Ladderane phospholipids in anammox bacteria comprise phosphocholine and phosphoethanolamine headgroups

Anammox bacteria present in wastewater treatment systems and marine environments are capable of anaerobically oxidizing ammonium to dinitrogen gas. This anammox metabolism takes place in the anammoxosome which membrane is composed of lipids with peculiar staircase-like 'ladderane' hydrocarbon chains that comprise three or four linearly concatenated cyclobutane structures. Here, we applied high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to elucidate the full identity of these ladderane lipids. This revealed a wide variety of ladderane lipid species with either a phosphocholine or phosphoethanolamine polar headgroup attached to the glycerol backbone. In addition, in silico analysis of genome data gained insight into the machinery for the biosynthesis of the phosphocholine and phosphoethanolamine phospholipids in anammox bacteria.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox)

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost‐effective and environment‐friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere.     

Biophysical properties of membrane lipids of anammox bacteria: II. Impact of temperature and bacteriohopanoids

Anammox bacteria possess unique membranes that are mainly comprised of phospholipids with extraordinary “ladderane” hydrocarbon chains containing 3 to 5 linearly concatenated cyclobutane moieties that have been postulated to form relatively impermeable membranes. In a previous study, we demonstrated that purified ladderane phospholipids form fluid-like mono- and bilayers that are tightly packed and relatively rigid. Here we studied the impact of temperature and the presence of bacteriohopanoids on the lipid density and acyl chain ordering in anammox membranes using Langmuir monolayer and fluorescence depolarization experiments on total lipid extracts. We showed that anammox membrane lipids of representatives of Candidatus “Kuenenia stuttgartiensis”, Candidatus “Brocadia fulgida” and Candidatus “Scalindua” were closely packed and formed membranes with a relatively high acyl chain ordering at the temperatures at which the cells were grown. Our findings suggest that bacteriohopanoids might play a role in maintaining the membrane fluidity in anammox cells.     

Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.     

Impact of Temperature on Ladderane Lipid Distribution in Anammox Bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria have the unique ability to synthesize fatty acids containing linearly concatenated cyclobutane rings, termed “ladderane lipids.” In this study we investigated the effect of temperature on the ladderane lipid composition and distribution in anammox enrichment cultures, marine particulate organic matter, and surface sediments. Under controlled laboratory conditions we observed an increase in the amount of C₂₀ [5]-ladderane fatty acids compared with the amount of C₁₈ [5]-ladderane fatty acids with increasing temperature and also an increase in the amount of C₁₈ [5]-ladderane fatty acids compared with the amount of C₂₀ [5]-ladderane fatty acids with decreasing temperature. Combining these data with results from the natural environment showed a significant (R² = 0.85, P = <0.0001, n = 121) positive sigmoidal relationship between the amounts of C₁₈ and C₂₀ [5]-ladderane fatty acids and the in situ temperature; i.e., there is an increase in the relative abundance of C₁₈ [5]-ladderane fatty acids at lower temperatures and vice versa, particularly at temperatures between 12°C and 20°C. Novel shorter (C₁₆) and longer (C₂₂ to C₂₄) ladderane fatty acids were also identified, but their relative amounts were small and did not change with temperature. The adaptation of ladderane fatty acid chain length to temperature changes is similar to the regulation of common fatty acid composition in other bacteria and may be the result of maintaining constant membrane fluidity under different temperature regimens (homeoviscous adaptation). Our results can potentially be used to discriminate between the origins of ladderane lipids in marine sediments, i.e., to determine if ladderanes are produced in situ in relatively cold surface sediments or if they are fossil remnants originating from the warmer upper water column.Anaerobic ammonium-oxidizing (anammox) bacteria possess the unique ability to oxidize NH₄⁺ with NO₂⁻ to N₂ under anoxic conditions (42). Since the discovery of the anammox process in a wastewater treatment plant in the Netherlands (21), studies have indicated that anammox bacteria are omnipresent in low-oxygen environments around the world. Anammox therefore forms an important link in both the oceanic (4, 7, 17, 18, 31) and freshwater (14, 33) nitrogen cycles. Unlike other Planctomycetes, anammox bacteria contain a unique “organelle” called the anammoxosome (19, 37, 44-46). The membrane of this compartment contains unusual “ladderane” lipids (37). The core ladderane lipids consist of C₁₈ and C₂₀ fatty acids containing either 3 or 5 linearly concatenated cyclobutane rings, which are ester bound to a glycerol backbone or ether bound as alkyl chains (35). In addition, the intact polar lipids containing the core lipid structures may have different types of polar head groups, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylglycerol (PG) (1, 22). In silico density simulation modeling experiments with a ladderane lipid-containing membrane (glycerol-bound mixed ether-ester containing both ladderane moieties) have indicated that ladderane lipids could provide a denser cell membrane than conventional membrane lipids (37). Since the anammoxosome appears to be impenetrable to fluorophores, the ladderane membrane could function in cell energy conservation (37, 44).Experimental evidence has shown that anammox bacteria isolated from wastewater treatment reactors grow over a wide range of temperatures (20 to 43°C) and have an optimum temperature of about 35°C (39). In the natural environment the anammox process has been reported to occur at temperatures as low as −2.5°C in sea ice (5, 26) and as high as 70°C in hot springs and hydrothermal vent areas (3, 12). Furthermore, “Candidatus Scalindua spp.” has been successfully enriched from marine sediment (Gullmarsfjord, Sweden) in sequencing batch reactors at temperatures of 15 and 20°C (43). In other bacteria containing common fatty acids temperature adaptation can be achieved by (among other things) modifying the composition of the membrane bilayers to deal with alterations in membrane viscosity due to changes in temperature. This process has been well documented and is termed “homeoviscous adaptation”; i.e., the fatty acid composition is changed to maintain membrane fluidity (23, 27, 34, 40). Currently, it is not known how anammox bacteria, with their highly unusual ladderane lipids, react to temperature. To investigate this, we analyzed the ladderane lipid composition of anammox bacteria grown at different temperatures in sequencing batch reactors and in samples from different natural environments covering a wide range of temperatures.     

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and experimental results developed in this study to elucidate the biosynthetic pathway that produces unique and intriguing ladderane lipids.     

The anammoxosome: an intracytoplasmic compartment in anammox bacteria

Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction.     

厌氧氨氧化菌脱氮机理及其在污水处理中的应用

厌氧氨氧化细菌(anammox)可以将亚硝酸盐和氨氮转化为氮气从而缩短氨氮转化的过程,它已经成为新型生物污水脱氮技术研究的热点之一。当前,有关厌氧氨氧化菌特有的生理结构特点、种群分类及其功能酶等方面的研究取得了一定突破,为实现其工业应用奠定了良好的理论基础;同时分子生物学技术在厌氧氨氧化细菌种群分布、群落多样性及其共生关系等方面的应用也大大促进了污水生物脱氮技术的革新和进步。总结了厌氧氨氧化菌主要的生理生化特点、细胞结构特点、脱氮机理、污水处理体系中的应用以及分子生物学方法对污水处理体系中厌氧氨氧化菌种群分析的研究现状,并指出未来anammox细菌在生物特性及在污水脱氮处理实际应用的研究中的热点问题。生物特性方面的主要研究热点有：（1）anammox细菌除厌氧氨氧化作用外,其它新陈代谢途径有待探索;（2）anammox细菌在不同环境中分布的倾向性问题;（3）新型anammox细菌的确定。污水处理的实际应用方面的主要研究热点有：（1）anammox污泥的快速高效富集问题;（2）设计高特异性引物;（3）anammox细菌和其他微生物的共生关系。     

Linking ultrastructure and function in four genera of anaerobic ammonium-oxidizing bacteria: cell plan, glycogen storage, and localization of cytochrome C proteins

Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes. Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.     

Bacterial lipid diversity

The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems.     

Biosynthesis of 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine in Caenorhabditis elegans.

Previously, we showed that lowering the growth temperature increased the level of eicosapentaenoic acid (EPA) in the phosphatidylcholine (PtdCho) of Caenorhabditis elegans. In this study, we investigated the molecular species composition of PtdCho of C. elegans, with an emphasis on EPA-containing species. C. elegans contained a substantial amount of 1,2-dipolyunsaturated fatty acid-containing PtdCho (1,2-diPUFA-PtdCho) species, such as arachidonic acid/EPA and EPA/EPA, which are unusual phospholipids in higher animals. The EPA/EPA-PtdCho content was significantly increased in C. elegans grown at a low temperature. To examine the possibility that the acyltransferase activity involved in the remodeling of phospholipids accounts for the production of 1,2-diPUFA-PtdCho, we investigated the substrate specificity of this enzyme in C. elegans and found that it did not exhibit a preference for saturated fatty acid for acylation to the sn-1 position of PtdCho. The efficacy of the esterification of EPA to the sn-1 position was almost equal to that of stearic acid. The lack of preference for a saturated fatty acid for acylation to the sn-1 position of PtdCho is thought to result in the existence of the unusual 1,2-diEPA-PtdCho in C. elegans.     

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, "Candidatus Brocadia anammoxidans" and "Candidatus Kuenenia stuttgartiensis", and one marine species, "Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. "Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle—the anammoxosome—in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.     

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g⁻¹ sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.     

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6.We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.     

Characterization of acyl-phosphatidylinositol from the opportunistic pathogen Corynebacterium amycolatum

The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M-H)-, observed at 1099 (the major one) 1113, and 1127. Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position. Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C. amycolatum. Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads. The present study reveals the existence of acyl-PI in C. amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.