The H+-ATPase in the Plasma Membrane of Saccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability

Saccharomyces cerevisiae plasma membrane H⁺-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pH_i). The cell population used as inoculum exhibited a significant heterogeneity of pH_i, and the fall of pH_i correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Cold Shock Syndrome in Anacystis nidulans

The phenomenon of cold shock in Anacystis nidulans has been explored further in terms of loss of viability and immediate and subsequent metabolic effects. Cold shock was observed also in two closely related strains in which unsaturated fatty acid contents are also known to be low and temperature-dependent. Loss of viability was maximum for cells grown at temperatures above 40 C (<10⁻⁴ survivors after 5 min at 0 C) but became negligibly small for cells grown below 34 C. Development of the cold-sensitive condition after transfer 25 → 39 C was slow and comparable to rate of growth; development of the insensitive condition after transfer 39 → 25 C was rapid, implying rapid in situ alteration. An immediate metabolic effect, observed as a decrease in rate of photosynthetic O₂ evolution measured at growth temperature, was less severe than loss of viability. Continued light incubation under growth conditions led to slow decay in rate of O₂ evolution accompanied by loss of membrane chlorophyll. The multiple effects which comprise the cold shock syndrome appear to be membrane-related phenomena and thereby provide an experimental probe of normal membrane function.     

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10⁶ cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

Understanding the acid tolerance response of bifidobacteria

Aims: To investigate the effect of pH on the viability and the acid tolerance response (ATR) of bifidobacteria. Methods and Results: The impact of low pH on the viability of five species of bifidobacteria was examined under conditions of strict anaerobiosis. Although differences in the ability to resist the lethal effects of low pH were apparent among the species, cell viability could be improved by the provision of fermentable substrate during an acidic pH stress or through the use of stationary phase cells. While a stationary phase ATR was found to occur in two species of bifidobacteria, there was no adaptive response in exponential phase cells. Proteomic analysis of exponential phase Bifidobacterium longum subjected to a mild acid pre‐exposure (pH 4·5, 2 h) prior to an acid challenge revealed a substantial loss in the total number of cellular proteins. In contrast, proteomic analysis of stationary phase cells revealed an increased abundance of proteins associated with the general stress response as well as the β‐subunit of the F₀F₁‐ATPase, known to be important in bifidobacteria acid tolerance. Conclusion: Neither Bif. longum or Bifidobacterium breve possesses an inducible exponential phase ATR. Significance and Impact of the Study: These findings provide further insights into the impact of pH on the viability of bifidobacteria and may partially explain the loss in viability associated with their storage in acid foods.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Isolation and Characterization of Mutants of Escherichia coli with Cellular Division Selectively Affected by Growth on Fatty Acids

Isolation and characterization of mutants of Escherichia coli that beta-oxidize fatty acids at normal rates, but which divide very slowly when grown on fatty acids, are described. These mutants grow normally on other carbon sources. By growth on oleate, experiments with radioactive precursors showed that the rates of incorporation into ribonucleic acid, protein, and cell wall were comparable to those observed with the parent, whereas the rate of incorporation into phospholipids was slightly decreased. Under these conditions the rate of incorporation of ³²P-orthophosphate into deoxyribonucleic acid was low. On the other hand, by growth on oleate, neither gross mass increase in the different macromolecules nor loss of viability was observed, whereas in the presence of inducer the derepression of the lac operon enzymes occurred. Therefore, extensive turnover of the macromolecules is involved when these mutants are grown on fatty acids. Studies of the crypticity and of the binding of 1-anilino-8-naphthalene sulfonate show differences in membrane structure between the mutants and the constitutive parent. Properties of these mutants, which are affected in the process of cellular division, are discussed.     

Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.     

The lipid composition of Acer pseudoplatanus cells grown in culture

Cells of Acer pseudoplatanus were grown in batch suspension culture for 22 days. The cultures were initiated at high cell density of 2 × 10⁵ cells per ml of culture. Growth was characterised by a short lag phase, an exponential phase of rapid cell division and growth, and finally a stationary phase. Quantitative but not qualitative changes were observed in total lipid content, fatty acids and phospholipids at different stages of growth. Total lipids, phospholipids and fatty acids showed maximum concentrations in 12 day old cells. The major phospholipids isolated were phosphatidylcholine and phosphatidylethanolamine with minor amounts of phosphatidic acid and lysophosphatides. Other lipid components present were mono- and digalactosyl diglycerides, cerebrosides, sterol glucosides, free fatty acids and esterified sterol glucosides. The major constituent fatty acids were myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3). During exponential cell growth the proportion of 16:0, 18:2 and 18:3 constituted nearly 90% of the total fatty acids. Triglycerides were the major repository of myristic acid (14:0) with substantial amounts of palmitic acid (16:0), whereas phospholipids contained 16:0, 18:2 and 18:3 in high amounts.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Survival of Low-pH Stress by Escherichia coli O157:H7: Correlation between Alterations in the Cell Envelope and Increased Acid Tolerance

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 × 10⁴ CFU ml⁻¹ were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.     

The effects of extreme hyposaline stress upon Polysiphonia lanosa (L.) Tandy from marine and estuarine sites

Marine and estuarine plants of the macroalga Polysiphonia lanosa (L.) Tandy (Rhodophyta: Ceramiales) were found to differ in their responses to extreme hyposaline stress. Marine plants showed evidence of cell mortality when maintained in dilute sea water media (<20%), in contrast to their estuarine counterparts. Measurements of thallus K⁺ upon transfer to freshwater medium demonstrated that marine cells show net loss of K ⁺ at a faster rate than estuarine cells. Conditions that led to an increased net K ⁺ efflux (e.g. increased temperature,.lower pH) also resulted in rapid loss of cell viability. Conversely, by reducing net K⁺ loss (e.g. decreasing temperature, increasing pH), cells could be maintained in hyposaline media that would otherwise have proved fatal. The sensitivity of marine cells to extreme hyposaline conditions was also found to be markedly affected by external Ca²⁺ concentration. Thus cell viability was maintained by the addition of CaCl₂ at > 2 mmol · dm ^?3. Addition of Ca²⁺ was also found to reduce net K ⁺ loss from marine cells in freshwater medium. The data indicate a direct relationship between the rate of net K⁺ efflux and cell mortality under conditions of extreme hyposaline stress. Cell death may result from rapid (K ⁺) ion loss, resulting in osmotic imbalance within the cell.     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

Macromolecular Synthesis and Degradation in Arthrobacter During Periods of Nutrient Deprivation

Cells of Arthrobacter atrocyaneus and A. crystallopoietes, harvested during their exponential phase, were starved in 0.03 M phosphate buffer (pH 7.0) for 28 days. During this time, the cells maintained 90 to 100% viability. Experimental results were similar for both organisms. Total cellular deoxyribonucleic acid was maintained. Measurable degradation rates for deoxyribonucleic acid as determined by radioisotope techniques were not observed, and only during the initial hours of starvation could a synthetic rate be determined. Total ribonucleic acid levels remained stable for the first 24 h of starvation, after which slow, continuous loss of orcinol-reactive material occurred. Synthetic and degradative rates of ribonucleic acid, as determined by radioisotope techniques, dropped quickly at the onset of starvation. Constant basal rates were attained after 24 h. In A. atrocyaneus, total cell protein was degraded continuously from the onset of starvation. In A. crystallopoietes, total cell protein remained stable for the first 24 h, after which slow continuous loss occurred. After 28 days, the total protein per cell was similar for both organisms. In the first week, amino acid pools stabilized at about 50% of the values characteristic of growth. Rates of degradation of protein decreased rapidly for the first 24 h for both organisms, but leveled to a constant basal rate thereafter. Rates of new protein synthesis dropped during the first 24 h and by 48 h achieved a constant basal rate.     

Unsaturated Fatty Acids Promote the Phagocytosis of P. aeruginosa and R. equi by RAW264.7 Macrophages

In the present study, using the murine monocyte/macrophage cell line RAW264.7 as a model system, we analyzed the phagocytosis rate and the bactericidal capacity of polyunsaturated fatty acids (PUFA)-enriched macrophages against Pseudomonas aeruginosa and Rhodococcus equi. The P. aeruginosa strain ATCC 10145, the virulent R. equi strain ATCC 33701, and the non-virulent R. equi strain ATCC 6939 were examined. Flow cytometric detection of intracellular microorganisms in combination with viability assays were used to determine the impact of PUFA on the number of engulfed, surviving as well as replicating bacteria. Macrophage enrichment with PUFA resulted in an increase of the internalization rate of the microorganisms by the immune cells. Moreover, an impeding action of the unsaturated fatty acids on the intracellular survival rates of the virulent strains P. aeruginosa ATCC 10145 and R. equi ATCC 33701 could be observed. The n-3 fatty acid docosahexaenoic acid (DHA) as well as the n-6 fatty acid arachidonic acid (AA) showed the most pronounced effects. Taken together, our data support the idea of supplementing PUFA to immunocompromised individuals as well as to people suffering from chronic infections with P. aeruginosa or R. equi to improve macrophage phagocytic and microbicidal activity.     

Relationship between cadmium sensitivity and degree of plasma membrane fatty acid unsaturation in Saccharomyces cerevisiae

The sensitivity of Saccharomyces cerevisiae to the redox-active metal copper has recently been found to be influenced by cellular fatty acid composition. This study sought to investigate whether fatty acid composition affected plasma membrane permeabilisation and whole-cell toxicity induced by the redox-inactive metal cadmium. S. cerevisiae NCYC 1383 was enriched with the polyunsaturated fatty acids linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Incorporation of the exogenous fatty acids resulted in them comprising more than 65% of the total fatty acids in plasma membrane lipids. Inhibition of cell division in the presence of Cd(NO₃)₂ was accentuated by growth in the presence of a polyunsaturated fatty acid. Furthermore, susceptibility to Cd²⁺-induced plasma membrane permeabilisation increased with the degree of fatty acid unsaturation. Thus, during exposure to Cd²⁺, K⁺ efflux from 18:2- and 18:3-enriched cells was up to 2.5-fold or 3-fold greater, respectively than that from unsupplemented cells. In addition, reductions in cell viability during exposure to Cd²⁺ were most marked in polyunsaturated-fatty-acid-supplemented cells. At certain times, unsupplemented Cd²⁺-exposed cells displayed up to 7-fold greater viability than supplemented Cd²⁺-exposed cells. The study demonstrates that the toxicity of the redox-inactive metal Cd²⁺ towards S. cerevisiae becomes markedly amplified with increased cellular and plasma membrane fatty acid unsaturation. Received: 14 March 1997 / Received revision: 4 June 1997 / Accepted: 7 June 1997     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.     

Enrichment with a polyunsaturated fatty acid enhances the survival of Saccharomyces cerevisiae in the presence of tributyltin

The toxicity of inorganic metal species towards Saccharomyces cerevisiae has been shown to be markedly dependent on cellular fatty acid composition. In this investigation, the influence of fatty acid supplementation on the toxicity of the lipophilic organometal, tributyltin was investigated. Growth of S. cerevisiae was increasingly inhibited when the tributyltin concentration was increased from 0 to 10 μM. However, the inhibitory effect was partly alleviated by supplementation of the medium with 1 mM linoleate (18:2), a treatment that leads to large-scale incorporation of this polyunsaturated fatty acid (to >60% of total fatty acids) in yeast membrane lipids. Cells that were previously enriched with 18:2 also showed reduced loss of vitality compared to cells grown in the absence of a fatty acid supplement, when exposed to tributyltin. For example, addition of tributyltin to a concentration of 0.1 μM was associated with an approximate 10% reduction in the H⁺ efflux activity of 18:2-enriched cells, but a 70% reduction in that of fatty acid-unsupplemented cells. Despite the increased tributyltin resistance of 18:2-enriched S. cerevisiae, the level of cell-associated tributyltin was found to be approximately two-fold higher in these organisms than in fatty acid-unsupplemented cells. These results demonstrate an increased resistance of 18:2-enriched membranes to the direct toxic action(s) of tributyltin. This is in contrast to the previously reported effect of 18:2 enrichment on sensitivity of S. cerevisiae to inorganic metal cations.