Defining Lipoprotein Localisation by Fluorescence Microscopy

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

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

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.

Original languageEnglish
Title of host publicationBacterial Protein Secretion Systems
Subtitle of host publicationMethods and Protocols
EditorsLaure Journet , Eric Cascales
Place of PublicationNew York
PublisherSpringer
Pages65-74
Number of pages10
ISBN (Electronic)9781493970339
ISBN (Print)9781493970315
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
PublisherSpringer
Volume1615
ISSN (Print)1064-3745

Keywords

  • Lipoprotein
  • Localisation
  • Cell envelope
  • Spheroplasts
  • Bacterial secretion
  • Fluorescence microscopy

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