biol Chem, 2024). The study also points out\nthat “although BamAE470K seemingly can assemble certain levels of OMPs to maintain a certain\ndegree of cell viability in the absence of BamBCDE under in vivo conditions, the OMP levels\nwere strongly reduced, indicating that the assembly function of BamAE470K alone in the absence\nof BamDE is severely defective.” It is also worth noting that during this study, BamA is present\nin the OM and thus not an example of de novo self-insertion.\nGiven that BamA is essential in all diderm bacteria, our discovery of a diderm cell that\novercomes the absence of BamA through de novo self-insertion is unprecedented.\nReviewer #2: SonA and SonB as Putative BamA Partners:\nThe identification of SonA and SonB as potential BamA partners is based on synteny. Since the\nauthors generated anti-BamA antibodies, they could perform co-immunoprecipitation or pull-\ndown experiments to test for physical interactions. In Proteobacteria, synteny between bamA,\nLPS biosynthesis demonstrated functional linkage, rather than physical protein partnerships.\nAuthors: To identify potential structural partners to BamA, we performed co-\nimmunoprecipitation (co-IP) and cross-linking experiments. Neither of these approaches\nidentified stable partners. These results have been added to the Supplementary Materials, new\nFigure 6, and the text has been modified to clarify this point. Based on these results, synteny,\noperon structure and co-expression profiles, we propose that BamA, SonA and SonB are\nfunctional partners that work together during OM biogenesis.\nReviewer #2: The Introduction is overly long and reads more like a review article on outer\nmembrane biogenesis. I suggest removing details that are not essential for understanding the\nmanuscript’s focus.\nAuthors: The introduction has been modified as suggested.\nReviewer #2: There is strong evidence suggesting non-overlapping functions (line 93-94).\nAuthors: This statement has been revised.\nReviewer #2: This statement is misleading. HxcQ does not self-transport; it contains a\nlipoprotein signal sequence and is transported via the Lol pathway.\nAuthors: This statement has been removed.\nReviewer #3: In vitro folding assay (Fig 6):\na): 6A: Where are the undigested controls?\nAuthors: Undigested controls have been added to the new Figure 7 and Supplementary Figure\n7.\n8\nb) A proportion of the unfolded BamA and SonA is resistant to proteolysis. How do the authors\nknow that retained bands are resistant to the protease due to folding and membrane insertion or\ninstead merely due to aggregation forming precipitates that are resistant to proteolysis? The\nexperiments should be repeated by running heated and unheated samples. Folded barrels are\nresistant to SDS denaturation and exhibit different electrophoretic migration than the unfolded\nform when the gel is run at 4C. Since the authors already have this assay established, repeating\nthis assay with the above conditions should be achievable in a short time frame.\nAuthors: We show that in the absence of liposomes, BamA and SonA are completely digested,\nindicating that the proteins are fully accessible for proteolysis. When the same experiments are\nset up in the presence of liposomes, discrete protein bands are observed, indicative of protected\nregions due to folding, rather than aggregation.\nAs described above in response to Reviewer #1, the folded and denatured states of BamA\ndisplayed a ~10 kDa shift in electrophoretic mobility at 4 °C (Figure R1B). Since reliable\nseparation of folded and denatured proteins requires immediate analysis, this approach was not\namenable to analyzing our protease assays, which were conducted for more than 6 hours.\nReviewer #3: The puncta of BamA is similar to the “assembly precincts” observed by the\nLithgow group by super resolution microscopy. I think that this observation should be discussed\nwith respect to this in the Discussion.\nGunasinghe SD, Shiota T, Stubenrauch CJ, Schulze KE, Webb CT, Fulcher AJ, Dunstan RA,\nHay ID, Naderer T, Whelan DR, Bell TDM, Elgass KD, Strugnell RA, Lithgow T. The WD40\nProtein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial\nOuter Membrane. Cell Rep. 2018 May 29;23(9):2782-2794. doi: 10.1016/j.celrep.2018.04.093.\nPMID: 29847806.\nAuthors: Following the reviewer’s suggestion, we performed a similar analysis to determine the\nnumber of BamA puncta in the vegetative cells of A. longum. We estimated ~8 ± 2 puncta of\nBamA per µm2, which is comparable to the ~13 precincts per µm2 estimated by the Lithgow\ngroup. This observation and data analysis have been added to the main text and as a\nSupplementary Table 5.\nReviewer #3: How were the anti-BamA and anti-OmpM1 antisera validated? Although I\nunderstand that this species is genetically intractable, it is little worrying that the potential for off-\ntarget binding cannot be excluded due to the lack of a negative control. It would be good to\nshow a WB against whole protein extracts of the various life stages. Any intensely detected off-\ntarget bands should be identified to determine their potential surface exposure (and therefore\nwhether there is the potential that the detected molecules by microscopy are a mix of BamA and\nother surface antigens).\nAuthors: We analyzed lysates from vegetative cells and spores using Western blot, which\ndetected BamA in vegetative and germinating cells but not in mature spores (new Figure 6). A\nsingle prominent band at ~63 kDa was detected in vegetative and germinating cells, indicating\ntight specificity for BamA. OmpM1 was also assessed and showed similar results but with lesser\nspecificity (new Supplementary Figure 6).\ndue to the presence of OmpM2 in germinating spores.\nReviewer #3: Alphafold structure predictions where mentioned several times for various\nproteins by where not shown or were tiny as for SonA, SonB, and BamA.\nAuthors: Alphafold structure predictions have been added to new Figure 3a and new\nSupplemental Figure 1.\nReviewer #3: line 80: Revise sentence. In E. coli the accessory subunits are BamB-E. These\nvary considerably between species (as is\nthen said later in the paragraph which contradicts with this sentence).\nAuthors: The paragraph has been revised as suggested.\nReviewer #3: line 87-89: The citation on this sentence is to a review published 8 years prior to\nthe primary articles in which the data establishing the existence of the described mechanistic\nsteps were published. Suggest updating references here and above.\nAuthors: The references have been updated.\nReviewer #3: lines 102-104: Revise. I am unaware of any review suggesting that TAM\ntransports lipoproteins. Neither of the reviews cited to this statement mention this idea. Indeed,\none of the two reviews cited do not mention TAM. There are no primary articles that I am aware\nof that provide any evidence of such a mechanism.\nAuthors: These statements have been removed from the introduction.\nReviewer #3: lines 106-108: A hypothesis is that assembly of substrates by TamA is similar to\nthe mechanism used by BamA…this has never been tested (at least no published articles). The\nsentence is constructed as a statement of truth and therefore needs to be revised.\nAuthors: The sentence has been removed.\nReviewer #3: line 496-498: An experiment to show that LPS is inserted into the OsM de novo\nwas not conducted. Revise sentence.\nAuthors: The sentence has been revised.\n10"}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of my concerns have been thoroughly addressed, and I recommend this manuscript for publication", "reviewer_2": "In the revised manuscript, the authors address most of my comments. One exception is that it remains inconclusive whether spores contain LPS. Considering that LPS biosynthesis was upregulated during sporulation, the lack of detectable LptD is not sufficient evidence to conclude LPS absence. In the referenced previous study, the experiments were not specifically designed to test for the absence of LPS. While LPS was not detectable by immunofluorescence in dormant spores, as far as I can tell, those spores were not decoated. Additionally, experiments involving direct purification and detection of LPS were not performed on dormant spores. The authors assume that the overproduced LPS ends up on the cell surface of the mother cell, but this has not been experimentally established. In E. coli, toxicity of LPS overproduction is believed to be a result of LPS accumulation at the cytoplasmic membrane. Therefore, at the very least, some discussion of the limitations of the study is warranted.", "reviewer_3": "The authors have done a good job in attempts to answer my queries. I still have concerns about the certainty that anti-OmpM1 is primarily detecting OmpM1 in the microscopy experiments as well as other over-statements. However, I believe these can be addressed by statements of caveats and by toning down other statements.\nNew Figure 6:\nThe non-specific band at 70kDa is more readily detected by the anti-OmpM1 than OmpM1 in the germinating sample. How did authors exclude the possibility that they are primarily detecting the 70kDa unknown protein during microscopy, which might be on the germination surface, rather than OmpM1? If this is not excluded, I think that the authors can simply state the ambiguity in interpretation of this experiment. Noting that the antiserum for BamA looks very clean for germinating lysate giving good confidence of specific labelling in microscopy.\nLines 110-112: “Together, these observations support a model in which BamA self\ninsertion initiates inner-to-outer membrane remodelling, a process functionally assisted by the novel proteins SonA and SonB.”\nI don’t see any experiment in the revised manuscript showing that SonA and SonB functionally assists BamA in remodelling the membrane. Actually, the new data seems to suggest that they are not involved in the function of BamA in OMP membrane insertion at all (figure 6: not part of a detectable complex with BamA). I also don’t see any experiment in the main or sup where SonA‘s or SonB’s function in a process related to outer membrane remodelling (I don’t think folding of these membrane proteins into lipid membranes in vitro constitutes “membrane remodelling”). I think this sentence needs revision and is an overstatement.\nResults subtitle: “Detection of accessory proteins associated with the BAM complex”. This subtitle needs revision because the section goes on to fail to show any accessory proteins that associate with BamA.\nAs an aside, it would have been nice to have a marked-up version of the manuscript with tracked changes with reference to changed lines in the rebuttal."}, "author_response": "Response to Reviewers\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nAll of my concerns have been thoroughly addressed, and I recommend this manuscript for\npublication\nAuthors: Thank you.\nReviewer #2 (Remarks to the Author): In the revised manuscript, the authors address most of\nmy comments. One exception is that it remains inconclusive whether spores contain LPS.\nConsidering that LPS biosynthesis was upregulated during sporulation, the lack of detectable\nLptD is not sufficient evidence to conclude LPS absence. In the referenced previous study, the\nexperiments were not specifically designed to test for the absence of LPS. While LPS was not\ndetectable by immunofluorescence in dormant spores, as far as I can tell, those spores were not\ndecoated. Additionally, experiments involving direct purification and detection of LPS were not\nperformed on dormant spores. The authors assume that the overproduced LPS ends up on the\ncell surface of the mother cell, but this has not been experimentally established. In E. coli,\ntoxicity of LPS overproduction is believed to be a result of LPS accumulation at the cytoplasmic\nmembrane. Therefore, at the very least, some discussion of the limitations of the study is\nwarranted.\nAuthors: The text has been revised as suggested by the reviewer. The following sentences\nhave been revised in the Results and Discussion sections, respectively:\n“The absence of the LPT periplasmic bridge proteins, LptC and LptA, further suggested that the\nspores also lacked LPS (Supplementary Table 2), although lipidomic analyses are required to\ndefinitively determine the presence or absence of LPS and its precursors.” (p. 11, lines 257-\n258)\n“However, the possibility remains that LPS precursors or intermediates may be present in\nmature spores or associated with the IsM. Lipidomic analyses of mature spores will be crucial in\ndetermining whether any LPS-related species are present at this stage.” (p. 19, lines 457-459)\nReviewer #3 (Remarks to the Author): The authors have done a good job in attempts to answer\nmy queries. I still have concerns about the certainty that anti-OmpM1 is primarily detecting\nOmpM1 in the microscopy experiments as well as other over-statements. However, I believe\nthese can be addressed by statements of caveats and by toning down other statements.\nNew Figure 6: The non-specific band at 70kDa is more readily detected by the anti-OmpM1 than\nOmpM1 in the germinating sample. How did authors exclude the possibility that they are\nprimarily detecting the 70kDa unknown protein during microscopy, which might be on the\ngermination surface, rather than OmpM1? If this is not excluded, I think that the authors can\nsimply state the ambiguity in interpretation of this experiment. Noting that the antiserum for\nmicroscopy.\nAuthors: The following sentences were added to the Results section to address this concern:\n“Western blot analysis detected some non-specific binding with the anti-OmpM1 serum, likely\ndue to cross-reactivity with abundant host proteins of similar molecular weight (Supplementary\nFigure 6). In contrast, the anti-BamA serum showed minimal background, especially in lysates\nfrom germinating spores, supporting the specificity of the labelling observed with both\nimmunofluorescence and immunoEM (Figure 4).” (p. 13, lines 316-320)\nThe figure legend of Supplemental Figure 6 was also updated to include this statement:\n“Non-specific binding was observed at ~70, 60, and 30 kDa, indicating that the anti-OmpM1\nserum was not as specific as the anti-BamA serum.”\nReviewer #3: Lines 110-112: “Together, these observations support a model in which BamA self\ninsertion initiates inner-to-outer membrane remodelling, a process functionally assisted by the\nnovel proteins SonA and SonB.” I don’t see any experiment in the revised manuscript showing\nthat SonA and SonB functionally assists BamA in remodelling the membrane. Actually, the new\ndata seems to suggest that they are not involved in the function of BamA in OMP membrane\ninsertion at all (figure 6: not part of a detectable complex with BamA). I also don’t see any\nexperiment in the main or sup where SonA‘s or SonB’s function in a process related to outer\nmembrane remodelling (I don’t think folding of these membrane proteins into lipid membranes in\nvitro constitutes “membrane remodelling”). I think this sentence needs revision and is an\noverstatement.\nAuthors: The sentence has been revised and now reads:\n“Together, these observations support a model in which BamA self-insertion initiates inner-to-\nouter membrane remodelling, a process that may be supported by the novel proteins SonA and\nSonB.” (p. 5, line 111)\nReviewer #3: Results subtitle: “Detection of accessory proteins associated with the BAM\ncomplex”. This subtitle needs revision because the section goes on to fail to show any\naccessory proteins that associate with BamA.\nAuthors: The subtitle has been changed to:\n“No accessory proteins detected in association with BamA”. (p. 15, line 354)\nWe also changed the related M&M subtitle to:\n“Co-immunoprecipitation using anti-BamA serum.” (p. 31, line 759)\nReviewer #3: As an aside, it would have been nice to have a marked-up version of the\nmanuscript with tracked changes with reference to changed lines in the rebuttal.\nAuthors: The previous version of the paper underwent numerous changes, resulting in a\nheavily marked-up text. With this submission, we are including a version of the manuscript that\ntracks all the changes (_withchanges) and a clean version (_clean).\n2"}, {"round": 3, "reviewer_comments": {"reviewer_3": "All of my concerns have been fully addressed, and I recommend this manuscript for publication. I believe this work will be of broad interest across several fields. I commend the authors for this great multidisciplinary effort!"}, "author_response": ""}]

Decision letter