Independence of deoxyribonucleic acid replication and initiation from membrane fluidity and the supply of unsaturated fatty acids in Escherichia coli.

Mutant derivatives of the unsaturated fatty acid auxotroph K1062 were employed to investigate whether the supposedly membrane-bound bacterial replication machinery requires for its replicatory functions a fluid membrane environment as is known for several membrane-associated protein functions. Temperatures Tt for fluid reversible nonfluid phase transitions of membrane phospholipids are raised from below 18 to 38 degrees C when mutant cells are supplemented with elaidate instead of with oleate. In this experimental system current or synchroneously initiated new rounds of DNA replication are shown in vivo to continue 8 degrees below Tt, provided appropriate corrections for the concurrent cellular metabolic breakdown are considered. Temperature rate profiles for in vitro deoxyribonucleic acid replication rates measured in lysates of either oleate- or elaidate-supplemented cells yield congruent Arrhenius plots without discontinuities at corresponding Tt positions. We conclude that neither the start nor the propagation of replication forks depends on a fluid membrane. The capacity for the assembly of new replication complexes was studied in replication-aligned cells either shifted from oleate to elaidate (at temperatures below Tt for newly synthesized phospholipids) or starved for oleate. Regardless of whether unsaturated fatty acids are exchanged or completely withheld, new replication complexes can be normally assembled and initiated. These results do not support the conclusions reached by Fralick and Lark (1973) that the availability of unsaturated fatty acids is a prerequisite for the assembly of a functional replication complex.     

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.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

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.     

Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli.

The photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate was shown to be taken up specifically by the fatty acid transport system expressed in Escherichia coli grown on oleate. This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E. coli. The fadL protein was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins. The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2. The denaturation was reversible depending upon the protein to detergent ratios. The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane. However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM. The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.     

FadD is required for utilization of endogenous fatty acids released from membrane lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.     

Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids

Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH.     

Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa

Prepupae of the Mediterranean arctiid moth Cymbalophora pudica spend hot spring and summer months in a summer diapause (aestivation). Although their cold-hardiness (survival after 1-day exposure to subzero temperatures) is relatively low, it may be moderately enhanced by prior cold acclimation at decreasing above-zero temperatures. In this study, fatty acids of phospholipids and triacylglycerols were analysed in five different tissues (body wall, midgut, fat body, silk glands and brain) dissected from both cold-acclimated and control aestivating prepupae. The five most abundant fatty acids (oleic, palmitic, stearic, linoleic and α-linolenic), found generally in both lipidic fractions and all five tissues, represent a typical fatty complement of Lepidoptera. The fatty acid profiles of individual tissues differed from each other and the response to cold acclimation was also tissue-specific. Moderate but significant increases in the proportion of unsaturated fatty acids after cold acclimation were observed in triacylglycerols of the body wall, fat body and silk glands. Additionally, significant rearrangements of fatty acid profiles were found in triacylglycerols of midgut and brain, without changing the unsaturation/saturation ratio. The adaptational value of enhanced fluidity of fat body triacylglycerols caused by their increased unsaturation remains unclear, because the lipidic energy depots are not utilized during aestivation of this insect. Minimal capacity to alter the membrane-bound fatty acids was found in all tissues except midgut, where the unsaturation/saturation ratio of phospholipids slightly increased after cold acclimation. A low ability to alter the composition of membrane lipids in response to low temperature, correlates well with the low capacity of C. pudica prepupae to enhance their cold-hardiness during cold-acclimation. This may be regarded as indirect support for the membrane lipid restructuring in insect cold adaptation. Accepted: 11 May 1998     

The effects of exposure to exogenous fatty acids and membrane fatty acid modification on the electrical properties of NG108-15 cells

The role of membrane lipid composition in determining the electrical properties of neuronal cells was investigated by altering the available fatty acids in the growth medium of cultured neuroblastoma X glioma hybrid cells, clone NG108-15. Growth of the cells for several days in the presence of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic) caused a pronounced decrease in the Na+ action-potential rate of rise (dV/dt) and smaller decreases in the amplitude, measured by intracellular recording. Oleic acid had no effect on the action potentials generated by the cells. In contrast, a saturated fatty acid (palmitate) and a trans monounsaturated fatty acid (elaidate) caused increases in both the rate of rise and the amplitude. No changes in the resting membrane potentials or Ca2+ action potentials of fatty acid-treated cells were observed. The membrane capacitance and time constant were not altered by exposure to arachidonate, oleate, or elaidate, whereas arachidonate caused a small increase in membrane resistance. Examination of the membrane phospholipid fatty acid composition of cells grown with various fatty acids revealed no consistent alterations which could explain these results. To examine the mechanism for arachidonate-induced decreases in dV/dt, the binding of 3H-saxitoxin (known to interact with voltage-sensitive Na+) channels was measured. Membranes from cells grown with arachidonate contained fewer saxitoxin binding sites, suggesting fewer Na+ channels in these cells. We conclude that conditions which lead to major changes in the membrane fatty acid composition have no effect on the resting membrane potential, membrane capacitance, time constant, or Ca2+ action potentials in NG108-15 cells. Membrane resistance also does not appear to be very sensitive to membrane fatty acid composition. However, changes in the availability of fatty acids and/or changes in the subsequent membrane fatty acid composition lead to altered Na+ action potentials. The primary mechanism for this alteration appears to be through changes in the number of Na+ channels in the cells.     

Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-acyl carrier protein transacylase

The microbial biosynthesis of free fatty acid, which can be used as precursors for the production of fuels or chemicals from renewable carbon sources, has attracted significant attention in recent years. Free fatty acids can be produced by introducing an acyl-carrier protein (ACP) thioesterase (TE) gene into Escherichia coli. The first committed step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl-CoA by an adenosine triphosphate (ATP)-dependent acetyl-CoA carboxylase followed by the conversion of malonyl-CoA to malonyl-ACP through the enzyme malonyl CoA-acyl carrier protein transacylase (MCT; FabD). The E. coli fabD gene encoding MCT has been cloned and studied. However, the effect of FabD overexpression in a fatty acid overproducing strain has not been examined. In this study, we examined the effect of FabD overexpression in a fatty acid overproducing strain carrying an acyl-ACP TE. Specifically, the effect of overexpressing a fabD gene from four different organisms on fatty acid production was compared. The strains carrying a fabD gene from E. coli, Streptomyces avermitilis MA-4680, or Streptomyces coelicolor A3(2) improved the free fatty acid production; these three strains produced more free fatty acids, about 11% more, than the control strain. The strain carrying a fabD gene from Clostridium acetobutylicum ATCC 824, however, produced similar quantities of free fatty acids as the control strain. In addition, the three FabD overexpressed strains also have higher fatty acid/glucose yields. The results suggested that FabD overexpression can be used to improve free fatty acid production by increasing the malonyl-ACP availability.     

Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli.

The lipid phase transition of Escherichia coli phospholipids containing cyclopropane fatty acids was compared with the otherwise homologous phospholipids lacking cyclopropane fatty acids. The phase transitions (determined by scanning calorimetry) of the two preparations were essentially identical. Infection of E. coli with phage T3 inhibited cyclopropane fatty acid formation over 98%, whereas infection with mutants which lack the phage coded S-adenosylmethionine cleavage enzyme had no effect on cyclopropane fatty acid synthesis. These data indicate that S-adenosylmethionine is the methylene in cyclopropane fatty acid synthesis.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.

One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditions contained only saturated and monounsaturated fatty acids, considerable incorporation of the diunsaturated fatty acid linoleate (18:2) (to more than 65% of the total fatty acids) was observed in both whole-cell homogenates and plasma membrane-enriched fractions from cells grown in linoleate-supplemented medium. Linoleate enrichment had no discernible effect on the growth of S. cerevisiae. However, linoleate-enriched cells were markedly more susceptible to copper-induced plasma membrane permeabilization. Thus, after addition of Cu(NO3)2, rates of cellular K+ release (loss of membrane integrity) were at least twofold higher from linoleate-supplemented cells than from unsupplemented cells; this difference increased with reductions in the Cu2+ concentration supplied. Levels of cellular Cu accumulation were also higher in linoleate-supplemented cells. These results were correlated with a very marked dependence of whole-cell Cu2+ toxicity on cellular fatty acid unsaturation. For example, within 10 min of exposure to 5 microM Cu2+, only 3% of linoleate-enriched cells remained viable (capable of colony formation). In contrast, 100% viability was maintained in cells previously grown in the absence of a fatty acid supplement. Cells displaying intermediate levels of linoleate incorporation showed intermediate Cu2+ sensitivity, while cells enriched with the triunsaturated fatty acid linolenate (18:3) were most sensitive to Cu2+. These results demonstrate for the first time that changes in cellular and plasma membrane fatty acid compositions can dramatically alter microbial sensitivity to copper.     

Effect of fatty acids on the proliferation of concanavalin A-stimulated rat lymph node lymphocytes

1. The effect of a range of fatty acids upon concanavalin A-stimulated [3H]thymidine incorporation into rat lymphocytes was investigated. 2. All fatty acids tested inhibited the response to mitogen but the extent of the inhibition was dependent upon the fatty acid concentration used, the time of addition of fatty acid and the duration of exposure of the cells to fatty acid. 3. All fatty acids were inhibitory at concentrations of 50 microM or above; at lower concentrations some were inhibitory and some were stimulatory. Above 50 microM the inhibitory effect was concentration dependent; the greater the fatty acid concentration, the greater the inhibition. 4. The longer the lymphocytes were exposed to the fatty acid the greater was the inhibitory effect. This was true if the fatty acids were added at the same time as the mitogenic stimulus or if they were added before or after the stimulus. Some fatty acids maintained their inhibitory effect when added 24 or 48 hr after the mitogenic stimulus. 5. Generally unsaturated fatty acids were more inhibitory than saturated fatty acids; the greatest inhibition of proliferation was caused by eicosapentaenoate and arachidonate and the least inhibition by myristate and palmitate. 6. Inhibition was greater in the absence of serum. 7. Inhibition by unsaturated fatty acids could be partially or totally relieved by addition in combination with myristate or palmitate, suggesting that the inhibitory effect of fatty acids may be due to alteration of membrane fluidity caused by an imbalance of fatty acids presented to the cells. 8. PGE2 levels were similar in the medium of cells grown in the presence of fatty acids with varying inhibitory effects, indicating that PGE2 production is not the sole mechanism of suppression of the proliferative response. 9. Although the mechanism by which fatty acids exert their effect remains to be determined, these results indicate that lymphocyte proliferation and so an immune response could be influenced by dietary lipid manipulation.     

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.     

Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids.

The phospholipid ester-linked fatty acids of 0-day-, 7-day-, and 30-day-starved cultures of Vibrio cholerae were compared. Statistically significant trends were noted in the fatty acid profiles as the cells starved. The amount of the cis-monoenoic fatty acids declined (e.g., 16:1 omega 7c: 0 day, 39%; 7 day, 18%; 30 day, 11%). In contrast, the saturated fatty acids, the cyclopropyl derivatives of the cis-monoenoic fatty acids, and trans-monoenoic fatty acids increased during starvation. For instance, the amounts of 16:1 omega 7t were: 0 day, 1%; 7 day, 13%; 30 day, 17%; which increased the trans/cis ratio for 16:1 omega 7 from 0.02 (0 day) to 0.70 (7 day) to 1.56 (30 day). This may be due to the reported high turnover rates of cis-monoenoic fatty acids of membrane phospholipids and the availability of enzymes for the metabolism of these isomers. During starvation-induced phospholipid loss, the cis-monoenoic fatty acids would, therefore, be preferentially utilized. The ability to either synthesize trans-monoenoic acids (which are not easily metabolized by bacteria) or modify the more volatile cis-monoenoic acids to their cyclopropyl derivatives may be a survival mechanism which helps maintain a functional (although structurally altered) membrane during starvation-induced lipid utilization. In addition, a trans/cis fatty acid ratio significantly greater than that reported for most bacterial cultures and environmental samples (less than 0.1) may be used as a starvation or stress lipid index. Such a ratio could help determine the nutritional status of ultramicrobacteria and other reported dormant cells in natural aquatic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.     

Cis-unsaturated fatty acids induce both lipogenesis and calcium binding in adipocytes

The addition of 0.4-3 mM of cis-unsaturated fatty acids such as oleic acid (18:1) or linoleic acid (18:2) to intact rat adipocytes stimulated lipogenesis at 37 degrees C. Saturated or trans-unsaturated fatty acids were ineffective. Fluorescence photobleaching recovery studies performed under similar conditions indicated that the cis-unsaturated fatty acids do not alter lateral mobility of either a lipid probe or a general protein marker in the plasma membrane. A high concentration (7 mM) of Ca2+, which by itself has some stimulatory effect on lipogenesis, significantly potentiated the effect of oleic acid on this insulin-like activity. Measurement of 45Ca2+ binding by fat cells has indicated that cis-unsaturated (but not saturated) fatty acids increased 12- to 20-fold the amount of Ca2+ associated with the cells. The dependence of this effect on the fatty acid concentration correlates well with the effect of the fatty acid on the induction of lipogenesis. Our results suggest that cis-unsaturated fatty acids affect membrane organization in a manner which induces a significant increase in membrane associated or intracellular Ca2+. This increase may be responsible for inducing exocytotic-like processes which facilitate translocation of glucose transport activity from storage sites to the plasma membrane and thus produce an insulin-like effect.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Effect of antibiotic AL-87 on the fatty acid composition of microorganisms in different taxonomic groups

The effect of antibiotic A1-87 on the fatty acid composition of S. aureus 209, E. coli O26 and M. luteus 169 significantly differing in this property was studied. The sub-bacteriostatic doses of the preparation induced the appearance of unsaturated fatty acids in S. aureus 209. These acids were not detected in the control cultures. They also significantly increased the content of the saturated branched fatty acid with 15 carbon atoms in this culture and decreased the content of the fatty acid of the same type with 19 carbon atoms. In E. coli O25 there was an almost two-fold increase in the content of the unsaturated straight chain fatty acids with a respective decrease in the content of cyclopropanoic acid and a markedly pronounced decrease in the content of nonadecanoic acid. In M. luteus 169 the content of the saturated branched fatty acid with 15 carbon atoms increased, while the content of the unsaturated straight chain fatty acids (C16:1, C18:1) decreased, the content of hexadecanoic acid being decreased almost two times. According to the present status the differences in the fatty acid composition of the above organisms are interpreted as one of the mechanisms increasing the membrane permeability.