Anionic bacterial sphingolipids increase membrane stiffness

Recent genetic and bioinformatic studies have led to the discovery that many bacterial species encode the genes required to produce sphingolipids. Shotgun lipidomic studies have identified numerous sphingolipid species with novel structures that do not exist in eukaryotic organisms. The impacts of these lipids on the biophysical properties of bacterial membranes have not yet been determined. In this study, we purify a novel anionic bacterial sphingolipid, ceramide phosphoglycerate (CPG), and investigate its effect on membrane zeta potential and bending stiffness. CPG and its precursor, ceramide 1-phosphate (C1P), are shown to increase the magnitude of the membrane zeta potential. These sphingolipids also increase the stiffness of these membranes, with CPG increasing rigidity more than C1P or ceramide. This work provides experimental and computational methods of lipid isolation and characterization that may be broadly applicable to a variety of uncharacterized bacterial sphingolipids. SIGNIFICANCEThe diversity of bacterial sphingolipids far exceeds those found in eukaryotes. However, the function and biophysical properties of these lipids are unknown. Characterization of these lipids is a challenge as they are not commercially available. In this study, we developed experimental methods to purify the anionic sphingolipid ceramide phosphoglycerate and incorporate it into liposomes for analysis. Furthermore, we built computational tools to determine the bending stiffness of sphingolipid-containing vesicles from thermal fluctuation data.