Lipidomic Analysis of Clostridium cadaveris and Clostridium fallax.

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin-layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk-1-enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd2 Gro), which is present in tetraacyl, monoalkenyl-triacyl, and dialkenyl-diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine-phosphate-modified diacylglycerol. The significance of these lipid compositions is discussed.

Dimerization and Long-Range Repulsion Established by Both Termini of the Microtubule-Associated Protein Tau.

Tau is a microtubule-associated protein found in neuronal axons that has several well-known functions, such as promoting microtubule polymerization, stabilizing microtubules against depolymerization, and spatially organizing microtubules in axons. Two contrasting models have been previously described to explain tau's ability to organize the spacing between microtubules: complementary dimerization of the projection domains of taus on adjacent microtubules or tau's projection domain acting as a polyelectrolyte brush. In this study, atomic force microscopy was used to interrogate intermolecular interactions between layers of tau protein immobilized on mica substrates and on silicon nitride atomic force microscope tips. On these surfaces, tau adopts an orientation comparable to that when bound to microtubules, with the basic microtubule binding domain immobilized and the acidic domains extending into solution. Force-distance curves collected via atomic force microscopy reveal that full length human tau, when assembled into dense surface-bound layers, can participate in attractive electrostatic interactions consistent with the previously reported dimerization model. However, modulating the ionic strength of the surrounding solution can change the structure of these layers to produce purely repulsive interactions consistent with a polyelectrolyte brush structure, thus providing biophysical evidence to support both the zipper and brush models. In addition, a pair of projection domain deletion mutants were examined to investigate whether the projection domain of the protein is essential for the dimerization and brush models. Force-distance curves collected on layers of these proteins demonstrate that the C-terminus can play a role analogous to that of the projection domain.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates lysocardiolipins.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690 acylates a variety of lysophospholipids such as lyso phosphatidylglycerol, lyso phosphatidylethanolamine and lyso phosphatidylserine. Despite di-acylate phosphatidylglycerol being a substrate, overexpression of At1g78690 in Escherichia coli leads to the accumulation of acyl-PG. Here we show that cardiolipin also accumulates in cells overexpressing At1g78690. To help try and explain this observation, we show, using a liquid chromatography mass spectrometry (LC-MS) based assay, that At1g78690 utilizes both mono- and di-lyso cardiolipin as an acyl acceptor. Because At1g78690 shares high homology (∼40%) with the cardiolipin remodeling enzyme tafazzin, we also tested whether At1g78690 was able to catalyze a tafazzin-like transacylation reaction. Di-linoleoyl phosphatidylcholine was used as the acyl donor and mono-lyso cardiolipin was used as the acyl acceptor in a reaction and the reaction was monitored by LC-MS. No transfer of the linoleoyl chains was detected in an At1g78690 dependent manner suggesting that, despite the strong homology, these enzymes catalyze unique reactions.

Major roles for minor bacterial lipids identified by mass spectrometry.

Mass spectrometry of lipids, especially those isolated from bacteria, has ballooned over the past two decades, affirming in the process the complexity of the lipidome. With this has come the identification of new and interesting lipid structures. Here is an overview of several novel lipids, from both Gram-negative and Gram-positive bacteria with roles in health and disease, whose structural identification was facilitated using mass spectrometry. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span.

In Saccharomyces cerevisiae, Pah1 phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a negative regulatory role in controlling de novo membrane phospholipid synthesis through its consumption of phosphatidate. We found that the pah1Δ mutant was defective in the utilization of non-fermentable carbon sources but not in oxidative phosphorylation; the mutant did not exhibit major changes in oxygen consumption rate, mitochondrial membrane potential, F1F0-ATP synthase activity, or gross mitochondrial morphology. The pah1Δ mutant contained an almost normal complement of major mitochondrial phospholipids with some alterations in molecular species. Although oxidative phosphorylation was not compromised in the pah1Δ mutant, the cellular levels of ATP in quiescent cells were reduced by 2-fold, inversely correlating with a 4-fold increase in membrane phospholipids. In addition, the quiescent pah1Δ mutant cells had 3-fold higher levels of mitochondrial superoxide and cellular lipid hydroperoxides, had reduced activities of superoxide dismutase 2 and catalase, and were hypersensitive to hydrogen peroxide. Consequently, the pah1Δ mutant had a shortened chronological life span. In addition, the loss of Tsa1 thioredoxin peroxidase caused a synthetic growth defect with the pah1Δ mutation. The shortened chronological life span of the pah1Δ mutant along with its growth defect on non-fermentable carbon sources and hypersensitivity to hydrogen peroxide was suppressed by the loss of Dgk1 diacylglycerol kinase, indicating that the underpinning of pah1Δ mutant defects was the excess synthesis of membrane phospholipids.

Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ϕ29 Appendage Protein GP12 Using Synthetic Substrate Analogs.

The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.

Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom.

In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates a variety of lysophospholipids including bis(monoacylglycero)phosphate.

When the lysoglycerophospholipid (GPL) acyltransferase At1g78690 from Arabidopsis thaliana is over-expressed in Escherichiacoli a headgroup acylated GPL, acyl phosphatidylglycerol (PG), accumulates despite that in vitro this enzyme catalyzes the transfer of an acyl chain from acyl-CoA to the sn-2 position of 1-acyl phosphatidylethanolamine (PE) or 1-acyl PG to form the sn-1, sn-2, di acyl PE and PG respectively; it does not acylate PG to form acyl PG. To begin to understand why the overexpression of a lyso GPL acyltransferase leads to the accumulation of a headgroup acylated GPL in E. coli we investigated the headgroup specificity of At1g78690. Using membranes prepared from E. coli overexpressing At1g78690, we assessed the ability of At1g78690 to catalyze the transfer of acyl chains from acyl-coenzyme A to a variety of lyso GPL acyl acceptors including lyso-phosphatidic acid (PA), -phosphatidylcholine (PC), -phosphatidylserine (PC), -phosphatidylinositol (PI) and three stereoisoforms of bis(monoacylglycero)phosphate (BMP). The predicted products were formed when lyso PI and lyso PC were used as the acyl acceptor but not with lyso PC or lyso PA. In addition, At1g78690 robustly acylates two BMP isoforms with sn-2 and/or sn-2' hydroxyls in the R-stereoconfiguration, but not the BMP isoform with the sn-2 and sn-2' hydroxyls in the S-stereoconfiguration. This strongly suggests that At1g78690 is stereoselective for hydroxyls with R-stereochemistry. In addition, this robust acylation of BMPs by At1g78690, which yields acyl PG like molecules, may explain the mechanism by which At1g78690 so strikingly alters the lipid composition of E. coli.

In vitro assembly of the outer core of the lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium.

There are five distinct core structures in the lipopolysaccharides of Escherichia coli and at least two in Salmonella isolates, which vary principally in the outer core oligosaccharide. Six outer core glycosyltransferases, E. coli K-12 WaaG, WaaB, and WaaO and Salmonella typhimurium WaaI, WaaJ, and WaaK, were cloned, overexpressed, and purified. A novel substrate for WaaG was isolated from ΔwaaG E. coli overexpressing the lipid A phosphatase lpxE and the lipid A late acyltransferase lpxM. The action of lpxE and lpxM in the ΔwaaG background yielded heptose2-1-dephospho Kdo2-lipid A, a 1-dephosphorylated hexa-acylated lipid A with the inner core sugars that is easily isolated by organic extraction. Using this structurally defined acceptor and commercially available sugar nucleotides, each outer core glycosyltransferases was assayed in vitro. We show that WaaG and WaaB add a glucose and galactose sequentially to heptose2-1-dephospho Kdo2-lipid A. E. coli K-12 WaaO and S. typhimurium WaaI add a galactose to the WaaG/WaaB product but can also add a galactose to the WaaG product directly without the branched core sugar added by WaaB. Both WaaI and WaaO require divalent metal ions for optimal activity; however, WaaO, unlike WaaI, can add several glucose residues to its lipid acceptor. Using the product of WaaG, WaaB, and WaaI, we show that S. typhimurium WaaJ and WaaK transfer a glucose and N-acetylglucosamine, respectively, to yield the full outer core. This is the first demonstration of the in vitro assembly of the outer core of the lipopolysaccharide using defined lipid A-oligosaccharide acceptors and sugar donors.

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis-specific lipid A.

Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram-negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate. Using a combined genetic and biochemical approach, we identified PA1343 as the PA gene encoding PagP. Although PA1343 lacks obvious primary structural similarity with known PagP enzymes, the ß-barrel tertiary structure with an interior hydrocarbon ruler appears to be conserved. PA PagP transfers palmitate to the 3' position of lipid A, in contrast to the 2 position seen with the enterobacterial PagP. Palmitoylated PA lipid A alters host innate immune responses, including increased resistance to some antimicrobial peptides and an elevated pro-inflammatory response, consistent with the synthesis of a hexa-acylated structure preferentially recognized by the TLR4/MD2 complex. Palmitoylation commonly confers resistance to cationic antimicrobial peptides, however, increased cytokine production resulting in inflammation is not seen with other palmitoylated lipid A, indicating a unique role for this modification in PA pathogenesis.

Comparative lipid profiling of the cnidarian Aiptasia pallida and its dinoflagellate symbiont.

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.

Christian Raetz: scientist and friend extraordinaire.

Chris Raetz passed away on August 16, 2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gram-negative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpinnings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiosity-driven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues.

Two-dimensional nuclear magnetic resonance structure determination module for introductory biochemistry: synthesis and structural characterization of lyso-glycerophospholipids.

In this laboratory module, introductory biochemistry students are exposed to two-dimensional (1) H-nuclear magnetic resonance of glycerophospholipids (GPLs). Working in groups of three, students enzymatically synthesized and purified a variety of 2-acyl lyso GPLs. The structure of the 2-acyl lyso GPL was verified using (1) H-correlation spectroscopy. Students scored significantly higher on an assessment of NMR knowledge after having participated in this lab module and in comparison to a similar cohort who did not participate. Inaddition, student confidence in their NMR knowledge and abilities increased 62% following the module and correlated with their ability to apply their NMR knowledge. Based on these results, the laboratory module was very effective at providing students with a more extensive understanding of the underlying concepts of NMR as a tool for structural determination.

Quantification of cardiolipin molecular species in Escherichia coli lipid extracts using liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Cardiolipin (CL), a glycerophospholipid containing four acyl chains, is found in most organisms including Gram-negative bacteria such as Escherichia coli. While CL composes only a fraction of the total glycerophospholipids, the four acyl chains lead to a large number of possible molecular species as defined by the total number of carbons and unsaturations in the acyl chains. Understanding the molecular composition of CL, and how it changes under different growth conditions, will aid in understanding the complex role of CL in E. coli. METHODS: Normal-phase liquid chromatography/electrospray ionization mass spectrometry was used to quantify the CL molecular species (as defined by the total number of carbons:unsaturations in the acyl chains) in lipid extracts prepared from E. coli grown at 15 °C, 30 °C, 37 °C and 42 °C. RESULTS: Fifty-six different CL species were identified as [M-2H](2-) ions in E. coli lipid extracts ranging from 60:0 to 72:4. CL species with an increased total number of unsaturations were more abundant in lipid extracts prepared from cells grown at 15 °C as compared to higher temperatures. CONCLUSIONS: This work characterizes the CL composition of E. coli cells grown at various temperatures. By quantifying CL species at a molecular level we have illuminated the molecular complexity of the CL in this relatively simple model organism. This data will be useful for understanding CL function in E. coli and other organisms.

Activity and crystal structure of Arabidopsis thaliana UDP-N-acetylglucosamine acyltransferase.

The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 Å resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an α-helical-rich C-terminus and characteristic N-terminal left-handed parallel ß-helix (LßH). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the LßH not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts.

Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli; phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of E. coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H](-) ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.

Putative N-acylphosphatidylethanolamine synthase from Arabidopsis thaliana is a lysoglycerophospholipid acyltransferase.

AT1G78690, a gene found in Arabidopsis thaliana, has been reported to encode a N-acyltransferase that transfers an acyl chain from acyl-CoA to the headgroup of phosphatidylethanolamine (PE) to form N-acylphosphatidylethanolamine (N-acyl-PE). Our investigation suggests that At1g78690p is not a PE-dependent N-acyltransferase but is instead a lysoglycerophospholipid O-acyltransferase. We overexpressed AT1G78690 in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphosphatidylglycerol (acyl-PG). Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-MS) analysis yielded [M - H](-) ions, corresponding by exact mass to acyl-PG rather than N-acyl-PE. Collision-induced dissociation mass spectrometry (MS/MS) yielded product ions consistent with acyl-PG. In addition, in vitro enzyme assays using both (32)P- and (14)C-radiolabeled substrates showed that AT1G78690 acylates 1-acyllysophosphatidylethanolamine (1-acyllyso-PE) and 1-acyllysophosphatidylglycerol (1-acyllyso-PG), but not PE or phosphatidylglycerol (PG), to form a diacylated product that co-migrates with PE and PG, respectively. We analyzed the diacylated product formed by AT1G78690 using a combination of base hydrolysis, phospholipase D treatment, ESI-MS, and MS/MS to show that AT1G78690 acylates the sn-2-position of 1-acyllyso-PE and 1-acyllyso-PG.

A mouse macrophage lipidome.

We report the lipidomic response of the murine macrophage RAW cell line to Kdo(2)-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations.

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli.

Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.

Phosphatidic acid and N-acylphosphatidylethanolamine form membrane domains in Escherichia coli mutant lacking cardiolipin and phosphatidylglycerol.

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.