Molecular Species Of Phosphatidylglycerol Research Articles

Coral Lipidome: Molecular Species of Phospholipids, Glycolipids, Betaine Lipids, and Sphingophosphonolipids.

Coral reefs are the most biodiversity-rich ecosystems in the world's oceans. Coral establishes complex interactions with various microorganisms that constitute an important part of the coral holobiont. The best-known coral endosymbionts are Symbiodiniaceae dinoflagellates. Each member of the coral microbiome contributes to its total lipidome, which integrates many molecular species. The present study summarizes available information on the molecular species of the plasma membrane lipids of the coral host and its dinoflagellates (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), ceramideaminoethylphosphonate, and diacylglyceryl-3-O-carboxyhydroxymethylcholine), and the thylakoid membrane lipids of dinoflagellates (phosphatidylglycerol (PG) and glycolipids). Alkyl chains of PC and PE molecular species differ between tropical and cold-water coral species, and features of their acyl chains depend on the coral's taxonomic position. PS and PI structural features are associated with the presence of an exoskeleton in the corals. The dinoflagellate thermosensitivity affects the profiles of PG and glycolipid molecular species, which can be modified by the coral host. Coral microbiome members, such as bacteria and fungi, can also be the source of the alkyl and acyl chains of coral membrane lipids. The lipidomics approach, providing broader and more detailed information about coral lipid composition, opens up new opportunities in the study of biochemistry and ecology of corals.

Lysophosphatidylglycerol (LPG) phospholipase D maintains membrane homeostasis in Staphylococcus aureus by converting LPG to lysophosphatidic acid

Lysophospholipids are deacylated derivatives of their bilayer forming phospholipid counterparts that are present at low concentrations in cells. Phosphatidylglycerol (PG) is the principal membrane phospholipid in Staphylococcus aureus and lysophosphatidylglycerol (LPG) is detected in low abundance. Here, we used a mass spectrometry screen to identify locus SAUSA300_1020 as the gene responsible for maintaining low concentrations of 1-acyl-LPG in S.aureus. The SAUSA300_1020 gene encodes a protein with a predicted amino terminal transmembrane α-helix attached to a globular glycerophosphodiester phosphodiesterase (GDPD) domain. We determined that the purified protein lacking the hydrophobic helix (LpgDΔN) possesses cation-dependent lysophosphatidylglycerol phospholipase D activity that generates both lysophosphatidic acid (LPA) and cyclic-LPA products and hydrolyzes cyclic-LPA to LPA. Mn2+ was the highest affinity cation and stabilized LpgDΔN to thermal denaturation. LpgDΔN was not specific for the phospholipid headgroup and degraded 1-acyl-LPG, but not 2-acyl-LPG. Furthermore, a 2.1Å crystal structure shows that LpgDΔN adopts the GDPD variation of the TIM barrel architecture except for the length and positioning of helix α6 and sheet β7. These alterations create a hydrophobic diffusion path for LPG to access the active site. The LpgD active site has the canonical GDPD metal binding and catalytic residues, and our biochemical characterization of site-directed mutants support a two-step mechanism involving a cyclic-LPA intermediate. Thus, the physiological function of LpgD in S.aureus is to convert LPG to LPA, which is re-cycled into the PG biosynthetic pathway at the LPA acyltransferase step to maintain membrane PG molecular species homeostasis.

Amino Acid Metabolism Controls Fatty Acid Structure in Staphylococcus aureus

Staphylococcus aureus produces a combination of branched and straight chain fatty acids to maintain membrane homeostasis. Recently, we showed that the branched:straight chain ratio in fatty acids is controlled by amino acid metabolism that supplies the branched‐chain acyl‐CoA primers for fatty acid biosynthesis. The fatty acid composition is determined when FabH catalyzes the initial condensation using the available short‐chain acyl‐CoA primers with malonyl‐acyl carrier protein. Branched‐chain ketoacid dehydrogenase (Bkd) is recognized as the primary enzyme responsible for the formation of branched‐chain acyl‐CoA primers. Most experiments with S. aureus are carried out in media with an abundant supply of branched‐chain amino acids where the Bkd pathway for the utilization of Ile dominates. However, branched‐chain fatty acids are still produced in bkd knockout strains and the enzyme(s) responsible for this alternate biosynthetic pathway is unknown. It is important to understand how S. aureus copes with limited availability of Ile because multiple laboratories conclude that isoleucine is a limiting nutrient at S. aureus infection sites that regulates metabolism and virulence.Mass tracing experiments with heavy amino acids show that Bkd is the key step in a pathway that selectively converts extracellular Ile into 2‐methyl‐butyryl‐CoA (anteiso‐C5‐CoA) that FabH uses to initiate anteiso fatty acid synthesis. The phosphatidylglycerol (PG) molecular species in wild‐type strains consists of a mixture of anteiso‐17:0 and ‐19:0 and 18:0 and 20:0 straight chain fatty acids in the 1‐position paired with anteiso‐15:0 in the 2‐position. If Ile is removed from the medium, Leu metabolism results in the introduction of iso‐15:0 into the 2‐position. Val is not significantly metabolized by the Bkd pathway. The analysis of the PG molecular species in strains lacking an active Bkd complex have a straight chain fatty acid in the 1‐position paired with either iso‐14:0 or anteiso‐15:0 in the 2‐position. This same switch in PG molecular species composition is observed when wild‐type strains are grown in defined medium lacking both Ile and Leu. Our metabolomics analyses show that C5‐CoA derived from the Bkd pathway is the most abundant acyl‐CoA primer available for FabH in wild‐type strains; however, when Ile and Leu are not present in the medium, iso‐C4‐CoA increases and anateiso‐C5‐CoA decreases. In the bkd knockout strains, the C4‐CoA pool is even more elevated, and the C5‐CoA pool is substantially lower. This means that in the absence of Bkd, a second pathway creates a supply of iso‐C4‐CoA for FabH to prime fatty acid synthesis and produce iso‐14:0 for the 2‐position. Thus, there are two pathways for the formation of branched‐chain acyl‐CoA substrates for FabH. The Bkd pathway is responsible for the utilization of extracellular Ile (and to a lesser extent, Leu) to initiate fatty acid synthesis, whereas the alternate, de novo biosynthetic pathway makes a significant contribution in wild‐type strain growth without exogenous Ile and is the only process operating in the bkd knockout strains. We are testing the hypothesis that the key enzyme in the de novo pathway to iso‐C4‐CoA is CidC, an enzyme related to pyruvate oxidase.

Crucial importance of length of fatty-acyl chains bound to the sn-2 position of phosphatidylglycerol for growth and photosynthesis of Synechocystis sp. PCC 6803

Phosphatidylglycerol (PG) in thylakoid membrane is essential for growth and photosynthesis of photosynthetic organisms. Although the sn-2 position of PG in thylakoid membrane is exclusively esterified with C16 fatty acids, the functional importance of the C16 fatty-acyl chains at the sn-2 position has not been clarified. In this study, we chemically synthesized non-metabolizable PG molecules: we introduced linoleic acid (18:2, fatty acid containing 18 carbons with 2 double bonds) and one of the saturated fatty acids with different chain length (12:0, 14:0, 16:0, 18:0 and 20:0) by ether linkage to the sn-1 and sn-2 positions, respectively. With the synthesized ether-linked PG molecules, we checked whether they could complement the growth and photosynthesis of pgsA mutant cells of Synechocystis sp. PCC 6803 to understand the importance of length of fatty chains at the sn-2 position of PG. The pgsA mutant is incapable of synthesizing PG, so it requires exogenous PG added to medium for growth. The growth rate and photosynthetic activity of mutant cells depended on the length of fatty chains: the PG molecular species binding 16:0 most effectively complemented the growth and photosynthesis of mutant cells, and other PG molecular species with fatty chains shorter or longer than 16:0 were less effective; especially, those binding 12:0 inhibited the growth and photosynthetic activity of the mutant cells. These data demonstrate that length of fatty chains bound to the sn-2 position of PG is critical for PG performance in growth and photosynthesis.

Competence-Associated Peptide BriC Alters Fatty Acid Biosynthesis in Streptococcus pneumoniae.

ABSTRACTMembrane lipid homeostasis is required for bacteria to survive in a spectrum of host environments. This homeostasis is achieved by regulation of fatty acid chain length and of the ratio of unsaturated to saturated fatty acids. In the pathogen Streptococcus pneumoniae, fatty acid biosynthesis is carried out by a cluster of fatty acid biosynthesis (fab) genes (FASII locus) whose expression is controlled by the FabT repressor. Encoded immediately downstream of the FASII locus is BriC, a competence-induced, cell-cell communication peptide that promotes biofilm development as well as nasopharyngeal colonization in a murine model of pneumococcal carriage. Here, we demonstrate that briC is cotranscribed with genes of the fab gene cluster and that a reduction of briC levels, caused by decoupling its transcription from fab gene cluster, negatively affects biofilm development. BriC elevates fabT transcription, which is predicted to alter the balance of unsaturated and saturated fatty acids produced by the pathway. We find that briC inactivation results in a decreased production of unsaturated fatty acids. This affects the membrane properties by decreasing the abundance of di-unsaturated phosphatidylglycerol molecular species. We propose that the link between BriC, FabT, and phospholipid composition contributes to the ability of S. pneumoniae to alter membrane homeostasis in response to the production of a quorum-sensing peptide.IMPORTANCE Adaptation of bacteria to their host environment is a key component of colonization and pathogenesis. As an essential component of bacterial membranes, fatty acid composition contributes to host adaptation. Similarly, cell-cell communication, which enables population level responses, also contributes to host adaptation. While much is known about the pathways that control the biosynthesis of fatty acids, many questions remain regarding regulation of these pathways and consequently the factors that affect the balance between unsaturated and saturated fatty acids. We find that BriC, a cell-cell communication peptide implicated in biofilm regulation and colonization, both is influenced by a fatty acid biosynthesis pathway and affects this same pathway. This study identifies a link between cell-cell communication, fatty acid composition, and biofilms and, in doing so, suggests that these pathways are integrated into the networks that control pneumococcal colonization and host adaptation.

Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on C[dbnd]C location level

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò–Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

Lipid Isolation Process and Study on Some Molecular Species of Polar Lipid Isolated from Seed of Madhuca ellitica

This study attempted the lipid extraction process from the seeds of Madhuca ellitica, a lipid-rich plant, and conducted a lipidomic analysis on molecular species of the obtained product. Total lipids of the crude seeds were found to contain 11.2% of polar lipids. The major fatty acids (FAs) of the polar lipids were palmitic (16:0), stearic (18:0), oleic (18:1n-9), and linoleic (18:2n-6) acids, which amounted to 28.5, 12.5, 44.8, and 13.2% of total FAs, respectively. The content and chemical structures of individual molecular species of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), and sulfoquinovosyldiacylglycerol (SQDG) were determined by HPLC with a tandem high-resolution mass spectrometry (HRMS). The major molecular species were 18:1/18:2 PE, 16:0/18:1 PC, 18:1/18:2 PC, 16:0/18:2 PG, 16:0/18:1 PG, 16:1/18:1 PI, 16:0/18:1 PI, 18:0/18:2 PI, 16:0/18:1 PA, 18:1/18:2 PA, 16:0/18:1 SQDG, and 18:0/18:1 SQDG. The application of a tandem HRMS allows us to determine the content of each isomer in pairs of the monoisotopic molecular species, for example, 18:0/18:2 and 18:1/18:1. The evaluation of the seed polar lipid profile will be helpful for developing the potential of this tree for nutritive and industrial uses.

Ion mobility and tandem mass spectrometry of phosphatidylglycerol and bis(monoacylglycerol) phosphate (BMP)

The tandem mass spectrometry, ion mobility, and normal phase HPLC of isomeric phosphatidylglycerol (PG) and bis(monoacylglycerol) phosphate (BMP) have been investigated in this study with the objective of differentiating these unique classes of lipids. Measurement of ion mobility using the traveling wave method for negative molecular and product ions from isomeric PG and BMP yielded identical results, but different ion mobilities were observed for positive product ions arising from collision-induced dissociation (CID). The fastest moving positive product ions from the ion mobility analysis of BMP(18:1/18:1) were monoglyceride-like, and the slowest moving product ions from this BMP corresponded to [M+H−2H2O]+, which were readily observed for BMP but were only at very low abundance in the CID spectra of PG. The major product ions observed from the sodium adduct of PG(18:1/18:1) were consistent with diglyceride-like ion formation, but for BMP(18:1/18:1) only monoglyceride-like product ions were formed. The usefulness of ion mobility separation was tested with the selection of positive product ions derived from the isomeric PG and BMP molecular species in the lipid extract of RAW 264.7 cells. The ion mobility spectra of monoglyceride-like ions derived from BMP species with various esterified fatty acyl groups displayed some separation in ion mobility based on fatty acyl chain length and presence of a double bond in the acyl chain. The mechanism of ion formation of the diglyceride- and monoglyceride-like ions from PG and BMP respectively was examined using deuterium-labeled species including PG(D3116:0/18:1) and PG and BMP labeled by deuterium exchange.

Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT).

A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58 %) and phosphatidylethanolamine (by 70 %). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.

FATTY ACID DESATURASE4 of Arabidopsis encodes a protein distinct from characterized fatty acid desaturases

Polar membrane glycerolipids occur in a mixture of molecular species defined by a polar head group and characteristic acyl groups esterified to a glycerol backbone. A molecular species of phosphatidylglycerol specific to chloroplasts of plants carries a Delta(3-trans) hexadecenoic acid in the sn-2 position of its core glyceryl moiety. The fad4-1 mutant of Arabidopsis thaliana missing this particular phosphatidylglycerol molecular species lacks the necessary fatty acid desaturase, or a component thereof. The overwhelming majority of acyl groups associated with membrane lipids in plants contains double bonds with a cis configuration. However, FAD4 is unusual because it is involved in the formation of a trans double bond introduced close to the carboxyl group of palmitic acid, which is specifically esterified to the sn-2 glyceryl carbon of phosphatidylglycerol. As a first step towards the analysis of this unusual desaturase reaction, the FAD4 gene was identified by mapping of the FAD4 locus and coexpression analysis with known lipid genes. FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic membrane bound fatty acid desaturases based on overall sequence conservation. However, the FAD4 protein contains two histidine motifs resembling those of metalloproteins such as fatty acid desaturases. FAD4 is targeted to the plastid. Overexpression of the cDNA in transgenic Arabidopsis led to increased accumulation of the Delta(3-trans) hexadecanoyl group in phosphatidylglycerol relative to wild type. Taken together these results are consistent with the hypothesis that FAD4 is the founding member of a novel class of fatty acid desaturases.

Relationships between Phosphatidylglycerol Molecular Species of Thylakoid Membrane Lipids and Sensitivities to Chilling‐induced Photoinhibition in Rice

In an attempt to explore the relationships between phosphatidylglycerol (PG) molecular species of thylakoid membrane lipids and sensitivities to chilling-induced photoinhibition, PG molecular species, D1 protein, electron transport activities of thylakoid membrane and the potential quantum yield (F(v)/F(m)) in rice treated under middle and low photon flux density (PFD) at 11 degrees C were analyzed by high performance liquid chromatography, enzyme hydrolysis, gas phase chromatography (GC) and so on. Results showed that the major molecular species of PGs in rice thylakoid membrane were 18:3/16:0, 18:3/16:1(3t), 18:2/16:0, 18:2/16:1(3t), 18:1/16:0, 18:1/16:1(3t), 16:0/16:0, 16:0/16:1(3t). There were large differences in the contents of unsaturated PG molecular species such as 18:1 approximately 3/16:0 approximately 16:1(3t) and saturated PG molecular species like 16:0/16:0 approximately 16:1(3t) among japonica cv 9516 (j-9516), japonica-indica hybrid F1 j-9516/i-SY63 (ji-95SY) and indica cv Shanyou 63 (i-SY63). J-9516 containing higher contents of unsaturated PG molecular species was manifest in stable D1 protein contents under chill and tolerant to chill-induced photoinhibition. In contrast to j-9516, i-SY63 with lower contents of unsaturated PG molecular species, exhibited unstable D1 protein contents under chill and was sensitive to chill-induced photoinhibition. ji-95SY containing middle contents of unsaturated PG molecular species between those of j-9516 and i-SY63, exhibited mid extent of sensitivity to chill-induced photoinhibition. The losses in D1 protein also account for the inhibition in electron transport activity of thylakoid membrane and the observed decline in F(v)/F(m). The PG molecular species that is efficient in raising chilling-resistant capacity were those containing unsaturated fatty acids, namely, unsaturated PG molecular species. These results implied that the substrate selectivity of the glycerol-3-phosphate acyltransferase in chloroplasts towards 16:0 or 18:1 displayed greatly the difference between japonica and indica rice. It was possible to enhance the capacity of resistance to chilling-induced photoinhibition by improving or modifying the GPAT gene.

Plasma lipids are altered in Gaucher disease: Biochemical markers to evaluate therapeutic intervention

Enzyme replacement therapy has been in clinical practice for the non-neuronopathic form of Gaucher disease for 15 years. However, the wide phenotypic variability in this disorder poses challenges to clinicians to assess patient severity and disease progression in order to effectively manage patients. Once therapy is initiated, methods to monitor the complex biochemical changes associated with the disease, and the response of these changes to therapy, are required in order to tailor therapy regimens to individual patients. We have evaluated the suitability of plasma sphingolipids and phospholipids as biochemical markers of disease burden and the efficacy of therapy to reduce that burden. Over 60 lipid species were measured using electrospray ionization-tandem mass spectrometry in plasma from controls and Gaucher patients, pre- and post-therapy. Glucosylceramide, molecular species of phosphatidylglycerol and G M3 ganglioside were elevated in Gaucher disease, whereas species of ceramide, dihexosylceramide and sphingomyelin were decreased. Multivariate analysis enabled us to calculate the combined response of these lipids to therapy in Gaucher patients and correlate them with patient severity. Plasma lipids are proposed to be useful biomarkers for Gaucher disease.

Assessment of the effects of hydrocarbon contamination on the sedimentary bacterial communities and determination of the polar lipid fraction purity: Relevance of intact phospholipid analysis

This in situ study reports on the relevance of intact phospholipids as new biomarkers and highlights the limits of the classical fatty acid analysis after the hydrolysis of the polar lipid fraction. The analysis of polar lipid fractions revealed significant contributions of marine macrophytes with either non-phospholipid compounds (sulfoquinovosyl diacylglycerol) or with both non-bacterial phospholipid classes (phosphatidylcholine) and non-bacterial phospholipid molecular species (some molecular species of phosphatidylglycerol). The analysis of intact phospholipids showed also the predominance of gram-negative bacteria in sediments whereas the analysis of fatty acids alone cannot confirm such information. Lastly, as regard to the effects of petroleum hydrocarbons on the sedimentary bacterial communities, we observed a higher degree of saturation of the fatty acyl chains of the phospholipids extracted from sediment exposed to a massive and chronic crude oil contamination. Such a result was interpreted as an adaptive response resulting in higher membrane rigidity of the bacterial communities.

The origin of phospholipids of the enveloped bacteriophage phi6

The phospholipid class and molecular species compositions of bacteriophage phi6 and its host Pseudomonas syringae were determined quantitatively using TLC and liquid-chromatography/electrospray ionization mass-spectrometry. In addition, the fatty acid compositions of the phospholipids were analyzed by gas-chromatography/mass-spectrometry. The phage contained significantly more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host cytoplasmic (CM) and outer (OM) membranes. In addition, the phospholipid molecular species composition of the viral membrane differed from those of the host membranes, but resembled that of CM more than OM as shown by principal component analysis (PCA). The membrane of phi6 contained more 34:1 and 34:2, and less 32:1 PE and PG molecular species than the host CM or OM. Also, phi6 contained negligible amounts of saturated phospholipid molecular species. These data provide the first biochemical evidence suggesting that phi6 obtains its lipids from the CM. This process is not unselective, but certain phospholipid species are preferentially incorporated in the phage membrane. Common factors leading to similar enrichment of PG in every membrane-containing bacterial virus system studied so far (phi6, PM2, PRD1, PR4, Bam35) are discussed.

Genetic modification of the fatty acid unsaturation of phosphatidylglycerol in chloroplasts alters the sensitivity of tobacco plants to cold stress

ABSTRACTThe cis‐unsaturated molecular species of phosphatidylglycerol (PG) in chloroplasts have been implicated in the chilling sensitivity of plants. Homozygous lines of transgenic tobacco (Nicotiana tabacum) that overexpressed the cDNA for glycerol‐3‐phosphate acyltransferase, a key enzyme in the determination of the extent of cis‐unsaturation of PG, were established from a chilling‐sensitive squash (Cucurbita moschata). In transgenic plants, the proportion of saturated plus trans‐monounsaturated molecular species of PG increased from 24 to 65%. However, this change did not affect the architecture of the chloroplasts. Chilling stress decreased the growth and biomass production of young seedlings of transgenic plants more severely than those of wild‐type plants, and this observation suggests that the changes in the proportion of cis‐unsaturated PG affected not only leaves but also developing plants. Chilling stress also damaged inflorescences. In particular, the abscission of flower buds and inflorescence meristems from transgenic plants occurred more frequently than that from wild‐type plants. Thus, it is likely that decreases in the proportion of cis‐unsaturated PG enhanced the sensitivity to chilling of reproductive organs.

Effect of various temperatures on phosphatidylglycerol biosynthesis in thylakoid membranes.

Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of phosphatidylglycerol (PG) in thylakoid membranes during spinach (Spinacia oleracea) and squash (Cucurbita pepo) cotyledon growth to investigate how the growth temperature affects the PG biosynthesis. Of the 10 molecular species detected, temperature affected mainly the relative content of molecular species containing linolenic acid (18:3) and those containing palmitic acid (16:0) at the sn-1 position of glycerol backbone. Lowering the temperature induced an increase in the former and a decrease in the latter. The relative content of molecular species containing 18:3 or 16:0 at the sn-1 position of the glycerol backbone were correlated with temperature. Our results indicate that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or oleic acid (18:1) and the activity of fatty acid desaturases are greatly affected by temperature. In addition, changes in the relative content of PG molecular species induced by variations in growth temperature depended mainly on the substrate selectivity of GPAT.

Cyclodextrins: A potential tool for studying the role of glycerolipids in photosynthetic membranes

A novel cyclodextrin derivative, i.e., permethylated-alpha-cyclodextrin (PM-alpha-CD), was used for removing glycerolipids from spinach thylakoid membranes and investigating their role in photosynthetic activities. A three-step selective removal of each lipid class was observed in treated membranes. Up to a concentration of 4 mM, PM-alpha-CD (in the presence of 75 microg chlorophyll a+b/mL), PM-alpha-CD displayed a marked selectivity for anionic lipids [sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)] in comparison with galactolipids. At this concentration, half of PG and SQDG were removed. Within this range of concentration, the volume response of treated thylakoids to variation of osmolarity, an indirect mean of verifying the structural integrity of the membrane, was not altered. Similarly, neither photosystem II (PSII) nor photosystem I (PSI) activity was affected. In contrast, the low-temperature fluorescence ratio F695/F740 drastically diminished from 1.45 to about 0.7, essentially due to the decrease of PSII fluorescence. The results derived from the fast fluorescence rise expressed in the form of a spider suggest that the fraction of inactivated (non-Q(A) reducing) reaction centers (RC) increases while the active (Q(A) reducing) RC remained intact. Raising the concentration of PM-alpha-CD from 4 to 7 mM resulted in a progressive but greater diminution of the galactolipid level than that of SQDG and PG. Within this concentration range, the integrity of the membrane was not altered, nor was either PSII or PSI activity, whereas the F695/F740 ratio decreased to about 0.45 as well as the fraction of inactivated RC. At concentrations above 7 mM of PM-alpha-CD, the integrity of the membrane was impaired, resulting in a decrease of both electron transport activities. At all concentrations, PM-alpha-CD did not show any selectivity toward either the acyl chains of the lipid molecules or the molecular species of PG. The results are discussed in terms of the role of glycerolipids in thylakoid membrane function and the relationship of the chemical structure of PM-alpha-CD and its lipid removal capacity.

Relationship between growth temperature and the levels of phosphatidylglycerol (PG) molecular species in thylakoid membranes of squash and spinach cotyledons

Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this study, we investigated the effect of temperature on PG molecular species in thylakoid membranes. Spinach (Spinacia oleracea), a chilling-resistant plant, and squash (Cucurbita pepo), a chilling-sensitive plant, were grown at different temperatures and PG molecular species isolated from developing cotyledons were analyzed by HPLC. The most abundant molecular species in spinach was those containing linolenic acid and that in squash was those containing palmitic acid at the sn-1 position of glycerol backbone. Temperature affected greatly the levels of these two types of molecular species in both plants. Lowering temperature induced an increase in the former at the expense of the latter. The levels of both types of molecular species were linearly correlated with growth temperatures. Our results indicate that the levels of these two molecular species can be regulated to adapt changes in growth temperature in spinach and squash. In addition, the molecular species containing trans-D 3-hexadecenoic acid rapidly decreased when spinach plants were grown at 30° C. It has been reported that these molecular species play a crucial role in the biogenesis and the trimerization of LHCII. Our results suggest that the process could be affected by high levels of linolenic acid containing molecular species at a relatively high temperature.

Do glycerolipids display lateral heterogeneity in the thylakoid membrane?

The lateral heterogeneity of lipids in the thylakoid membrane has been questioned for over 20 yrs. It is generally believed that glycerolipids are asymmetrically distributed within the plane of the membrane. In the present investigation, we isolated several thylakoid membrane domains by using sonication followed by separation in an aqueous dextran-polyethylene glycol two-phase system. This technique, which avoids detergent treatments, allowed us to obtain stroma and grana lamellae vesicles as well as grana central core and grana margin vesicles from thylakoids. The relative distribution of the four lipid classes, i.e., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol, was found to be statistically identical in all four thylakoid fractions and in whole thylakoids. Similarly, the relative amount of fatty acids in each individual lipid and the eight main phosphatidylglycerol molecular species was identical in all thylakoid membrane fractions tested as well as in the intact thylakoid membrane. Based on presently available procedures for obtaining thylakoid subfractions that are unable to discriminate microdomains within the membrane, it is concluded that glycerolipids are evenly distributed within the plane of the thylakoid membrane. These data are discussed in terms of "bulk" and "specific" lipids.

Bacterial cell membrane hydrolysis by secreted phospholipases A 2: a major physiological role of human group IIa sPLA 2 involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A 2 (human sPLA 2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA 2s. This specificity is due to selective penetration of the highly cationic human sPLA 2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA 2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA 2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA 2s. Overall, the results highlight the potential of the human sPLA 2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.