Defining Lipoprotein Localisation by Fluorescence Microscopy
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.
Key wordsLipoprotein Localisation Cell envelope Spheroplasts Bacterial secretion Fluorescence microscopy
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).
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