Dark Side of Escherichia coli Biogenic Inner Membrane: Overabundance of Three Main Phospholipids on Cytoplasmic Leaflet.

Transmembrane (TM) asymmetry of glycerophospholipids in the plasma membrane is well established, yet still a complex and evolving area of study regarding the exact mechanisms, magnitude, and composition. The emerging controversy challenges "Fluid Mosaic Sandwich" model simplicity by combining compositional (different types of lipids on each side) and numerical (different total amounts of phospholipids) asymmetries. While Gram-negative Escherichia coli phosphatidylethanolamine (PE) distribution is asymmetric (mostly cytoplasmic), the TM distribution of phosphatidylglycerol (PG) and cardiolipin (CL) across the inner (cytoplasmic) membrane (IM) is unknown. By integrating novel in vivo mapping techniques for PE, PG, and CL, we made the unexpected observation that their TM distribution does not correspond to the distribution of their total mass between the two IM leaflets. Instead, the underpopulated periplasmic leaflet can be occupied by lipid A, diacylglycerol (DAG), lipoproteins, and glycolipid MPase, whereas CL is found predominantly in the cytoplasmic leaflet of the IM. While PG is significantly enriched in the cytoplasmic leaflet of the IM in logarithmically growing E. coli, its asymmetrical distribution is inverted in stationary-grown cells, cells lacking CL, or cells subjected to growth in a hypotonic medium. These data suggest that cells can minimize the numerical differences in TM distribution by triggering a biosynthetic TM "cleanup" response, preventing membrane collapse. The numerical asymmetric interleaflet distribution of phospholipids may serve specific functional roles, potentially governed by critical threshold levels of lipid A and DAG that determine their compatibility for colocalization within the same leaflet. The overabundance of glycerophospholipids in the cytoplasmic leaflet of the IM may be driven by packing and localization constraints imposed during cell division and impacted by the rate of phospholipid biosynthesis and intermembrane flows. This imbalance may generate or result from the lipid concentration gradients and downhill flows across IM and between IM and outer membrane (OM), and the necessity to maintain appropriate electrostatic balance across the IM.