Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

Abstract

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 wildtype 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.A unique lipid composition and lipid-protein interactions appear to exist at the transient membrane domain that defines the division site in bacterial cells (1). Using the cardiolipin (CL) 4 -specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2). Subsequently others reported similar CL domains in Bacillus subtilis (3) and Pseudomonas putida (4). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ⌬minCDE mutant (5). We suggested that formation of CL domains at cell pole/division sites plays an important role in selection and recognition of the division site by amphitropic cell cycle and cell division proteins, such as DnaA (initiation of DNA replication at oriC), MinD (a part of MinCDE system preventing positioning of the divisome at cell poles in E. coli), and FtsA (bacterial actin, which is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin), responsible for targeting the Z-ring to the mid-cell membrane domain). They interact directly with membrane phospholipids through specific amphipathic motifs enriched in basic amino acids, which confers the preference for anionic lipids (for references see Ref. 1). In E. coli the ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP bound to MinD resulting in release of MinD, and consequently MinC, from the membrane into the cytoplasm. As a result, all three proteins oscillate between the cell poles maintaining the maximum concentration of the inhibitor MinC at the cell p...

Quantitative analysis of N‐acylphosphatidylethanolamine molecular species in rat brain using solid‐phase extraction combined with reversed‐phase chromatography and tandem mass spectrometry

“…1A), similarly to N ‐acylphosphatidylserine 42. While other approaches to NAPE determination rely on either full scan, SIM, or a single SRM transition per analyte 6, 7, 25, 26, 27, 28, 29, 30, 31, our method uses these two N ‐acyl specific transitions for each analyte, leading to a more selective identification and minimizing the risk of false positive results, as shown in Supporting Information Fig. S1.…”

Focal targeting by human β-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis

“…Furthermore, it is established that secretion proceeds more efficiently in the presence of anionic lipids (17). Numerous bacteria, including E. faecalis, possess anionic lipid microdomains in their membranes, as visualized by incorporation of the fluorescent dye NAO (18,(36)(37)(38). Anionic lipids are specifically enriched at focal sites of secretion in S. pyogenes (18), and depletion of anionic lipids results in the mislocalization of translocon foci in B. subtilis (19).…”

Putative N-Acylphosphatidylethanolamine Synthase from Arabidopsis thaliana Is a Lysoglycerophospholipid Acyltransferase

“…3Ϫ (8500 Ci/mmol, 10 mCi/ml) and palmitoyl- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]CoA (60 mCi/mmol, 0.02 mCi/ml) were from PerkinElmer Life Sciences. Palmitoyl-CoA and arachidonyl-CoA, porcine pancreas phospholipase A 2 , and Streptomyces chromofuscus phospholipase D were from Sigma.…”

“…This overnight was used to start a 10-ml culture in LB-amp containing 100 Ci of [ 32 P]PO 4 . This culture, as well as an unlabeled culture for monitoring growth, was incubated at 37°C.…”

“…The minor product ion at m/z 426.242 corresponds to N-arachidonylphosphoethanolamine (Fig. 8F, inset) (4). This may indicate that the in vitro product is N-acylated; however, negative ion MS/MS analysis of 16:0, 18:1 PE standard also yielded a similar minor product ion (supplemental Fig.…”