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β-Barrel proteins tether the outer membrane in many Gram-negative bacteria

Nature Microbiology volume 6, pages 19–26 (2021)Cite this article

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Abstract

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

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Fig. 1: β-barrel OM proteins are covalently attached to PG and form a tight tether in C. burnetii.
Fig. 2: β-barrel tethering in C. burnetii is abundant in stationary phase.
Fig. 3: The L,D-transpeptidase ldt2 is required for covalent attachment of BbpA and BbpB to PG.
Fig. 4: Model depicting attachments of OM proteins to PG in C. burnetii.

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Data availability

The MS datasets are available through the GlycoPOST repository (GPST000124). The cryo-EM micrographs used as data in Fig. 3c are available at Figshare (https://doi.org/10.6084/m9.figshare.12792506). The molecular dynamics simulation data and additional raw data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank M. Suzuki, A. E. Acosta Martin and M. Collins for their contribution to developing the MS method and V. Nair for his help with cryo-EM. We thank R. Kissinger for his three-dimensional modelling, with assistance from A. Athman, and A. Mora for graphics support. We thank X. De Bolle and P. Godessart for stimulating discussion and J. Zupan, P. Zambryski, D. Kelly, A. Taylor, J. Shaw, E. F. Diaz Parga, R. Wheeler, I. Boneca, M.-K. Taha and E. Hoiczyk for the bacterial cultures used for peptidoglycan extraction. The PG MS analyses were performed by the biOMICS Facility of the Faculty of Science Mass Spectrometry Centre at the University of Sheffield, UK and the Research Technologies Branch at the National Institutes of Health (NIH) in Rockville, USA. The cryo-EM work was performed at the Research Technologies Branch at the NIH in Hamilton, USA. This work was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (R.A.H. and S.A.P.), a Medical Research Council grant no. MR/S009272/1 (S.M.) and a Biotechnology and Biological Sciences Research Council grant no. BBSRC BB/N000951/1 (S.M.). R.E.S. and A.V.P. are supported by a Biotechnology and Biological Sciences Research Council studentship (doctoral training program grant no. BB/M011151/1). J.C.G. acknowledges support from the NIH under grant no. R01-GM123169. C.J.C. was supported by a National Science Foundation (NSF) Graduate Research Fellowship under grant no. 2017219379. J.M.P. was supported by the Laboratory Directed Research and Development program at Oak Ridge National Laboratory, which is managed by UT-Battelle for the US Department of Energy under contract no. DE-AC05-00OR22725. This work used resources of the Compute and Data Environment for Science at Oak Ridge National Laboratory as well as the Extreme Science and Engineering Discovery Environment (allocation no. TG-MCB130173), which is supported by an NSF grant no. ACI-1548562.

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Author notes

  1. Kelsi M. Sandoz

    Present address: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

  2. These authors contributed equally: Roger A. Moore, Paul A. Beare, Ankur V. Patel, Robert E. Smith.

Authors and Affiliations

  1. Rocky Mountain Laboratories, Laboratory of Bacteriology, National Institute of Allergy & Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Kelsi M. Sandoz, Paul A. Beare & Robert A. Heinzen

  2. Rocky Mountain Laboratories, Laboratory of Persistent Viral Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Roger A. Moore & Suzette A. Priola

  3. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

    Ankur V. Patel, Robert E. Smith & Stéphane Mesnage

  4. Protein Metrics Inc., Cupertino, CA, USA

    Marshall Bern

  5. School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Hyea Hwang

  6. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

    Connor J. Cooper & Jerry M. Parks

  7. Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN, USA

    Connor J. Cooper & Jerry M. Parks

  8. School of Physics, Georgia Institute of Technology, Atlanta, GA, USA

    James C. Gumbart

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Contributions

All authors contributed instrumentally. K.M.S., R.E.S., A.V.P., M.B., R.A.M. and S.M. designed and performed the experiments and MS/MS data analysis. P.A.B. generated the recombinant and knockout strains. J.M.P., C.J.C., J.C.G. and H.H. performed the protein modelling and molecular dynamics simulation. S.M., S.A.P. and R.A.H. supervised the study. K.M.S., S.M. and R.A.H. prepared the manuscript.

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Correspondence to Kelsi M. Sandoz, Stéphane Mesnage or Robert A. Heinzen.

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Extended data

Extended Data Fig. 1 PG modifications identified on proteobacterial OM proteins.

Proteins from A. tumefaciens, C. burnetii, and L. pneumophila are covalently attached to PG. Proteins identified in MS/MS data that contained a PG modification scoring higher than the first decoy and containing greater than 40% of the expected b- and y- ions were established as cut-off criteria. Predicted signal peptide cleavage sites of OM proteins covalently attached to PG from C. burnetii, L. pneumophila, and A. tumefaciens are shown. Amino acid numbering is based on the predicted signal peptide cleavage site. Peptide sequences that were found covalently attached to PG are bolded. Residues with a PG tripeptide modification are also highlighted in red.

Extended Data Fig. 2 Automated spectrum assignment identifies PG modifications on C. burnetii OM proteins.

Representative MS/MS spectra for C. burnetii BbpA, ala-BbpA, BbpB β-barrel proteins and the lipoprotein LimB, covalently attached to mDAP (m) residues of PG. Spectra are shown as annotated by Byonic with manual annotations corresponding to internal fragments in black. J[ + 72.0848] has been replaced by m to represent mDAP. The PG tripeptide (AEm) is highlighted in red in the HCD spectra showing covalent attachment of PG to BbpA, ala-BbpA, and BbpB. The LimB Lys21 residue with PG tripeptide modification is highlighted in red in the ETD spectra showing covalent attachment of PG to LimB.

Extended Data Fig. 3 Automated spectrum assignment identifies PG modifications on β-barrel proteins from A. tumefaciens.

Representative MS/MS spectra for A. tumefaciens β-barrel proteins covalently attached to mDAP (m) residues of PG. Residues with PG tripeptide modification are highlighted in red. Spectra are shown as annotated by Byonic using an unbiased search approach.

Extended Data Fig. 4 Automated spectrum assignment identifies PG modifications on β-barrel proteins from L. pneumophila.

Representative MS/MS spectra for L. pneumophila Major Outer Membrane Protein (MOMP) covalently attached to mDAP (m) residues of PG. Residues with PG tripeptide modification are highlighted in red. Spectra are shown as annotated by Byonic using an unbiased search approach.

Extended Data Fig. 5 Structural topology of C. burnetii BbpA and BbpB.

Predicted periplasmic, transmembrane, and extracellular domains of BbpA and BbpB using PRED-TMBB. BbpA and BbpB are depicted with periplasmic N-terminal regions. Similar topology is predicted in β-barrel proteins from A. tumefaciens and L. pneumophila that are covalently attached to PG.

Extended Data Fig. 6 Structural modeling of C. burnetii OM proteins.

a, The protein sequences of BbpA, BbpB, and LimB are shown without N-terminal signal peptides. The disordered N-terminal domains of BbpA and BbpB were excluded from structural models but were included in subsequent molecular dynamics simulations (blue). Residues bound to PG in molecular dynamics simulations are highlighted in red. b, The mature LimB was modeled as a random coil lacking any appreciable secondary structure. The three acyl tails at the N-terminus are shown in grey as is Lys21. c, Contact map predicted from coevolution analysis of BbpA. Contacts are shown in shades of blue (darker blue = higher probability) and contacts from E. coli OmpA (PDB entry 1QJP) are shown in gray. d, Predicted contacts mapped onto the final Rosetta model of BbpA. Contacts are color coded by Cα-Cα distance: green (<5 Å), yellow (5-10 Å), red (>10 Å).

Extended Data Fig. 7 β-barrels form a tight tether between the OM and PG.

Structural model of BbpA (red) in the C. burnetii OM. Structures of the cell envelope are colored as follows: inner leaflet of OM (grey), lipid A of LPS (orange), core oligosaccharides (yellow), glycan chains of PG (blue) peptide stems of PG (green). A similar model was generated for BbpB. b, Molecular dynamics simulation of C. burnetii OM-PG protein-tethered models. The distance in angstroms between the phosphorus atoms of the inner leaflet of the OM and PG layers was measured for three runs for BbpA, ala-BbpA, BbpB, LimB, and Lpp from E. coli. The solid lines are running averages. Distances measured in angstroms for run 1, run 2, and run 3 are shown.

Extended Data Fig. 8 Predicted or annotated Ldts in Proteobacteria used in this study.

Predicted or annotated Ldts in E. coli (lpp + ), C. burnetii, L. pneumophila, A. tumefaciens, M. xanthus, N. gonorrhoeae, H. pylori, and C. jejuni. The latter seven organisms lack lpp homologs. The C. burnetii ldt genes that were successfully inactivated in this study are denoted with an asterisk. C. burnetii ldts upregulated during stationary phase are in bold. The following ldts were previously annotated as enhanced entry genes (enh); cbu0053, cbu0318, cbu1122, cbu1138, and cbu1394.

Extended Data Fig. 9 The L,D transpeptidase ldt2 is required for covalent attachment of BbpA and BbpB to PG.

XIC analysis was performed on PG that was extracted from WT and ∆ldt mutant strains and analyzed by MS/MS. XIC’s of precursor masses (m/z) corresponding to PG-bound BbpA (AEmGGPDYVPAPS, m/z = 906.909, z = 2), ala-BbpA (AEmAGGPDYVPAPS, m/z = 942.427, z = 2), and BbpB (AEmGGPDIPM, m/z = 769.843, z = 2) are shown.

Extended Data Fig. 10 Onset of replication is delayed in the ∆ldt2 deletion mutant.

Growth of wild-type (WT) C. burnetii and a ∆ldt2 mutant strain in ACCM-D was assessed using qPCR to quantitate genome equivalents (GE) during a 21-day incubation. The results are expressed as the means from n = 3 independent experiments. Error bars indicate the standard error of the mean, and asterisks indicate a statistically significant difference from WT C. burnetii. **P at 3, 6 and 9 days post-inoculation = 0.0046, 0.0027, and 0.0001, respectively. Statistical significance was calculated using a two-sided Student’s t test.

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Unprocessed western blots.

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Unprocessed western blots.

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Sandoz, K.M., Moore, R.A., Beare, P.A. et al. β-Barrel proteins tether the outer membrane in many Gram-negative bacteria. Nat Microbiol 6, 19–26 (2021). https://doi.org/10.1038/s41564-020-00798-4

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  • DOI: https://doi.org/10.1038/s41564-020-00798-4

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