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 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.

FIGURE 1.

UE54 lacking CL and PG undergoes relatively normal cell division. A, UE54 cells are shorter then parental strains (UE53 and MG1665) with wild-type phospholipids composition. The cell length distribution is shown. B, mid-cell FtsZ-GFP localization in the UEM542 mutant cells lacking PG and CL; fluorescence (top) and DIC images (bottom). C, GFP-MinD oscillation in the UEM541 mutant cells lacking PG and CL. Time-lapse images were taken at ∼6- to 8-s intervals.

FIGURE 2.

Anionic lipid membrane domains in the UE54 cells lacking PG and CL as revealed by NAO staining. A, fluorescence microscopy of liposomes prepared from CL (left) and PA (right) and stained with NAO; green (top) and red (bottom) fluorescence images. B and C, UE54 cells stained with anionic phospholipid specific dye NAO; B, UE54 cell pole and division site green fluorescence; C, several individual cells. Images were processed in Photoshop and converted to a grayscale with inversion resulting in the black contour-white background images. D, UE54 cells stained with lipophilic dye FM4-64. Staining procedures were performed as descried under “Experimental Procedures.” Images were processed as described in C; E, UE53 cells stained with 100 nm NAO; green fluorescence was recorded.

FIGURE 3.

Negative-ion ESI-MS of lipid extracts from MG1655 and UE54. The mass spectra were obtained as described under “Experimental Procedures.” A, MG1655; B, UE54.

FIGURE 4.

Reversed-phase LC negative-ion ESI-MS of UE54 and MG1655 lipid extracts reveals novel ions in the m/z 900–1000 range. The mass spectrum of the material eluting between min 9.0 and 11.0 for UE54 (A) and MG1655 (B).

FIGURE 5.

Negative-ion MS/MS analysis of [M-H]^- ion at m/z 980. 83 and proposed structures. A, MS/MS analysis of m/z 980.83. B, expansion of spectrum from 350 to 480 atomic mass units. C, proposed structures of N-acyl-PE for the [M-H]^- species at m/z 980.83. Although the acyl chain positions can vary, the acyl chains contain a total of 52 carbons with 2 unsaturations. D, negative-ion MS/MS analysis of synthetic N-acyl-PE. The inset shows the structure and proposed fragmentation pattern.

FIGURE 6.

Quantification of N-acyl-PE species in MG1655 and UE54 lipid extracts. The peak area of the extracted ion current for an individual N-acyl-PE species was normalized to the peak area of the extracted ion current for the 55:2 N-acyl-PE synthetic standard. This ratio is used to calculate the picomoles of each N-acyl-PE per mg of protein. Error bars represent deviation among three separate experiments.

FIGURE 7.

The minicells relative PE to parent cells UE543 are enriched in anionic phospholipids, PA and N-acyl-PE. A, DIC images of UE534 minicell-producing cells (on the left) and fluorescent images of DAPI-stained nucleoids of the same cells (in the middle). Division of UE534 at cell poles (indicated by arrows) resulted in the production of anucleated minicells and nucleoid containing short filaments; right image, DIC, purified minicells after the sixth sucrose gradient. B, comparison of the ratios of PA species to total PE in the minicells and large cells. C, comparison of the ratios of N-acyl-PE species to total PE in the minicells and large cells. The peak area of the extracted ion current for an individual PA, PE, or N-acyl-PE species was normalized to the peak area of the extracted ion current of the corresponding standard (12:0, 13:0 PA, 12:0, 13:0 PE, 55:2 N-acyl-PE). The ratio for each PA (B) or N-acyl-PE (C) species was then divided by the sum of the ratios for all of the PE species to yield PA/total PE (B) or N-acyl-PE/total PE (C) ratios. Error bars represent deviation among three separate experiments.