Detecting Lipoproteins Sneaking Out of the Lipopolysaccharide Leaflet

The envelope of Gram-negative bacteria is an essential compartment which is in direct contact with the environment; the envelope maintains cellular integrity and functions as a permeability barrier protecting the cell from toxic compounds. The outer layer of the envelope is an asymmetric membrane whose external leaflet is mainly composed of lipopolysaccharide molecules. Recently, there has been growing evidence that lipoproteins (i.e., soluble proteins anchored to a membrane by a lipid moiety) decorate the lipopolysaccharide leaflet in the model bacterium Escherichia coli, challenging the current paradigm that lipoproteins remain in the periplasm in this organism. However, assessing the surface exposure of lipoproteins is challenging. Here, we describe an optimized and reproducible dotblot protocol to assess the presence of lipoproteins at the surface of E. coli and other bacterial models. We added all necessary controls to reduce the possibility of artifacts giving rise to false-positive results. We selected the stress sensor RcsF as a model lipoprotein to illustrate the method, which can be used for any other lipoprotein.