Phospholipid composition of methane-utilizing bacteria.

The phospholipids of Methylococcus capsulatus, Methylosinus trichosporium, La Paz, and OBT were examined in relation to their qualitative and quantitative composition. M. capsulatus exhibited a phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and phosphatidyl-choline. The esterified fatty acids were predominantly C16:0 and C16:1. M. trichosporium, La Paz, and OBT exhibited an essentially identical phospholipid composition consisting of phosphatidylmonomethylethanolamine, phosphatidyl-dimethylethanolamine, phosphatidylcholine, and phosphatidylglycerol. Only trace amounts (less than 1%) of cardiolipin were found in these organisms. The major esterified fatty acid in these organisms was C18:1 (87 to 90%). The monounsaturated fatty acids from all four organisms consisted of both cis and trans isomers, each of which contained delta8, delta9, delta10, and delta11 double-bond positional isomers.     

Incorporation of dietary cis and trans isomers of octadecenoate in lipid classes of liver and hepatoma.

Groups of rats bearing Morris minimal deviation hepatoma 7288CTC were fed a fat-free diet supplemented with either 0.5% safflower oil (diet A), 15% safflower oil or free acids (diets Band C), or 15% safflower oil or free safflower fatty acids (diet D) for 4 weeks. A group of normal rats was also fed diet D. Triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines isolated from livers and hepatomas of animals on each diet were analyzed quantitatively for positional isomers in the cis- and trans-octadecenoate fractions. When sufficient samples could be obtained, the cis- and trans-hexadecenoate fractions were also analyzed. Plasma from normal rats on diet D was analyzed in the same manner. The octadecenoate fractions of all hepatoma and liver lipid classes from animals fed diets A, B, and C were greater than 95% the cis isomers. Trans isomers accounted for approximately 15, 30, 50, and 70% of the octadecenoate fractions isolated from liver triglycerides, cholesteryl esters, phosphatidylcholines, and phosphatidylethanolamines, respectively, of animals fed diet D. In contrast, all hepatoma lipid classes from animals on diet D contained the same approximate percentage of trans isomers (15 to 20%). Oleic and vaccenic acids were the major positional cis-octadecenoate isomers of all liver and hepatoma lipid classes from animals fed diets A, B, and C. The ratios of oleic to vaccenic, unaffected by diets A, B, and C, differed for each lipid class in liver, but the ratios were similar for the two hepatoma neutral lipid classes and for the two phospholipid classes. The cis-octadecenoate fractions from all liver and hepatoma lipid classes of animals fed diet D consisted predominantly of the delta9, delta11, and delta12 isomers. The cis delta10 isomer, which was a major isomer of the diet, was almost excluded from liver, hepatoma, and plasma lipids. The positional isomers of the trans-octadecenoate fractions from liver and hepatoma triglycerides and cholesteryl esters exhibited the same approximate distribution as the trans fatty acids of diet D. In contrast, the 10-trans-octadecenoate, like 10-cis-octadecenoate, was almost excluded from the phospholipids of liver and plasma. Unlike liver, the hepatoma phospholipids contained 10-trans-octadecenoate at approximately half the percentage of neutral lipids. Because diet D contained no hexadecenoic fatty acids, the occurrence of trans-hexadecenoate isomers in liver and plasma lipids indicated a chain shortening process. Predominance of the 8-trans-hexadecenoate isomer indicated a preference of the 10-trans-octadecenoate isomer for chain shortening.     

The Bacillus subtilis acyl lipid desaturase is a delta5 desaturase

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions delta5, delta7, and delta9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the delta5 position, regardless of the growth temperature and the length chain of the fatty acids.     

Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopropane fatty acids and alk-1'-enyl ethers by capillary column chromatography

The positional isomers of the cyclopropane fatty acids of Clostridium butyricum phospholipids have been analyzed by capillary column gas-liquid chromatography. Greater than 95% of the methylenehexadecanoic acids was the 9,10 isomer. On the other hand, 60-70% of the hexadecenoic acid precursors was the Delta(7) isomer, and the remainder was the Delta(9) isomer. Of the methyleneoctadecanoic acids 75-80% was the 11,12 isomer, with the remainder being the 9,10 isomer. There were approximately equal amounts of the Delta(9)- and Delta(11)-octadecenoic acids in the phospholipids. This study reveals a surprisingly strong specificity of the cyclopropane synthetase for the (n-7) series of monoenoic fatty acids. An analysis by capillary column chromatography of the monoenoic and cyclopropane aldehyde dimethylacetals derived from the plasmalogens (1-alk-1'-enyl-2-acyl-glycero-phosphatides) of C. butyricum revealed the presence of the same positional isomeric mixtures of the 16- and 18-carbon monoenoic residues in approximately the same ratios as were found in the fatty acids. In the formation of the cyclopropane alk-1'-enyl ethers there was also specificity for the (n-7) series, but it was not as strong as that seen in the fatty acids. The ratio of the 7,8 isomer to the 9,10 isomer was higher in the methyl-enehexadecanals than in the corresponding fatty acids. This paper extends the use of Golay capillary columns to the analysis of the positional isomers of plasmalogen aldehydes as their dimethylacetal derivatives.     

Nonesterified fatty acids inhibit iron-dependent lipid peroxidation

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.     

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions.

Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids.     

Cyclic fatty esters: Stereochemistry of monounsaturated products from the hydrogenation and reduction of 9-(6-propyl-3-cyclohenxenyl)-8-nonenoic acid or its ester

Diunsaturated, C-18 cyclic fatty acid methyl esters (CFAME) were previously synthesized as model derivatives for characterization and biological evaluation of cyclic fatty acids (CFA) formed in heat-abused vegetable oils. The propyl substituted, diunsaturated CFMAE (I) was selectively reduced to prepare two monounsaturated, positional isomers with the double bond located either in the ester substituent (alkene isomer II) or in the ring (cyclohexene isomer III). The stereochemistry of these monounsaturated products was investigated by capillary GLC and NMR. Capillary GLC showed that each positional isomer was a mixture of two ‘ring’ isomers (i.e. a mixture of two isomers with side chains either cis or trans). The ring double bond in diene I was readily hydrogenated with various metal catalysts, and no cyclohexene isomer III was detected in the product. Platinum oxide poisoned with Ph₃P was the most selective catalyst examined to convert diene I to monoene II. Diimide reduction was the only method foud to reduce selectively the double bond in the ester side chain of diene I. This diimide reduction was facilitated when the Z-double bond in the side chain was isomerized to E-double bond with p-toluenesulfinic acid. Cyclohexene isomer III and alkene isomer II were separated by argentation HPLC. These two isomeric monoenes were characterized by GC-MS, capillary GLC, micro-ozonolysis, IR and NMR. Catalytic hydrogenation with Ph₃P-poisoned PtO₂ and diimide reduction of the diunsaturated cyclic ester may provide useful methods to synthesize and label monounsaturated cyclic fatty esters.     

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis.

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.     

Chlorinated fatty acid distribution in Mycobacterium convolutum phospholipids after growth on 1-chlorohexadecane.

The composition of phospholipids from Mycobacterium convolutum R22 was determined after growth at two temperatures (20 and 30 degrees C) with 1-chlorohexadecane as the substrate. Comparisons were made with the phospholipids of cells grown on n-hexadecane. Phosphatidylinositolmannosides and phosphatidylethanolamine (PE) were the major phospholipids in n-hexadecane-grown cells. In 1-chlorohexadecane-grown cells, phosphatidylinositolmannosides were approximately half of the total phospholipids, with lesser amounts of PE and cardiolipin (CL). The relative level of PE was greater at 20 degrees C (versus that at 30 degrees C) after growth on either substrate. A determination was made of structure and positional distribution of constituent fatty acid in both CL and PE. The relative amount of unsaturated fatty acid was higher at 20 degrees C. There were two C16:1 fatty acids (C16:1 delta 9 and C16:1 delta 11), and these had positional preferences in both CL and PE. The positional sites of chlorinated fatty acids differed in both CL and PE at the two temperatures. The results confirm that microorganisms can specifically distribute chlorinated fatty acids into cellular phospholipids.     

Mechanism of ethanol-induced changes in lipid composition of Escherichia coli: inhibition of saturated fatty acid synthesis in vivo.

The in vivo effects of ethanol on lipid synthesis in Escherichia coli have been examined. Under conditions which uncoupled fatty acid synthesis from phospholipid synthesis, ethanol decreased the amount of saturated fatty acids synthesized but had little effect on the selectivity of their incorporation into phospholipids. In the absence of fatty acid degradation and unsaturated fatty acid synthesis, E. coli was still able to adapt its membrane lipids to ethanol, while the inhibition of total fatty acid synthesis eliminated this response. During growth in the presence of ethanol, strain K1060 (an unsaturated fatty acid auxotroph) incorporated an increased amount of exogenous heptadecanoic acid (17:0) to compensate for the reduction in palmitic acid (16:0) available from biosynthesis. Thus, our results indicate that the reduced levels of saturated fatty acids observed in the phospholipids of E. coli following growth in the presence of ethanol result primarily from a decrease in the amounts of saturated fatty acids available for phospholipid synthesis.     

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.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds

The polyenoic fatty acid isomerase from Propioniumbacterium acnes (PAI) was expressed in E. coli and biochemically characterized. PAI catalyzes the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating (10E,12Z)-conjugated linoleic acid (CLA). PAI accepted a wide range of free polyunsaturated fatty acids as substrates ranging from 18:2 fatty acids to 22:6, converting them to fatty acids with two or three conjugated double bonds. For expression of PAI in yeast the PAI-sequence encoding 20 N-terminal amino acid residues was altered for optimal codon usage, yielding codon optimized PAI (coPAI). The percentage of 10,12-CLA of total esterified fatty acids was 8 times higher in yeast transformed with coPAI than in cells transformed with PAI. CLA was detected in amounts up to 5.7% of total free fatty acids in yeast transformed with coPAI but none was detected in yeast transformed with PAI. PAI or coPAI under the control of the constitutive CaMV 35S promoter or the seed-specific USP promoter was transformed into tobacco plants. CLA was only detected in seeds in coPAI-transgenic plants. The amount of CLA detected in esterified fatty acids was up to 0.3%, in free fatty acids up to 15%.     

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.     

19F nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Orientational order in the presence of a series of positional isomers of cis-octadecenoic acid

The 19F nuclear magnetic resonance (NMR) spectra of membranes of Acholeplasma laidlawii B enriched with one of a series of positional isomers of cis-octadecenoic acid plus small amounts of one of a number of isomers of monofluoropalmitic acid were interpreted in terms of an orientational order parameter (Smol). The variation of Smol with the position of the fluorine label in the liquid-crystalline state yielded an "order profile" with characteristics similar to those obtained via 2H NMR and which was relatively invariant regardless of the site of cis unsaturation. In the gel state, values of Smol approached the theoretical maximum, and the order profiles in the presence of different isomeric cis-octadecenoic acids displayed distinct dissimilarities. When the cis double bond was located proximal to the methyl terminus of the fatty acyl chain, a gradient of order across the bilayer was still evident in the gel state. When the cis double bond was located near the carbonyl head group, values of Smol were approximately equal at all chain positions. These observations were interpreted as indicating that in the gel state the stringency of packing restrictions is still subject to variation across the width of the bilayer. Relative overall orientational order among all isomers examined (specifically, 18:1c delta 4, delta 5, delta 6, delta 7, delta 8, delta 9, delta 10, delta 11, delta 12, delta 13, delta 14, and delta 15) varied directly as a function of proximity to the lipid gel to liquid-crystalline phase transition (Tm) (determined via differential scanning calorimetry) when compared at a constant temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.