Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures.

Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4-28°C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10°C. On temperature drop to 4°C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice. A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7°C and the difference between PO-PE and OP-PE is some 10°C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions.

Yeast Tok1p channel is a major contributor to membrane potential maintenance under chemical stress.

Tok1p is a highly specific yeast plasma membrane potassium channel with strong outward directionality. Its opening is induced by membrane depolarization. Although the biophysical properties of Tok1p are well-described, its potentially important physiological role is currently largely unexplored. To address this issue, we examined the Tok1p activity following chemically-induced depolarization by measuring changes of plasma membrane potential (ΔΨ) using the diS-C3(3) fluorescence assay in a Tok1p-expressing and a Tok1p-deficient strain. We report that Tok1p channel activity in response to chemical stress does not depend solely on the extent of depolarization, as might have been expected, but may also be negatively influenced by accompanying effects of the used compound. The stressors may interact with the plasma membrane or the channel itself, or cause cytosolic acidification. All of these effects may negatively influence the Tok1p channel opening. While ODDC-induced depolarization exhibits the cleanest Tok1p activation, restoring an astonishing 75% of lost ΔΨ, higher BAC concentrations reduce Tok1p activity, probably because of direct interactions with the channel and/or its lipid microenvironment. This is not only the first study of the physiological role of Tok1p in ΔΨ maintenance under chemical stress, but also the first estimate of the extent of depolarization the channel is able to counterbalance.

Regioisomeric and enantiomeric analysis of triacylglycerols.

A survey of useful methods for separation and identification of regioisomers and enantiomers of triacylglycerols. Gas chromatography, gas chromatography-mass spectrometry, 13C NMR determination of regioisomers by enzymatic methods, and supercritical fluid chromatography are briefly surveyed, whereas a detailed description is given of the analysis of triacylglycerols by liquid chromatography, especially with silver ion (Ag+; argentation), and nonaqueous reversed phase liquid chromatography. Special attention is paid to chiral chromatography. Details of mass spectrometry of triacylglycerols are also described, especially the identification of important triacylglycerol ions such as [M + H-RCOOH]+ in atmospheric pressure chemical ionization mass spectra.

Resveratrol suppresses ethanol stress in winery and bottom brewery yeast by affecting superoxide dismutase, lipid peroxidation and fatty acid profile.

Mid-exponential cultures of two traditional biotechnological yeast species, winery Saccharomyces cerevisiae and the less ethanol tolerant bottom-fermenting brewery Saccharomyces pastorianus, were exposed to different concentrations of added ethanol (3, 5 and 8%) The degree of ethanol-induced cell stress was assessed by measuring the cellular activity of superoxide dismutase (SOD), level of lipid peroxidation products, changes in cell lipid content and fatty acid profile. The resveratrol as an antioxidant was found to decrease the ethanol-induced rise of SOD activity and suppress the ethanol-induced decrease in cell lipids. A lower resveratrol concentration (0.5 mg/l) even reduced the extent of lipid peroxidation in cells. Resveratrol also alleviated ethanol-induced changes in cell lipid composition in both species by strongly enhancing the proportion of saturated fatty acids and contributing thereby to membrane stabilization. Lower resveratrol concentrations could thus diminish the negative effects of ethanol stress on yeast cells and improve their physiological state. These effects may be utilized to enhance yeast vitality in high-ethanol-producing fermentations or to increase the number of yeast generations in brewery.

Stratification of yeast cells during chronological aging by size points to the role of trehalose in cell vitality.

Cells of the budding yeast Saccharomyces cerevisiae undergo a process akin to differentiation during prolonged culture without medium replenishment. Various methods have been used to separate and determine the potential role and fate of the different cell species. We have stratified chronologically-aged yeast cultures into cells of different sizes, using centrifugal elutriation, and characterized these subpopulations physiologically. We distinguish two extreme cell types, very small (XS) and very large (L) cells. L cells display higher viability based on two separate criteria. They respire much more actively, but produce lower levels of reactive oxygen species (ROS). L cells are capable of dividing, albeit slowly, giving rise to XS cells which do not divide. L cells are more resistant to osmotic stress and they have higher trehalose content, a storage carbohydrate often connected to stress resistance. Depletion of trehalose by deletion of TPS2 does not affect the vital characteristics of L cells, but it improves some of these characteristics in XS cells. Therefore, we propose that the response of L and XS cells to the trehalose produced in the former differs in a way that lowers the vitality of the latter. We compare our XS- and L-fraction cell characteristics with those of cells isolated from stationary cultures by others based on density. This comparison suggests that the cells have some similarities but also differences that may prove useful in addressing whether it is the segregation or the response to trehalose that may play the predominant role in cell division from stationary culture.

Influencing fatty acid composition of yeasts by lanthanides.

The growth of microorganisms is affected by cultivation conditions, concentration of carbon and nitrogen sources and the presence of trace elements. One of the new possibilities of influencing the production of cell mass or lipids is the use of lanthanides. Lanthanides are biologically non-essential elements with wide applications in technology and industry and their concentration as environmental contaminants is therefore increasing. Although non-essential, lanthanides have been proposed (and even used) to produce beneficial effects in plants but their mechanisms of action are unclear. Recently, it was suggested that they may replace essential elements or operate as potent blockers of Ca(2+) channels. We tested the effect of low concentrations of lanthanides on traditional biotechnologically useful yeast species (Kluyveromyces polysporus, Saccharomyces cerevisiae, Torulospora delbrueckii), and species capable of high accumulation of lipids (Rhodotorula glutinis, Trichosporon cutaneum, Candida sp., Yarrowia lipolytica). Low concentrations of lanthanum and monazite were conducive to an increase in cell mass and lipids and also higher production of palmitoleic acid, commonly used in cosmetics and medicine, and ω6-linoleic acid which is a precursor of thromboxanes, prostaglandins and leucotrienes.

Biotransformation of volatile fatty acids by oleaginous and non-oleaginous yeast species.

The possibility of utilizing volatile fatty acids (VFA)-containing waste substrates from biotechnological and industrial processes was investigated by cultivating both oleaginous (Candida sp., Rhodotorula glutinis, Trichosporon cutaneum, Yarrowia lipolytica) and non-oleaginous (Kluyveromyces polysporus, Saccharomyces cerevisiae, Torulaspora delbrueckii) yeast species on acetic acid, propionic acid and a combination of either acid with glucose as carbon and energy sources. Both oleaginous and non-oleaginous yeasts grew on VFA. Oleaginous yeasts accumulated lipids to 15-48% of dry cell weight, non-oleaginous yeasts also grew on VFA and showed comparable biomass yields but the lipid content was only 2-5%. Biomass and lipid yield increased in cultivations on VFA plus glucose. The lipid composition was comparable to plant-derived oils and therefore might be exploitable in biodiesel production; nearly all species, when cultured on propionate, showed a high content of the desirable odd-chain unsaturated FA, especially 17:1 acid. This study points at the wide array of possible applications of many yeasts, even non-oleaginous strains, for biovalorization of industrial wastes. Despite their low lipid content these species are useful because they can readily utilize VFA from waste products and, since they are not biologically hazardous, their biomass can be afterwards used, e.g. as livestock fodder.

Vital mitochondrial functions show profound changes during yeast culture ageing.

During a 10-day culture ageing, cells of the wild-type Saccharomyces cerevisiae strain JC 482 retain their viability, while mitochondrial function and morphology change. Cell routine and uncoupled respiration rates increase to a maximum on day 4 and then decline to near zero. The decline, which occurs also in mitochondria isolated from cells of different age, is not due to increasing proportion of petites. Rhodamine 123 fluorescence intensity reporting on mitochondrial membrane potential appears to drop slightly for 4 days and then more sharply at the time when respiration rate also decreases. The MitoTracker Green fluorescent signal related to the mitochondrial content per cell also decreases. The branched tubular mitochondrial network of 1-day-old cells dissolves into short fragments; during the first 4 days, this fragmentation is associated with increasing function of mitochondria, while later on, it accompanies functional decline, which is also indicated by the decreasing ratio of Rhodamine 123 fluorescence to MitoTracker Green fluorescence. As shown by cell counting, microscopy and flow cytometry, the cell size distribution in the population broadens, and the population thus becomes more heterogeneous. The changes in respiration rate, mitochondrial membrane potential, mass and structure point to changes in the mitochondrial status during ageing.

Alcohols are inhibitors of Saccharomyces cerevisiae multidrug-resistance pumps Pdr5p and Snq2p.

The effect of alcohols on cell membrane proteins has originally been assumed to be mediated by their primary action on membrane lipid matrix. Many studies carried out later on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using our fluorescence diS-C3 (3) diagnostic assay for multidrug-resistance pump inhibitors in a set of isogenic yeast Pdr5p and Snq2p mutants, we found that n-alcohols (from ethanol to hexanol) variously affect the activity of both pumps. Beginning with propanol, these alcohols have an inhibitory effect that increases with increasing length of the alcohol acyl chain. While ethanol does not exert any inhibitory effect at any of the concentration used (up to 3%), hexanol exerts a strong inhibition at 0.1%. The alcohol-induced inhibition of MDR pumps was detected even in cells whose membrane functional and structural integrity were not compromised. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins.

Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed- and chiral-phase LC-MS.

LC with atmospheric pressure chemical ionization (ACPI) MS with RP and chiral phase was used for separation of triacylglycerols (TAGs) from yeasts of the genera Candida, Kluyveromyces, Rhodotorula, Saccharomyces, Torulospora, Trichosporon, and Yarrowia. Chiral LC-APCI-MS is based on using two columns in series packed with a 3,5-dimethylphenyl carbamate modified ß-cyclodextrin chiral phase. All regioisomers and enantiomers of TAGs containing one to five double bonds were separated. Molecular species of TAGs, i.e. regioisomers and enantiomers, were identified and quantified by MS/MS. Among the 94 identified TAGs, the most abundant were triolein, oleopalmitoleoolein, and dipalmitoleoolein. In strains producing palmitoleic acid in amounts >25% of total fatty acids (FAs), this acid, or unsaturated FA is bound in sn-1. In strains containing palmitoleic acid at 10-25% total FAs this acid is mainly bound in sn-3, saturated FA being bound in sn-1. Strains containing <10% palmitoleic acid form preferentially symmetrical TAGs.

Biosynthesis and metabolic pathways of pivalic acid.

Occurrence, biosynthesis, and biodegradation of pivalic acid and other compounds, having a quaternary carbon atom by different bacteria, are described. We have summarized the relevant data that have so far been published, presenting them in a graphical form, i.e., as biodegradation pathways including B12-dependent isomerization and desaturation that lead to the degradation of pivalic acid and similar compounds to products with other than quaternary carbon atoms, i.e., compounds whose catabolism is well known.

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces.

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.

Rhamnolipid-producing thermophilic bacteria of species Thermus and Meiothermus.

Novel rhamnolipid-producing strains of three thermophilic bacteria, Thermus sp., T. aquaticus and Meiothermus ruber were identified that have not been previously described as rhamnolipid producers. Rhamnolipids were extracted from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed 77 rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy-fatty acids, saturated, monounsaturated or diunsaturated, even- or odd-chain, up to unusual long chains with 24 carbon atoms. The stereochemistry of rhamnose was L and that of 3-hydroxy-fatty acids was R, the position of double bonds in monoenoic acids was cis ω-9. All three strains produced a rhamnolipid that differs in structure from Pseudomonas aeruginosa rhamnolipids and exhibits excellent surfactant properties. Importantly, in comparison to P. aeruginosa both strains, i.e., Thermus and Meiothermus, are Biosafety level 1 microorganisms and are not pathogenic to humans.

Biosynthesis of ω-alicyclic fatty acids induced by cyclic precursors and change of membrane fluidity in thermophilic bacteria Geobacillus stearothermophilus and Meiothermus ruber.

Two thermophilic strains belonging to Geobacillus stearothermophilus and Meiothermus ruber, which naturally do not synthesize ω-alicyclic fatty acids (ω-FAs) were cultivated with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl carboxylic acids. Gas chromatography-mass spectrometry analysis of fatty acid methyl and picolinyl esters showed that both strains are able to synthesize ω-FAs when cultivated with the appropriate precursor. The incorporation of cyclic acids influenced the whole FA composition as well as membrane fluidity. Membrane fluidity of intact cells was studied by measuring the fluorescence polarisation of the probe l,6-diphenyl-1,3,5-hexatriene incorporated into membrane lipid bilayers. Cytoplasmic membrane became more fluid with increasing content of ω-FAs. This is caused by considerable changes in lipid packing within the membrane induced by the presence of ω-FAs not found in the natural environment of Geobacillus and Meiothermus strains.

Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p.

We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C(3)(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C(3)(3) by both pumps and confirmed the activation of membrane H(+)-ATPase by CCCP.

Arsenolipids in the green alga Coccomyxa (Trebouxiophyceae, Chlorophyta).

Lipid-like compounds containing a dimethylarsinoyl group, i.e. Me2As(O)-, have been identified by liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICP-MS) and non-aqueous reversed-phase high-performance liquid chromatography (positive and/or negative high-resolution tandem electrospray ionization mass spectrometry (NARP-HPLC/HR-ESI+(-)-MS/MS) from three strains of green algae of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta). The algae were cultivated in a medium containing 10 g arsenic/L, i.e. 133.5 mmol/L of Na2HAsO4.7H2O. After extraction by methyl-tert-butyl ether (MTBE), total lipids were analyzed by ICP-MS or ESI-MS without any further separation or fractionation. A total of 39 molecular species of arsenic triacylglycerols (AsTAG), 15 arsenic phosphatidylcholines (AsPC), 8 arsenic phosphatidylethanolamines (AsPE), 6 arsenic phosphatidylinositols (AsPI), 2 arsenic phosphatidylglycerols (AsPG) and 5 unknown lipids (probably ceramides) were identified. The structures of all molecular species were confirmed by tandem MS. Dry matter of the individual strains contained different amounts of total arsenolipids, i.e. C. elongata CCALA 427 (0.32 mg/g), C. onubensis (1.48 mg/g), C. elongata S3 (2.13 mg/g). On the other hand, there were only slight differences between strains in the relative abundances of individual molecular species. Possible biosynthesis of long-chain lipids with the end group Me2As(O) has also been suggested.

The influence of N and S poles of static magnetic field (SMF) on Candida albicans hyphal formation and antifungal activity of amphotericin B.

Due to the increasing number of Candida albicans' infections and the resistance of this pathogenic fungus to drugs, new therapeutic strategies are sought. One of such strategies may be the use of static magnetic field (SMF). C. albicans cultures were subjected to static magnetic field of the induction 0.5 T in the presence of fluconazole and amphotericin B. We identified a reduction of C. albicans hyphal length. Also, a statistically significant additional effect on the viability of C. albicans was revealed when SMF was combined with the antimycotic drug amphotericin B. The synergistic effect of this antimycotic and SMF may be due to the fact that amphotericin B binds to ergosterol in plasma membrane and SMF similarly to MF could influence domain orientation in plasma membrane (PM).

Kocuria Bacterial Isolates from Radioactive Springs of Jáchymov spa (Joachimsthal) as Sources of Polyunsaturated Fatty Acids.

Four bacterial isolates, which produced polyunsaturated fatty acids (PUFA), were isolated from water samples of radioactive springs collected from Jáchymov spa. Jáchymov (Sankt Joachimsthal) is a city in northwestern Bohemia, where Marie and Pierre Curie isolated radium in 1898 from the mineral uraninite. To date, four springs (Agricola, Behounek, C1, and Curie) have been used for spa purposes, that is for the treatment of nervous and rheumatic disorders by constantly produced radioactive gas radon (222 Rn) dissolved in the water. The radioactivity reaches 24 kBq/L. Using 16S rRNA gene sequence analysis, all four isolates were identified as members of the genus Kocuria, with two isolates designated 208 and 401 affiliated with Kocuria kristinae, while isolates 101 and 301 most likely with K. rhizophila. The content of fatty acids in polar lipids was determined by gas chromatography-mass spectrometry (GC-MS) and two PUFA, that is arachidonic and eicosapentaenoic, were identified. The position of double bonds was confirmed by GC-MS of 3-pyridylcarbinol (formerly picolinyl) esters. We assume that all four isolates of Kocuria produce PUFA to increase the stability of cell membranes, which may be impaired by the reaction of the reactive oxygen species. These can arise, for example, because of α radiation during 222 Rn decay.

Length of the hypermutation motif DGYW/WRCH in the focus of statistical limits. Implications for a double-motif or extended motif recognition models.

The motif DGYW/WRCH (Mh) and its frequently discussed simplified derivative GYW/WRC (Mhs) are involved in immunoglobulin (Ig) hypermutation. Both these motifs appear to be markedly shorter than the corresponding conventionally predicted minima of valid sequence lengths (MVSL). The same conclusion concerning both Mh and Mhs can also be obtained in the combined case including a less strict semi-empirically defined w-value and one nucleotide length tolerance related to MVSL. Such disagreement indicates considerably low information content in Mh and Mhs when evaluating these motifs as alphabetical structures (words). This fact raises a question of actually recognized structures (presumably longer than Mh and Mhs). Interestingly, both Mh and Mhs dimers or pairs of closely located Mh or Mhs achieve confirmation of length validity in the case of w=0.05, suggesting thus double-motif recognition as one of statistically consistent explanations. This possibility is also in agreement with the results of our model sequence study of mRNA derived from variable Ig gene sequences (rIgV) with respect to the most frequently occurring structures formed by motif overlaps in all model sequence sets. On the other hand, additional superior occurrence of motif pairs at a structurally important distance of a single DNA thread was found in the conserved domain (cd00099) related sequences of Elasmobranchii origin and less markedly in the corresponding human rIgV, but not in a randomly selected human subset of rIgV. The data are discussed with respect to statistical evaluation and structural properties of hypermutation motifs or the competent enzyme, i.e. activation-induced cytidine deaminase.

Biologically active compounds of semi-metals.

Semi-metals (boron, silicon, arsenic and selenium) form organo-metal compounds, some of which are found in nature and affect the physiology of living organisms. They include, e.g., the boron-containing antibiotics aplasmomycin, borophycin, boromycin, and tartrolon or the silicon compounds present in "silicate" bacteria, relatives of the genus Bacillus, which release silicon from aluminosilicates through the secretion of organic acids. Arsenic is incorporated into arsenosugars and arsenobetaines by marine algae and invertebrates, and fungi and bacteria can produce volatile methylated arsenic compounds. Some prokaryotes can use arsenate as a terminal electron acceptor while others can utilize arsenite as an electron donor to generate energy. Selenium is incorporated into selenocysteine that is found in some proteins. Biomethylation of selenide produces methylselenide and dimethylselenide. Selenium analogues of amino acids, antitumor, antibacterial, antifungal, antiviral, anti-infective drugs are often used as analogues of important pharmacological sulfur compounds. Other metalloids, i.e. the rare and toxic tellurium and the radioactive short-lived astatine, have no biological significance.