Membrane staining and phospholipid tracking in Pseudomonas aeruginosa PAO1 using the phosphatidylcholine mimic propargyl-choline

The use of membrane-specific dyes for in vivo fluorescent microscopy is commonplace. However, most of these reagents are non-specific and cannot track specific lipid species movement, instead often acting as non-covalent lipid-associated probes or requiring the uptake of whole lipids and acyl tails into the membrane. This issue has been solved in eukaryotic cell biology by the use of click-chemistry-liable phospholipid headgroup pulse labels. Here, we describe a method for in vivo phospholipid labelling by fluorescent imaging in Pseudomonas aeruginosa using a phosphatidylcholine mimic, ‘propargyl-choline’ (PCho). This click-chemistry-liable headgroup mimic is visible by microscopy and allows the covalent labelling of lipids. Fluorescence of the cell membranes, visible in heterogeneous patches, is dependent on PCho concentration and is localized in the membrane fraction of cells, demonstrating that it is suitable for membrane labelling and cell imaging.