Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for maintenance of bacterial outer membrane lipid asymmetry

Nature Microbiology volume 2, pages 1616–1623 (2017)Cite this article

Subjects

Abstract

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds1 , 2. The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet1,2,3. This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane4. The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C5,6, but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

Three systems are known to maintain OM lipid asymmetry: the OM phospholipase A2 PldA7, the lipopolysaccharide (LPS) palmitoyl transferase PagP8 and the Mla (maintenance of outer membrane lipid asymmetry) system. Only the Mla system maintains asymmetry directly via phospholipid extraction, while PldA and PagP both generate lysophospholipids in the outer leaflet that still require removal. The Mla system is conserved in Gram-negative bacteria and plant chloroplasts9,10, consisting of the inner membrane (IM) ABC transporter MlaBDEF, the periplasmic protein MlaC and the OM lipoprotein MlaA4,6,11. Phenotypes from defects in the Mla system are mild under laboratory conditions and require small-molecule OM stressors such as SDS/EDTA or antibiotics4,5,6,11. However, MlaA has been identified as a virulence factor in various bacteria12,13,14,15. In addition, the Mla system was recently shown to regulate outer membrane vesicle formation16. The deleterious effects of Mla knockouts probably result from phospholipid accumulation in the OM outer leaflet, forming bilayer patches that are portals for entry of lipophilic small molecules17. So far, structural information on Mla components is available only for MlaC and MlaD5. MlaC probably accepts a phospholipid from MlaA and shuttles it to the ABC transporter for ATP-dependent plasma membrane insertion5. MlaA is the most enigmatic Mla component, particularly since its identity as a periplasmically exposed lipoprotein appears hard to reconcile with its proposed activity on outer leaflet phospholipids.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: MlaA is a monomeric α-helical OM protein with a central channel.
Fig. 2: MlaA binds OM phospholipids.
Fig. 3: Functional analyses of MlaA.
Fig. 4: MlaA is an outer leaflet phospholipid translocation channel.

References

  1. Henderson, J. C. et al. The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer. Annu. Rev. Microbiol. 70, 255–278 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. May, K. L. & Silhavy, T. J. Making a membrane on the other side of the wall. Biochim. Biophys. Acta. 1862, 1386-1393 (2017).

    Google Scholar 

  3. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Malinverni, J. C. & Silhavy, T. J. An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane. Proc. Natl Acad. Sci. USA 106, 8009–8014 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ekiert, D. C. et al. Architectures of lipid transport systems for the bacterial outer membrane. Cell 169, 273–285 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Chong, Z.-S., Woo, W.-F. & Chng, S.-S. Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli. Mol. Microbiol. 98, 1133–1146 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Snijder, H. J. et al. Structural evidence for dimerization-regulated activation of an integral membrane phospholipase. Nature 401, 717–721 (1999).

    Article  CAS  PubMed  Google Scholar 

  8. Bishop, R. E. et al. Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria. EMBO J. 19, 5071–5080 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Casali, N. & Riley, L. W. A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics 8, 60 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Awai, K., Xu, C., Tamot, B. & Benning, C. A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc. Natl Acad. Sci. USA 103, 10817–10822 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Thong, S. et al. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry. eLife 5, e19042 (2016).

    Article  Google Scholar 

  12. Zhao, L. et al. Deletion of the vacJ gene affects the biology and virulence in Haemophilus parasuis serovar 5. Gene 603, 42–53 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Carpenter, C. D. et al. The Vps/VacJ ABC transporter is required for intercellular spread of Shigella flexneri. Infect. Immun. 82, 660–669 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Suzuki, T. et al. Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri. Mol. Microbiol. 11, 31–41 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Shen, L. et al. PA2800 plays an important role in both antibiotic susceptibility and virulence in Pseudomonas aeruginosa. Curr. Microbiol. 65,601–609 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Roier, S. et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat. Commun. 7, 10515 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nikaido, H. Restoring permeability barrier function to outer membrane. Chem. Biol. 12, 507–509 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Li, G.-W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624–635 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Arunmanee, W. et al. Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis. Proc. Natl Acad. Sci. USA 113, E5034–E5043 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Takeuchi, Y. & Nikaido, H. Persistence of segregated phospholipid domains in phospholipid-lipopolysaccharide mixed bilayers: studies with spin-labeled phospholipids. Biochemistry 20, 523–529 (1981).

    Article  CAS  PubMed  Google Scholar 

  22. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nichols, R. J. et al. Phenotypic landscape of a bacterial cell. Cell 144,143–156 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Sutterlin, H. A. et al. Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway. Proc. Natl Acad. Sci. USA 113, E1565–E1574 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115, 717–722 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Steeb, B. et al. Parallel exploitation of diverse host nutrients enhances Salmonella virulence. PLoS Pathog. 9, e1003301 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chandran, V. et al. Structure of the outer membrane complex of a type IV secretion system. Nature 462, 1011–1015 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hankins, H. M., Baldridge, R. D., Xu, P. & Graham, T. R. Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. Traffic 16, 35–47 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Daleke, D. L. Regulation of transbilayer plasma membrane phospholipid asymmetry. J. Lipid Res. 44, 233–42 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Miroux, B. & Walker, J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Cronan, J. E. A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55, 152–157 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Prilipov, A., Phale, P. S., Van Gelder, P., Rosenbusch, J. P. & Koebnik, R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol. Lett. 163,65–72 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Waterman, D. G. et al. Diffraction-geometry refinement in the DIALS framework. Acta Crystallogr. D Struct. Biol. 72, 558–575 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  39. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  43. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. The PyMOL molecular graphics system, Version 1.8 Schrödinger, LLC.

  46. Bond, C. S. & Schüttelkopf, A. W. ALINE: aWYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr. 65, 510–512 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8,49 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. Electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65,1074–1080 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gotfredsen, M. & Gerdes, K. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Mol. Microbiol. 29, 1065–1076 (1998).

    Article  CAS  PubMed  Google Scholar 

  50. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Cherepanov, P. P. & Wackernagel, W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14 (1995).

    Article  CAS  PubMed  Google Scholar 

  52. Hoiseth, S. K. & Stocker, B. A. D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

    Article  CAS  PubMed  Google Scholar 

  53. Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    Article  Google Scholar 

  54. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Herzog, F. A., Braun, L., Schoen, I. & Vogel, V. Improved side chain dynamics in MARTINI simulations of protein–lipid interfaces. J. Chem. Theory Comput. 12, 2446–2458 (2016).

    Article  CAS  PubMed  Google Scholar 

  56. Yesylevskyy, S. O., Schäfer, L. V., Sengupta, D. & Marrink, S. J. Polarizable water model for the coarse-grained MARTINI force field. PLoS Comput. Biol. 6, e1000810 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Periole, X., Cavalli, M., Marrink, S.-J. & Ceruso, M. A. Combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009).

    Article  CAS  PubMed  Google Scholar 

  58. Hsu, P.-C., Jefferies, D. & Khalid, S. Molecular dynamics simulations predict the pathways via which pristine fullerenes penetrate bacterial membranes. J. Phys. Chem. B 120, 11170–11179 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. Bennett, W. F. D. & Tieleman, D. P. Water defect and pore formation in atomistic and coarse-grained lipid membranes: pushing the limits of coarse graining. J. Chem. Theory Comput. 7, 2981–2988 (2011).

    Article  CAS  PubMed  Google Scholar 

  60. Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985).

    Article  CAS  Google Scholar 

  61. Andersen, H. C. & C., H. Molecular dynamics simulations at constant pressure and/or temperature. J. Chem. Phys. 72, 2384–2393 (1980).

    Article  CAS  Google Scholar 

  62. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

    Article  CAS  Google Scholar 

  63. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    Article  CAS  Google Scholar 

  64. Vermeer, L. S., de Groot, B. L., Réat, V., Milon, A. & Czaplicki, J. Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments. Eur. Biophys. J. 36, 919–931 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 14101–14107 (2007).

    Article  Google Scholar 

  66. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

    Article  CAS  Google Scholar 

  67. Jo, S., Vargyas, M., Vasko-Szedlar, J., Roux, B. & Im, W. PBEQ-Solver for online visualization of electrostatic potential of biomolecules. Nucleic Acids Res. 36, W270–W275 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996).

    Article  CAS  PubMed  Google Scholar 

  69. Humphrey, W. F., Dalke, A. & Schulten, K. VMD – visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the staff at beam lines I24, I04 and I04-1 of the Diamond Light Source UK for beam time (proposal mx13587) and assistance with data collection. The research of S.S.K., U.K., D.B. and B.v.d.B. has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources that are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in-kind contribution.

Author information

Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

    Javier Abellón-Ruiz, Arnaud Baslé & Bert van den Berg

  2. Department of Physics & Earth Sciences, Jacobs University Bremen, 28759, Bremen, Germany

    Shreyas S. Kaptan & Ulrich Kleinekathöfer

  3. Focal Area Infection Biology, University of Basel, CH-4056, Basel, Switzerland

    Beatrice Claudi & Dirk Bumann

Authors
  1. Javier Abellón-Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shreyas S. Kaptan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arnaud Baslé
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beatrice Claudi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dirk Bumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ulrich Kleinekathöfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bert van den Berg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.v.d.B and J.A-R. purified MlaA proteins, crystallized MlaA–OmpF complexes, and determined crystal structures. J.A-R. constructed MlaA variant proteins and conducted in vitro functional assays. S.S.K carried out and analysed molecular dynamics simulations. U.K. supervised the computational studies. A.B. collected X-ray diffraction data and maintained the Newcastle Structural Biology Laboratory. B.C. carried out competitive fitness experiments, supervised by D.B. B.v.d.B and J.A-R designed experiments and B.v.d.B wrote the paper, with input from all co-authors.

Corresponding author

Correspondence to Bert van den Berg.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References.

Life Sciences Reporting Summary

Supplementary Video 1

Atomistic molecular dynamics simulation (200 ns) of the KpMlaA–OmpF complex, showing the movement of the POPE molecule interacting with MlaA (oxygens, red; nitrogens, blue). MlaA is shown as a cartoon and coloured light blue (OmpF; grey). Phosphate head groups are shown as olive and green balls for the outer and inner leaflets respectively, water molecules as small blue spheres. The centre of the channel is indicated by the orange line. Residue Asp152, interacting with the POPE head group is shown.

Supplementary Video 2

Coarse-grained molecular dynamics simulation (2 μs) of the KpMlaA–OmpF complex.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abellón-Ruiz, J., Kaptan, S.S., Baslé, A. et al. Structural basis for maintenance of bacterial outer membrane lipid asymmetry. Nat Microbiol 2, 1616–1623 (2017). https://doi.org/10.1038/s41564-017-0046-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-017-0046-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing