Recent data support the hypothesis that Gram-positive bacteria (monoderms) arose from Gram-negative ones (diderms) through loss of the outer membrane (OM), but how this happened remains unknown. As tethering of the OM is essential for cell envelope stability in diderm bacteria, its destabilization may have been involved in this transition. In the present study, we present an in-depth analysis of the four known main OM-tethering systems across the Tree of Bacteria (ToB). We show that the presence of such systems follows the ToB with a bimodal distribution matching the deepest phylogenetic divergence between Terrabacteria and Gracilicutes. Whereas the lipoprotein peptidoglycan-associated lipoprotein (Pal) is restricted to the Gracilicutes, along with a more sporadic occurrence of OmpA, and Braun’s lipoprotein is present only in a subclade of Gammaproteobacteria, diderm Terrabacteria display, as the main system, the OmpM protein. We propose an evolutionary scenario whereby OmpM represents a simple, ancestral OM-tethering system that was later replaced by one based on Pal after the emergence of the Lol machinery to deliver lipoproteins to the OM, with OmpA as a possible transition state. We speculate that the existence of only one main OM-tethering system in the Terrabacteria would have allowed the multiple OM losses specifically inferred in this clade through OmpM perturbation, and we provide experimental support for this hypothesis by inactivating all four ompM gene copies in the genetically tractable diderm Firmicute Veillonella parvula. High-resolution imaging and tomogram reconstructions reveal a non-lethal phenotype in which vast portions of the OM detach from the cells, forming huge vesicles with an inflated periplasm shared by multiple dividing cells. Together, our results highlight an ancient shift of OM-tethering systems in bacterial evolution and suggest a mechanism for OM loss and the multiple emergences of the monoderm phenotype from diderm ancestors.
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The authors declare that all data supporting the findings of the present study are available within the paper and its supplementary information files (source data for Figs. 2 and 3a, Extended Data Figs. 1, 2a, 3, 5a and 6a and supplementary data for Supplementary Figs. 1a,b and 2a,b) or, otherwise, are available from the corresponding author on request. Source data are provided with this paper.
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This work was supported by funding from the French National Research Agency (ANR) (no. Fir-OM ANR-16-CE12-0010), the Institut Pasteur’s Programmes Transversaux de Recherche (no. PTR 39-16), the French government’s Investissement d’Avenir Program, Laboratoire d’Excellence’s Integrative Biology of Emerging Infectious Diseases (grant no. ANR-10-LABX-62-IBEID) and the Fondation pour la Recherche Médicale (grant no. DEQ20180339185). N.P. is funded by a Pasteur-Roux Postdoctoral Fellowship from the Institut Pasteur. We thank A. Jiménez-Fernández for help with Veillonella genetics techniques. We thank the UTechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur (Paris, France) and the France BioImaging infrastructure network supported by the ANR (no. ANR-10–INSB–04; Investments for the Future), and the Région Ile-de-France (program Domaine d’Intérêt Majeur-Malinf) for the use of the Zeiss LSM 780 Elyra PS1 microscope. We thank S. Tachon from the NanoImaging Core facility of the Center for Technological Resources and Research of Institut Pasteur for assistance with the tomography acquisitions at the Titan Krios microscope. We thank the French Government Programme Investissements d’Avenir France BioImaging (FBI, no. ANR-10-INSB-04-01) for equipment support. We thank M. Nilges and the Equipex CACSICE (Centre d’analyse de systèmes complexes dans les environnements complexes) for providing the Falcon II direct detector. We thank the IT department at Institut Pasteur, Paris, for providing computational and storage services (TARS cluster).
All the authors declare that they have no competing interests.
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Extended Data Fig. 1 Number of ompM copies per genome plotted on a reference Negativicutes tree containing 135 genomes.
Selenomonadales are labelled in green, Acidaminococcales in red and Veillonellales in blue. The phylogeny is based on the concatenation of RNA polymerase subunits β, β’ and elongation factor IF-2 (3,027 amino-acid positions). The tree was calculated using IQTREE version 1.6.3 with the model LG + R5. Colours of branches represent bootstrap values from 80 (red) to 100 (green). The ompM copies present inside the OM gene cluster are marked by full circles, those outside the cluster by empty ones. Source data is provided in the file “Source Data for Figure ED1.newick”.
Extended Data Fig. 2 Phenotypic complementation of the ∆ompM1-3 mutant by production of native or HA-tagged OmpM1.
(a) Growth curves. All data points represent the mean of a pentaplicate, error bars were omitted for sake of visibility. Cultures were made in BHILC medium in presence of 250 µg.l–1 of anhydrotetracycline in 96-well plates at 37 °C under anaerobic conditions. pRPF185 – empty vector, pJW34 – a vector expressing native OmpM1 under the control of tet promoter, pJW35 – a vector expressing HA-tagged OmpM1 under the control of tet promoter. (b) – Epifluorescence observation of cells labelled with the biological membrane staining dye FM 4-64 (Thermo Fisher Scientific). Overnight cultures were launched at 1/10th dilution of a stationary phase culture in SK medium at 37 °C in anaerobic conditions in presence of aTc at 250 µg.l–1, before being labeled with the biological membrane staining dye FM 4-64 (Thermo Fisher Scientific) and observed by epifluorescence microscopy. Expression of either the native version of OmpM1 or its HA-tagged version led to complementation of the strong phenotype (formation of OM “bubbles” observed in the ∆ompM1-3 mutant). Growth default of this mutant was also fully or partly complemented by native or HA-tagged OmpM1. All scale bars represent 5 µm. The samples presented are representative of multiple (n > 20) experiments.
Extended Data Fig. 3 The drastic phenotype of the ∆ompM1-3 mutant cannot be complemented by expressing in trans OmpM4 or OmpA.
In (a) the ∆ompM1-3 was transformed with a plasmid expressing either native (pJW63) or HA-tagged (pJW62) OmpM4 from an ATC inducible promoter. In (b) the ∆ompM1-3 was transformed with a plasmid expressing either native (pJW65) or HA-tagged (pJW66) OmpA from an ATC inducible promoter. Both OmpM4-HA and OmpA-HA were readily detected by western blot using an anti-HA primary antibody coupled to horse radish peroxydase when ATC was added at 250 µg.l–1 (left panels). However, the strong phenotype of the ∆ompM1-3 was not complemented by expressing either native or HA-tagged version of OmpM4, as well as native or HA-tagged version of OmpA, as shown on the right panels following FM 4-64 epifluorescence. Scale bars represent 5 µm. The samples presented are representative of multiple (n > 10) experiments.
Extended Data Fig. 4 Detailed characterization of the strong phenotype of the ∆ompM1-3 mutant using cryoelectron tomography.
Cells of the ∆ompM1-3 mutant were grown for 48 h in SK medium before being prepared, deposited and observed using a 300 kV Titan Krios (Thermo Fisher Scientific) transmission electron microscope. Different slices of cryoelectron tomography of different positions of a mesh grid containing the ΔompM1-3 mutant are shown. Scale bars represent 0.5 µM, blue arrows OM, red arrows peptidoglycan, green arrows IM, magenta arrows fimbriae, yellow asterisks indicate empty vesicles. The CryoEM acquisitions were performed twice, but the phenotype of the presented samples, as assessed by conventional fluorescence microscopy, is representative of multiple (n > 20) experiments.
Comparison of WT SKV38 strain of V. parvula containing pRPF185 (empty vector), pJW34, a vector expressing untagged OmpM1, and pJW35, a vector expressing HA-tagged OmpM1 both under the control of tet promoter. All cultures were induced overnight with 250 µg.l–1 anhydrotetracycline. In (a), we used an anti HA primary and antibodies coupled to horse radish peroxidase (HRP) to detect the expression of OmpM1-HA (arrow) from pJW35 by Western blot. M corresponds to SeeBlue Plus Prestained Protein Standard (Invitrogen). In (b), we performed immunofluorescence microscopy of WT cells expressing either OmpM1-HA (pJW35) or native OmpM1 (pJW34) using an anti-HA antibody coupled to fluorescein. Cells were non-permeabilized. While native OmpM1 was not detectable, OmpM1-HA that contains the HA tag in one of the predicted outer loops of its beta-barrel could be detected as all around the cell, confirming that it located in the outer membrane and is surface exposed. Surface exposition of tagged OmpM1 expressed from pJW35 was confirmed using Scanning Electron Microscopy in (c). We used anti-HA primary antibodies and secondary antibodies coupled to 20 nm gold particles on non-permeabilized cells. Images were obtained using a JEOL JSM 6700 F field emission scanning electron microscope and with a backscattered electrons detector (BSE) to detect the gold labelling and with a secondary electron detector (SEI) to image the surface of the sample. The acquisitions were performed once.
As for supplementary Fig. S10 we compared E. coli cells expressing containing pRPF185 (empty vector), pJW34, a vector expressing untagged OmpM1, and pJW35, a vector expressing HA-tagged OmpM1 both under the control of tet promoter. All cultures were induced overnight with 250 µg.l–1 anhydrotetracycline. In (a), we used an anti-HA antibodies coupled to HRP to detect, in E. coli, the expression of OmpM1-HA (arrow) from pJW35 by Western blot, while the non-tagged native OmpM1 expressed from pJW34 could not be detected. M corresponds to SeeBlue Plus Prestained Protein Standard (Invitrogen). In (b), we performed immunofluorescence microscopy of E. coli cells expressing no OmpM1 (empty vector pRPF185) or OmpM1-HA (pJW35) using an anti-HA antibody couple to fluorescein. E. coli cells were non-permeabilized. OmpM1-HA that contains the HA tag in one of the predicted outer loops of its beta-barrel could be detected as all around E. coli cells, confirming that it located in the outer membrane and is surface exposed also in this bacterium, therefore suggesting that it could be transported and inserted in the outer membrane of a classical diderm bacterium. Surface exposition of tagged OmpM1 expressed from pJW35 in E. coli was confirmed using Scanning Electron Microscopy in (c). We used anti-HA primary antibodies and secondary antibodies coupled to 20 nm gold particles on non-permeabilized cells. Images were obtained using a JEOL JSM 6700 F field emission scanning electron microscope and with a backscattered electrons detector (BSE) to detect the gold labelling and with a secondary electron detector (SEI) to image the surface of the sample. The acquisitions were performed once.
Supplementary Results and Discussion, Figs. 1–5, Tables 1–3, Material and Methods, References.
Cryo-ET of V. parvula WT. The animation shows all tomogram slices, then a 3D rendering of the cell envelope structure based on manual segmentation of tomogram slices. Blue marking, OM; red marking, PG; green marking, IM. The video corresponds to Fig. 5a.
Cryo-ET of V. parvula ΔompM1–3 mutant; detached OM forming blebs and vesicles is clearly visible. The animation shows all tomogram slices, then a 3D rendering of the cell envelope structure based on manual segmentation of tomogram slices. Blue marking, OM; red marking, PG; green marking, IM. The video corresponds to Fig. 5b.
Cryo-ET of V. parvula ΔompM1–3 mutant; detached OM forming blebs and vesicles is clearly visible. The animation shows all tomogram slices, then a 3D rendering of the cell envelope structure based on manual segmentation of tomogram slices. Blue marking, OM; red marking, PG; green marking, IM. The video corresponds to Fig. 5c.
Cryo-ET of V. parvula ΔompM1–3 mutant complemented with pJW35 vector (expressing OmpM1-HA under control of tet promoter) induced overnight with 250 µg l−1 of ATC. The animation shows all tomogram slices, then a 3D rendering of the cell envelope structure based on manual segmentation of tomogram slices. Blue marking, OM; red marking, PG; green marking, IM. The video corresponds to Fig. 5d.
All the accession nos. of the sequences used to infer the information presented throughout the study. The Excel file contains a detailed legend describing each sheet inside.
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Witwinowski, J., Sartori-Rupp, A., Taib, N. et al. An ancient divide in outer membrane tethering systems in bacteria suggests a mechanism for the diderm-to-monoderm transition. Nat Microbiol 7, 411–422 (2022). https://doi.org/10.1038/s41564-022-01066-3