Role of autocide AMI in development of Myxococcus xanthus.

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.     

Cell-density-dependent killing of Myxococcus xanthus by autocide AMV.

Autocide AMV of Myxococcus xanthus was purified and identified as phosphatidylethanolamine. Alkaline hydrolysis of AMV yielded a high proportion of mono- and diunsaturated fatty acids. The bactericidal activity of AMV on M. xanthus depended upon the density of target cells: the greater the cell density, the greater the killing by AMV. For example, at 2 U of AMV per ml, 0, 50, and 99% killing was measured with 2 X 10(4), 2 X 10(5), and 2 X 10(7) target cells per ml, respectively. The cell-density-dependent activity of AMV was also observed on solid medium. Studies with model lipid compounds suggest that the inhibitory activity of AMV is due to the fatty acid moiety, released from phosphatidylethanolamine by the concerted (enzymatic) activity of many cells. Mutants of M. xanthus selected for resistance to AMI (a mixture of fatty acids) were also resistant to AMV. The possible role of AMV in developmental lysis is discussed.     

Lysophosphatidylethanolamine is a substrate for the short-chain alcohol dehydrogenase SocA from Myxococcus xanthus

Short-chain alcohol dehydrogenases (SCADHs) synthesize a variety of intercellular signals and other chemically diverse products. It is difficult to predict the substrate of a SCADH on the basis of amino acid sequence homology, as the substrates are not known for most SCADHs. In Myxococcus xanthus, the SCADH CsgA is responsible for C signaling during fruiting body development, although the mechanism is unclear. Overexpression of the SCADH SocA compensates for the lack of CsgA and restores development and C signaling in csgA mutants. The potential of SocA in generating the C signal enzymatically was explored by developing a dehydrogenase assay-based screen to purify the SocA substrate(s). A SocA substrate was extracted from M. xanthus cells with acidified ethyl acetate and sequentially purified by solid-phase extraction on silica gel and by reverse-phase high-performance liquid chromatography. The fraction with the highest SocA dehydrogenase activity contained the lysophospholipid 1-acyl 2-hydroxy-sn-glycerophosphoethanolamine (lyso-PE) as indicated by the fragment ions and a phosphatidylethanolamine-specific neutral loss scan following liquid chromatography coupled to mass spectrometry. The abundant lysophospholipid with the mass m/z 450 (molecular ion [M-H]-) had a monounsaturated acyl chain with 16 carbons. SocA oxidizes lyso-PE containing either saturated or unsaturated fatty acids but exhibits poor activity on l-alpha-glycerophosphorylethanolamine, suggesting that an acyl chain is important for activity. Of the five different head groups, only ethanolamine showed appreciable activity. The apparent Km and Vmax for lyso-PE 18:1 were 116 microM and 875 micromol min(-1) mg(-1), respectively. The catalytic efficiency (k(cat)/Km) was 1 x 10(8) M(-1) s(-1). The proposed product, 1-acyloxy-3-(2-aminoethylphosphatyl) acetone was unstable, and the fragmented products were unable to rescue csgA mutant development. The active fraction from thin-layer chromatography also contained an unidentified SocA substrate that had morphogenic properties.     

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Heart phospholipid content and fatty acid composition in the rat after feeding different lipid supplemented diets.

The heart phospholipid content and fatty acid composition were examined in adult rats after four weeks of feeding lipid-supplemented diets (20 g % w/w) containing sunflower oil-lard (1:1) mixture (SL group) or margarine (M group). Our results showed a decreased cardiolipin content and distribution in both experimental groups and an increased lysophosphatidylcholine and phosphatidylcholine content and distribution in the SL group with a tendency to lower phosphatidylcholine/phospatidylethanolamine ratio in both experimental groups. In the SL group, the content of saturated fatty acids was higher and that of monounsaturated fatty acids was lower than in the control group. The M group showed inverse results. The content of saturated fatty acids was lower and that of monounsaturated was higher than in the control group. Polyunsaturated n-6 fatty acids were decreased in both experimental groups and n-3 fatty acids were increased in the M group. Feeding lipid-supplemented diets reduced n-6/n-3 and 20:4/22:6 ratios in the M group. The polyunsaturated/saturated fatty acid ratio was lower in the SL and higher in indicating the M group than in the control group. Our results are in agreement with the other reports indicating that the heart is sensitive to diet-induced lipid alterations.     

Concentrates of DHA from fish oil by selective esterification of cholesterol by immobilized isoforms of lipase from Candida rugosa

Two lipases (Lip A and Lip B), were purified from a commercial lipase preparation produced by Candida rugosa and partially characterized. The purified lipases were immobilized on Duolite A 568 and used in the selective esterification of cholesterol with free fatty acids from sardine fish oil. The results showed that Lip A and Lip B preferentially esterified saturated and monounsaturated fatty acids allowing a 3.4-fold (Lip B, 24 h) and 4-fold (Lip A, 10 h) enrichment of docosahexaenoic acid in the remaining free fatty acid fraction. Selectivity towards eicosapentaenoic acid was less pronounced. By this selective esterification docosahexaenoic acid was concentrated from 7.4 to 32% with a recovery of 95% of its initial content in sardine fish oil.     

Isolation and Anti-Fatty Liver Activity of a Novel Cerebroside from the Sea Cucumber Acaudina molpadioides

Cerebrosides are a kind of important bioactive substance in sea cucumber. A novel cerebroside, AMC-2, was purified from the less-polar lipid fraction of the sea cucumber Acaudina molpadioides by repeated column chromatography. The major structure of AMC-2 was analyzed by gas chromatography-mass spectra. The amide-linked fatty acid unit was confirmed to be four saturated and monounsaturated α-hydroxy fatty acids, the long-chain base was dihydroxy sphingoid base with one double bond, and the glycosyl group was glucose. We also investigated the anti-fatty liver activity of AMC-2 in rats with fatty liver induced by orotic acid. AMC-2 significantly reduced hepatic triglyceride (TG) and total cholesterol (TC) levels at a diet supplement of 0.03% and 0.006%. The indexes of stearoyl-CoA desaturase (SCD) activity and mRNA expression were significantly decreased by AMC-2. This indicates that AMC-2 ameliorated nonalcoholic fatty liver disease (NAFLD) through suppression of SCD activity and impaired the biosynthesis of monounsaturated fatty acids in the livers of the rats.     

Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.     

Characterization of trimethylsilyl derivatives of cerebrosides by direct inlet-chemical ionization mass spectrometry

Submicrogram quantities of trimethylsilyl derivatives of cerebrosides obtained from the spleen of a patient with Gaucher's disease and from bovine brain were analyzed by direct probe inlet-chemical ionization mass spectrometry, using isobutane as the reagent gas. Quasimolecular ions (QM+, M + 73) and other recognizable fragment ions produced by the successive elimination of trimethylsilanol and sugar residue gave useful information about fatty acid compositions. These ions could also be utilized for qualitative analyses of the molecular species of cerebrosides. Cerebrosides with non-hydroxy and hydroxy fatty acids could be discriminated from each other by comparing the intensities of their quasimolecular ions. Cerebrosides with saturated and monounsaturated fatty acids could also be discrimnated from each other, because the mass number decreased by two mass units in cerebrosides with monounsaturated fatty acids. It was concluded that structural information and molecular species determination could be obtained from small amounts of purified cerebrosides.     

Lipid composition of the nitrogen starved green alga Neochloris oleoabundans

Neochloris oleoabundans has been cultivated in mineral medium deficient in nitrogen. The yield of lipids was 35–54% of cell dry weight. Triglycerides comprised 80% of the total lipids. Aliphatic hydrocarbons, sterols, pigments, glycolipids and phospholipids comprised the remaining lipid fraction. Saturated, monounsaturated and diunsaturated octodecanoic acid represented approximately one-half of the total fatty acids.     

Fatty Acid Composition of Endoneurium and Perineurium from Adult Rat Sciatic Nerve

Reports on lipid composition of peripheral nervous system have generally been restricted to the saturated fatty acids of the endoneurium. In this work we attempt to determine the fatty acid composition of the different lipid classes in both endo- and perineurium from sciatic nerve microdissection on adult rats. Unsaturated fatty acids were found to make up around 60% of total fatty acids in samples of endoneurium and perineurium, with monounsaturated fatty acids forming 40-50% of total unsaturated fatty acid content. Although the same fatty acids were present in both tissues there was a striking difference in C 18:1 (n-9) and C 18:2 (n-6) ratio between endoneurium and perineurium, which is particularly rich in linoleic acid. The nonpolar perineurial lipids were found to be richest in linoleic acid. Phospholipids were present in the perineurium, and they contained high proportions of saturated and medium-chain monounsaturated fatty acids.     

Manipulation of Plasma Membrane Fatty Acid Composition of Fetal Rat Brain Cells Grown in a Serum-Free Denned Medium

Modifications of plasma membrane acyl-linked phospholipid fatty acid composition were produced by supplementing the culture medium with essential fatty acids. The plasma membrane fraction was purified by Percoll gradient centrifugation from dissociated fetal rat brain cells grown in a serum-free culture medium. Both the concentration dependence and the time course of the modifications were examined. Supplementation of the medium with essential polyunsaturated fatty acid, linolenic acid (18:3 omega 3) or linoleic acid (18:2 omega 6), produced incorporation of the elongated and desaturated products of omega 3 or omega 6 class, respectively, i.e., the incorporation was class specific. Within each class, the most unsaturated and elongated members, i.e., terminal members, were preferentially incorporated until they reached a maximum concentration within 6-7 days. At higher concentrations of supplemented fatty acids, additional class specific incorporation in plasma membrane was produced by an increase in the concentration of intermediate members. At the same time, the concentration of monounsaturated fatty acids declined and that of saturated fatty acids remained unchanged. The modifications in fatty acid composition were reversible, with the time course similar to that of incorporation. The total plasma membrane phospholipid and sterol contents did not change with alterations of fatty acid composition, but did change with time in culture. This preparation should prove useful for investigating the role of polyunsaturated fatty acids in brain cell functions, including neuronal excitability.     

Lipophilic Antioxidants in Human Sebum and Aging

Skin surface lipids (SSL), a very complex mixture of sebum mixed to small amounts of epidermal lipids, mantle the human epidermis, thus representing the outermost protection of the body against exogenous oxidative insults. The present work is a systematic and quantitative analysis of upper-chest SSL and their content in antioxidants in 100 healthy volunteers, divided into five age groups using TLC, HPLC, and GC-MS methods. Further, the effect of exposing SSL in vitro to increasing doses of UV irradiation was examined. Straight monounsaturated and diunsaturated as well as branched monounsaturated fatty acids of triglycerides and pooled fractions were found to be higher at maturity than in childhood and in advancing age. Diunsaturated fatty acids were below 3% of the total and constituted exclusively of C18:2 &#106 5,8 , C20:2 &#106 7,10 , C18:2 &#106 9,12 . Squalene, vitamin E (vit. E) and Coenzyme Q 10 (CoQ 10 ) were found to increase from childhood to maturity to decrease again significantly in old age. Vitamin E and CoQ 10 were the only known lipophilic antioxidants present in SSL. In spite of their low levels they were found to synergically inhibit the UV induced depletion of squalene, cholesterol and of unsaturated fatty acids of SSL. In fact, exposure of SSL to increasing amounts of UV irradiation led preferentially to lowering of the levels of vit. E and CoQ 10 . Four minimal erythema dose (MED) (5.6 J/cm 2 ) were able to deplete 84% vit. E and 70% ubiquinone, and only 13% squalene. Diunsaturated and monounsaturated fatty acids as well as cholesterol were unaffected even following 10 MED UV exposures, which produced a 26% loss of squalene. The same UV dose when applied in the absence of vit. E and CoQ 10 produced a 90% decrease of squalene.     

Lipophilic antioxidants in human sebum and aging

Skin surface lipids (SSL), a very complex mixture of sebum mixed to small amounts of epidermal lipids, mantle the human epidermis, thus representing the outermost protection of the body against exogenous oxidative insults. The present work is a systematic and quantitative analysis of upper-chest SSL and their content in antioxidants in 100 healthy volunteers, divided into five age groups using TLC, HPLC, and GC-MS methods. Further, the effect of exposing SSL in vitro to increasing doses of UV irradiation was examined. Straight monounsaturated and diunsaturated as well as branched monounsaturated fatty acids of triglycerides and pooled fractions were found to be higher at maturity than in childhood and in advancing age. Diunsaturated fatty acids were below 3% of the total and constituted exclusively of C18:2 Δ5,8 , C20:2 Δ7,10 , C18:2 Δ9,12 . Squalene, vitamin E (vit. E) and Coenzyme Q 10 (CoQ 10 ) were found to increase from childhood to maturity to decrease again significantly in old age. Vitamin E and CoQ 10 were the only known lipophilic antioxidants present in SSL. In spite of their low levels they were found to synergically inhibit the UV induced depletion of squalene, cholesterol and of unsaturated fatty acids of SSL. In fact, exposure of SSL to increasing amounts of UV irradiation led preferentially to lowering of the levels of vit. E and CoQ 10 . Four minimal erythema dose (MED) (5.6 J/cm 2 ) were able to deplete 84% vit. E and 70% ubiquinone, and only 13% squalene. Diunsaturated and monounsaturated fatty acids as well as cholesterol were unaffected even following 10 MED UV exposures, which produced a 26% loss of squalene. The same UV dose when applied in the absence of vit. E and CoQ 10 produced a 90% decrease of squalene.     

Determination of double bond positions of unsaturated fatty acids by a chemical ionization mass spectrometry computer system

After stereospecific oxidation, trimethylsilylated methyl esters of mono- and diunsaturated fatty acids were analyzed by combined gas-liquid chromatography-chemical ionization mass spectrometry. The positions of original double bonds were deduced from the fragment ions produced by the cleavage of the carbon-carbon bond between two trimethylsilyl ethers. These fragment ions were recorded at m/e 187 and 259 in the case of 16:1(n-7), at m/e 187 and 287 in the case of 18:1(n-7), at m/e 215 and 259 in the case of 18:1(n-9), and at m/e 172 and 259 in the case of 18:2(n-6), respectively. The diastereoisomers of monounsaturated fatty acids can be discriminated by comparing the intensities of the fragment ions at m/e 253 and 285 in the case of 16:1 and at m/e 281 and 313 in the case of 18:1. The diastereosiomers of diunsaturated fatty acids may also be distinguished from each other by comparing the intensities of the fragment ions formed by the loss of trimethylsilyl function from the characteristic ions. Mono- and diunsaturated fatty acids in liver may be dominantly in cis- 18:1(n-1) and cis-cis-18:2(n-6) as determined by a mass chromatographic technique.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

Cutins of Malus pumila fruits and leaves

The cutins of fruits and leaves of four apple cultivars have been analysed using TLC, GLC and GC-MS. They are similarly composed of saturated, monounsaturated and diunsaturated fatty, hydroxy-fatty and epoxyhydroxy-fatty acids. The most abundant monomers are 18-hydroxyoctadeca-9,12-dienoic, 10,16-dihydroxyhexadecanoic, 9,10-epoxy-18-hydroxyoctadec-12-enoic, 9,10-epoxy-18-hydroxyoctadecanoic and 9,10,18-trihydroxyoctadecanoic acids. The fruit cutins have high contents of epoxides (35–40%) and unsaturated components ( > 40%) and C₁₈ compounds predominate over C₁₆. The leaf cutins contain smaller amounts of unsaturated components than the fruits and higher proportions of C₁₆ compounds. The adaxial leaf cutin differs in composition from the abaxial. 10,16-Dihydroxyhexadecanoic and 9,10-epoxy-18-hydroxoctadecanoic acids are the major constituents (each ca. 30%) of the adaxial leaf cutin and 10,16-dihydroxyhexadecanoic acid (55–65%) predominates in the abaxial.     

The identification of aminoalkylphosphonyl cerebrosides in the marine gastropod, Monodonta labio.

From Monodonta labio a mixture of aminoalkylphosphonyl cerebrosides was isolated in a yield of 1.0% of the total complex lipids. The aminoalkylphosphonyl cerebrosides were found to be composed of a major portion of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactopyranosyl]ceramide and a minor portion of 1-O-[6'-O-(aminoethylphosphonyl)galactopyranosyl]ceramide. Palmitic (49.7%) and 2-hydroxy palmitic (12.2%) acids were the main fatty acid constituents and a small amount of oleic acid was also detected. The long chain bases had a characteristic pattern with 16-22 carbon chain lengths, and were classified into four groups: normal dihydroxy monounsaturated bases (40.6%), branched dihydroxy monounsaturated bases (34.0%), dihydroxy diunsaturated bases (22.0%), and trihydroxy bases (2.2%). Among them dihydroxy 18 : 1 and 18 : 2 and branched 19 : 1 bases were predominant.     

Biosynthesis of C30 to C56 fatty acids by an extract of Mycobacterium tuberculosis H37Ra.

A 10,800 X g supernatant fraction from disrupted cells of Mycobacterium tuberculosis H37Ra was incubated with [2-14C]malonate to produce labeled long-chain fatty acids upon saponification. These acids were derivatized to the p-bromophenacyl ester and separated into the nonmycolic saturated, monounsaturated, and multiunsaturated esters by argentation thin-layer chromatography. Each of these fractions was then analyzed by high-performance liquid chromatography by using a C18-bonded silica cartridge and a mobile phase of a linear gradient of 0 to 70% p-dioxane in acetonitrile. The results showed that the cell-free system is able to synthesize both saturated and monounsaturated fatty acids of the sizes C30 to C40 and C48 to C56. This latter series was strikingly similar to meromycolic acid, a putative precursor of mycolic acid. When acetate or oleate was used as the labeled substrate, the major products were no longer than C32. When palmitate was used as the labeled substrate, the saturated acids ranged in size from C18 to C32, whereas the monounsaturated products contained C18, C26 to C30 and C40 fatty acids. Fatty acids greater than C40 were also detected. When methyl-labeled S-adenosyl-L-methionine was used as the substrate, the methyl group was incorporated into short-chain and C48 to C56 fatty acids. Unlabeled malonyl-coenzyme A was included in all of these reactions. This cell-free system was not able to synthesize mycolic acid (final product) or its keto derivative (intermediate product). However, since the meromycolate-like C48 to C56 fatty acids were synthesized, we suggest that the present system is able to take the synthesis to a point before the alpha-alkyl condensation reaction.