Influence of unsaturated fatty acid membrane component on sensitivity of an Escherichia coli fatty acid auxotroph to conditions of nutrient depletion

The unsaturated fatty acid auxotroph Escherichia coli AK7 was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18:3) to vary the degree of unsaturation of cell membrane lipids. The susceptibility of oleic acid- and linolenic acid-grown cells to starvation at 37 degrees C in 154 mM NaCl was compared following the decline in the number of CFU by plating the cells on agar medium. The decline in CFU was faster for linolenic acid-than for oleic acid-grown cells, but it was not indicative of cell death, since culturable CFU was recovered after respirable substrate was added to the starved cell suspension. Cell envelope microviscosity (determined by fluorescence polarization) of oleic acid- and linolenic acid-grown cells was equal in the presence of a respirable substrate, but in its absence the microviscosity of linolenic acid-grown cells was lower than that of oleic acid-grown cells. The results suggest that cell envelope microviscosity is an important factor in determining the sensitivity of E. coli to conditions of nutrient depletion.     

Effect of di-n-octyl phthalate on fatty acid composition of phosphatidylcholine in Tetrahymena

We examined the effect of di-n-octyl phthalate (DOP) on fatty acid composition of phosphatidylcholine (PC) in Tetrahymena pyriformis NT-1. When Tetrahymena cells were grown in DOP-containing proteose peptone medium, the cell growth was repressed. This repression was attended by decreases in the PC content of the cells and decreases in oleic (18:1), linoleic (18:2) and linolenic (18:3) acids of PC and an increase in palmitoleic acid (16:1). The ratio of 18:1/stearic acid (18:0) of PC in cells grown in DOP-containing medium was lower than that of control cells, while the ratio of 16:1/palmitic acid (16:0) was higher than that of control. On the other hand, no changes in the ratios of 18:2/18:1 and 18:3/18:2 were observed. The activity of microsomal stearoyl-CoA desaturase from cells grown with DOP (0.63 mumol/ml medium) decreased to 27% of that from control cells, while the microsomal palmitoyl-CoA desaturase activity increased to 210% of the control value. By the addition of dioleoyl glyceride to the DOP-containing medium, the effects of DOP on Tetrahymena cells were completely blocked. These results suggest that the changes in fatty acid composition of PC may be due to the alteration of the substrate specificity of microsomal delta 9-desaturase, and the decrease in stearoyl-CoA desaturase activity may be a cause for the cell growth repression.     

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.     

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 (     

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.     

Multivariate approach to evaluating the fatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassica napus L.)

The fatty acid composition of oil of the zero erucic acid commercial Brassica napus L. is typical for this species. It is rich in oleic acid and contains moderate levels of linoleic and linolenic acid. For human nutrition, it is advantageous primarily to obtain the highest possible content of oleic acid and to maintain the 2:1 ratio of linoleic to linolenic acid, while preserving the average total content of saturated acids. Uni- and multivariate analyses of variance were used for evaluation of doubled haploid lines of winter oilseed rape in respect of five fatty acids: palmitic (C16:0), stearic (C18:0) oleic (C18:1), linoleic (C18:2) and linolenic (C18:3). Some proposals of studying doubled haploid (DH) lines with the use of canonical transformation were also given. In MANOVA, the five original variables (individual fatty acids) were replaced by three 'new' variables (combinations of these acids) and used to evaluate DH lines with respect to the requirements concerning the nutritional role of fatty acids. The first variable was the total content of the saturated acids (C16:0 + C18:0), the second (unchanging) was the content of the monounsaturated acid C18:1, and the third was the difference between polyunsaturated acids, i.e. between linoleic acid, and the doubled content of linolenic acid (C18:2 - 2 x C18:3).     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Starved cells of the fatty acid auxotroph Escherichia coli AK7 develop abnormal sensitivity to media with low osmolarity.

The unsaturated fatty acid auxotroph Escherichia coli AK7 supplied with linolenic acid, while appearing normal during logarithmic growth, showed a fast decline in CFU during starvation as a result of an osmotic downshift when transferred to standard agar plates unsupplemented with an osmolyte such as 300 mosM sucrose or salt (NaCl or KCl). The starved cells could recover their osmoresistance when an energy source was added to the starvation medium.     

The hydrogenation of unsaturated fatty acids by five bacterial isolates from the sheep rumen, including a new species.

Five strictly anaerobic bacteria able to hydrogenate unsaturated fatty acids were isolated from sheep rumen. One was characterized as Ruminococcus albus, two as Eubacterium spp. and two as Fusocillus spp., one of which is named as a new species. The Fusocillus organisms were able to hydrogenate oleic acid and linoleic acid to stearic acid, and linolenic acid to cis-octadec-15-enoic acid. The R. albus and the two Eubacteria did not hydrogenate oleic acid but converted linoleic and linolenic acids to a mixture of octadecenoic acids; trans-octadec-II-enoic acid predominated but several isomeric cis and trans octadecenoic acids were produced together with isomers of non-conjugated octadecadienoic acids. The intermediate and final products of hydrogenation by each organism were compatible with the results from mixed rumen bacteria.     

Lipid utilization in adult Pieris brassicae with special reference to the rôle of linolenic acid

Fatty acids of adult Pieris brassicae and the incorporation of dietary linolenic acid-1-¹⁴C into adult (and egg) lipids were analysed 1 and 9 days after ecdysis. Females grown on a leaf diet retained palmitic, palmitoleic, and oleic acids but lost linoleic and linolenic acids during adult life, while males utilized their fatty acids more evenly. On an artificial diet both sexes retained palmitic acid but utilized palmitoleic and oleic acids. In both cases females laid eggs with a high palmitic and oleic acid content.Analysis of thorax flight muscles (artificial diet) revealed that 67·9% of the lipids in 1-day females and 83·6% in 9-day females was phospholipid (PL). During adult life linolenic acid increased in thorax neutral lipids (NL) from 14·6 to 20·0% in females and from 18·5 to 30·0% in males. Males incorporated more linolenic acid-1-¹⁴C especially in fat body and flight muscle PL than females. The majority of this was recovered from phosphatidyl cholines (PTC) in 1-day adults whereas in 9-day adults phosphatidyl ethanolamines (PTE) and another compound, most likely cardiolipin, contained more label (29·0% in PTC, 33·1% in PTE, 34·9% in cardiolipin, and 2·0% in sphingomyelin in the thorax of females). The females also incorporated the label into egg lipids (42·2% in PL, 57·8% in NL). There was recovered from PTC 54·5% of the label in egg PL.Most of the label in thorax NL was found to be in free fatty acids (FFA). The label disappeared from triglycerides during adult life and tended to accumulate in FFA (82·7% in 9-day females) while in diglycerides the label did not vary during adult life (17·2% in 9-day post-emergence females). PTC apparently is a fairly labile PL type which is utilized in muscle whereas PTE and cardiolipin may be more structural in function and accumulated more label from linolenic acid with increasing adult age. Linolenic acid, then, essentially is a structural fatty acid and its rôle appears to be mainly in the structures of flight muscle membranes and organelles.     

The effect of fluoride on the growth and fatty acid composition of Sphagnum fimbriatum at two temperatures

The fatly acid composition of different lipid fractions (neutral, glyco- and phospholipids) was studied in Sphagnum fimbriatum Wils, gametophytes grown in aseptic cultures at two temperatures (15°C and 25°C). The effect of a growth-retarding concentration (0.1 mM) of KF was also investigated. Fifteen-day treatment with KF affected the fatty acid composition more strongly at the higher than at the lower temperature. The proportion of palmitic acid (16:0) increased but the proportion of linoleic (18:2) decreased in all the lipid fractions, and that of linolenic (18:3) acid decreased in the fractions containing glyco- and neutral lipids. This indicates that the fluoride ions inhibit lengthening of the fatty acid chain. Compared with gametophytes grown at 25°C, material cultivated at 15°C had a much higher proportion of a highly unsaturated fatty acid, linolenic acid (18:3), in all the lipid fractions, but a lower proportion of oleic acid (18:1) in the neutral and phospholipids, and a lower proportion of linoleic (18:2) acid in all three fractions.     

Differential effects of cis and trans fatty acid isomers, oleic and elaidic acids, on the cholesteryl ester transfer protein activity.

In a previous publication (Lagrost, L. and Barter, P.J. (1991) Biochim. Biophys. Acta 1085, 209-216), saturated and cis unsaturated non-esterified fatty acids have been shown to modulate the rate at which cholesteryl esters are transferred from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the presence of the human cholesteryl ester transfer protein (CETP). In the present report, the effects of cis (oleic acid) and trans (elaidic acid) monounsaturated isomers on the CETP-mediated transfer of cholesteryl esters between HDL and LDL were compared. Mixtures of human LDL and HDL3, containing or not radiolabelled cholesteryl esters, were incubated at 37 degrees C with CETP in the presence or in the absence of either stearic (18:0), oleic (18:1 cis) or elaidic (18:1 trans) acids. It was observed that oleic acid and elaidic acid had different effects on the CETP-mediated redistribution of radiolabelled cholesteryl esters as well as on the net mass transfer of cholesterol from HDL3 to LDL. In particular, at high non-esterified fatty acid/lipoprotein ratio, the transfer of cholesteryl esters was significantly inhibited by the cis isomer and increased by the trans isomer.     

Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.     

Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations ([Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in [Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the [Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the [Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.     

Relationship of changes in the fatty acid compositions and fruit softening in peach (Prunus persica L. Batsch)

Fatty acid compositions of peach (Prunus persica L. Batsch) mesocarp tissues from ‘Kawanakajima Hakuto’ and its firm-fleshed mutant ‘Shuangjiuhong’ were examined by gas chromatography during the developmental stages from 20 days before to 20 days after fruit ripening. Fruits were harvested at 4-day intervals from July to September. The predominant fatty acids were linoleic, palmitic and linolenic acids with 27.66–48.93 %, 23.59–31.65 %, and 12.08–28.35 % in ‘Shuangjiuhong’, and 32.64–42.79 %, 23.53–28.95 %, 16.14–39.15 % in ‘Kawanakajima Hakuto’, respectively. Saturated fatty acids (palmitic and stearic acids) remained relatively constant throughout the ripeness period. On the contrast, from 15 days before ripening, notable decline in oleic acid and increase of linoleic and linolenic acids were observed in both cultivars. In addition, from 10 days before ripening, much lower levels of oleic and linolenic acids and higher proportion of linoleic acid were observed in ‘Shuangjiuhong’ than those found in ‘Kawanakajima Hakuto’. And notably higher SFA level, lower levels of UFA and IUFA in the firm-fleshed peach were investigated during those stages. Correlation analysis showed that oleic acid and SFA had very significantly positive, whereas linolenic acid, UFA and IUFA had significantly negative correlation with fruit firmness. These results above suggest that lower levels of oleic and linolenic acids, UFA and IUFA, and higher linoleic acid and SFA content represent fruits with firmer flesh and help to retain the fruit texture.     

Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.     

Changes in the fatty acid composition of Drosophila melanogaster during development and ageing.

In Drosophila melanogaster the saturated fatty acids increase in amount early in pupal development relative to the concentrations in late third instar larvae, then decline to the levels characteristic of one-day-old adults. Conversely, the monounsaturated fatty acids decline in content early in pupal development, then increase late in the pupal period. Lauric acid (12:0), myristic acid (14:0) and palmitoleic acid (16:1) become more prominent and oleic acid (18:1) and palmitic acid (16:0) less prominent as the adult ages. At about 40 days of adult age myristic acid (14:0) begins to decrease and oleic acid (18:1) to increase. Within 20 days of eclosion males and females contain different amounts of myristic acid (14:0), palmitic acid (16:0) and oleic acid (18:1).     

Analysis of survival rates and cellular fatty acid profiles of Listeria monocytogenes treated with supercritical carbon dioxide under the influence of cosolvents

In the present study, we identified several process variables that significantly affect the efficiency of supercritical carbon dioxide inactivation of the food-borne pathogen Listeria monocytogenes. Treatment with SC-CO(2) completely disabled the colony-forming activity of the cells (8-log reduction) within specific treatment time (10-50 min), pressure (80-150 bar), and temperature ranges (35-45 degrees C). Microorganism inactivation rates increased proportionally with pressure and temperature, but the inactivation rate decreased significantly when cells were suspended in phosphate-buffered saline rather than in physiological saline. Additionally, when the microbial cell suspension was 80-100% (w/w) of water, the SC-CO(2)-mediated reduction in CFU ml(-1) was 4-8 log higher at the same treatment conditions than in typical cell suspensions (a water content of 800-4000% [w/w]) or dry preparations that had only 2-10% (w/w) of water. The addition of a fatty acid, oleic acid, decreased the effectiveness of the microbial inactivation by SC-CO(2), but the addition of a surfactant, sucrose monolaurate, increased the effectiveness. Therefore, cosolvents for SC-CO(2), including water, a fatty acid, and a surfactant in this study, were found to greatly influence on the inactivation effectiveness. The extraction of cellular substances, such as nucleic acid- and protein-like materials and fatty acids, was monitored by spectrophotometry and GC/MS and increased with SC-CO(2) treatment time. Additionally, using scanning and transmission electron microscopies, we investigated morphological changes in the SC-CO(2)-treated cells. The effects of the variables we have described herein represent a significant contribution to our current knowledge of this method of inactivating food-borne pathogens.     

Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen

AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation. METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan. From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei. During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid. Unlike previously studied ruminal biohydrogenating bacteria, B. hungatei Su6 was able to convert alpha-linolenic acid to stearic acid. Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates. Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid. An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid. CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B. hungatei plays an important role in unsaturated fatty acid transformations in the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years. Access to B. hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.