Streptococcus pneumoniae, S. mitis, and S. oralis Produce a Phosphatidylglycerol-Dependent, ltaS-Independent Glycerophosphate-Linked Glycolipid.

Lipoteichoic acid (LTA) is a Gram-positive bacterial cell surface polymer that participates in host-microbe interactions. It was previously reported that the major human pathogen Streptococcus pneumoniae and the closely related oral commensals S. mitis and S. oralis produce type IV LTAs. Herein, using liquid chromatography/mass spectrometry-based lipidomic analysis, we found that in addition to type IV LTA biosynthetic precursors, S. mitis, S. oralis, and S. pneumoniae also produce glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a biosynthetic precursor of type I LTA. cdsA and pgsA mutants produce DHDAG but lack (Gro-P)-DHDAG, indicating that the Gro-P moiety is derived from phosphatidylglycerol (PG), whose biosynthesis requires these genes. S. mitis, but not S. pneumoniae or S. oralis, encodes an ortholog of the PG-dependent type I LTA synthase, ltaS By heterologous expression analyses, we confirmed that S. mitisltaS confers poly(Gro-P) synthesis in both Escherichia coli and Staphylococcus aureus and that S. mitisltaS can rescue the growth defect of an S. aureusltaS mutant. However, we do not detect a poly(Gro-P) polymer in S. mitis using an anti-type I LTA antibody. Moreover, Gro-P-linked DHDAG is still synthesized by an S. mitisltaS mutant, demonstrating that S. mitis LtaS does not catalyze Gro-P transfer to DHDAG. Finally, an S. mitisltaS mutant has increased sensitivity to human serum, demonstrating that ltaS confers a beneficial but currently undefined function in S. mitis Overall, our results demonstrate that S. mitis, S. pneumoniae, and S. oralis produce a Gro-P-linked glycolipid via a PG-dependent, ltaS-independent mechanism.IMPORTANCE The cell wall is a critical structural component of bacterial cells that confers important physiological functions. For pathogens, it is a site of host-pathogen interactions. In this work, we analyze the glycolipids synthesized by the mitis group streptococcal species, S. pneumoniae, S. oralis, and S. mitis We find that all produce the glycolipid, glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a precursor for the cell wall polymer type I lipoteichoic acid in other bacteria. We investigate whether the known enzyme for type I LTA synthesis, LtaS, plays a role in synthesizing this molecule in S. mitis Our results indicate that a novel mechanism is responsible. Our results are significant because they identify a novel feature of S. pneumoniae, S. oralis, and S. mitis glycolipid biology.

Phosphatidylglycerophosphate phosphatase is required for root growth in Arabidopsis.

Phosphatidylglycerol (PG) is an indispensable lipid class in photosynthetic activity. However, the importance of PG biosynthesis in non-photosynthetic organs remains elusive. We previously identified phosphatidylglycerophosphate phosphatase 1 (PGPP1), which catalyzes the last step of PG biosynthesis in Arabidopsis thaliana. In the present report, we noted considerably shorter roots of the pgpp1-1 mutant compared to the wild type. We observed defective order of columella cells in the root apices, which was complemented by introducing the wild-type PGPP1 gene. Although PGPP1 is chloroplast-localized in leaf mesophyll cells, we observed mitochondrial localization of PGPP1 in root cells, suggesting possible dual targeting of PGPP1. Moreover, we identified previously uncharacterized 2 protein tyrosine phosphatase-like proteins as functional PGPPs. These proteins, designated PTPMT1 and PTPMT2, complemented growth and lipid phenotypes of Δgep4, a Saccharomyces cerevisiae mutant of PGPP. The ptpmt1-1 ptpmt2-1 exhibited no visible phenotype; however, the pgpp1-1 ptpmt1-1 ptpmt2-1 significantly enhanced the root phenotype of pgpp1-1 without further affecting the photosynthesis, suggesting that these newly found PGPPs are involved in the root phenotype. Radiolabeling experiment of mutant roots showed that decreased PG biosynthesis is associated with the mutation of PGPP1. These results suggest that PG biosynthesis is required for the root growth.

The influence of mild acidity on lysyl-phosphatidylglycerol biosynthesis and lipid membrane physico-chemical properties in methicillin-resistant Staphylococcus aureus.

The increased biosynthesis of lysyl-phosphatidylglycerol in Staphylococcus aureus when cultured under conditions of mild acidity and the resultant increased proportion of this lipid in the plasma membrane of the bacterium, alters the physico-chemical properties of lipid bilayers in a manner which is itself dependent upon environmental pH. Clinically relevant strains of S. aureus, both methicillin susceptible and resistant, all exhibited increased lysyl-phosphatidylglycerol biosynthesis in response to mild environmental acidity, albeit to differing degrees, from ∼30% to ∼55% total phospholipid. Polar lipid extracts from these bacteria were analysed by 31P NMR and reconstituted into vesicles and monolayers, which were characterised by zeta potential measurements and Langmuir isotherms respectively. A combination of increased lysyl-phosphatidylglycerol content and mild environmental acidity were found to synergistically neutralise the charge of the membranes, in one instance altering the zeta potential from -56mV to +21mV, and induce closer packing between the lipids. Challenge of reconstituted S. aureus lipid model membranes by the antimicrobial peptide magainin 2 F5W was examined using monolayer subphase injection and neutron diffraction, and revealed that ionisation of the headgroup α-amine of lysyl-phosphatidylglycerol at pH 5.5, which reduced the magnitude of the peptide-lipid interaction by 80%, was more important for resisting peptide partitioning than increased lipid content alone. The significance of these results is discussed in relation to how colonising mildly acidic environments such as human mucosa may be facilitated by increased lysyl-phosphatidylglycerol biosynthesis and the implications of this for further biophysical analysis of the role of this lipid in bacterial membranes.

Bacterial aminoacyl phospholipids - Biosynthesis and role in basic cellular processes and pathogenicity.

The bacterial cell membrane accomplishes the controlled exchange of molecules with the extracellular space and mediates specific interactions with the environment. However, the cytoplasmic membrane also includes vulnerable targets for antimicrobial agents. A common feature of cationic antimicrobial peptides (CAMPs) produced by other bacteria or by the host immune system is to utilize the negative charge of bacterial phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) for initial adherence and subsequent penetration into the membrane bilayer. To resist cationic antimicrobials many bacteria integrate positive charges into the membrane surface. This is accomplished by aminoacylation of negatively charged (PG) or (CL) with alanine, arginine, or lysine residues. The Multiple Peptide Resistance Factor (MprF) of Staphylococcus aureus is the prototype of a highly conserved protein family of aminoacyl phosphatidylglycerol synthases (aaPGSs) which modify PG or CL with amino acids. MprF is an oligomerizing membrane protein responsible for both, synthesis of lysyl phosphatidylglycerol (LysPG) in the inner leaflet of the cytoplasmic membrane and translocation of LysPG to the outer leaflet. This review focuses on occurrence, synthesis and function of bacterial aminoacyl phospholipids (aaPLs) and on the role of such lipids in basic cellular processes and pathogenicity. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

In Vivo and in Vitro Synthesis of Phosphatidylglycerol by an Escherichia coli Cardiolipin Synthase.

Phosphatidylglycerol (PG) makes up 5-20% of the phospholipids of Escherichia coli and is essential for growth in wild-type cells. PG is synthesized from the dephosphorylation of its immediate precursor, phosphatidylglycerol phosphate (PGP) whose synthase in E. coli is PgsA. Using genetic, biochemical, and highly sensitive mass spectrometric approaches, we identified an alternative mechanism for PG synthesis in E. coli that is PgsA independent. The reaction of synthesis involves the conversion of phosphatidylethanolamine and glycerol into PG and is catalyzed by ClsB, a phospholipase D-type cardiolipin synthase. This enzymatic reaction is demonstrated herein both in vivo and in vitro as well as by using the purified ClsB protein. When the growth medium was supplemented with glycerol, the expression of E. coli ClsB significantly increased PG and cardiolipin levels, with the growth deficiency of pgsA null strain also being complemented under such conditions. Identification of this alternative mechanism for PG synthesis not only expands our knowledge of bacterial anionic phospholipid biosynthesis, but also sheds light on the biochemical functions of the cls gene redundancy in E. coli and other bacteria. Finally, the PGP-independent PG synthesis in E. coli may also have important implications for the understanding of PG biosynthesis in eukaryotes that remains incomplete.

Arabidopsis phosphatidylglycerophosphate phosphatase 1 involved in phosphatidylglycerol biosynthesis and photosynthetic function.

Phosphatidylglycerol (PG) is an indispensable lipid constituent of photosynthetic membranes, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. Here, we report the identification and characterization of AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth phenotype and PG-producing activity, suggesting that AtPGPP1 encodes a functional PGPP. The GUS reporter assay showed that AtPGPP1 was preferentially expressed in hypocotyl, vasculatures, trichomes, guard cells, and stigmas. A subcellular localization study with GFP reporter indicated that AtPGPP1 is mainly localized at chloroplasts. A T-DNA-tagged knockout mutant of AtPGPP1, designated pgpp1-1, showed pale green phenotype with reduced PG and chlorophyll contents but no defect in embryo development. In the pgpp1-1 mutant, ultrastructure of plastids indicated defective development of chloroplasts and measurement of photosynthetic parameters showed impaired photosynthetic activity. These results suggest that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.

Phosphatidylglycerol-derived phospholipids have a universal, domain-crossing role in stress responses.

Phosphatidylglycerol and phospholipids derived from it are widely distributed throughout the three domains of life. Cardiolipin is the best characterized of these phospholipids, and plays a key role in the response to environmental variations. Phosphatidylglycerol-derived phospholipids confer cell membranes with a wide range of responses, including changes in surface charge, fluidity, flexibility, morphology, biosynthesis and remodeling, that adapt the cell to these situations. Furthermore, the synthesis and remodeling of these phospholipids is finely regulated, highlighting the importance of these lipids in cell homeostasis and responses during stressful situations. In this article, we review the most important roles of these anionic phospholipids across domains, focusing on the biophysical basis by which these phospholipids are used in stress responses.

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine.

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.

Unconventional membrane lipid biosynthesis in Xanthomonas campestris.

All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N-methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non-canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis.

Isolation and characterization of a phosphatidylglycerophosphate phosphatase1, PGPP1, in Chlamydomonas reinhardtii.

Phosphatidylglycerol (PG) is the exclusive phospholipid synthesized in chloroplasts and plays important roles in photosynthesis. However, phosphatidylglycerophosphate phosphatase (PGPP), which catalyzes the final step of PG biosynthesis, is a missing piece in photosynthetic eukaryotes. Here, we isolated a previously uncharacterized haloacid dehalogenase-like phosphatase, designated CrPGPP1, as a putative PGPP in Chlamydomonas reinhardtii. CrPGPP1 complemented growth and lipid compositional defects in Δgep4, a yeast mutant of PGPP, which indicates that CrPGPP1 is a functional PGPP. Two aspartic acid residues, which are both essential for the yeast PGPP (Gep4p) activity, are also conserved in the putative catalytic motif of CrPGPP1. Site-specific mutagenesis showed that the first but not the second aspartic acid residue was required for CrPGPP1 to complement the growth defect of Δgep4 mutant, which highlights the distinct molecular features of CrPGPP1. Our results suggest that CrPGPP1 is a functional PGPP in C. reinhardtii, for the first PGPP in photosynthetic eukaryotes.

Preparation of primary cultures of mouse epidermal keratinocytes and the measurement of phospholipase D activity.

In this chapter information is provided about the outer layer of the skin, the epidermis, and the predominant cells comprising this epithelium, the keratinocytes. The evidence supporting a possible role for the lipid-metabolizing enzyme phospholipase D in regulating keratinocyte differentiation is also discussed. A detailed protocol for the preparation of primary cultures of epidermal keratinocytes from neonatal mice is described, to allow other investigators to obtain data concerning these important cells involved in forming and maintaining the mechanical and water permeability of the skin. Finally, a complete protocol for monitoring phospholipase D activity in intact cells is supplied in the hope that additional research will result in a better understanding of the role of phospholipase D in controlling keratinocyte proliferation and differentiation.

Phosphatidylglycerol biosynthesis is required for the development of embryos and normal membrane structures of chloroplasts and mitochondria in Arabidopsis.

Phosphatidylglycerophosphate (PGP) synthase, encoded by PGP1 and PGP2 in Arabidopsis, catalyzes a committed step in the biosynthesis of phosphatidylglycerol (PG). In this study, we isolated a pgp1pgp2 double mutant of Arabidopsis to study the function of PG. In this mutant, embryo development was delayed and the majority of seeds did not germinate. Thylakoid membranes did not develop in plastids, mitochondrial membrane structures were abnormal in the mutant embryos, and radiolabeling of phospholipids showed that radioactivity was not significantly incorporated into PG. These results demonstrated that PG biosynthesis is essential for the development of embryos and normal membrane structures of chloroplasts and mitochondria.

Formation of oxidized phosphatidylinositol and 12-oxo-phytodienoic acid containing acylated phosphatidylglycerol during the hypersensitive response in Arabidopsis.

Plant membranes are composed of a wide array of polar lipids. The functionality of these extends far beyond a pure structural role. Membrane lipids function as enzyme co-factors, establish organelle identity and as substrates for enzymes such as lipases and lipoxygenases. Enzymatic degradation or oxidation (enzymatic or non-enzymatic) of membrane lipids leads to the formation of a diverse group of bioactive compounds. Plant defense reactions provoked by pathogenic microorganisms are often associated with substantial modifications of the lipidome. In this study, we profiled changes in phospholipids during the hypersensitive response triggered by recognition of the bacterial effector protein AvrRpm1 in Arabidopsis thaliana. A simple and robust LC-MS based method for profiling plant lipids was designed to separate all the major species of glycerolipids extracted from Arabidopsis leaf tissue. The method efficiently separated several isobaric and near isobaric lipid species, which otherwise are difficult to quantify in direct infusion based profiling. In addition to the previously reported OPDA-containing galactolipids found to be induced during hypersensitive response in Arabidopsis, three OPDA-containing sulfoquinovosyl diacylglycerol species, one phosphatidylinositol species as well as two acylated OPDA-containing phosphatidylglycerol species were found to accumulate during the hypersensitive response in Arabidopsis. Our study confirms and extends on the notion that the hypersensitive response in Arabidopsis triggers a unique profile of Allene Oxide Synthase dependent oxidation of membrane lipids. Primary targets of this oxidation seem to be uncharged and anionic lipid species.

The lack of L-PG production and the repercussions of it in regards to M. Tuberculosis interactions with mononuclear phagocytes.

The lysine connection with phosphatidylglycerol (PG) alters the M. tuberculosis(Mtb) surface charge, and consequently it may decrease the bacterial vulnerability to antimicrobial action of the immune cells. The aim of the study was to assess the significance of PG lysinylation in the Mtb interactions with mononuclear phagocytes. Both the Mtb strain with deletion of lysX gene (Mtb-lysX) which is responsible for PG lysinylation as well as the complemented strain (Mtb-compl) was used to infect human blood monocytes or THP-1 cells. The monocytes were obtained by MACS technique, or THP-1 cells. The Mtb-lysX strain has exhibited the enhanced sensitivity to HNP 1-3. However, it was not susceptible to bactericidal action of cathepsin G. The LysX deletion did not influence the Mtb ability of monocyte induction to IL-10 secretion. The intra- and extracellular expression of MHC-II was similarly reduced after the Mtb-lysX or Mtb-Rv infections. Noticeably significant is that the Mtb strain with deleted lysX has not affected the intensity of the gene expression of cathepsin G compared to the uninfected monocytes. That is the clear contrast to what the Mtb-Rv strain has proved. The obtained results suggest that the Mtb ability to lysinylate PG is a participatory element in mycobacterial strategy of survival inside phagocytic cells. However, the extended studies are needed to determine its influence on the other immune cells and define its role in the developing of Mtb infection.

Mechanisms of glycerophospholipid homeostasis in mammalian cells.

The membranes of mammalian cells contain hundreds of different phospholipid species, a variety of glycolipids and cholesterol. While the reasons for such compositional diversity are not well established, they probably relate to a multitude of membrane-associated functions each of which sets specific requirements for the chemical and physical properties of membranes. The lipid composition of membranes must therefore be accurately controlled. The maintenance of phospholipid homeostasis in a mammalian cell is a daunting task due to presence of many phospholipid (and other lipid) classes and hundreds of different molecular species. In addition, the phospholipid composition of the cellular membranes depends on several different phenomena including biosynthesis, remodelling, degradation and interorganelle trafficking. Accordingly, it is not surprising that phospholipid homeostasis in mammalian cells is poorly understood. Particularly little is known about the regulation and coordination of processes contributing to homeostasis. Nevertheless, it has become obvious that selective degradation plays a major role, albeit the enzymes involved remain to be discovered. Beside the complexity of the phenomenon, methodological limitations have hampered the progress in this field. Here, we review the key features of the processes contributing to phospholipid homeostasis in mammalian cells, with a particular emphasis on the regulation and coordination of biosynthesis and degradation.

Group B Streptococcus, phospholipids and pulmonary hypertension.

OBJECTIVE: Group B Streptococcus is the most common cause of bacterial infection in the newborn. Our aim was to purify and identify molecules produced by the bacterium, which cause pulmonary hypertension. STUDY DESIGN: Guided by bioassays performed in neonatal lambs, we utilized standard biochemical techniques for the purification of these bioactive compounds. The compounds were identified by mass spectrometry. Fully synthetic compounds were then tested using the bioassay to confirm their ability to induce pulmonary hypertension. RESULT: The purified bacterial components causing pulmonary hypertension were the phospholipids cardiolipin and phosphatidylglycerol. Synthetic cardiolipin or phosphatidylglycerol also induced pulmonary hypertension in lambs. CONCLUSION: Bacterial phospholipids are capable of causing pulmonary hypertension. This finding opens new avenues for therapeutic intervention in persistent pulmonary hypertension of the newborn and generates hypotheses regarding the etiology of respiratory distress in the newborn and the possible effect of antibiotic therapy.

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis.

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo.

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.

Amino acid-containing membrane lipids in bacteria.

In the bacterial model organism Escherichia coli only the three major membrane lipids phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin occur, all of which belong to the glycerophospholipids. The amino acid-containing phosphatidylserine is a major lipid in eukaryotic membranes but in most bacteria it occurs only as a minor biosynthetic intermediate. In some bacteria, the anionic glycerophospholipids phosphatidylglycerol and cardiolipin can be decorated with aminoacyl residues. For example, phosphatidylglycerol can be decorated with lysine, alanine, or arginine whereas in the case of cardiolipin, lysine or d-alanine modifications are known. In few bacteria, diacylglycerol-derived lipids can be substituted with lysine or homoserine. Acyl-oxyacyl lipids in which the lipidic part is amide-linked to the alpha-amino group of an amino acid are widely distributed among bacteria and ornithine-containing lipids are the most common version of this lipid type. Only few bacterial groups form glycine-containing lipids, serineglycine-containing lipids, sphingolipids, or sulfonolipids. Although many of these amino acid-containing bacterial membrane lipids are produced in response to certain stress conditions, little is known about the specific molecular functions of these lipids.

A novel function of the human CLS1 in phosphatidylglycerol synthesis and remodeling.

Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1>C18:2>C18:0>C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.