Rapid Transmembrane Movement of Newly Synthesized Phosphatidylethanolamine across the Inner Membrane of Escherichia coli

Abstract

Phospholipids are the major lipid component of the cell envelope of the Gram-negative bacterium Escherichia coli. Phospholipids are the only lipid constituent in the inner membrane and they build the inner leaflet of the outer membrane. The lipid matrix of the extracellular leaflet of the outer membrane consists of lipopolysaccharide (1, 2). The phospholipid content of the E. coli envelope comprises 75% phosphatidylethanolamine (PE), 1 which is a zwitterionic phospholipid, 20% phosphatidylglycerol, and 5% cardiolipin, both anionic phospholipids (3). Phospholipids are synthesized at the cytosolic side of the inner membrane (4). Therefore, in growing cells trans-and intermembrane transport of phospholipids must occur from the endofacial leaflet of the inner membrane to the other destinations in the expanding envelope.In this study the transmembrane movement of PE across the inner membrane of E. coli was investigated. Besides being the most abundant phospholipid in E. coli, PE has several specific functions in the membrane, recently reviewed by Dowhan (5). PE as a non-bilayer-preferring lipid is essential for the polymorphic regulation of the membrane lipid organization in E. coli (6). It also stimulates several membrane transport systems (7,8) and is involved as a chaperone in the correct folding of the lac permease in the inner membrane of E. coli (9, 10).A study done by Donohue-Rolfe and Schaechter (11) demonstrated transport of phospholipids from the inner to the outer membrane of E. coli in vivo, using pulse labeling of phospholipids followed by subfractionation of the cell envelope. PE appeared in the outer membrane with a t 1/2 of 2.8 min at 37°C, whereas phosphatidylglycerol and cardiolipin were transported with half-times shorter than 30 s. Transport of the phospholipids was severely impaired when the proton motive force (pmf) was absent, whereas inhibitors of ATP, protein, or lipid synthesis did not have an effect on the process. Similar pulse labeling studies were performed in the Gram-positive bacterium Bacillus megaterium (12, 13). Rapid transmembrane movement (t 1/2 of 3 min at 37°C) of newly synthesized PE across the plasma membrane was demonstrated that was completely independent of the synthesis of lipid or protein and also independent of sources of metabolic energy.To investigate the mechanisms of phospholipid transport in more detail, in vitro systems have been used. Recently, transmembrane movement of short chain NBD-labeled phospholipid analogs was characterized in vitro in inner membrane vesicles from the above two prokaryotes. In right-side out vesicles from B. megaterium an inward movement of the analogs was observed after they had been incorporated in the outer leaflet of the membrane (14). In inside-out inner membrane vesicles isolated from E. coli, transmembrane movement of the fluorescent phospholipids was detected after their incorporation in the cytoplasmic leaflet of this membrane (15). Both prokaryotic in vitro systems have in common that the process of transmembrane movement is not ...

ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

“…Recent experiments designed to study phospholipid flip-flop in bacterial membranes and model liposomes have suggested that this process might be energy-independent in bacteria (58,59). In model membranes, lipid flip-flop occurs at near physiological rates in the absence of ATP or a proton motive force, provided that synthetic membrane-spanning peptides are reconstituted together with the lipids (60).…”

Lipid A Modification Systems in Gram-Negative Bacteria

“…Why phospholipid transport to the outer membrane is blocked in msbA mutants is also unclear (143). There is evidence that phospholipid flip-flop is not ATP dependent in bacterial inner membrane vesicles (153). Although purified MsbA is a lipid-activated ATPase (154), phospholipid flip-flop could not be demonstrated in liposomes containing MsbA alone (155).…”

Lipid II: A central component in bacterial cell wall synthesis and a target for antibiotics

“…Indeed, all experimental studies that addressed this issue reported halftimes for transport of phospholipids in the order of minutes or less [12][13][14][15]. Biosynthesis and transport were found to be mechanistically not coupled and transport in-out was comparably rapid to transport out-in [13,16].…”