The presence of odd-chain fatty acids in Drosophila phospholipids

ABSTRACT

Most fatty acids in phospholipids and other lipid species carry an even number of carbon atoms. Also odd-chain fatty acids (OCFAs), such as C15:0 and C17:0, are widespread throughout the living organism. However, the qualitative and quantitative profiles of OCFAs-containing lipids in living organisms remain unclear. Here, we show that OCFAs are present in Drosophila phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and that their level increases in accordance with progression of growth. Furthermore, we found that food-derived propionic acid/propanoic acid (C3:0) is utilized for production of OCFA-containing PC and PE. This study provides the basis for understanding in vivo function of OCFA-containing phospholipids in development and lipid homeostasis.     

Microbial assimilation of hydrocarbons: cellular distribution of fatty acids

The distribution of cellular fatty acids in defined lipid classes was analyzed in Micrococcus cerificans after growth on specified hydrocarbons. Neutral lipid, phospholipid, and cell residue fatty acids were qualitatively and quantitatively determined for M. cerificans grown on nutrient broth, tetradecane (C(14)), pentadecane (C(15)), hexadecane (C(16)), and heptadecane (C(17)), respectively. Percentage of total cellular fatty acid localized in defined lipid classes from cells grown on the above growth substrates was (i) neutral lipid-11.8, 1.81, 7.74, 23.1, and 2%; (ii) phospholipid-74.5, 65, 66.43, 62.1, and 86%; (iii) cell residue lipid-13.5, 33.29, 25.82, 14.78, and 11.9%. Phospholipid fatty acid chain length directly reflected the carbon number of the alkane substrate, with 40, 84, 98, and 77% of the fatty acids being 14, 15, 16, and 17 carbons when cells were grown on C(14), C(15), C(16), and C(17)n-alkanes, respectively. The bound lipids of the cell residue after chloroform-methanol extraction were characterized by 2-hydroxydodecanoic and 2-hydroxytetradecanoic acids plus a broad spectrum of fatty acids ranging from C(10) to C(17) chain length. An increase in total unsaturated fatty acid localized in the phospholipids was noted from cells grown on alkanes greater than 15 carbons long. An extracellular accumulation of free fatty acid (FFA) was demonstrated in hexadecane-grown cultures that was not apparent in non-hydrocarbon-grown cultures. Identification of extracellular FFA demonstrated direct derivation from hexadecane oxidation. Studies supporting inhibition of de novo fatty acid biosynthesis in relationship to extracellular FFA and hexadecane oxidation are described. The ability to alter the fatty acid composition of membrane polar lipids in a predictable manner by the alkane carbon source provides an excellent model system for the investigation of membrane structure-function relationships in M. cerificans.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

Storage and oxidation of long-chain fatty acids in the C57/BL6 mouse heart as measured by NMR spectroscopy

Triglyceride turnover in the isolated C57/BL6 mouse heart was measured by dynamic 13C edit-(1)H observe NMR and the rate of fatty acid oxidation was determined by 13C NMR isotopomer analysis. In the presence of a physiological mixture of substrates, energy was produced in the citric acid cycle by oxidation of long-chain fatty acids (18%), ketones (34%), lactate (24%), pyruvate (7%), and other sources (17%). Exogenous fatty acids appeared in the triglyceride pool at 0.24 micromol/g dry wt/min, similar to the rate of oxidation of long-chain fatty acids, 0.16 micromol/g dry wt/min. Isoproterenol decreased the rate of de novo triglyceride synthesis and increased the rate of fatty acid oxidation.     

Fatty acids in the species of several zygomycete taxa

The composition of fatty acids synthesized de novo by thirty strains of zygomycetes from various taxa was studied. The qualitative fatty acid compositions of the fungal lipids were found to be virtually identical, but there were significant differences in the contents of individual acids. Highly active producers of essential C18 fatty acids, with their content exceeding 30-40% of total fatty acids, were discovered among the fungi of the families Mucoraceae, Pilobolaceae, and Radiomycetaceae. Linoleic acid was found to predominate in the fungi of the genera Radiomyces, Mycotypha, and Circinella, and linolenic acid (identified as its gamma-isomer by gas-liquid chromatography), in the fungi of the genera Absidia, Circinella, Pilaira, and Hesseltinella. The total yield (mg/l) of bioactive acids (C18:3, C18:2, C18:1) varied from 761.4 in Pilaira anomala to 3477.9 in Syncephalastrum racemosum; the total yield of essential acids, from 520.7 in Pilaira anomala to 1154.5 in Hesseltinella vesiculosa; of linoleic acid, from 279.7 in Pilaira anomala to 836.3 in Mycotypha indica; and of linolenic acid, from 120.8 in Mycotypha indica to 708.0 in Hesseltinella vesiculosa. The data on the efficient synthesis of these acids make the actively producing strains promising for biotechnological synthesis of commercially valuable lipids. Linderina pennispora VKM F-1219, a zygomycete of the family Kickxellaceae, which was earlier singled out into the order Kickxellales [12], was shown to differ from zygomycetes of the order Mucorales in having a high content of cis-9-hexadecenoic (palmitoleic) acid, reaching 37.0% of the fatty acid total.     

Fatty acids are precursors of alkylamines in Deinococcus radiodurans.

Deinococcus radiodurans contains novel phospholipids of which the structures of three have been previously described. These three lipids contain both fatty acids and alkylamines. Both the fatty acid and alkylamine constituents were found to be composed of a mixture of species, of which C15, C16, and C17 saturated and monounsaturated alkyl chains predominated. Alkylamines contained a relatively higher proportion of saturated species. Progression of bacterial growth through the mid-log to stationary phases was accompanied by an increase in the proportions of C15 and C17 alkyl chains in both fatty acid and alkylamine constituents. Radiolabeled palmitic acid was found to be rapidly incorporated into both fatty acid and alkylamine components of phosphatidylglyceroylalkylamine, which is the precursor of the more-complex phosphoglycolipids found in major amounts in D. radiodurans. After culturing D. radiodurans in the presence of a mixture of palmitic acids labeled with 14C and 3H in the 1 and 9,10 positions, respectively, the same 14C/3H ratio was recovered in both fatty acid and alkylamine constituents, strongly suggesting that alkylamines are derived from intact fatty acids rather than by a de novo pathway. The results identify a novel product of fatty acid metabolism which has not to date been observed in any other organism.     

Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432

Nocardia globerula strain 432 was able to synthesize triacylglycerols (TAG) during cultivation on 2,6,10,14-tetramethyl pentadecane (pristane) under nitrogen-limiting conditions. Within these cells, 4,8,12-trimethyl tridecanoic acid was the major fatty acid detected. Fatty acids with an odd number of carbon atoms and minor amounts of even-numbered fatty acids were also observed. Experiments carried out with acrylic acid, an inhibitor of beta-oxidation, suggested that odd-numbered fatty acids such as C15:0, C17:0 and 10-methyl C17:0 were synthesized de novo using propionyl-CoA, the beta-oxidation product, as precursor. Although N. globerula 432 incorporated mainly straight chain fatty acids into TAG, the branched fatty acid 4,8,12-trimethyl tridecanoic acid also appeared, to some extent, in the acylglycerols. The importance of TAG biosynthesis by pristane-grown cells of N. globerula strain 432 is discussed.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

Mitochondrial fatty acid synthesis in Trypanosoma brucei

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [(14)C]pyruvate or [(14)C]threonine, either of which is catabolized to [(14)C]acetyl-CoA in the mitochondrion. Although some of the [(14)C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.     

Synthesis of fatty acids in the perfused mouse liver

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of (3)H from (3)H(2)O (1-7mumol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-(14)C]lactic acid and [U-(14)C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of (3)H(2)O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12-16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with (3)H(2)O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.     

Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.     

Molecular and Functional Analysis of Three Fatty Acyl-CoA Reductases with Distinct Substrate Specificities in Copepod Calanus finmarchicus

The marine copepod Calanus finmarchicus constitutes the substantial amount of biomass in the Arctic and Northern seas. It is unique in that this small crustacean accumulates a high level of wax esters as carbon storage which is mainly comprised of 20:1n−9 and 22:1n−11 alcohols (Alc) linked with various kinds of fatty acids, including n−3 polyunsaturated fatty acids. The absence of 20:1n−9 Alc and 22:1n−11 Alc in diatoms and dinoflagellates, the primary food sources of copepods, suggests the existence of de novo biosynthesis of fatty alcohols in C. finmarchinus. Here, we report identification of three genes, CfFAR1, CfFAR2, and CfFAR3, coding for fatty acyl-CoA reductases involved in the conversion of various fatty acyl-CoAs to their corresponding alcohols. Functional characterization of these genes in yeast indicated that CfFAR1 could use a wide range of saturated fatty acids from C18 to C26 as substrates, CfFAR2 had a narrow range of substrates with only very-long-chain saturated fatty acid 24:0 and 26:0, while CfFAR3 was active towards both saturated (16:0 and 18:0) and unsaturated (18:1 and 20:1) fatty acids producing corresponding alcohols. This finding suggested that these three fatty acyl-CoA reductases are likely responsible for de novo synthesis of a series of fatty alcohol moieties of wax esters in C. finmarchicus.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Temperature-induced Changes in the Synthesis of Unsaturated Fatty Acids by Acanthamoeba castellanii

ABSTRACT. Major fatty acid components of Acanthamoeba castellanii lipids extracted after growth at 30°C include myristate, palmitate, stearate and the polyunsaturates linoleate, eicosadienoate, eicosatrienoate and arachidonate, with oleate as the sole major monounsaturated fatty acid. By comparison, growth at 15°C gave increased linoleate, eicosatrienoate and arachidonate, but decreased oleate and palmitate. When the growth temperature was shifted downwards from 30°C to 15°C, increased lipid unsaturation occurred over a period of 24 h; thus decreases of oleate and eicosadienoate were accompanied by increases in linoleate, eicosatrienoate, arachidonate and eicosapentaenoate. An upwards shift from 15°C to 30°C gave negligible alterations in fatty acid composition over a similar period. At 15°C organisms rapidly use [1-¹⁴C] acetate for de novo fatty acid synthesis; stearate is converted via oleate to further desaturation and chain elongation products. Similar short term experiments at 30°C indicate only de novo synthesis and Δ9-desaturation; synthesis of polyunsaturates was a much slower process. Rapid incorporation of [1-¹⁴C] oleate at 30°C was not accompanied by metabolic conversion over two hours, whereas at 15°C n-6 desaturation to linoleate was observed. Temperature shift of organisms from 15°C to 30°C in the presence of [1-¹⁴C] acetate revealed that over half of the fatty acids in newly-synthesised lipids were saturated, but the proportions of unsaturated fatty acids increased with time until the total polyenoate components reached 17% after 22 h. A shift of temperature in the reverse direction gave a corresponding figure of 60% for polyunsaturated fatty acids. These results emphasize the importance of n-6 desaturation in the low temperature adaptation of Acanthamoeba castellanii .     

Regulation of Osteoblast Gene Expression and Phenotype by Polylactide-fatty Acid Surfaces

Cell function is influenced by surface structure and molecules. Molecules that enhance cellular differentiation can be applied to tissue scaffold surfaces to stimulate endogenous tissue regeneration. The application of this approach to bone implants yields surfaces coated with factors (proteins, peptides, etc...) that promote the differentiation of osteoblasts, the cells that make bone. Increased bone formation leads to increased healing and union of the implant with endogenous bone. To obtain better control over surface coating we developed PLLA copolymers with allyl (PLLA-co-DAG) and 3-hydroxypropyl (PLLA-co-HP) side chains to which we can attach functional groups. Given the potential of fatty acids being able to incorporate into lipid bilayers and/or influence gene expression, we grafted different fatty acid side chains to PLLA-co-HP by esterifying the corresponding fatty acids with the PLLA-co-HP 3-hydroxypropyl side chains. The effects of the polymer modifications on osteoblasts were then evaluated. While cellular morphology differed between surface coatings, they did not reflect changes in cellular phenotype. Changes in gene expression were most evident with arachidonate and 3-hydroxypropyl side-chains which exhibited osteoblast differentiating capabilities. Linoleate, myristate, oleate, and stearate ester side-chains did not have a significant influence on osteoblast phenotype. Growth characteristics of osteoblasts did not differ between the fatty acid copolymer films, although cells grown on PLLA-co-HP exhibited a trend toward increased growth. Taken together our findings demonstrate that surface fatty acid composition can impact osteoblast phenotype.     

Metabolism of fatty acids and their incorporation into phospholipids of the mitochondria and endoplasmic reticulum in isolated hepatocytes determined by isolation of fluorescence derivatives

Isolated hepatocytes were incubated in the presence of [14C]palmitic, [14C]linoleic or [14C]linolenic acid and the time-courses of incorporation of radioactivity into phosphatidylcholine and phosphatidylethanolamine of microsomes and mitochondria were followed. For this purpose a procedure was developed for HPLC separation of 9-diazomethylanthracene (ADAM) derivatives of fatty acids. When [14C]palmitic acid was used, the major product of elongation and desaturation was octadecadienoic acid, which accounted for 35-65% of the total radioactivity. Labeled palmitoleic, stearic and oleic acids could also be isolated. In fatty acids which do not participate to any large extent in deacylation-reacylation reactions, the pattern of incorporation was characteristic: a high rate of incorporation into microsomal and a low rate of incorporation into mitochondrial phospholipids during the first 40 min, followed by a decrease in the former and an increase in mitochondrial labeling. This pattern is consistent with the fact that de novo synthesis of these two phospholipids occurs in the endoplasmic reticulum in vivo. When cells were incubated in the presence of [14C]linoleic acid, 70-90% of the radioactivity recovered in phospholipids was in this same form, whereas the remaining label was mainly in arachidonic acid and, to some extent, in eicosatrienoic acid. When hepatocytes were incubated in the presence of [14C]linolenic acid, 70-85% of the radioactivity in isolated phospholipids was associated with linolenic acid. As much as 20% of the label was recovered in docosahexanoic acid and 5-10% in arachidonic acid. In the case of the two latter labeled substrates the exchange reactions seem to dominate over de novo synthesis. For phospholipids synthesized de novo the transfer from the endoplasmic reticulum to mitochondria requires about 3 h.     

Lipid composition and metabolism in megakaryocytes at different stages of maturation

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

Fatty acid composition and primer specificity of de novo fatty acid synthetase in Bacillus globispores, Bacillus insolitus, and Bacillus psychrophilus

The fatty acid compositions of three psychrophilic species of Bacillus were determined by gas--liquid chromatography. The proportions of straight-chain fatty acids, branched-chain fatty acids, and unsaturated fatty acids were found to be 13.3, 86.7, and 26.1% of the total cellular fatty acids for Bacillus globispores, 36.6, 63.4, and 25.1% for Bacillus insolitus, and 6.9, 93.1, and 18.4% for Bacillus psychrophilus, respectively. In all three organisms the de novo fatty acid synthetase specificity towards acyl-CoA primers was butyryl-CoA greater than propionyl-CoA much greater than acetyl-CoA. This shows that B. insolitus, which has an unusually large proportion of straight-chain fatty acids for Bacillus, does not possess a different de novo fatty acid synthetase than the other two organisms. Therefore, the greater proportion of straight-chain fatty acids in B. insolitus may be explained by a large supply of straight-chain primer.