Modulating Membrane Composition Alters Free Fatty Acid Tolerance in Escherichia coli

Microbial synthesis of free fatty acids (FFA) is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10) that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.     

Changes in Membrane Fatty Acid Composition of Pseudomonas aeruginosa in Response to UV-C Radiations

The changes in lipid composition enable the micro-organisms to maintain membrane functions in the face of environmental fluctuations. The relationship between membrane fatty acid composition and UV-C stress was determined for mid-exponential phase and stationary phase Pseudomonas aeruginosa. The total lipids were obtained by dichloromethane/methanol (3:1) and were quantified by GC. The TLC analysis of phospholipids showed the presence of three major fractions phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Significant modifications, as manifested by an increase of UFA, were obtained. Interestingly, this microorganism showed a remarkable capacity for recovery from the stressful effects of UV-C.     

低温对茶树叶片膜脂脂肪酸和蛋白质的影响

本文研究低温胁迫过程中龙井43和大叶云峰叶片膜脂脂肪酸的变化。结果发现,低温下不同品种茶树叶片膜脂脂肪酸配比变化趋势不同,较抗寒的龙井43,不饱和脂肪酸指数(IUFA)和亚麻酸(183)比例随低温期间不同阶段呈现出"低-高-低"的变化趋势;而不抗寒的大叶云峰的变化趋势无明显规律性。此外,还研究了越冬过程中龙井43叶片可溶性蛋白和膜蛋白的变化,发现低温期间龙井43叶片可溶性蛋白含量和组分基本稳定,而膜蛋白含量在低温胁迫时大幅度上升,且经低温诱导出现了46KD、38KD两种新的蛋白组分,并在温度升高后消失。     

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

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

Small Changes in Environmental Parameters Lead to Alterations in Antibiotic Resistance,Cell Morphology and Membrane Fatty Acid Composition in Staphylococcus lugdunensis

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6–8), temperature (35–39°C) and osmotic pressure (0–5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.     

菜豆叶片衰老期间叶绿体被膜膜脂与脂肪酸组分的变化

菜豆叶片叶绿体总脂和被膜膜脂中均含有单半乳糖甘油二脂和双半乳糖甘油二脂,在整个衰老期间两种糖脂的比值变化不大。叶绿体总脂中含有5种磷脂,脂肪酸以不饱和的亚麻酸为主,而被膜膜脂中仅含磷脂酰胆碱和磷脂酰甘油,脂肪酸以饱和的棕榈酸为主,不饱和亚油酸为次。叶片衰老过程中被膜所含两种磷脂比值明显降低,脂肪酸的不饱和指数也因亚麻酸相对含量显著减少、棕榈酸相对含量增加而降低。     

Thermal Tolerance of Zymomonas mobilis: Temperature-Induced Changes in Membrane Composition

The membrane composition of Zymomonas mobilis changed dramatically in response to growth temperature. With increasing temperature, the proportion of vaccenic acid declined with an increase in myristic acid, the proportion of phosphatidylcholine and cardiolipin increased with decreases in phosphatidylethanolamine and phosphatidylglycerol, and the phospholipid/protein ratio of the membrane declined. These changes in membrane composition were correlated with changes in thermal tolerance and with changes in membrane fluidity. Cells grown at 20°C were more sensitive to inactivation at 45°C than were cells grown at 30°C, as expected. However, cells grown at 41°C (near the maximal growth temperature for Z. mobilis) were hypersensitive to thermal inactivation, suggesting that cells may be damaged during growth at this temperature. When cells were held at 45°C, soluble proteins from cells grown at 41°C were rapidly lost into the surrounding buffer in contrast to cells grown at lower temperatures. The synthesis of phospholipid-deficient membranes during growth at 41°C was proposed as being responsible for this increased thermal sensitivity.     

Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.     

Correlation between the Circadian Rhythm of Resistance to Extreme Temperatures and Changes in Fatty Acid Composition in Cotton Seedlings

Fluctuations in fatty acid composition were examined in cotton (Gossypium hirsutum L. cv Deltapine 50) leaves during light-dark cycles of 12:12 h and under continuous light and were correlated to the rhythmic changes in chilling (5[deg]C) resistance (CR) and heat (53[deg]C) resistance (HR). The chilling-resistant and chilling-sensitive phases developed in the dark or the light period, respectively, and this rhythm persisted under continuous light for three cycles. The heat-resistant phase developed in the light period and an additional peak of HR occurred in the middle of the dark period. Under continuous light, only one peak of HR developed, lasting from the middle of the subjective night to the middle of the subjective day. The amounts of palmitic and oleic acids were constant during the light-dark cycle and under continuous light, but those of linoleic and linolenic acids fluctuated, attaining a high level in the middle of the dark period or the subjective night, and a low level in the middle of the light period or the subjective day. A low temperature of 20[deg]C induced CR and affected changes in fatty acid composition similar to those that occurred during the daily CR phase. A high temperature of 40[deg]C induced HR but did not affect changes in fatty acid composition. The results in their entirety show that the CR that develops rhythmically as well as the low-temperature-induced CR coincide with increased levels of polyunsaturated fatty acids. No correlation is found between changes in fatty acid composition and the HR that develops rhythmically or the high-temperature-induced HR.     

酸适应对一株乳酸乳球菌膜脂肪酸组成和膜蛋白表达的影响

[目的]筛选具有较强酸适应能力的菌株,研究酸适应对其膜脂肪酸组成和膜蛋白表达的影响.[方法]从20株菌中筛选出一株具有较强酸适应能力的乳酸乳球菌KLDS4.0312,以GC-MS法测定该菌酸适应前后膜脂肪酸组成变化;对酸适应前后该菌膜蛋白的差异表达进行双向电泳分析.[结果]酸适应后,该菌膜不饱和脂肪酸含量从30.77%上升到42.93%,饱和脂肪酸含量从69.23%下降到57.07%,且有一种新的长链单不饱和脂肪酸C<,19:1>-n6被诱导产生.酸适应过程中至少有65个蛋白质点表达出现显著差异,其中上调的蛋白质点有43个,减弱表达的蛋白质点有22个.而添加氯霉素后,菌株的酸适应能力消除,可能与氯霉素抑制新蛋白的合成有关.[结论]说明细胞膜脂肪酸组成的适应性改变和应激蛋白的诱导产生是该菌主要的酸适应机制.     

随气温下降大头茶膜脂脂肪酸组分及膜保护系统的适应性变化

 本实验以自然生长的大头茶为材料,研究了其叶片膜脂脂肪酸组分及膜保护系统随气温下降的变化。结果表明,膜脂脂肪酸不饱和度增加;同时,SOD活性增强,Vc含量增加,POX出现了新的同工酶谱带,而使保护系统清除自由基的能力增强。这两方面的变化有着密切的关系,且都与大头茶的抗冷性发展相适应。     

逆境对真菌膜脂肪酸成分的影响

利用气相色谱对真菌膜脂肪酸在逆境下的变化进行研究,发现低温胁迫时膜上的不饱和脂肪酸较多,低碳源浓度、低氧浓度和高盐浓度胁迫时,膜上不饱和脂肪酸的含量反而出现降低。表明不同逆境胁迫时,真菌中膜的脂肪酸含量不一样。     

Failure of Escherichia coli to Alter Its Fatty Acid Composition in Response to Cholesterol-Induced Changes in Membrane Fluidity

Substantial amounts of exogenously supplied cholesterol were incorporated into the membranes of Escherichia coli during growth and caused a large decrease in membrane fluidity. Although no compensatory changes in fatty acid composition were observed, the incorporation of cholesterol did not affect the rate of growth of E. coli or interfere with the changes in fatty acid composition which normally occur during growth at different temperatures.     

Organic Solvent Tolerance of Escherichia coli Is Independent of OmpF Levels in the Membrane

The organic solvent tolerance of Escherichia coli was measured under conditions in which OmpF levels were controlled by various means as follows: alteration of NaCl concentration in the medium, transformation with a stress-responsive gene (marA, robA, or soxS), or disruption of the ompF gene. It was shown that solvent tolerance of E. coli did not depend upon OmpF levels in the membrane.     

Changes in Chloroplast Membrane Lipids during Adaptation of Barley to Extreme Salinity

During adaptation of barley (Hordeum vulgare L.) seedlings to extremely high concentrations of sodium chloride in the root space, the content of galactolipids of chloroplast membranes decreased considerably. Alterations in membrane lipids were due to the high concentration of ions rather than to the increase in the water potential. Sodium chloride was accumulated in the leaf cells and affected lipid-synthesizing enzymes such as galactosyl transferase and acylase which are attached to the chloroplast envelope. The return of salt-adapted barley seedlings to a nutrient solution with low salt concentration resulted in a reversal of the observed changes. It is suggested that the decrease in content of galactolipids in biomembranes is one of the factors causing increased salt resistance in barley plants which are adapted to extreme salinity.     

Fatty Acid and Aliphatic Hydrocarbon Composition of Sarcina lutea Grown in Three Different Media

Sarcina lutea was grown in Trypticase Soy Broth, Nutrient Broth, and a chemically defined medium. Gas chromatographic analysis of lipid components demonstrated that the composition of the medium had an effect on the relative per cent composition of the aliphatic hydrocarbons and fatty acids present in the cells. The branched olefinic hydrocarbons from the organisms grown in Trypticase Soy Broth showed no predominance or only a slight predominance of odd-numbered carbon chains, whereas the hydrocarbons from cells grown in the other two media showed an obvious predominance of odd-numbered carbon chains. The monocarboxylic fatty acid content and distribution showed only minor differences, with all normal saturated fatty acids present in relatively small quantities for cells grown in Nutrient Broth and in a chemically defined medium.     

细胞膜磷脂棕榈酸含量对自絮凝颗粒酵母耐酒精能力的影响及作用机制

研究揭示细胞膜磷脂脂肪酸组成与酵母菌耐酒精能力的一种新颖关系及其机制。分别培养于添加 0 6mmol L棕榈酸、亚油酸或亚麻酸不同条件下的自絮凝颗粒酵母 ,其细胞膜富含各自所添加的脂肪酸。细胞膜富含棕榈酸、亚油酸或亚麻酸的三种菌体于 30℃经 2 0 %(v v)酒精冲击 6h的存活率分别为 5 2 %、1 8%和 0。通过考察三种菌体于 30℃在 1 5 %(v v)酒精冲击下的细胞膜透性发现 ,细胞膜富含棕榈酸的菌体的胞外核苷酸平衡浓度分别仅为细胞膜富含亚油酸或亚麻酸菌体的 48%和 32 %,其细胞膜透性系数 (P′)分别仅为后者的 37%和 2 0 %,且三者的胞外核苷酸浓度和P′由小到大的排列顺序均与它们的存活率由高到低的排列顺序完全一致。因此 ,细胞膜富含棕榈酸的菌体具有较强的耐酒精能力是与其在高浓度酒精冲击下可维持较低的细胞膜透性密切相关的 的。     

细菌在逆境条件下膜脂肪酸变化的研究进展

细胞膜是控制细菌细胞进行物质交换的屏障。在逆境条件下,细菌通过改变细胞膜脂肪酸的组分和含量,以调整适当的膜流动性和适应性,保护细胞膜免受不利和多变逆境条件的影响。有些细菌在逆境胁迫的条件下会进入活的但不可培养的(Viable but non-culturable, VBNC)状态。总结了细菌几种逆境胁迫及其诱导因子,并论述了细菌和部分具有VBNC态细菌在逆境胁迫下膜脂肪酸的种类及含量的变化、以及脂肪酸检测方法的研究进展,为进一步解析细菌逆境胁迫机制提供参考。