NORMAL GENOMIC

• Parsons JB et al.
• Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.
• Proc Natl Acad Sci U S A. 2014; 111: 10532-7
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• Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The DeltafakA and DeltafakB1 DeltafakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the DeltafakA strain. The severe deficiency in alpha-hemolysin protein secretion in DeltafakA and DeltafakB1 DeltafakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.

• Camus G et al.
• Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core.
• J Biol Chem. 2013; 288: 9915-23
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• The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy.

• Parsons JB, Frank MW, Subramanian C, Saenkham P, Rock CO
• Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors.
• Proc Natl Acad Sci U S A. 2011; 108: 15378-83
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• The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target for antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular fatty acids. The regulation of FASII by extracellular fatty acids was examined in Staphylococcus aureus and Streptococcus pneumoniae, representing two important groups of pathogens. Both bacteria use the same enzymatic tool kit for the conversion of extracellular fatty acids to acyl-acyl carrier protein, elongation, and incorporation into phospholipids. Exogenous fatty acids completely replace the endogenous fatty acids in S. pneumoniae but support only 50% of phospholipid synthesis in S. aureus. Fatty acids overcame FASII inhibition in S. pneumoniae but not in S. aureus. Extracellular fatty acids strongly suppress malonyl-CoA levels in S. pneumoniae but not in S. aureus, showing a feedback regulatory system in S. pneumoniae that is absent in S. aureus. Fatty acids overcame either a biochemical or a genetic block at acetyl-CoA carboxylase (ACC) in S. aureus, confirming that regulation at the ACC step is the key difference between these two species. Bacteria that possess a stringent biochemical feedback inhibition of ACC and malonyl-CoA formation triggered by environmental fatty acids are able to circumvent FASII inhibition. However, if exogenous fatty acids do not suppress malonyl-CoA formation, FASII inhibitors remain effective in the presence of fatty acid supplements.

• Ahlawat S, Morrison DA
• ClpXP degrades SsrA-tagged proteins in Streptococcus pneumoniae.
• J Bacteriol. 2009; 191: 2894-8
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• Bacterial proteins that are abnormally truncated due to incomplete mRNA or the presence of rare codons are extended by an SsrA tag during ribosome rescue in a trans-translation process important for maintaining protein quality. In Escherichia coli, the SsrA-tagged proteins become the target of the Tsp, Lon, FtsH, ClpXP, and ClpAP proteases. Here we show that degradation of model SsrA-tagged proteins in Streptococcus pneumoniae depends primarily or exclusively on ClpXP in vivo. In addition, we show the E. coli SsrA tag is also a target of S. pneumoniae ClpXP in vivo, even though the N-terminal portions of the tags differ significantly between the two species, suggesting there may be no adaptor protein for SsrA in S. pneumoniae.

• Kim Y, Li H, Binkowski TA, Holzle D, Joachimiak A
• Crystal structure of fatty acid/phospholipid synthesis protein PlsX from Enterococcus faecalis.
• J Struct Funct Genomics. 2009; 10: 157-63
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• PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids. The plsX gene is co-localized with a bacterial fab gene cluster which encodes several key fatty acid biosynthetic enzymes. The protein is a member of a large, conserved protein family (Pfam02504) found exclusively in bacteria. The PlsX sequence homologues include both phosphate acetyltransferases and phosphate butaryltransferases that catalyze the transfer of an acetyl or butaryl group to orthophosphate. We have determined the crystal structure of PlsX from the human pathogen Enterococcus faecalis. PlsX is a alpha/beta/alpha sandwich that resembles a Rossmann fold and forms a dimer. A putative catalytic site has been identified within a deep groove on the interface between monomers. This site showed strong surface similarity to epimerases and reductases. It was recently proposed that PlsX is a phosphate acyltransferase that catalyzes the formation of acyl-phosphate from the acyl-acyl carrier protein; however the specific biochemical function of the PlsX protein awaits further experimental scrutiny.

• Kuruma Y, Stano P, Ueda T, Luisi PL
• A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells.
• Biochim Biophys Acta. 2009; 1788: 567-74
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• Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity--inside liposomes--of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within - which corresponds to the autopoietic growth mechanism.

• Ghosh AK, Ramakrishnan G, Rajasekharan R
• YLR099C (ICT1) encodes a soluble Acyl-CoA-dependent lysophosphatidic acid acyltransferase responsible for enhanced phospholipid synthesis on organic solvent stress in Saccharomyces cerevisiae.
• J Biol Chem. 2008; 283: 9768-75
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• One of the major determinants of organic solvent tolerance is the increase in membrane phospholipids. Here we report for the first time that an increase in the synthesis of phosphatidic acid is responsible for enhanced phospholipid synthesis that confers tolerance to the organic solvent in Saccharomyces cerevisiae. This increase in phosphatidic acid formation is because of the induction of Ict1p, a soluble oleoyl-CoA:lysophosphatidic acid acyltransferase. YLR099C (ICT1) was reported to be maximally expressed during solvent tolerance (Miura, S., Zou, W., Ueda, M., and Tanaka, A. (2000) Appl. Environ. Microbiol. 66, 4883-4889); however, its physiological significance was not understood. In silico analysis revealed the absence of any transmembrane domain in Ict1p. Domain analysis showed that it has a hydrolase/acyltransferase domain with a distinct lipid-binding motif and a lysophospholipase domain. Analysis of ict1Delta strain showed a drastic reduction in phosphatidic acid suggesting the role of Ict1p in phosphatidic acid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in phosphatidic acid and other phospholipids on organic solvent exposure. To understand the biochemical function of Ict1p, the gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was found to specifically acylate lysophosphatidic acid. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared with palmitoyl- and stearoyl-CoAs. This study provides a mechanism for organic solvent tolerance from the point of membrane dynamics in S. cerevisiae.

• Schujman GE, de Mendoza D
• Regulation of type II fatty acid synthase in Gram-positive bacteria.
• Curr Opin Microbiol. 2008; 11: 148-52
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• Bacterial cells stringently regulate the synthesis of their membrane phospholipids but the responsible mechanisms are incompletely understood. Recent biochemical, genetic and structural analyses have greatly expanded the knowledge of lipid metabolism in Gram-positive bacteria, revealing that these organisms use novel mechanisms to regulate this essential pathway. A remarkable progress was the identification of a new pathway for the initiation of phospholipid biosynthesis that uncovered a mechanism that coordinates fatty acid and phospholipid biosynthesis. Recent advances in structure determination of a global transcription factor have led to significant insights of the underlying complexities and functional elegance of membrane lipid homeostasis in Gram-positive bacteria.

• Ohta Y, Kondo Y, Kawada K, Teranaka T, Yoshino N
• Synthesis and antibacterial activity of quaternary ammonium salt-type antibacterial agents with a phosphate group.
• J Oleo Sci. 2008; 57: 445-52
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• Quaternary ammonium salts are frequently used as antibacterial agent that disrupts cell membrane through the binding of their ammonium cations to anionic sites in the outer layer tissue of bacteria. This article describes the synthesis of quaternary ammonium salt-type antibacterial agents with a phosphate group that strongly binds to hydroxyapatite and bromide ion as counterion. Evaluation of the antibacterial activity of the synthesized compounds in terms of minimum inhibitory concentration (MIC test) showed that the compounds exhibit an excellent antibacterial activity on a variety of bacteria including Gram-positive and Gram-negative bacteria, yeast, and fungi.

• Soupene E, Fyrst H, Kuypers FA
• Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.
• Proc Natl Acad Sci U S A. 2008; 105: 88-93
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• The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.

• Hara Y, Seki M, Matsuoka S, Hara H, Yamashita A, Matsumoto K
• Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.
• Genes Genet Syst. 2008; 83: 433-42
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• The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

• Aygun-Sunar S, Bilaloglu R, Goldfine H, Daldal F
• Rhodobacter capsulatus OlsA is a bifunctional enzyme active in both ornithine lipid and phosphatidic acid biosynthesis.
• J Bacteriol. 2007; 189: 8564-74
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• The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

• Oursel D, Loutelier-Bourhis C, Orange N, Chevalier S, Norris V, Lange CM
• Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization.
• Rapid Commun Mass Spectrom. 2007; 21: 1721-8
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• A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.

• Oxley A, Jutfelt F, Sundell K, Olsen RE
• Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine.
• Comp Biochem Physiol B Biochem Mol Biol. 2007; 146: 115-23
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• Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.

• Schujman GE, de Mendoza D
• Solving an old puzzle in phospholipid biosynthesis.
• Nat Chem Biol. 2006; 2: 573-4

• Rodionov DG, Pisabarro AG, de Pedro MA, Kusser W, Ishiguro EE
• Beta-lactam-induced bacteriolysis of amino acid-deprived Escherichia coli is dependent on phospholipid synthesis.
• J Bacteriol. 1995; 177: 992-7
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• The penicillin tolerance of amino acid-deprived relA+ Escherichia coli is attributed to the stringent response; i.e., relaxation of the stringent response suppresses penicillin tolerance. The beta-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic sn-glycerol 3-phosphate dehydrogenase). Therefore, beta-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. The lysis process during amino acid deprivation can be experimentally dissociated into two stages designated the priming stage (during which the interaction between the beta-lactam and the penicillin-binding proteins occurs) and the beta-lactam-independent lysis induction stage. Both stages were shown to require phospholipid synthesis. It has been known for some time that the inhibition of phospholipid synthesis is among the plethora of physiological changes resulting from the stringent response. These results indicate that the inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response are both secondary consequences of the inhibition of phospholipid synthesis.

• Cooper CL, Jackowski S, Rock CO
• Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.
• J Bacteriol. 1987; 169: 605-11
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• Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.

• Rampini C, Dubois C, Barbu V
• Phosphate depletion decrease mitogen-mediated stimulation of phospholipid synthesis in human peripheral lymphocytes.
• Biochem Biophys Res Commun. 1984; 118: 371-7
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• Concanavalin A-mediated stimulation of 32P-phosphate incorporation into phospholipids of human peripheral lymphocytes is comparatively studied in normal and phosphate-depleted media. In the phosphate-depleted medium, 2 hours after the start of cell activation, the stimulation sharply decreases for phosphatidylinositol (6.5-fold) and for phosphatidylcholine (in the latter case, the stimulation is even replaced by a slight inhibition of the incorporation). These results must be related to the rate-limiting effect of inorganic phosphate on ATP formation and thus on phospholipid synthesis, an effect which may be particularly pronounced when there is both phosphate depletion and cell activation.

• Rock CO
• Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli.
• J Biol Chem. 1984; 259: 6188-94
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• Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.

• Green PR, Merrill AH Jr, Bell RM
• Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase.
• J Biol Chem. 1981; 256: 11151-9

• Herrmann H, Gercken G
• Synthesis of phospholipids in Leishmania donovani.
• Hoppe Seylers Z Physiol Chem. 1980; 361: 1735-42

• Bauza MT, De Loach JR, Aguanno JJ, Larrabee AR
• Acyl carrier protein prosthetic group exchange and phospholipid synthesis in synchronized cultures of a pantothenate auxotroph Escherichia coli.
• Arch Biochem Biophys. 1976; 174: 344-9

• Merlie JP, Pizer LI
• Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate.
• J Bacteriol. 1973; 116: 355-66
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• Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with (32)P-inorganic phosphate and (32)P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.

• Lapidot Y, Barzilay I, Hajdu J
• The synthesis of diacyl-dl-(and-L-) alpha-glycerol phosphate.
• Chem Phys Lipids. 1969; 3: 125-34

• Prottey C, Hawthorne JN
• The biosynthesis of phosphatidic acid and phosphatidylinositol in mammalian pancreas.
• Biochem J. 1967; 105: 379-92
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• 1. The labelling of guinea-pig pancreas phospholipids in vivo after intraperitoneal injection of [(32)P]orthophosphate is described. 2. Acyl-CoA synthetase activity in pancreas homogenates has been studied. There is no absolute requirement for added fatty acids, indicating an adequate supply of endogenous fatty acids in these preparations. 3. Phosphatidic acid is formed in guinea-pig pancreas preparations by two distinct routes, namely the acylation of l-3-glycerophosphate and the phosphorylation of 1,2-diglyceride. Phosphatidic acid formed by either mechanism is converted into phosphatidylinositol by guinea-pig pancreas in vitro. 4. The enzymes of pancreas that convert phosphatidic acid into phosphatidylinositol via CDP-diglyceride have been characterized. 5. Addition of bovine serum albumin is necessary in assaying certain of these enzymes.

• GRISOLIA S, AMELUNXEN R, RAIJMAN L
• Acetyl and carbamyl phosphate utilization with asparatate transcarbamylase kinase.
• Biochem Biophys Res Commun. 1963; 11: 75-8

• KANFER JN, KENNEDY EP
• Synthesis of phosphatidylserine by Escherichia coli.
• J Biol Chem. 1962; 237: 270271-270271

• NIKAIDO H
• Phospholipid as a possible component of carrier system in beta-galactoside permease of Escherichia coli.
• Biochem Biophys Res Commun. 1962; 9: 486-92

• TARDIEUX-ROCHE A
• [Attempts at a quantitative evaluation of the dissolving capacity of bacteria for natural phosphates].
• Ann Inst Pasteur (Paris). 1962; 103: 314-7

• NISHI A
• Role of polyphosphate and phospholipid in germinating spores of Aspergillus niger.
• J Bacteriol. 1961; 81: 10-9

• CHOJNACKI T
• [Biosynthesis of phospholipids in insects. II. Studies on the incorporation of orthophosphate-P32 in the moth Celerio euphorbiae].
• Acta Biochim Pol. 1961; 8: 167-75

• BAER E, CIPLIJAUSKAS LJ, VISSER T
• Synthesis of vinyl phosphate, a possible intermediate in carbohydrate metabolism and photosynthesis.
• J Biol Chem. 1959; 234: 1-4

• DAVIS BD, KALAN EB, SPRINSON DB, SRINIVASAN PR
• The enzymatic conversion of sedoheptulose-1, 7-diphosphate to shikimic acid.
• J Biol Chem. 1956; 223: 913-20

• HOKIN LE, HOKIN MR
• Effects of acetylcholine on phosphate turnover in phospholipides of brain cortex in vitro.
• Biochim Biophys Acta. 1955; 16: 229-37

• MITCHELL P, MOYLE J
• The Gram reaction and cell composition: nucleic acids and other phosphate fractions.
• J Gen Microbiol. 1954; 10: 533-40

• TOLBERT ME, OKEY R
• The relative rates of renewal of choline and phosphate in liver phospholipide in the rat.
• J Biol Chem. 1952; 194: 755-68

• GLOCK GE
• Formation and breakdown of pentose phosphates by liver fractions.
• Nature. 1952; 170: 162-3

• KING TE, NEAL AL, STRONG FM
• Synthesis of pantolactone phosphate and its effect on growth of microorganisms.
• J Biol Chem. 1951; 189: 307-14