Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS

This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion. An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8. The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol. Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.     

Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.     

Polar lipids from the radiation resistant bacterium Deinococcus radiodurans: structural investigations on glucosaminyl and N-acetyl glucosaminyl lipids

Deinococcus radiodurans, although a gram-positive bacterium, has a complex cell wall with multiple layers and associates to this structural particularity, a quite unusual lipid composition for gram-positive bacteria. The conventional phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol...) are absent. Among the nine polar lipids detected in the R1 Anderson strain, three are glycolipids only one is a phospholipid, the other ones are glycophospholipids. One of the latter compounds contains one free amino group. Analysis by aminoacid autoanalyser enables to identify glucosamine in one glycolipid and in two glycophospholipids. Sugar analysis by gas-liquid chromatography after acid methanolysis and trifluoroacetylation, reveals the occurrence of N-acetyl glucosaminyl residues in one glycolipid and in one phospholipid. The following identification for the two lipids of D. radiodurans is proposed: phosphatidyl glucosaminyl glycerol and phosphatidyl N-acetyl glucosaminyl glycerol.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

Human fetal and adult spleen phospholipids and their fatty acid composition

Total phospholipid contents and the individual phospholipid components of human adult and fetal spleens from 17--18 and 23--24 week's pregnancies composition of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol and phosphatidic acid studied in human adult, 17--18 and 23--24 week fetal spleens.     

Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C

The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine: diphosphatidylglycerol: phosphatidylinositol, 90:5:5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70–75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–7}\text{days}$) appeared to be greater than that for phosphatidylinositol ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8–60}\text{days}$). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.     

CEREBRAL PHOSPHOLIPIDS IN ''QUAKING'' MICE

The brain of the adult ‘quaking’ mutant mouse is characterized by a reduced phospholipid content which can be ascribed not only to the myelin deficiency but also to a well-defined hydrocephalus. The mutant brain shows highly significant decreases in the percentage of ethanolamine plasmalogen, triphosphoinositide and phosphatidic acid in the whole phospholipid fraction, while the proportions of phosphatidylcholine, phosphatidyl-ethanolamine, diphosphatidylglycerol and phosphatidylinositol increase. The changes are consistent with the selective absence of ‘myelinic’ phospholipid although phosphatidylserine, sphingomyelin and diphosphoinositide are unchanged.     

Diversity of the culturable lichen-derived actinobacteria and the taxonomy of Streptomyces parmotrematis sp. nov.

A total of 37 actinobacteria were isolated from eighteen lichen samples collected in Thailand. Based on the 16S rRNA gene sequences, they were identified into five genera including Actinoplanes (1 strain), Actinomadura (1 strain), Pseudosporangium (1 strain), Wangella (1 strain) and Streptomyces (33 strains). Among these isolates, strain Ptm05^T, Ptm01 and Ptm12 showed low 16S rRNA gene similarity and was selected for the further taxonomic study using the polyphasic approach. These strains showed the highest 16S rRNA gene sequence similarity with Streptomyces sparsogenes ATCC 25498^T (97.44–97.72%). Strain Ptm05^T was selected for the type strain. The chemical cell composition of the strain was similar to the members of Streptomyces genus. LL-diaminopimelic acids were detected in the peptidoglycan. Menaquinones were MK-9(H₈) and MK-9(H₆). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid, one unidentified glycolipid and one unidentified lipid were detected as the polar lipids. The predominant cellular fatty acids are anteiso-C_15:0, iso-C_15:0, iso-C_16:0, iso-C_17:0 and C_16:0. The dDNA-DNA hybridization values among strain Ptm05^T and its closely related Streptomyces type strains were 17.2–18.0%. In addition, the ANIb and ANIm between strain Ptm05^T and related Streptomyces type strains were ranged from 75.69 to 76.13% and 85.21 to 85.35%, respectively. Based on phenotypic and genomic evidence, strain Ptm05^T (=?TBRC 14546^T?=?NBRC 115203^T) represents the novel species of the genus Streptomyces for which the name Streptomyces parmotrematis sp. nov. is proposed. This study showed that the lichens are the promising source of the novel actinobacterial taxa.

     

Effect of acute thioacetamide administration on rat brain phospholipid metabolism

Brain phospholipid composition and the [³²P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.     

Effect of cold stress and norepinephrine on the turnover of phospholipids in brown adipose tissue of the golden hamster (Mesocricetus auratus)

The brown adipose tissue of cold adapted hamsters has an approximately 35% higher phospholipid content than that of the controls. The basal turnover rate of phosphatidylinositol is higher in cold-adapted animals than in the controls, whereas 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and diphosphatidylglycerol is not changed. Administration of norepinephrine increases the incorporation of 32P into both control and cold-adapted animals by 200% and 220% respectively. The 32P incorporation into other phospholipid fractions is not modified.     

The lipid composition of Mycoplasma laidlawii strain B

1. Total lipid was extracted from Mycoplasma laidlawii strain B with chloroform-methanol mixtures and fractionated into neutral lipid, glycolipid and phospholipid components by chromatography on silicic acid. 2. Saponification of the glycolipid fraction, which represented nearly half of the total lipid, yielded two glycosides for which the structures O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol were established. 3. The ratio of monoglucosyl diglyceride to diglucosyl diglyceride increased with the age of the culture, though the total glycolipid concentration remained virtually constant. The glycolipid concentration was unaffected by the addition of cholesterol to the culture medium. 4. The phospholipid fraction consisted of two components, phosphatidylglucose and phosphatidylglycerol. Organisms harvested at acidic pH also contained O-amino acyl esters of phosphatidylglycerol. No lipids containing inositol could be detected.     

Acyl phosphatidylglycerol: a major phospholipid of Corynebacterium amycolatum

The type strain and several clinical isolates of Corynebacterium amycolatum were examined for lipid composition as a chemotaxonomic character for routine identification. The phospholipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides, together with various unidentified compounds. One of them, accounting for 20–29% of total phospholipids, was purified and characterized as acyl phosphatidylglycerol by chromatographic and spectrometric techniques. The acyl substituents on the phosphatidyl moiety were characterized as tetradecanoyl, pentadecanoyl, hexadecenoyl, hexadecanoyl, heptadecenoyl, heptadecanoyl, octadecenoyl (the major one), and octadecanoyl. The acyl group on the polar head (glycerol) was only octadecenoyl. Phospholipid analysis by thin-layer chromatography of a collection of Corynebacterium strains proved that this compound is widely distributed, although it only represents a minor (2–9%) component among mycolic acid-containing species. Acyl phosphatidylglycerol can be considered as a useful chemical marker for the identification of C. amycolatum in addition to the absence of mycolic acids.     

Lipids of Leishmania promastigotes.

A chromatographic analysis of lipids of cultured promastigotes of Leishmania donovani, L. braziliensis, L. mexicana, L. tropica, L. enriettii, L. hertigi, L. adleri, and L. tarentolae showed that total lipids were 2--15% of dry wt, and neutral and polar lipids were 14--55% and 45--86% of total lipids. Major lipid classes were as follows: sterol ester, triacylglycerol, sterol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine. Predominant fatty acids were 18:2 (n - 6) greater than 18:3 (n - 3) greater than 18:1 (n - 9) greater than 18:0 greater than 22:6 (n - 3) greater than 22:5 (n - 6) greater than 16:0 greater than 14:0 greater than 18:4 (n - 3) greater than 20:3 (n - 3). Some remarkable distributions of fatty acids among the phospholipid fractions were observed, as follows: diphosphatidylglycerol 18--33% 22:6 (n - 3); phosphatidylinositol 31--68% 18:1 (n - 9); phosphatidylcholine 13--41% 18:3 (n - 3). Alk-l-enyldiacyl glycerols, and alk-l-enylacyl and alkylacyl forms of phosphatidylethanolamine and of phosphatidylinositol were found, and their glyceryl ethers and fatty adehydes analyzed. Notable in the phosphatidylethanolamine of some leishmanias was a cyclopropane fatty acid (4--11%), identified as cis-9,10-methylene octadecanoic acid by chromatographic, and by mass and proton resonance spectrometric analyses. The comparative biochemistry of the cyclopropane fatty acid, characteristic of many prokaryotes, and of alpha-linolenic acid, characteristic of photosynthetic plants, are commented upon.     

Structural analysis of a glycosylphosphatidylinositol glycolipid ofLeishmania donovani

A glycosylphosphatidylinositol (GPI) glycolipid antigen recognized by sera from patients with visceral leishmaniasis was isolated from Leishmania donovani promastigotes. The carbohydrate moiety was cleaved from the lipid part by digestion with specific phosphatidylinositol phospholipase C. After separation, structural analysis was carried out on the phosphorylated inositol oligosaccharide and the alkylacyl glycerol. The following major structures were found: [formula: see text] The presence of the conserved sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN-PI of glycosyl phosphatidylinositol protein anchors in this antigen may be consistent with a precursor role of Leishmania glycosyl phosphatidylinositol anchored proteins for this glycolipid.     

Phospholipid composition of a plasma membrane-enriched fraction from developing soybean roots

Phospholipid polar head group and fatty acid composition were determined for plasma membrane enriched fractions from developing soybean root (Glycine max [L.] Merr. cult. Wells II). Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots at pH 7.8 and in the presence of 5 millimolar ethylenediaminetetraacetate, 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N tetraacetic acid, and 10 millimolar NaF. Lipid extracts analyzed for phospholipid composition revealed two major phospholipid components: phosphatidylcholine and phosphatidylethanolmine. Minor phospholipid components identified were phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol. Lipid degradation by endogenous phospholipase D during membrane isolation at pH 6.5 and in the absence of chelating agents and NaF resulted in the recovery of large amounts of phosphatidic acid. Phosphatidylcholine was the principal substrate for phospholipase D.     

Activation of D-beta-hydroxybutyrate apodehydrogenase using molecular species of mixed fatty acyl phospholipids

D-beta-Hydroxybutyrate apodehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for enzymatic function. The purified enzyme which is devoid of lipid can be reactivated with lecithin or mixtures of natural phospholipid-containing lecithin. However, it is mitochondrial phospholipid which activates the enzyme optimally and with kinetic parameters similar to that of the native membrane-bound enzyme. Mitochondrial phospholipid consists of three classes of phospholipid (lecithin:phosphatidylethanolamine:diphosphatidylglycerol in a ratio of approximately 2:2:1 by phosphorus); each class consists of a multiplicity of different molecular species due to diversity in the fatty acyl substituents. In this study, we have synthesized defined molecular species of mixed fatty acyl phospholipids to evaluate whether multiplicity of phospholipid molecular species are essential for optimal reactivation. We find that: 1) ternary mixtures of single molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylpropan-1,3-diol in the liquid crystalline state mimic the optimal reactivation of the enzyme obtained with mitochondrial phospholipids; 2) although some negatively charged phospholipid appears necessary for optimizing the efficiency of activation, diphosphatidylglycerol can be replaced by phosphatidylpropan-1,3-diol, another negatively charged phospholipid; and 3) biphasic Arrhenius plots can be correlated with the liquid crystalline and gel states of the phospholipid.     

A novel glycolipid and phospholipid in the purple membrane

A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl-Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3-HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2, 3-di-O-phytanylglycerol.     

<Emphasis Type="Italic">Promicromonospora viridis</Emphasis> sp. nov., a novel actinomycete isolated from soil

Two Gram-stain positive, aerobic actinomycete strains, designated NEAU-JGR1^T and NEAU-JGC41, were isolated from soil collected from Fairy Lake Botanical Garden in Shenzhen, Guangdong Province, south of China. The 16S rRNA gene sequences analysis showed that the two strains exhibited 99.5% 16S rRNA gene sequence similarity with each other and were closely related to Promicromonospora thailandica JCM 17130^T (99.4, 99.3%) and Promicromonospora citrea DSM 43110^T (99.2, 99.2%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains clustered together and formed a cluster with P. thailandica JCM 17130^T and P. citrea DSM 43110^T. Both strains were observed to contain MK-9(H₄) and MK-9(H₂) as predominant menaquinones. Their whole cell sugar profiles were found to main contained rhamnose, ribose, glucose and galactose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycophosphatidylinositol, phosphatidylinositol mannoside, an unidentified glycolipid and an unidentified phospholipid. The predominant cellular fatty acids for the two strains were identified as anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0. The DNA–DNA hybridization value between strains NEAU-JGR1^T and NEAU-JGC41 was 85.1?±?0.3%, and the values between the two strains and their close phylogenetic relatives were well below 70%, supporting the conclusion that they represent a distinct genomic species. An array of phenotypic characteristics also differentiated the isolates from closely related species. On the basis of the genetic and phenotypic properties, strains NEAU-JGR1^T and NEAU-JGC41 can be classified as representatives of a novel species of the genus Promicromonospora, for which the name Promicromonospora viridis sp. nov., is proposed. The type strain is NEAU-JGR1^T (=?DSM 105536^T?=?CGMCC 4.7473^T).     

Description of <Emphasis Type="Italic">Geodermatophilus bullaregiensis</Emphasis> sp. nov.

The taxonomic position of an aerobic actinobacterial strain, BMG841^T, isolated from the Bulla Regia monument (Tunisia) and exhibiting a high resistance to gamma-radiation (D10 ~9 kGy) was determined using polyphasic approach. The optimal growth range was found to be 25–35 °C at pH of 7.0–8.5. The strain was observed to form black dry colonies. Chemotaxonomic characteristics of the isolate showed a cell wall type III, with galactose and glucose as diagnostic sugars; phosphatidylcholine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid as main polar lipids; and MK-9(H₄) as the predominant menaquinone. The major cellular fatty acids were identified as iso-C_16:0 and iso-C_15:0. Phylogenetic analysis indicated that strain BMG841^T represents a novel member of the genus Geodermatophilus with high 16S rRNA gene sequence identity with Geodermatophilus saharensis (98.28 %). Based on phylogenetic and phenotypic analysis, strain BMG841^T is proposed as the type strain (=DSM 46841^T = CECT 8821^T) of a novel species, Geodermatophilus bullaregiensis.     

Phospholipids in the developing soybean seed

The distribution of phospholipids in developing soybean seeds [Glycine max (L.) Merr., var. “Chippewa 64,” “Harosoy 63,” “Wayne,” and “Clark 63”] was followed. From 30 to 60 days after flowering expressed as mole per cent of phospholipid phosphorus phosphatidic acid decreased from 14.8 to 9.1; phosphatidylinositol increased from 0 to 9.1; phosphatidylcholine increased from 8.2 to 9.8; phosphatidylethanolamine increased from 5.3 to 8.6; phosphatidylglycerol increased from 3.2 to 4.8; diphosphatidylglycerol increased from 2.7 to 4.1; and N-acylphosphatidylethanolamine decreased from 65.8 to 54.6. However, from 60 days after flowering to maturity, phosphatidic acid decreased to 0; phosphatidylinositol increased roughly 2-fold; phosphatidylcholine increased roughly 4.7-fold; phosphatidylethanolamine increased 3-fold; N-acylphosphatidylethanolamine decreased 11-fold; whereas phosphatidylglycerol and diphosphatidylglycerol remained essentially constant. Percentages of individual phospholipid species were not statistically different between any two varieties at a given time period.