Lipid-engineered Escherichia coli Membranes Reveal Critical Lipid Headgroup Size for Protein Function

Abstract

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco-and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayerprone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayerprone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyldiacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

Identification of Specific Lipid-binding Sites in Integral Membrane Proteins

“…Note that in diglucosyldiacylglycerol (DGlcDAG), the O 2 atom is involved in the connection between the two sugar rings and would therefore not be available for binding, although binding of the second sugar ring to the motif would be prohibited by the bulkiness of DGlcDAG as a whole. Although full-fledged MD simulations of LacY in GlcDAG and DGlcDAG bilayers should be done (a technically challenging process that goes beyond the scope of this paper), these results provide an explanation on why GlcDAG appears to allow LacY function and not DGlcDAG (64). In contrast, and …”

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

“…Light Microscopy-Ordinary phase contrast microscopy of various cultures, and fluorescence microscopy of cells labeled with 1 M membrane stain FM4-64 (Molecular Probes), was performed as described (36).…”

“…The latter seems lower in intact whole cells, potentially because of binding or less efficient extraction here. Note that GlcDAG is synthesized from diacylglycerol released from the normal turnover of PG (36). Hence, all GlcDAG here initially comes from PG; this indicates that the large amounts of alMGS enzyme may up-regulate PG synthesis substantially.…”

“…Wild-type cells contain ϳ75 mol % PE (zwitterionic) and 25 mol % PG plus CL (both anionic); the latter is increased in the stationary growth phase. The foreign GlcDAG is synthesized by glucosylation of diacylglycerol, released from the turnover of PG (36). alDGS-expressing cells have wild-type lipid composition, because its substrate GlcDAG is not synthesized here (data not shown).…”

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

“…The structural organization and function of LacY, as well as several other secondary transporters, is sensitive to the membrane lipid composition (2,8). The topological organization and function of LacY in vivo has been extensively studied as a function of lipid headgroup composition in wild type E. coli cells containing the normal levels of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) as well as in mutants either lacking PE (4,9) or in which PE was substituted by either phosphatidylcholine (PC) (10), monoglucosyl diacylglycerol (GlcDAG) (11) or diglucosyl diacylglycerol (GlcGlcDAG) (12).…”