Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.     

Changes in fatty acid composition of lipid classes in developing mustard seed

Maturation of mustard (Sinapis alba) seed proceeds with a sharp decrease in the amounts of palmitic and linoleic acids in the total lipids up to 6 weeks after flowering (WAF). Concomitantly, the concentration of oleic acid increases, reaching a plateau at 4 WAF, which is followed by chain elongation of oleic acid to gadoleic and erucic acids. Compositional changes in constituent fatty acids of individual lipid classes indicate that the very long-chain monounsaturated fatty acids (C₂₀ and C₂₂), as opposed to common long-chain fatty acids (C₁₆ and C₁₈), are metabolized to triacylglycerols mainly by esterification to preformed diacylglycerols and monoacylglycerols, rather than via esterification to glycerol-3-phosphate or lysophosphatidic acids.     

Docosahexaenoic,arachidonic, palmitic,and oleic acids are differentially esterified into phospholipids of frog retina

Docosahexaenoic acid (22:6n-3) is highly enriched in the retina. To determine if retinal cells take up and metabolize fatty acids in a specific manner, retinas from Rana pipiens were incubated for 3 h with an equimolar mixture of tritiated 22:6n-3, arachidonic acid (20:4n-6), palmitic acid, and oleic acid. The radiolabeling of retinal lipids was determined and compared to the endogenous fatty acid content of the lipids. The results showed that in most, but not all, cases, the relative labeling with the four precursor fatty acids was similar to their relative abundance in each glycerolipid. Thus, during retinal glycerolipid synthesis, either through de novo or acyl exchange reactions, fatty acids are incorporated in proportions reflecting their steady-state mass levels. Since other studies with labeled glycerol have shown greater differences between early labeling patterns and molecular species mass, the final incorporation we report may be due primarily to acyl exchange reactions.     

Seasonal Changes of Fatty Acid Compositions in Overwintering Larvae of Rice Stem Borer,Chilo suppressalis (Lepidoptera: Pyralidae)

Cold acclimation and overwintering state can affect fatty acid compositions of insects. To determine compositional change of fatty acids during nondiapause and diapause stages, an experiment was conducted to investigate fatty acid constituents from whole body of C. suppressalis larvae. Five most abundant fatty acids were found to be palmitoleic (35–58%), palmitic (18–44%), oleic (14–23%), stearic (0.5–2.5%) and linoleic acid (0.4–2%). However, linolenic, erucic, lauric and myristic acid were found at lower level. Saturated fatty acids significantly decreased and conversely unsaturated fatty acids increased from August (pre-diapause) to October (initiation of diapause). The increase in seasonal cold hardiness during cold acclimation, exposed at −15°C for 24 h, was related to degree of fatty acid unsaturation. The elevation of palmitoleic acid content at low temperature resulted in an increase in the overall degree of unsaturation in the whole body. These results indicated the importance of unsaturated fatty acids composition to prepare larvae entering diapause phase.     

Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A:1,2-diacylglycerol acyltransferase

The mechanism for the reduced hepatic production of triacylglycerol in the presence of eicosapentaenoic acid was explored in short-term experiments using cultured parenchymal cells and microsomes from rat liver. Oleic, palmitic, stearic, and linoleic acids were the most potent stimulators of triacyl[3H]glycerol synthesis and secretion by hepatocytes, whereas erucic, alpha-linolenic, gamma-linolenic, arachidonic, docosahexaenoic, and eicosapentaenoic acids (in decreasing order) were less stimulatory. There was a linear correlation (r = 0.85, P less than 0.01) between synthesis and secretion of triacyl[3H]glycerol for the fatty acids examined. The extreme and opposite effects of eicosapentaenoic and oleic acids on triacylglycerol metabolism were studied in more detail. With increasing number of free fatty acid molecules bound per molecule of albumin, the rate of synthesis and secretion of triacyl[3H]glycerol increased, most markedly for oleic acid. Cellular uptake of the two fatty acids was similar, but more free eicosapentaenoic acid accumulated intracellularly. Eicosapentaenoic acid caused higher incorporation of [3H]water into phospholipid and lower incorporation into triacylglycerol and cholesteryl ester as compared to oleic acid. No difference was observed between the fatty acids on incorporation into cellular free fatty acids, monoacylglycerol and diacylglycerol. The amount of some 16- and 18-carbon fatty acids in triacylglycerol was significantly higher in the presence of oleic acid compared with eicosapentaenoic acid. Rat liver microsomes in the presence of added 1,2-dioleoyl-glycerol incorporated eicosapentaenoic acid and eicosapentaenoyl-CoA into triacylglycerol to a lesser extent than oleic acid and its CoA derivative. Decreased formation of triacylglycerol was also observed when eicosapentaenoyl-CoA was given together with oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linoleoyl-CoA, linolenoyl-CoA, and arachi-donoyl-CoA had no inhibitory effect. In conclusion, inhibition of acyl-CoA:1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) by eicosapentaenoic acid may be important for reduced synthesis and secretion of triacylglycerol from the liver.     

Fatty Acid synthesis in endosperm of young castor bean seedlings

Enzyme assays on organelles isolated from the endosperm of germinating castor bean (Ricinus communis) by sucrose density gradient centrifugation showed that fatty acid synthesis from [¹⁴C]malonyl-CoA was localized exclusively in the plastids. The optimum pH was 7.7 and the products was mainly free palmitic and oleic acids. Both NADH and NADPH were required as reductants for maximum activity. Acetyl-CoA, and acyl-carrier protein from Escherichia coli increased the rate of fatty acid synthesis, while low O₂ levels suppressed synthesis. In the absence of NADPH or at low O₂ concentration, stearic acid became a major product at the expense of oleic acid. Fatty acid synthesis activity was highest during the first 3 days of germination, preceding the maximum development of mitochondria and glyoxysomes. It is proposed that the plastids are the source of fatty acids incorporated into the membranes of developing organelles.     

High rates of [1-14C]acetate incorporation into the lipid of isolated spinach chloroplasts.

Spinach chloroplasts, isolated by techniques yielding preparations with high O2- evolving activity, showed rates of light-dependent acetate incorporation into lipids 3-4 fold higher than any previously reported. Incorporation rates as high as 500 nmol of acetate/h per mg of chlorophyll were measured in buffered sorbitol solutions containing only NaHCO3 and [1-14C]acetate, and as high as 800 nmol/h per mg of chlorophyll when 0.13 mM-Triton X-100 was also included in the reaction media. The fatty acids synthesized were predominantly oleic (70-80% of the total fatty acid radioactivity) and palmitic (20-25%) with only minor amounts (1-5%) of linoleic acid. Linolenic acid synthesis was not detected in the system in vitro. Free fatty acids accounted for 70-90% of the radioactivity incorporated and the remainder was shared fairly evenly between 1,2-diacylglycerols and polar lipids. Oleic acid constituted 80-90% of the free fatty acids synthesized, but the diacylglycerols and polar lipids contained slightly more palmitic acid than oleic acid. Triton X-100 stimulated the synthesis of diacylglycerols 3-6 fold, but stimulated free fatty acid synthesis only 1-1.5-fold. Added glycerol 1-phosphate stimulated both the synthesis of diacylglycerols and palmitic acid relative to oleic acid, but did not increase acetate incorporation into total chloroplast lipids. CoA and ATP, when added separately, stimulated acetate incorporation into chloroplast lipids to variable extents and had no effect on the types of lipid synthesized, but when added together resulted in 34% of the incorporated acetate appearing in long-chain acyl-CoA. Pyruvate was a much less effective precursor of chloroplast fatty acids than was acetate.     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Acyl-CoA Synthetase in Oilseeds: Fatty Acid Structural Requirements for Activity and Selectivity

The substrate specificities and selectivities of acyl-CoA synthetasesfrom maturing oilseeds were investigated to reveal fatty acidstructures that the enzymes recognize. The synthetases fromrapeseed (Brassica nap us) and castor bean (Ricinus communis)activated palmitic acid 16:0 most rapidly among the saturatedfatty acids tested. Native unsat-urated fatty acids, oleic 18:1cis-9, linoleic 18:2 cis-9,12 and linolenic acid 18:3 m-9,12,15,were all effectively utilized. Palmitoleic acid 16:1 cis-9 wasalso a good substrate, while myristoleic acid 14:1 cis-9 wasa poor substrate. The activation of erucic acid 22:1 cis-13was very slow. Elaidic acid 18:1 trans-9 was utilized at ratessimilar to those of the cis isomer. The efficiencies of petroselinicacid 18:1 cis-6 were half the efficiencies of oleic acid, whilethe rates of activation of m-vaccenic acid 18:1 cw-11 were comparableto those for oleic acid. These findings suggest that acyl-CoAsynthetases of oilseeds producing long-chain fatty acids strictlyrecognize the molecular structures of fatty acids, i.e., thecarbon-chain length between C¹⁶-C¹⁸ and the position of thefirst double bond (     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids

The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after ¹⁴CO₂ feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as “carriers” for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after ¹⁴CO₂ and ¹⁴C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids.     

Palmitic acid metabolism in hepatopancreas of the freshwater shrimp Macrobrachium borellii

• 1.1. The palmitic acid fate as substrate for the synthesis of either glycerides or other fatty acids was studied in vivo and in the microsomal fraction from hepatopancreas of Macrobrachium borellii.
• 2.2. Most of the palmitic acid administered in vivo circulated to the hepatopancreas, being incorporated mainly in the triacylglycerol (TG) fraction.
• 3.3. Palmitic acid transformations into palmitoleic, stearic and oleic acids were observed in the hepatopancreas.
• 4.4. The in vitro biosynthesis of TG in hepatopancreas was more active than in other tissues. In the microsomal fraction, palmitic acid was also incorporated mainly in TG, and followed the α-glycerophosphate pathway.

     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

Trypanosoma cruzi: Fatty acid metabolism in vitro

Trypanosoma cruzi populations, composed primarily of trypomastigote forms, readily converted palmitic acid, linoleic acid, oleic acid, and stearic acid to CO₂. Appreciable amounts of carbon from these four fatty acids were also incorporated into neutral and phospholipid lipids by these parasites. Palmitic acid, a 16 carbon saturated fatty acid, was converted at rates greater than those of the other three fatty acids.     

Changes in the composition of winter rape oil during seed maturation

The course of biosynthesis of fatty acids in the seeds of winter rape (Brassica napus L. ssp.oleifera, f.biennis cv. T?ebí?ská) was investigated. After the termination of flowering seed samples were taken at five intervals, the seeds were divided into 4 fractions according to size, and their weight, water content, oil content and fatty acid composition were determined. The oil content was found to increase in all size categories with time, with the exception of a minute drop when complete maturity is reached. Larger seeds contained more oil. The fatty acid composition changes with time in the individual size fractions almost continuously. The same holds for differences between seed sizes of the same sample. The main change in oil composition consists in the decrease of C₁₈ acids in favour of C₂₂ acids. Greatest decrements during maturation were found with oleic acid, less with linoleic acid. In absolute amounts the quantity of all synthesized acids rises, the greatest rise being observed with C₂₂ acids (i.e. predominantly erucic acid). It follows from the mean rates of synthesis of the individual groups (C₁₆, C₁₈, C₂₀, C₂₂) of fatty acids that the fraction of C₂₂ rate of synthesis increases, while that of the C₁₈ acids decreases with the same speed. The results indicate that the fatty acid synthesis is most intense during the second half of seed maturation, the main role being played by accelerating the synthesis of higher acids, especially of erucic acid.     

Biological Control of Phytopathogenic Fungi by Fatty Acids

The aim of the present study was to evaluate the antifungal activity of fatty acids against phytopathogenic fungi. Two pot experiments were conducted by mixing palmitic and oleic acids in the soil in which poor plant growth was observed. In addition, the antifungal activities of nine fatty acids (butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and linoleic acid) against four phytopathogenic fungi: Alternaria solani, Colletotrichum lagenarium, Fusarium oxysporum f. sp. Cucumerinum, and Fusarium oxysporum f. sp. lycopersici, were assessed by measuring mycelial growth and spore germination via Petri dish assay. The results of the pot experiments showed that the mixture of palmitic and oleic acids enhanced the growth of the seedlings of continuous-tomato and continuous-cucumber. Except for oleic acid, in the Petri dish assay, the fatty acids tested were observed to inhibit the mycelial growth of one or more tested fungi. In addition to the suppression of mycelial growth, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, and palmitic acid showed an inhibitory effect against spore germination and the extent of inhibition varied with both the type of fatty acids, and the fungi. In particular, capric acid displayed strong inhibitory effect against C. lagenarium on the mycelial growth and spore germination. The saturated fatty acids, i.e. palmitic acids, showed stronger antifungal activity than the unsaturated fatty acids, i.e. oleic acid. It suggests that fatty acids might be applicable to exploring for alternative approaches to integrated control of phytopathogens.     

Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteria

The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans Δ11 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis Δ9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria.     

Cellular fatty acid profiles for the differentiation of Penicillium species

Cellular fatty acid composition of eighteen species of Penicillium was studied to investigate its taxonomic usefulness. Many fungi included in this study displayed the same fatty acid composition but differed in relative concentration. Several test species presented the same fatty acid composition but differed in relative concentration. The principal fatty acids were palmitic (16:0), oleic (18:1) and linoleic (18:2). The amount of unsatured fatty acids varied between 68.5% and 78.5%. Multivariate analyses of data showed that it is possible to differentiate some species that belonged to different Penicillium groups. The level of agreement of long chain fatty acids with morphological taxonomy was acceptable.     

Stereospecific distribution of plamitic acid in the triacylglycerols of rat adipocytes. Effects of varying the composition of the substrate fatty acid in vitro

The effects of inclusion of different fatty acids in the medium on the rate of esterification of palmitic acid and its stereospecific distribution among the three positions of the triacyl-sn-glycerols by preparations of rat adipocytes in vitro have been determined. Myristic acid, stearic acid, oleic acid and linoleic acid were used as diluents and the concentration of the combined unesterified fatty acids in the medium was held constant; only the proportion of palmitic acid was varied. The amount of palmitic acid esterified was always linearly related to its relative concentration in the medium and was not significantly affected by the nature of the diluent fatty acid chosen. Constant relative proportions were recovered in triacylglycerols and in intermediates in each instance. The amount of palmitic acid esterified to each of the positions of the triacyl-sn-glycerols was linearly dependent on the relative proportion in the medium but the nature of the relationship was markedly influenced by which fatty acid was present. When stearic acid was present, simple relationships were found over the whole range tested. When either myristic acid, oleic acid or linoleic acid was present, abrupt changes in the manner of esterification of palmitic acid were observed in position sn-1 when the relative concentrations of palmitic acid and the diluent reached critical values, which differed with each fatty acid. In position sn-2 when oleic acid or linoleic acid was present, a similar change was observed, and in position sn-3 it was obtained with myristic acid as diluent. The results are discussed in terms of changes in the relative affinities of the acyltransferases for palmitic acid. Palmitic acid was esterified into various molecular species in proportions that indicated acylation with non-correlative specificity at higher relative concentrations but not at lower.