Hydrogenous and deuterated phospholipid extracts from Escherichia coli as biomimetic cytoplasmic bacterial membranes

Model lipid bilayers, reconstituted by using bacterial lipid extracts, are reliable systems to investigate the physical properties of bacterial membranes, and can be used, for example, to aid the design of new antibiotics. Here, we discuss the optimisation of a protocol for the production of hydrogenous and deuterated glycerophospholipid (GPL) extracts from Escherichia coli, and their reconstitution into model membranes. This protocol stands apart from state-of-the-art methods by introducing an additional purification step, which ensures a better separation of the GPL molecules from other membrane components such as neutral lipids. The composition of these extracts was characterised with different analytical methods. Experimental conditions were optimised for producing bacterial membrane models in the form of vesicles, lipid monolayers at the air/water interface and supported lipid bilayers. A combination of biophysical techniques, including Langmuir isotherms, neutron reflectometry, quartz crystal microbalance with dissipation monitoring, and small angle X-ray scattering provided detailed information on the self-assembled structures, and highlighted interesting differences between hydrogenous and deuterated extracts. Altogether, we report a detailed description of extraction and characterisation of hydrogenous and deuterated E. coli GPL extracts. The study of such complex lipid mixtures is important to recreate highly biologically relevant bacterial membrane models for studies aimed at understanding the biological function of bacterial membranes.