A retrospective: use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Abstract

Although the study of individual phospholipids and their synthesis began in the 1920s first in plants and then mammals, it was not until the early 1960s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960s. In 1970s and 1980s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Figure 1

The Kennedy “Clan” on the occasion of his 90^th birthday. Eugene Kennedy (1919–2011) is 4^th from the left in the front row. The gathering was in October 2009 at Harvard Medical School. Pictured are former graduate students, postdoctoral fellows and scientific associates of Eugene Kennedy.

Figure 2

Pathways for phospholipid biosynthesis in mammalian cells. The zig-zig lines in each red structure represent the long chain fatty acids on the phosphatidyl moiety. These are mostly saturated at the sn-1 position of unsaturated at the sn-2 position of the glycerol backbone. The components of each fatty acid are color coded as to the building blocks from which they are derived. The enzymes are located in the cytoplasm and the endoplasmic reticulum. The “Kennedy Pathway” generally refers to the pathways beginning with ethanolamine and choline. Not shown are the formation of PS from PE or PC by head group exchange with serine, the decarboxylation of PS to form PE, the methylation of PE by S-adenosyl methionine to form PC, and the formation of PG and CL in the mitochondria.

Figure 3

Pathways for synthesis of phospholipids in E. coli. The following enzymes with their respective genes named carry out: 1. CDP-diacylglycerol synthase (CdsA); 2. phosphatidylserine synthase (PssA); 3. phosphatidylserine decarboxylase (Psd); 4. phosphatidylglycerophosphate synthase (PgsA); 5. phosphatidylglycerophosphate phosphatase (Pgp) encoded by three genes; 6. cardiolipin synthase (Cls) encoded by three genes with one substrate being PG in all three cases and the second substrate for {ClsA} and [ClsC] indicated by the brackets. Definitive identification of the second substrate for ClsB has not been established; 7. Phosphatidylglycerol:pre-membrane derived oligosaccharide (MDO) sn-glycerol-1-P transferase (MDO synthase); 8. diacylglycerol kinase (DgkA).

Figure 4

Synthesis of foreign lipids in E. coli. See Fig. 3 for the native pathways (blue arrows) and native lipids (blue and grey). The following enzymes with their respective genes named carry out (red arrows): 1. phosphatidylcholine synthase (Legionella pneumophila [185, 248]); 2. phosphatidylinositol synthase (Saccharomyces cerevisiae [186]); 3. glucosyl diacylglycerol synthase (Acholeplasma laidlawii [212]); 4. diglucosyl diacylglycerol synthase (Acholeplasma laidlawii [211]); 5) lysyl t-RNA:phosphatidylglyerceol lysine transferase (Staphococcus aureus [187]).

Figure 5

Structure, physical and chemical properties of lipids native to E. coli and those introduced from other sources. See Fig. 4 for pathways of synthesis and genes encoding enzymes responsible for the synthesis of each lipid except N-acyl PE, which is discussed under section 7.2.2. The glycerol backbone (red) shown in ester linkage to fatty acids (aliphatic chains R₁-R₄) at the sn-1 and sn-2 positions and in either phosphodiester linkage for phospholipids or in a glycosidic linked for glycolipids at the sn-3 position. Head groups are color coded to indicate the charge nature of each head group.

Figure 6

Topological organization of LacY as a function of the presence or absence of PE. Cytoplasm is at the top of each figure, TMs are noted by rectangles, NT and CT refer to the N and C terminus respectively, and extramembrane domains oriented to the cytoplasm (C) or periplasm (P) in PE-containing cells are indicated. (A) LacY topology as determined in PE-containing (+PE) cells is depicted. The approximate position of D325 in TM X is indicated. (B) LacY topology after assembly in PE-lacking cells (−PE) cells. The exposure of TM VII (red) to the periplasm results in the loss of salt bridges of TM VII with TM X and TM XI as noted by the charges. (C) LacY topology determined after induction of PE synthesis in cells where assembly of LacY initially occurred in the absence of PE.