ntrB基因对Agrobacterium sp.ATCC31749合成胞外多糖的调控研究

利用三亲本接合方法将含有ntrB基因两端同源序列的自杀载体pJQ-ntrB-cat导入土壤杆菌(Agrobacterium sp.ATCC 31749)中,获得了ntrB基因突变株.结果表明,ntrB突变株对NH4Cl和KNO3的利用能力有所下降.当分别以谷氨酸和谷氨酰胺为氮源时,ntrB突变株生长状况与野生菌相同.n...     

Characterization of Pyoverdin(pss), the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin(pss), in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin(pss) of approximately 50 mug/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of >/= 10 muM. Pyoverdin(pss) was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin(pss) and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin(pss) was structurally unique since amino acid analysis of reductive hydrolysates yielded beta-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin(pss) exhibited a relatively high affinity constant for Fe(III), with values of 10 at pH 7.0 and 10 at pH 10.0. Iron uptake assays with [Fe]pyoverdin(pss) demonstrated rapid active uptake of Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin(pss). The chemical and biological properties of pyoverdin(pss) are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Phosphatidylethanolamine is not essential for growth of Sinorhizobium meliloti on complex culture media

In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE), Sinorhizobium meliloti also possesses phosphatidylcholine (PC) as a major membrane lipid. The biosynthesis of PC in S. meliloti can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly. Therefore, for S. meliloti, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis. In this study, we investigated the first step of PE biosynthesis in S. meliloti catalyzed by phosphatidylserine synthase (PssA). A sinorhizobial mutant lacking PE was complemented with an S. meliloti gene bank, and the complementing DNA was sequenced. The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases. Inactivation of the sinorhizobial pssA gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type. A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions. On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation. Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype.     

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.     

Topological characterization of an inner membrane (1-->3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749

The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of beta-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31-41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of approximately 60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of approximately 420 kDa and approximately 500 kDa.     

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing Claviceps purpurea strains

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.     

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.     

The major phospholipid of Escherichia coli, phosphatidylethanolamine, is required for efficient production and secretion of alkaline phosphatase

The major phospholipid of the Escherichia coli membranes--the zwitterion phosphatidylethanolamine (PE)--is the only phospholipid involved in the formation of non-bilayer structure of membrane lipids, which is supposed to be necessary for efficient translocation of secreted proteins across the cytoplasmic membrane. The effect of PE on the production and secretion of alkaline phosphatase has been studied in this work using the mutant strain E. coli AD93, which is unable to synthesize PE. It was shown that this phospholipid is required for the efficient production and secretion of alkaline phosphatase. The anionic phospholipid cardiolipin in combination with divalent cations Mg2+ functionally replaces PE in these processes, participating in the regulation of lipid polymorphism.     

Effects of dietary eritadenine on the liver microsomal Delta6-desaturase activity and its mRNA in rats

Eritadenine, a hypocholesterolemic factor of Lentinus edodes mushroom, has a wide range of effects on lipid metabolism such as an increase in the liver microsomal phosphatidylethanolamine (PE) concentration, a decrease in the liver microsomal Delta6-desaturase activity, and an alteration of the fatty acid and molecular species profile of liver and plasma lipids. In this study, the time-dependent effects of dietary eritadenine on several variables concerning lipid metabolism were investigated in rats to clarify the sequence of metabolic changes caused by eritadenine, with special interest in the association of the liver microsomal phospholipid profile and the activity of Delta6-desaturase. The effect of dietary eritadenine on the abundance of mRNA for Delta6-desaturase was also investigated. When the time required for a half-change of variables was estimated during the first 5 days after the change from the control diet to the eritadenine-supplemented (50 mg/kg) diet, the change rates of the variables were fastest in the following order: alteration of the liver microsomal phospholipid profile>decrease in liver microsomal Delta6-desaturase activity>alteration of the fatty acid and molecular species profiles of microsomal and plasma phosphatidylcholine (PC)>decrease in the plasma cholesterol concentration. There was a significant correlation between the Delta6-desaturase activity and liver microsomal PE concentration, but not PC concentration, or the proportion of PC and PE or the PC/PE ratio. The suppression of Delta6-desaturase activity by dietary eritadenine was accompanied by a significant reduction in the abundance of mRNA for the enzyme. These results suggest that dietary eritadenine might suppress the activity of liver microsomal Delta6-desaturase by altering the microsomal phospholipid profile, as represented by an increase in PE concentration, and that the effect of eritadenine is mediated by the regulation of gene expression.     

Docosahexaenoic acid-containing phosphatidylethanolamine in the external layer of liposomes protects docosahexaenoic acid from 2,2'-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation

We have proposed that incorporation of docosahexaenoic acid (DHA) into phosphatidylethanolamine (PE) might enhance resistance to lipid peroxidation in vivo. In this study, we examined the relationship between the transbilayer distribution of PE and the oxidative stability of DHA in PE. Liposomes composed of a phospholipid mixture were used as models for biological membranes. To modulate the transbilayer distribution of PE obtained from the liver of rats fed DHA (PE-DHA), we used phosphatidylcholine (PC) with two types of acyl chain region: dipalmitoyl (PC16:0) or dioleoyl (PC18:1). The proportion of PE-DHA in the liposomal external layer was significantly higher in liposomes containing PC18:1 than in those containing PC16:0. This tendency was more pronounced in liposomes extruded using a polycarbonate filter with smaller pore sizes. Additionally, PE-DHA in the external layer of liposomes prepared using a filter with smaller pore sizes could protect DHA itself from 2,2(')-azobis(2-aminopropane)dihydrochloride-mediated lipid peroxidation.     

Cardiolipin Accumulation in the Inner and Outer Membranes of Escherichia coli Mutants Defective in Phosphatidylserine Synthetase

Mutants of Escherichia coli defective in phosphatidylserine synthetase (pss) make less phosphatidylethanolamine than normal cells, and they are temperature sensitive for growth. We have isolated a new mutant, designated RA2021, which is better than previously available strains in that the residual phosphatidylethanolamine level approaches 25% after 4 h at 42 degrees C. The total amount of phospholipid normalized to the density of the culture is about the same in RA2021 (pss-21) as in the isogenic wild-type RA2000 (pss(+)). Consequently, there is a net accumulation of polyglycerophosphatides in the mutant, particularly of cardiolipin. The addition of 10 to 20 mM MgCl(2) to a culture of RA2021 prolongs growth under nonpermissive conditions and prevents loss of cell viability, but it does not eliminate the temperature-sensitive phenotype. Divalent cations, like Mg(2+), do not correct the phospholipid composition of the mutant, but may act indirectly by balancing the negative charges of phosphatidylglycerol and cardiolipin. To determine the effects of the pss mutation on membrane composition, we have examined the subcellular distribution of the polyglycerophosphatides that accumulate in these strains. All of the excess anionic lipids of RA2021 are associated with the envelope fraction and are distributed equally between the inner and outer membranes. The protein compositions of the isolated membranes do not differ significantly in the mutant and wild type. The fatty acid composition of RA2021 is almost the same as wild type at 30 degrees C, but there is more palmitic and cyclopropane fatty acid at 42 degrees C. These results demonstrate that the modification of the polar lipid composition observed in pss mutants affects both membranes and that cardiolipin, which is not ordinarily present in large quantities, can accumulate in the outer membrane when it is overproduced by the cell. The altered polar headgroup composition of the outer membrane in pss mutants may account, in part, for their hypersensitivity to the aminoglycoside antibiotics.     

Genome sequence of the curdlan-producing Agrobacterium sp. strain ATCC 31749

Agrobacterium sp. ATCC 31749 is an industrial strain for the commercial production of curdlan, an important exopolysaccharide with food and medical applications. Here we report the genome sequence of the curdlan-producing strain ATCC 31749. Genome sequencing is the first step toward the understanding of regulation of curdlan biosynthesis.     

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.     

Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)

Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans. In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells.     

Sequential synthesis and methylation of phosphatidylethanolamine promote lipid droplet biosynthesis and stability in tissue culture and in vivo

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase

Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway. This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype. An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype. Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC. Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases. S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline. Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA. In accordance with this the gene has been named AtNMT1. The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability

Phosphatidylethanolamine is the only zwitterionic phospholipid in Escherichia coli and accounts for 70-80% of the total glycerophospholipids of this organism. To investigate the function of phosphatidylethanolamine in E. coli, we constructed an inactivated allele (pss93::kan) of the gene encoding the phosphatidylserine synthase which catalyzes the committed step to the synthesis of phosphatidylethanolamine. Growth of this mutant was dependent on a plasmid-borne copy of the wild type gene. After curing the mutant of the wild type gene, growth stopped when the content of phosphatidylethanolamine reached 30% of the total phospholipid. Divalent metal ions at millimolar concentrations suppressed the growth phenotype of the mutant in the following order of efficiency: Ca2+ greater than Mg2+ greater than Sr2+. Although phosphatidylserine synthase activity was not detectable, phosphatidylethanolamine was still present at 0.007% of the total phospholipid after growth for many generations in rich medium containing 20 mM Mg2+. The remainder of the phospholipid was primarily phosphatidylglycerol and cardiolipin with no other unique phosphate-containing chloroform-soluble material present. The phospholipid to protein ratio and the fatty acid composition were very similar to the parental strain. The broad divalent metal ion auxotrophy brought about by the lack of phosphatidylethanolamine suggests a primarily structural role for this phospholipid in E. coli.