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When oxylipin and mitochondrion probes, i.e., fluorescing antibodies specific for 3-hydroxy fatty acids (3-OH oxylipins) and rhodamine 123 (Rh123), were added to yeast cells, these probes accumulated mainly in the sexual cells (i.e., both associated with ascospores) and not in the vegetative cells. This suggests increased mitochondrial activity in asci, since 3-OH oxylipins are mitochondrially produced and it is known that Rh123 accumulates selectively in functional mitochondria that maintain a high transmembrane potential (Delta Psi m). This increased activity may be necessary for the production and effective release of the many spores found in single-celled asci. These results may be useful in the rapid identification of asci and in yeast sexual spore mechanics, which may find application in yeast systematics as well as hydro-, aero-, and nano-technologies.

In this paper we report the influence of acetylsalicylic acid on oxylipin migration in Cryptococcus neoformans var. neoformans UOFS Y-1378, previously isolated from human bone lesion. Transmission electron microscopy suggests that osmiophilic material originates in mitochondria and is deposited inside the yeast cell wall, from which it is excreted into the environment, along capsule protuberances, or through capsule detachments. Previous studies using immunogold labeling indicate that these osmiophilic layers contain 3-hydroxy oxylipins. In this study, the addition of acetylsalicylic acid (an inhibitor of mitochondrial function) in increasing amounts to the cells abrogated the migration of osmiophilic material, as well as capsule detachment from cell walls, and hence, oxylipin excretion. Consequently, we hypothesize that 3-hydroxy oxylipins are produced in mitochondria, probably via incomplete beta-oxidation or fatty acid synthesis, from which they are deposited inside the cell wall and excreted through tubular protuberances attached to the surrounding capsules and (or) through detachment of these oxylipin-containing capsules.

Using a well tested antibody specific for 3-hydroxy oxylipins, we mapped the presence of these oxylipins in selected Cryptococcus (Filobasidiella) species. Immunofluorescence microscopy studies revealed that these compounds are deposited on cell wall surfaces, appendages, and collarettes. In vitro studies revealed that growth of Cryptococcus species was inhibited by acetylsalicylic acid (which is known to inhibit mitochondrial function, including the production of 3-hydroxy oxylipins) at concentrations as low as 1 mmol/L. The results suggest that acetylsalicylic acid is effective in controlling the growth of tested pathogens, probably by targeting their mitochondria. This study further expands the known function of this anti-inflammatory drug as anti-fungal agent.

Using immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fatty acids in Cryptococcus neoformans. Our results suggest that these oxylipins accumulate in capsules where they are released as hydrophobic droplets through tubular protuberances into the surrounding medium.

Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium--an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete beta-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as a target for ASA antifungal action.

Yeasts use different mechanisms to release ascospores of different lengths from bottle-shaped asci. Round to oval-shaped ascospores are enveloped in oxylipin-coated compressible sheaths, enabling ascospores to slide past each other when they reach the narrowing ascus neck. However, more elongated ascospores do not contain sheaths, but are linked by means of oxylipin-coated interlocked hooked ridges on the surfaces of neighboring ascospores, thereby keeping them aligned while they are pushed towards the ascus tip by turgor pressure. In this study, we found elongated, oxylipin-coated sheathed ascospores in Dipodascus geniculatus that are released effectively from bottle-shaped asci without alignment. This is possible because the ascus neck and opening have a diameter that is the same as the length of the ascospore, thus allowing the ascospores to turn sideways without blocking the ascus when they are released. We found that increased concentrations of acetylsalicylic acid inhibit both ascospore release and 3-hydroxy oxylipin production in this yeast, thereby implicating this oxylipin in sexual reproduction.

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.

The distribution of 3-hydroxy oxylipins in Saturnispora saitoi was mapped using immunofluorescence microscopy. Fluorescence was observed on aggregating ascospores, indicating the presence of 3-hydroxy oxylipins on the surface or between ascospores. The oxylipin was identified as 3-hydroxy 9:1 using gas chromatography mass spectrometry. Furthermore, ultrastructural studies using scanning and transmission electron microscopy on ascospores revealed a clear equatorial ledge surrounding oval-shaped ascospores.

Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography-mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall.

Yeasts utilize different mechanisms to release ascospores of different lengths from bottle-shaped asci. Using electron microscopy, confocal laser scanning microscopy, gas chromatography-mass spectrometry and digital live imaging, the individual release of oval ascospores from tight-fitting narrow bottle-necks, is reported in the yeast Dipodascus albidus. These ascospores are surrounded by compressible, oxylipin-coated sheaths enabling ascospores to slide past each other when forced by turgor pressure and by possible sheath contractions towards the narrowing ascus-neck. In this paper, the release mechanisms of ascospores of various lengths from bottle-shaped asci and produced by different yeasts are compared. We suggest that different release mechanisms, utilizing compressible sheaths or geared-alignment, have possibly evolved to compensate for variation in ascospore length. Alternatively, sheaths and ridges might be two evolutionary solutions to the same biomechanical problem, i.e. to release ascospores irrespective of length from bottle-shaped asci.

Through gas chromatography - mass spectrometry, the presence of oxylipins, mainly 3-hydroxy 9:1 and 3-hydroxy 10:1, was detected in Saccharomycopsis fermentans, Saccharomycopsis javanensis, and Saccharomycopsis vini. The distribution of these compounds was mapped using immunofluorescence microscopy, and they were found to be closely associated with the surfaces of aggregating ascospores.

Immunofluorescence microscopy exposed the presence of novel 3-hydroxy oxylipins on the surfaces of aggregated hat-shaped ascospores of Ascoidea africana. These compounds were confirmed by gas chromatography - mass spectrometry analysis. Only the complete structure of a novel 3-hydroxy 10:1 could be determined.

3-Hydroxy oxylipins were uncovered on ascospores of Eremothecium sinecaudum using immunofluorescence microscopy. This was confirmed by gas chromatography mass spectrometry. These oxylipins were observed only on ascospore parts characterised by nano-scale surface ornamentations simulating a corkscrew as demonstrated by scanning electron microscopy. Conventional ascospore staining further confirms its hydrophobic nature. Using confocal laser scanning microscopy we found that the corkscrew part with spiky tip of needle-shaped ascospores may play a role in rupturing the ascus in order to affect its release. Through oxylipin inhibition studies we hypothesise a possible role for 3-hydroxy oxylipins in facilitating the rupturing process.

A novel cascade of 3-hydroxy fatty acids was discovered in the yeast Saccharomycopsis synnaedendra. The cascade, probably derived from incomplete beta-oxidation, comprises both even and uneven carbon numbered as well as saturated and unsaturated 3-hydroxy oxylipins. This yeast may now be used as model to further study the metabolism of these compounds as well as their biotechnological production.

It was discovered that the addition of 10 g/l acetate to a medium containing 30 g/l sunflower oil caused a drastic increase in citric acid production by Yarrowia lipolytica UOFS Y-1701 i.e. from 0.5 g/l in the absence of acetate to 18.7 g/l in the presence of acetate. Similarly, the ratio of citric acid:isocitric acid increased significantly from 1.7:1 in the absence of acetate to 3.7:1 in the presence of acetate after 240 h of growth.

Immunofluorescence microscopy was used to assess members of the yeast genus Dipodascus for the presence of 3-hydroxy oxylipins. Fluorescence was associated with the aggregating ascospores in all species tested, thus suggesting the association of 3-hydroxy oxylipins with these cells, especially the surrounding slime sheaths. An ultrastructural study of the ascospores revealed sheaths with indentations, probably caused by the close packing of the ascospores to form clusters. In addition, an increase in the neutral and glycolipid fractions as well as a decrease in the phospholipid fraction during ascosporogenesis in D. ambrosiae was found.

Electron microscopy studies indicated that the major oxylipin 3-hydroxy palmitic acid (16:0) was associated with aggregating vegetative cells and formed a web-like structure around these cells. Cross sections through this structure showed a hydrophilic outer layer and a more hydrophobic inner layer suggesting that the web-like structure is in fact tube-like micelles. This information sheds more light on the role of these hydroxyoxylipins in fungi.

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium--in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.

Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel 'ghosting' phenomenon observed during assumed lectin-mediated aggregation. We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a 'ghostlike' fashion without visually affecting the cell wall structure or the layers. This migration resulted in the binding of these layers to cell walls of adjacent cells. Consequently, 'ghosting' seems a prerequisite for flocculation to occur. However, 'ghosting' alone may not be sufficient to ensure flocculation.

The soil fungus Mucor genevensis was shown to convert exogenous arachidonic acid to the oxylipin 3-hydroxy-5Z,8Z-tetradecadienoic acid (3-HTDE) as determined by gas chromatography/mass spectrometry. This metabolite was only found in the aqueous supernatant together with free linoleic acid, but not in the final fungal biomass. In contrast, the corresponding primary arachidonic acid metabolite (3R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE), which has been earlier shown to be produced by the yeast Dipodascopsis uninucleata, could not be detected. These observations may be plausibly explained by a retroconversion by M. genevensis of arachidonic acid to linoleic acid before the latter is metabolised to 3-HTDE.

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.

In this study, the lipid fractions i.e. neutral (NL), phospho-(PL) and glycolipids (GL) with associated fatty acids (FAs) of 54 strains, representing the Schizosaccharomycetales, were analyzed during stationary growth phase and compared. Trace amounts of linoleic acid (18:2) were present in most of the strains representing Schizosaccharomyces. An increased percentage 18:2 was observed in the PL fraction when compared to the NL fraction. This is possibly related to membranes requiring polyunsaturated FAs for fluidity. On the basis of the percentage oleic acid (18:1) and 18:2 FAs in the different lipid fractions, the Schizosaccharomycetales can clearly be divided into two groups i.e. Group 1 (represented by the genus Hasegawaea) comprising strains producing relatively large amounts of 18:2 and relatively low amounts of 18:1 when compared to Group 2 (represented by the genus Schizosaccharomyces comprising Schizosaccharomyces octosporus and Schizosaccharomyces pombe). These results are in accordance with 18S and 26S rRNA base sequence analyses and emphasize the difference between the genera Hasegawaea and Schizosaccharomyces. Utilizing gas chromatography-mass spectrometry analyses, it was found that these strains were all capable of producing gamma-linolenic acid. This further emphasizes the uniqueness of this order in the Dikaryomycota.

In this study, seven fungal strains, representing different phylogenetic groups within the Dikaryomycota, were tested for the presence of gamma-linolenic acid [18:3(omega 6)], when grown in synthetic liquid media devoid of fatty acids, on a series of 40 different carbon sources. The fungal strains represented the species Dipodascopsis uninucleata, Eurotium rubrum, Galactomyces geotrichum, Neurospora crassa, Saccharomyces cerevisiae, Spongipellis unicolor and Talaromyces flavus. Cultures were periodically harvested during growth and the fatty acids in the total lipids analysed as methyl esters, using gas chromatography and mass spectrometry. It was found that 18:3(omega 6) is present in E. rubrum CBS 350.65, S. unicolor CBS 117.16 and in T. flavus CBS 310.38NT, when these strains were grown on certain carbon sources. No correlation between the growth phase of the organism and the presence of 18:3(omega 6) could be detected. In order to confirm the production of 18:3(omega 6), the lipid metabolism of two unrelated dikaryomycotan fungi (S. unicolor CBS 117.16 and E. rubrum CBS 350.65) grown on two different carbon sources each, was examined. Cultures of E. rubrum CBS 350.65 were grown on glucose and sorbose and cultures of S. unicolor CBS 117.16 on glucose and sucrose in synthetic liquid media with a C:N ratio of 50:1 (w/w). The total lipids of these cultures were fractionated and the fatty acids in the fractions analysed as methyl esters, using gas chromatography and mass spectrometry. The lipid metabolism of both E. rubrum CBS 350.65 and S. unicolor CBS 117.16 differed on the two carbon sources used. The ab initio production of 18:3(omega 6) by E. rubrum CBS 350.65 in synthetic liquid media was confirmed. In contrast, the ab initio production of 18:3(omega 6) by S. unicolor CBS 117.16 in synthetic liquid media could not be confirmed.

Sporangiospores of Mucor circinelloides f. circinelloides CBS108.16 could germinate and grow on a wide variety of carbon sources in synthetic liquid media. Growth was supported by aldoses which have the same configuration at carbon atom number two as glucose. Di- and trisaccharides consisting of D-glucopyranosyl moieties were assimilated, while polysaccharides like inuline and starch were also utilised. Various alcohols and organic acids could be assimilated, while the phenolic compounds tested could not support aerobic growth. The fungus was able to ferment carbohydrates consisting of D-glucopyranosyl moieties, grow in the absence of vitamins and in the presence of cycloheximide. It also liquefied gelatin and produced lipases and cellulolytic enzymes. It was found that the highest percentages polyunsaturated fatty acids were produced when acetic acid, glucose, mannitol, soluble starch or trehalose was used as carbon source. The absence of vitamins in the medium lowered the percentage of these fatty acids.

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or p-coumaric acid/I could be detected and estimated in < 1 h. The method is specific for the two phenolic acids sice no interference by other components was observed.

Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:0) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGs) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SUS-TAGs and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% 18:0 in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGs.