Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase. We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.     

Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase.

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.     

Inactivation of the gene for phospholipid N-methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth

In phosphatidylcholine (PC)-containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline-dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP-diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N-methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S-adenosylmethionine-dependent N-methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt-deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt-deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild-type-like growth. Double knock-out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.     

Cloning and characterization of the gene for phosphatidylcholine synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.     

Brucella abortus synthesizes phosphatidylcholine from choline provided by the host

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-beta-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process.     

Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions

Membrane lipids in most bacteria generally consist of the glycerophospholipids phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE). A subset of bacteria also possesses the methylated derivatives of PE, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine (PC). In Sinorhizobium meliloti, which can form a nitrogen-fixing root nodule symbiosis with Medicago spp., PC can be formed by two entirely different biosynthetic pathways, either the PE methylation pathway or the recently discovered PC synthase pathway. In the latter pathway, one of the building blocks for PC formation, choline, is obtained from the eukaryotic host. Under phosphorus-limiting conditions of growth, S. meliloti replaces its membrane phospholipids by membrane-forming lipids that do not contain phosphorus; namely, the sulfolipid sulfoquinovosyl diacylglycerol, ornithine-derived lipids, and diacylglyceryl-N,N,N-trimethylhomoserine. Although none of these phosphorus-free lipids is essential for growth in culture media rich in phosphorus or for the symbiotic interaction with the legume host, they are expected to have major roles under free-living conditions in environments poor in accessible phosphorus. In contrast, sinorhizobial mutants deficient in PC show severe growth defects and are completely unable to form nodules on their host plants. Even bradyrhizobial mutants with reduced PC biosynthesis can form only root nodules displaying reduced rates of nitrogen fixation. Therefore, in the cases of these microsymbionts, the ability to form sufficient bacterial PC is crucial for a successful interplay with their host plants.     

Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway

Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes. Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt). Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline. The CDP-choline then condenses with diacylglycerol (DAG) to form PC. In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs). PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in Sinorhizobium meliloti was characterized. The completed release of the Pseudomonas aeruginosa PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from Rhodobacter sphaeroides. Moreover, the P. aeruginosa PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of S. meliloti. Both the pcs and pmtA homologues were cloned from PAO1, and homologous sequences were found in almost all of the P. aeruginosa strains examined. Although the pathway for synthesizing PC by use of Pcs is functional in P. aeruginosa, it does not appear that this organism uses the PmtA pathway for PC synthesis. We demonstrate that the PC synthesized by P. aeruginosa PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects

Biochemical studies in the human malaria parasite, Plasmodium falciparum, indicated that in addition to the pathway for synthesis of phosphatidylcholine from choline (CDP-choline pathway), the parasite synthesizes this major membrane phospholipid via an alternative pathway named the serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway using host serine and ethanolamine as precursors. However, the role the transmethylation of phosphatidylethanolamine plays in the biosynthesis of phosphatidylcholine and the importance of the SDPM pathway in the parasite's growth and survival remain unknown. Here, we provide genetic evidence that knock-out of the PfPMT gene encoding the phosphoethanolamine methyltransferase enzyme completely abrogates the biosynthesis of phosphatidylcholine via the SDPM pathway. Lipid analysis in knock-out parasites revealed that unlike in mammalian and yeast cells, methylation of phosphatidylethanolamine to phosphatidylcholine does not occur in P. falciparum, thus making the SDPM and CDP-choline pathways the only routes for phosphatidylcholine biosynthesis in this organism. Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites.     

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3-3H]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of L-[3-3H]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2-14C]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.     

Multiple phospholipid N-methyltransferases with distinct substrate specificities are encoded in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.     

Disruption of choline methyl group donation for phosphatidylethanolamine methylation in hepatocarcinoma cells

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.     

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.     

Comparative study of the effects of short- and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes

This study describes the effects of short- and long-term ethanol treatment and withdrawal on the biosynthesis of the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in hepatocytes isolated from rats, using isotopically labelled choline and ethanolamine as exogenous precursors. Our results demonstrate that short-term ethanol consumption increases the incorporation of exogenous polar bases into PC and PE, whereas long-term ethanol administration provokes a differential effect in both PC and PE biosynthesis via cytidine diphosphate derivatives (CDP-derivatives), decreasing PC synthesis and increasing the biosynthesis of PE. We suggest that the increased biosynthesis of PE after ethanol treatment results from changes in lipogenic substrates produced as a consequence of ethanol metabolism, whilst the specific inhibition of PC biosynthesis seems to be a consequence of alterations of enzymes involved in the CDP-choline pathway. With regard to the influence of ethanol on PE methylation to give PC, our results demonstrate that ethanol activates this pathway in short-term, as well as chronic ethanol treatment. Ethanol withdrawal returns the activity of the PC and PE pathways to control levels. The alterations in the biosynthesis of the main phospholipids, PC and PE, demonstrated in this study could be of a great physiological interest in determining the pathology of alcoholism.     

Choline Deficiency in Cultured Adrenal Medullary Cells: Effect on Phosphatidylcholine Biosynthesis

The effect of choline deficiency on the composition and biosynthesis of the major membrane phospholipids was examined in adrenal medullary cells maintained in suspension cultures. The amount and proportions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in these cells were not affected by the removal of choline from the culture media. However, the rate of biosynthesis of choline at the phosphatide level by the stepwise methylation of PE increased twofold within 24 h after choline was removed from the culture media, while ethanolamine incorporation into PE was increased by 50%. In contrast, the rate of incorporation of labeled choline into PC, presumably via CDP-choline, was virtually identical in cells that had been preincubated in the presence or absence of 1 mM choline. These results demonstrate that cultured cells of neural origin are capable of compensating for lack of exogenous choline by forming choline at the phosphatide level through the sequential methylation of PE. The hypolipidemic drug, DH-990, when added to the culture media, inhibited conversion of phosphatidylmonomethylethanolamine (PME) to PC, but had no effect on the N-methylation of PE. This differential effect indicates that the initial N-methylation of PE is catalyzed by an enzyme that is distinguishable from the enzyme(s) catalyzing the conversion of PME to PC.     

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)‐choline to diacylglycerol. Phosphocholine, the direct precursor to CDP‐choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [³²P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [³²P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.     

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.