Lipid profiles of conidia of Aspergillus niger and a fatty acid auxotroph

Conidial lipids of the wild-type (V35) Aspergillus niger and its unsaturated fatty acid auxotroph (UFA2) were compared. The wild type contained lower levels (7.6%) of phospholipids and higher levels (28.4%) of glycolipids than the mutant (16.5 and 22.2%, respectively). Oleic (33.4%), linoleic (22.5%), palmitic (12.8%), stearic (7.4%), and linolenic (6.2%) were the main fatty acids of the wild type (V35). The mutant grew only in the presence of unsaturated fatty acid having at least one delta 9cis double bond, and its conidial fatty acid profile was influenced by the exogenous acid. Analyses of the fatty acids of UFA2 grown in the presence of different fatty acid supplements support the original view that the mutant is defective in delta 9-desaturase activity.     

An unsaturated fatty acid mutant of Aspergillus niger with partially defective delta 9-desaturase

The wild-type Aspergillus niger (V35) does not require fatty acids for growth. Four unsaturated fatty acid auxotrophs designated as UFA1, UFA2, UFA3, and UFA4 have been produced from this organism by treating the conidia of the wild-type strain with a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, followed by isolation on media containing monounsaturated fatty acids and the nonionic detergent, Brij 58. Optimal growth of the mutants comparable with that of the wild type was achieved with medium supplemented with C16 or C18 unsaturated fatty acids containing at least one cis double bond at the delta 9 position. Some other fatty acids (18:1 delta 11 cis and 16:1 delta 9 trans) support growth to some extent. The mutants do not grow at all in the presence of saturated fatty acids. Fatty acid analyses of the mutant, UFA2, grown in the presence of different fatty acid supplements reveal that it may be defective in a desaturase system. Experiments with unlabeled and [1-14C]palmitoyl-CoA have shown that the microsomes of the mutant (UFA2) contain a partially defective delta 9-desaturase system.     

Lipid production by an unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum grown in single-stage continuous culture

An unsaturated fatty acid auxotroph of the oleaginous yeast Apiotrichum curvatum, named UfaM3, blocked in the conversion of stearic to oleic acid was cultivated in single-stage continuous culture. The influence of consumed carbon to nitrogen ratios (C/N ratios, g g^–1) obtained at various dilution rates (D) on fatty acid (FA) accumulation and its profiles were studied. In continuous culture in N-limited medium a maximum FA accumulation of 45.6% (g g^–1 of dry biomass) was obtained at an optimal D of 0.049 h^–1, recording an efficiency of substrate conversion of 0.48 g g^–1 and 0.22 g g^–1 for biomass and lipids, respectively. The quality of lipid approached cocoa butter at an optimal C/N ratio of between 20 and 30. The C/N ratio in the incoming medium was 38.5 g g^–1 with 30 g l^–1 of glucose and both C and N sources were completely consumed at a critical D of 0.07 h^–1. The stability of the mutant was demonstrated in the steady-state conditions of the chemostat with regard to the FA composition of its lipids. Correspondence to: P. J. Blanc     

Heterogeneous amino acid transport rate changes in an E. coli unsaturated fatty acid auxotroph.

Amino acid transport rates in an $\text{E. coli}$ unsaturated fatty acid auxotroph were non-uniformly affected by enrichment of membrane lipids in various unsaturated fatty acids. Proline and threonine transport rates were depressed much more than lysine and asparagine rates by trans unsaturated acids. Myristoleate and linolenate enrichment also produced non-uniform but lesser rate reductions. Although changes in the relative numoer of effective transport catalysts could account for these findings, comparisons of proline and lysine transport rates over a broad temperature range indicated that non-uniform alterations in transport catalyst reaction rates account at least partly for the activity changes associated with membrane lipid alterations.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli.

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFAts) grown at 28degrees C (PL28), and at 42degrees C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19degrees C for PL28 and at 43degrees C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42 degrees C) are in the state where the gel and liquid crystalline phases coexist.     

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.     

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.     

Growth in high osmotic medium of an unsaturated fatty acid auxotroph of Escherichia coli K-12

A temperature-sensitive mutant of Escherichia coli K-12, which is impaired in the biosynthesis of the unsaturated fatty acids at 42 C, continues to grow and remains viable in a medium with a high osmotic pressure. Under these growth conditions, 50% of the fatty acids of the parental strain and 80% of the fatty acids of the mutant strain are saturated.     

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.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Lipid analysis and fatty acid profiles of individual arterial atherosclerotic plaques

A protocol for the analysis of the lipid profile of microsamples of aortic tissue was developed. Lipid extraction was from intact tissue using acetone and chloroform/methanol (2/1, v/v). The extract was analyzed for total lipid, esterified cholesterol, cholesterol, triacylglycerol, and phospholipid. The extract was then processed to separate cholesteryl esters, triacylglycerol, and phospholipid which were hydrolyzed and the fatty acid composition was determined by GLC of pentafluorobenzyl ester derivatives. A lipid profile could be obtained on samples with a wet weight of 5 mg.     

Influence of unsaturated fatty acids, membrane fluidity and oxygenation on the survival of an E. coli fatty acid auxotroph following gamma-irradiation.

Escherichia coli K1060, a fatty acid auxotroph unable to either synthesize or degrade unsaturated fatty acids (uFAs), was used to study the effect of membrane fluidity on survival after exposure to ionizing radiation. Using this strain of E. coli, significant alterations in the fatty acid composition of the membrane have been produced and verified by gas chromatography. Linolenic, oleic, elaidic and palmitelaidic acids were the uFAs used. Survival above the transition temperature (Tt) (liquid crystal in equilibrium gel) was comparable for these fatty-acid-supplemented membranes after exposure to gamma-irradiation, whereas gamma-irradiation below Tt resulted ina significant decrease in survival. An oxygen enhancement effect was observed for each experimental condition employed.     

Lipid production by a cellulolytic strain of Aspergillus niger

Studies on lipid formation by a cellulolytic mould. Aspergillus niger AS-101, were carried out in shake flasks. The maximum amount of lipid accumulated comprised 17% and 14·9% of the mycelial dry weight on glucose and cellulose media, respectively. Unsaturated fatty acids comprised around 80% of the total fatty material, with linoleic acid predominating.     

Inhibition of medium-chain fatty acid beta-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid

Valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, were found to inhibit strongly the metabolism of decanoic acid in homogenates of rat liver. Reductions in decanoate consumption in response to inhibitors were paralleled by decreases in the formation of octanoic and hexanoic acids, two products of decanoate beta-oxidation. In contrast, 4-pentenoic acid, an established inhibitor of long-chain fatty acid beta-oxidation, had little effect on the metabolism of decanoate. It is concluded that the title compounds are potent, broad-spectrum inhibitors of fatty acid beta-oxidation, a property which may be of key toxicological importance in the pathology of valproate-induced liver injury.     

Use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes

KD115 (ol1), an unsaturated fatty acid auxotroph of S. cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids. The parent strain S288C was studied as a control. The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement. The fatty acid supplement represented 70-80% of the yeast fatty acids. Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1. The mitochondrial morphology of yeast in each growth supplement was notably different. The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures. As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above. The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane.