Glucose enhances catecholamine-stimulated lipolysis via increased glycerol-3-phosphate synthesis in 3T3-L1 adipocytes and rat adipose tissue.

BACKGROUND: During lipolysis, triglyceride (TG) are hydrolyzed into a glycerol and fatty acids in adipocyte. A significant portion of the fatty acids are re-esterificated into TG, and this is a critical step in promoting lipolysis. Although glycerol-3-phosphate (G3P) is required for triglyceride synthesis in mammalian cell, the substrate for G3P synthesis during active lipolysis is not known. A recent study showed that the inhibition of glucose uptake reduces catecholamine-stimulated lipolysis, suggesting that glucose availability is important in lipolysis in adipocytes. We hypothesized that glucose might play an essential role in generating G3P and thereby promoting catecholamine-stimulated lipolysis in adipocytes. Therefore, we determined the effect of glucose availability on catecholamine-stimulated lipolysis in 3T3-L1 adipocytes and rat adipose tissue. METHODS AND RESULTS: 3T3-L1 adipocytes and rat epididymal fat pads were cultured in a medium with/without glucose during stimulation by isoproterenol. Glycerol release was higher when adipocytes were cultured in a glucose-containing medium than that in a medium without glucose. Measurement of glucose uptake during catecholamine-stimulated lipolysis showed a slight, but significant increase in glucose uptake. We also compared glucose metabolism-related protein, such as glucose transporter 4, hexokinase, glycerol-3-phosphate dehydrogenase and lipase contents between fat tissues that play a critical role in active lipolysis. Epididymal fat exhibited higher lipolytic activity than inguinal fat because of higher lipase and glucose metabolism-related protein contents. CONCLUSION: We demonstrated that catecholamine-stimulated lipolysis is enhanced in the presence of glucose, and suggests that glucose is one of the primary substrates for G3P in adipocytes during active lipolysis.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

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.

Structural and Functional Insights into an Archaeal Lipid Synthase.

The UbiA superfamily of intramembrane prenyltransferases catalyzes an isoprenyl transfer reaction in the biosynthesis of lipophilic compounds involved in cellular physiological processes. Digeranylgeranylglyceryl phosphate (DGGGP) synthase (DGGGPase) generates unique membrane core lipids for the formation of the ether bond between the glycerol moiety and the alkyl chains in archaea and has been confirmed to be a member of the UbiA superfamily. Here, the crystal structure is reported to exhibit nine transmembrane helices along with a large lateral opening covered by a cytosolic cap domain and a unique substrate-binding central cavity. Notably, the lipid-bound states of this enzyme demonstrate that the putative substrate-binding pocket is occupied by the lipidic molecules used for crystallization, indicating the binding mode of hydrophobic substrates. Collectively, these structural and functional studies provide not only an understanding of lipid biosynthesis by substrate-specific lipid-modifying enzymes but also insights into the mechanisms of lipid membrane remodeling and adaptation.

Plastid Glycerol-3-phosphate Acyltransferase Enhanced Plant Growth and Prokaryotic Glycerolipid Synthesis in Brassica napus.

Plastid-localized glycerol-3-phosphate acyltransferase (ATS1) catalyzes the first-step reaction in glycerolipid assembly through transferring an acyl moiety to glycerol-3-phosphate (G3P) to generate lysophosphatidic acid (LPA), an intermediate in lipid metabolism. The effect of ATS1 overexpression on glycerolipid metabolism and growth remained to be elucidated in plants, particularly oil crop plants. Here, we found that overexpression of BnATS1 from Brassica napus enhanced plant growth and prokaryotic glycerolipid biosynthesis. BnATS1 is localized in chloroplasts and an in vitro assay showed that BnATS1 had acylation activity toward glycerol 3-phosphate to produce LPA. Lipid profiling showed that overexpression of BnATS1 led to increases in multiple glycerolipids including phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), phosphatidylcholine (PC), and phosphatidylinositol (PI), with increased polyunsaturated fatty acids. Moreover, increased MGDG was attributed to the elevation of 34:6- and 34:5-MGDG, which were derived from the prokaryotic pathway. These results suggest that BnATS1 promotes accumulation of polyunsaturated fatty acids in cellular membranes, thus enhances plant growth under low-temperature conditions in Brassica napus.

Reduced SCD1 activity alters markers of fatty acid reesterification, glyceroneogenesis, and lipolysis in murine white adipose tissue and 3T3-L1 adipocytes.

White adipose tissue (WAT) has a critical role in lipid handling. Previous work demonstrated that SCD1 is an important regulator of WAT fatty acid (FA) composition; however, its influence on the various interconnected pathways influencing WAT lipid handling remains unclear. Our objective was to investigate the role of SCD1 on WAT lipid handling using Scd1 knockout (KO) mice and SCD1-inhibited 3T3-L1 adipocytes by measuring gene, protein, and metabolite markers related to FA reesterification, glyceroneogenesis, and lipolysis. Triacylglycerol (TAG) content was higher in inguinal WAT (iWAT) from KO mice compared with wild-type, but significantly lower in epididymal WAT (eWAT). The SCD1 desaturation index was decreased in both WAT depots in KO mice. FA reesterification, as measured with a NEFA:glycerol ratio, was reduced in both WAT depots in KO mice, as well as SCD1-inhibited 3T3-L1 adipocytes. Pck1, Atgl, and Hsl gene expression was reduced in both WAT depots of KO mice, while Pck2 and Pdk4 gene expression showed depot-specific regulation. Pck1, Atgl, and Hsl gene expression was reduced, and phosphoenolpyruvate carboxykinase protein content was ablated, in SCD1-inhibited adipocytes. Our data provide evidence that SCD1 has a broad impact on WAT lipid handling by altering TAG composition in a depot-specific manner, reducing FA reesterification, and regulating markers of lipolysis and glyceroneogenesis.

Structures and genetics of biosynthesis of glycerol 1-phosphate-containing O-polysaccharides of Escherichia coli O28ab, O37, and O100.

O-polysaccharides of E. coli O28ab, O37, and O100 were found to contain glycerol 1-phosphate and the following structures of their oligosaccharide repeats were established by sugar analysis, Smith degradation (for O28ab), 1D and 2D (1)H, (13)C, and (13)P NMR spectroscopy: [Formula: see text]. Functions of putative glycosyltransferases genes in the O-antigen gene clusters of the strains studied were tentatively assigned based on similarities to genes of other E. coli O-serogroups available from GenBank and taking into account the O-polysaccharide structures established.

Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.

Increased glyceride-glycerol synthesis in liver and brown adipose tissue of rat: in-vivo contribution of glycolysis and glyceroneogenesis.

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (~70 %) as well as in BAT and EAT (~80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

The sympathetic nervous system regulates the three glycerol-3P generation pathways in white adipose tissue of fasted, diabetic and high-protein diet-fed rats.

The aim of the present study was to investigate the participation of the sympathetic nervous system (SNS) in the control of glycerol-3-P (G3P) generating pathways in white adipose tissue (WAT) of rats in three situations in which the plasma insulin levels are low. WAT from 48 h fasted animals, 3 day-streptozotocin diabetic animals and high-protein, carbohydrate-free (HP) diet-fed rats was surgical denervated and the G3P generation pathways were evaluated. Food deprivation, diabetes and the HP diet provoke a marked decrease in the rate of glucose uptake and glycerokinase (GyK) activity, but a significant increase in the glyceroneogenesis, estimated by the phosphoenolpyruvate carboxykinase (PEPCK) activity and the incorporation of 1-[(14)C]-pyruvate into glycerol-TAG. The denervation provokes a reduction (~70%) in the NE content of WAT in fasted, diabetic and HP diet-fed rats. The denervation induced an increase in WAT glucose uptake of fed, fasted, diabetic and HP diet-fed rats (40%, 60%, 3.2 fold and 35%, respectively). TAG-glycerol synthesis from pyruvate was reduced by denervation in adipocytes of fed (58%) and fasted (36%), saline-treated (58%) and diabetic (23%), and HP diet-fed rats (11%). In these same groups the denervation reduced the PEPCK mRNA expression (75%-95%) and the PEPCK activity (35%-60%). The denervation caused a ~35% decrease in GyK activity of control rats and a further ~35% reduction in the already low enzyme activity of fasted, diabetic and HP diet-fed rats. These data suggest that the SNS plays an important role in modulating G3P generating pathways in WAT, in situations where insulin levels are low.

Improvement and validation of ¹²5I-high-performance liquid chromatography method for determination of total human serum choline and ethanolamine plasmalogens.

BACKGROUND: Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimer's disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography ((125)I-HPLC method). The present study reports the improvement and validation of (125)I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter. METHODS: 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l-α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery. RESULTS: Linearity was established over the concentration range 5-300 µmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses. CONCLUSIONS: The improved (125)I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.

Glycerol-3-phosphate and systemic immunity.

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.

Functional assignment of an enzyme that catalyzes the synthesis of an archaea-type ether lipid in bacteria.

An archaea-type ether lipid in bacteria: PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. The product becomes dephosphorylated and acetylated in vivo.

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.

Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.

Mice deficient in phosphofructokinase-M have greatly decreased fat stores.

Synthesis of triacylglycerol requires the glucose-derived glycerol component, and glucose uptake has been viewed as the rate-limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK-M have greatly reduced fat stores. Mice with disrupted activity of the PFK-M distal promoter were obtained from Lexicon Pharmaceuticals, developed from OmniBank OST#56064. Intra-abdominal fat was measured by magnetic resonance imaging of the methylene proton signal. Lipogenesis from labeled glucose was measured in isolated adipocytes. Lipolysis (glycerol and free fatty acid release) was measured in perifused adipocytes. Intra-abdominal fat in PFK-M-deficient female mice (5-10 months old) was 17 +/- 3% of that of wild-type littermates (n = 4; P < 0.02). Epididymal fat weight in 15 animals (7-9.5 months) was 34 +/- 4% of control littermate (P < 0.002), with 10-30% lower body weight. Basal and insulin-stimulated lipogenesis in PFK-M-deficient epididymal adipocytes was 40% of the rates in cells from heterozygous littermates (n = 3; P < 0.05). The rate of isoproterenol-stimulated lipolysis in wild-type adipocytes declined approximately 10% after 1 h and 50% after 2 h; in PFK-M-deficient cells it declined much more rapidly, 50% in 1 h and 90% in 2 h, and lipolytic oscillations appeared to be damped (n = 4). These results indicate an important role for PFK-M in adipose metabolism. This may be related to the ability of this isoform to generate glycolytic oscillations, because such oscillations may enhance the production of the triacylglycerol precursor alpha-glycerophosphate.

Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes.

Lipoteichoic acid (LTA) is an important cell wall polymer in gram-positive bacteria and often consists a polyglycerolphosphate backbone chain that is linked to the membrane by a glycolipid. In Listeria monocytogenes this glycolipid is Gal-Glc-DAG or Gal-Ptd-6Glc-DAG. Using a bioinformatics approach, we have identified L. monocytogenes genes predicted to be involved in glycolipid (lmo2555 and lmo2554) and LTA backbone (lmo0644 and lmo0927) synthesis. LTA and glycolipid analysis of wild-type and mutant strains confirmed the function of Lmo2555 and Lmo2554 as glycosyltransferases required for the formation of Glc-DAG and Gal-Glc-DAG. Deletion of a third gene, lmo2553, located in the same operon resulted in the production of LTA with an altered structure. lmo0927 and lmo0644 encode proteins with high similarity to the staphylococcal LTA synthase LtaS, which is responsible for polyglycerolphosphate backbone synthesis. We show that both proteins are involved in LTA synthesis. Our data support a model whereby Lmo0644 acts as an LTA primase LtaP and transfers the initial glycerolphosphate onto the glycolipid anchor, and Lmo0927 functions as LTA synthase LtaS, which extends the glycerolphosphate backbone chain. Inactivation of LtaS leads to severe growth and cell division defects, underscoring the pivotal role of LTA in this gram-positive pathogen.

Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.

Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells.

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus Delta ltaS mutant could grow at 30 degrees C but not at 37 degrees C. Even at the permissive temperature, Delta ltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Delta ltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Delta ltaS mutant at 37 degrees C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Delta ltaS mutation was found to be synthetically lethal with the Delta tagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30 degrees C, suggesting that LTA and WTA compensate for one another in an essential function.

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet.

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-14C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-14C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.