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 nonbilayer-prone 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 (nonbilayer-prone) 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 digalactosyl-diacylglycerol 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.