Skip to main content
SpringerLink
Log in

Defining Lipoprotein Localisation by Fluorescence Microscopy

  • Protocol
  • First Online:
Bacterial Protein Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

Abstract

In recent years it has become evident that lipoproteins play crucial roles in the assembly of bacterial envelope-embedded nanomachineries and in the processes of protein export/secretion. In this chapter we describe a method to determine their precise localisation, for example inner versus outer membrane, in Gram-negative bacteria using human opportunistic pathogen Pseudomonas aeruginosa as a model. A fusion protein between a given putative lipoprotein and the red fluorescent protein mCherry must be created and expressed in a strain expressing cytoplasmic green fluorescent protein (GFP). Then the peripheral localisation of the fusion protein in the cell can be examined by treating cells with lysozyme to create spheroplasts and monitoring fluorescence under a confocal microscope. Mutants in the signal peptide can be engineered to study the association with the membrane and efficiency of transport. This protocol can be adapted to monitor lipoprotein localisation in other Gram-negative bacteria.

†To the memory of Didier Grunwald, who passed away recently.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Farris C, Sanowar S, Bader MW, Pfuetzner R, Miller SI (2010) Antimicrobial peptides activate the Rcs regulon through the outer membrane lipoprotein RcsF. J Bacteriol 192(19):4894–4903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Leverrier P, Declercq JP, Denoncin K, Vertommen D, Hiniker A, Cho SH et al (2011) Crystal structure of the outer membrane protein RcsF, a new substrate for the periplasmic protein-disulfide isomerase DsbC. J Biol Chem 286(19):16734–16742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD et al (1999) Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285(5428):736–739

    Article  CAS  PubMed  Google Scholar 

  4. Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57(1):50–108

    PubMed  PubMed Central  CAS  Google Scholar 

  5. Zuckert WR (2014) Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond. Biochim Biophys Acta 1843(8):1509–1516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Konovalova A, Silhavy TJ (2015) Outer membrane lipoprotein biogenesis: Lol is not the end. Philos Trans R Soc Lond Ser B Biol Sci 370(1679)

    Google Scholar 

  7. Babu MM, Priya ML, Selvan AT, Madera M, Gough J, Aravind L et al (2006) A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J Bacteriol 188(8):2761–2773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Madan Babu M, Sankaran K (2002) DOLOP--database of bacterial lipoproteins. Bioinformatics 18(4):641–643

    Article  CAS  PubMed  Google Scholar 

  9. Remans K, Vercammen K, Bodilis J, Cornelis P (2010) Genome-wide analysis and literature-based survey of lipoproteins in Pseudomonas aeruginosa. Microbiology 156(Pt 9):2597–2607

    Article  CAS  PubMed  Google Scholar 

  10. Casabona MG, Vandenbrouck Y, Attree I, Coute Y (2013) Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane. Proteomics 13(16):2419–2423

    Article  CAS  PubMed  Google Scholar 

  11. Fernandez D, Dang TA, Spudich GM, Zhou XR, Berger BR, Christie PJ (1996) The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol 178(11):3156–3167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fernandez D, Spudich GM, Zhou XR, Christie PJ (1996) The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol 178(11):3168–3176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Christie PJ, Cascales E (2005) Structural and dynamic properties of bacterial type IV secretion systems (review). Mol Membr Biol 22(1–2):51–61

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Collin S, Guilvout I, Nickerson NN, Pugsley AP (2011) Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin. Mol Microbiol 80(3):655–665

    Article  CAS  PubMed  Google Scholar 

  15. Izore T, Perdu C, Job V, Attree I, Faudry E, Dessen A (2011) Structural characterization and membrane localization of ExsB from the type III secretion system (T3SS) of Pseudomonas aeruginosa. J Mol Biol 413(1):236–246

    Article  CAS  PubMed  Google Scholar 

  16. Perdu C, Huber P, Bouillot S, Blocker A, Elsen S, Attree I et al (2015) ExsB is required for correct assembly of the Pseudomonas aeruginosa type III secretion apparatus in the bacterial membrane and full virulence in vivo. Infect Immun 83(5):1789–1798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Guilvout I, Chami M, Engel A, Pugsley AP, Bayan N (2006) Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin. EMBO J 25(22):5241–5249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Viarre V, Cascales E, Ball G, Michel GP, Filloux A, Voulhoux R (2009) HxcQ liposecretin is self-piloted to the outer membrane by its N-terminal lipid anchor. J Biol Chem 284(49):33815–33823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Aschtgen MS, Bernard CS, De Bentzmann S, Lloubes R, Cascales E (2008) SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli. J Bacteriol 190(22):7523–7531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Casabona MG, Silverman JM, Sall KM, Boyer F, Coute Y, Poirel J et al (2013) An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas Aeruginosa. Environ Microbiol 15(2):471–486

    Article  CAS  PubMed  Google Scholar 

  21. Durand E, Nguyen VS, Zoued A, Logger L, Pehau-Arnaudet G, Aschtgen MS et al (2015) Biogenesis and structure of a type VI secretion membrane core complex. Nature 523(7562):555–560

    Article  CAS  PubMed  Google Scholar 

  22. Felisberto-Rodrigues C, Durand E, Aschtgen MS, Blangy S, Ortiz-Lombardia M, Douzi B et al (2011) Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. PLoS Pathog 7(11):e1002386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rao VA, Shepherd SM, English G, Coulthurst SJ, Hunter WN (2011) The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system. Acta Crystallogr Sect D 67(Pt 12):1065–1072

    Article  CAS  Google Scholar 

  24. Basler M, Ho BT, Mekalanos JJ (2013) Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152(4):884–894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Alcock F, Baker MA, Greene NP, Palmer T, Wallace MI, Berks BC (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. Proc Natl Acad Sci U S A 110(38):E3650–E3659

    Article  PubMed  PubMed Central  Google Scholar 

  26. Guillon L, El Mecherki M, Altenburger S, Graumann PL, Schalk IJ (2012) High cellular organization of pyoverdine biosynthesis in Pseudomonas aeruginosa: clustering of PvdA at the old cell pole. Environ Microbiol 14(8):1982–1994

    Article  CAS  PubMed  Google Scholar 

  27. Imperi F, Visca P (2013) Subcellular localization of the pyoverdine biogenesis machinery of Pseudomonas aeruginosa: a membrane-associated “siderosome”. FEBS Lett 587(21):3387–3391

    Article  CAS  PubMed  Google Scholar 

  28. Lewenza S, Mhlanga MM, Pugsley AP (2008) Novel inner membrane retention signals in Pseudomonas aeruginosa lipoproteins. J Bacteriol 190(18):6119–6125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682

    Article  CAS  PubMed  Google Scholar 

  30. De Bentzmann S, Giraud C, Bernard CS, Calderon V, Ewald F, Plesiat P et al (2012) Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB. PLoS Pathog 8(11):e1003052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Thibault J, Faudry E, Ebel C, Attree I, Elsen S (2009) Anti-activator ExsD forms a 1:1 complex with ExsA to inhibit transcription of type III secretion operons. J Biol Chem 284(23):15762–15770

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hoang TT, Kutchma AJ, Becher A, Schweizer HP (2000) Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43(1):59–72

    Article  CAS  PubMed  Google Scholar 

  33. Newman JR, Fuqua C (1999) Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227(2):197–203

    Article  CAS  PubMed  Google Scholar 

  34. Chuanchuen R, Narasaki CT, Schweizer HP (2002) Benchtop and microcentrifuge preparation of Pseudomonas aeruginosa competent cells. Biotechniques 33(4):760. 2–3

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. K. M. Sall for initiating studies on TagQ and TssJ1 fusion proteins and Dr. S. Elsen for help in plasmid generation. MGC was supported by a PhD grant from the French Cystic Fibrosis Association Vaincre la Mucovisidose. The microscopy facility is supported by the Biosciences and Biotechnology Institute of Grenoble (BIG), CEA-Grenoble and the grant to Laboratoire of Excellence, LabEx GRAL (ANR-10-LABX-49-01).

Author information

Authors and Affiliations

  1. Bacterial Pathogenesis and Cellular Responses, Centre National pour la Recherche Scientifique (CNRS), University Grenoble Alpes, INSERM, Biosciences and Biotechnology Institut, CEA Grenoble, Grenoble, France

    Maria Guillermina Casabona, Mylène Robert-Genthon, Didier Grunwald & Ina Attrée

  2. Division of Molecular Microbiology, School of Life Sciences, University of Dundee, DD1 5EH, Dundee, Scotland, UK

    Maria Guillermina Casabona

Authors
  1. Maria Guillermina Casabona
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mylène Robert-Genthon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Didier Grunwald
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ina Attrée
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ina Attrée .

Editor information

Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Casabona, M.G., Robert-Genthon, M., Grunwald, D., Attrée, I. (2017). Defining Lipoprotein Localisation by Fluorescence Microscopy. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation