Triacylglycerols (TAGs) containing positional isomers of hypogeic (Hy), palmitoleic (Po), and palmitvaccenic (Pv) acids from three microorganisms (top-fermenting brewer's yeast Saccharomyces cerevisiae, green alga Coccomyxa elongata, and arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis) were analyzed. Dozens of regioisomers and enantiomers of TAGs containing one, two or three hexadecenoic acids have been identified by means of reversed phase chromatography/mass spectrometry (RP-HPLC/MS). The regioisomers of TAGs containing two palmitic acids and any hexadecenoic acid were separated. Analysis of regioisomers of TAGs having one Pv residue showed that asymmetric molecular species such as PvPP or PPPv were dominant in Rhizophagus. TAGs were also analyzed on a chiral phase column and nine molecular species of TAGs containing two palmitic and any of three hexadecenoic acids were separated and identified. In the case of TAGs containing one palmitic and two hexadecenoic acids, the separation was successful only if the hexadecenoic acids were identical. Separation of TAGs containing three hexadecenoic acids was successful only if all three hexadecenoic acids were identical. Regardless of the type of TAG, it was found that TAGs in the AM fungus and containing palmitvaccenic acid bound at the sn-1 position of the glycerol backbone were dominant, suggesting similarity in the biosynthesis of the different TAGs. The covalent adduct chemical ionization method was used for identification of TAGs as adduct with (1-methyleneimino)-1-ethenyl ion, which reacted with double bond of the unsaturated fatty acid. Tandem MS thus makes it possible to identify TAGs containing various hexadecenoic acids.
Melting summer snow in the Austrian Alps exhibited a yellowish bloom that was mainly comprised of an unidentified unicellular chrysophyte. Molecular data (18S rRNA and rbcL genes) showed a close relationship to published sequences from an American pond alga formerly identified as Kremastochrysis sp. The genera Kremastochrysis and Kremastochrysopsis are morphologically distinguished by the number of flagella observed with the light microscope, and therefore we assigned the Austrian snow alga and an American pond alga to the genus Kremastochrysopsis. Transmission and scanning electron microscopy revealed that swimming cells had two flagella oriented in opposite directions, typical for the Hibberdiales. Molecular phylogenetic analyses showed that both new species were closely related to Hibberdia. Kremastochrysopsis ocellata, the type species and only known species, has two chloroplasts per cell and the zoospores have red eyespots. Our two organisms had only a single chloroplast and no zoospore eyespot, but their gene sequences differed substantially. Therefore, we described two new species, Kremastochrysopsis austriaca sp. nov and Kremstochrysopsis americana sp. nov. When grown in culture, both taxa showed a characteristic hyponeustonic growth (hanging below the water surface), whereas older immotile cells grew at the bottom of the culture vessel. Ecologically, Kremastochrysopsis austriaca sp. nov., which caused snow discolorations, had no close phylogenetic relationships to other psychrophilic chrysophytes, for example, Chromulina chionophilia, Hydrurus sp., and Ochromonas-like flagellates.
- MeSH
- chloroplasty * MeSH
- Chrysophyceae * MeSH
- fylogeneze MeSH
- RNA ribozomální 16S MeSH
- RNA ribozomální 18S MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Rakousko MeSH
The green algal genus Cylindrocystis is widespread in various types of environments, including extreme habitats. However, very little is known about its diversity, especially in polar regions. In the present study, we isolated seven new Cylindrocystis-like strains from terrestrial and freshwater habitats in Svalbard (High Arctic). We aimed to compare the new isolates on a molecular (rbcL and 18S rDNA), morphological (light and confocal laser scanning microscopy), and cytological (Raman microscopy) basis. Our results demonstrated that the Arctic Cylindrocystis were not of a monophyletic origin and that the studied strains clustered within two clades (tentatively named the soil and freshwater/glacier clades) and four separate lineages. Morphological data (cell size, shape, and chloroplast morphology) supported the presence of several distinct taxa among the new isolates. Moreover, the results showed that the Arctic Cylindrocystis strains were closely related to strains originating from the temperate zone, indicating high ecological versatility and successful long-distance dispersal of the genus. Large amounts of inorganic polyphosphate (polyP) grains were detected within the chloroplasts of the cultured Arctic Cylindrocystis strains, suggesting effective luxury uptake of phosphorus. Additionally, various intracellular structures were identified using Raman microscopy and cytochemical and fluorescent staining. This study represents the first attempt to combine molecular, morphological, ecological, and biogeographical data for Arctic Cylindrocystis. Our novel cytological observations partially explain the success of Cylindrocystis-like microalgae in polar regions.
- MeSH
- Chlorophyta * MeSH
- fylogeneze MeSH
- polyfosfáty MeSH
- Streptophyta * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Arktida MeSH
- Svalbard MeSH
In this study, a unicellular soil alga isolated from farmland in Germany was surveyed. The investigation of the hypervariable molecular markers ITS1 rDNA and ITS2 rDNA identified strain E71.10 as conspecific with Vischeria sp. SAG 51.91 (Eustigmatophyceae). The culture was tested for biomass generation and for the yield of fatty acids and amino acids. The survey included four different culture conditions (conventional, elevated CO2, nitrogen depletion, or sodium chloride stress) at room temperature. The best yield of dry biomass was achieved applying 1% CO2, whereas nitrogen-free medium resulted into least growth. The fatty acid content peaked in nitrogen-free medium at 59% per dry mass. Eicosapentaenoic acid was the most abundant fatty acid in all treatments (except for nitrogen free), accounting for 10.44 to 16.72 g/100 g dry mass. The highest content of amino acids (20%) was achieved under conventional conditions. The results show that abiotic factors strongly influence to which extent metabolites are intracellularly stored and they confirm also for this yet undescribed strain of Vischeria that Eustigmatophyceae are promising candidates for biotechnology.
- MeSH
- aminokyseliny metabolismus MeSH
- biomasa MeSH
- biotechnologie MeSH
- dusík metabolismus MeSH
- Heterokontophyta klasifikace genetika růst a vývoj metabolismus MeSH
- kultivační média chemie MeSH
- kyselina eikosapentaenová analogy a deriváty metabolismus MeSH
- mastné kyseliny metabolismus MeSH
- mikrořasy metabolismus MeSH
- půda * MeSH
- ribozomální DNA MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Německo MeSH
The analysis of triacylglycerols and phospholipids - phosphatidylcholines allowed the use of shotgun lipidomics to identify very long-chain fatty acids and very long-chain polyunsaturated fatty acids in microalgae. These fatty acids were determined in triacylglycerols by positive electrospray ionization of neutral loss scans of different fatty acids, e.g. 24:0, 24:1ω9, 24:6ω3, 26:0, 26:1ω9, 28:0, 28:1ω9, 28:2ω6, and 28:8ω3. Likewise, very long-chain fatty acids in phosphatidylcholines were identified by negative electrospray ionization mass spectrometry in the selected ion-monitoring of the two most important ions (R1COO- and R2COO-). The limit of detection was determined at 10 nmol/L (∼11 pg/μL) in triacylglycerols and 8.6 nmoles/L (∼8 pg/μL) in phosphatidylcholines. The use of liquid chromatography-mass spectrometry is suitable for very long-chain polyunsaturated fatty acids with up to 8 double bonds due to the time of analysis as well as for reasons of lower thermal stability of polyunsaturated fatty acids towards saturated fatty acids, but gas chromatography-mass spectrometry is better suited for the analysis of saturated very long-chain fatty acids.
- MeSH
- Bacteria metabolismus MeSH
- Chlorophyta metabolismus MeSH
- fosfatidylcholiny analýza MeSH
- limita detekce MeSH
- mastné kyseliny analýza MeSH
- nenasycené mastné kyseliny analýza MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- referenční standardy MeSH
- triglyceridy analýza MeSH
- Publikační typ
- časopisecké články MeSH
Melting snowfields in polar and alpine regions often exhibit a red and orange colouration caused by microalgae. The diversity of these organisms is still poorly understood. We applied a polyphasic approach using three molecular markers and light and electron microscopy to investigate spherical cysts sampled from alpine mountains in Europe, North America and South America as well as from both polar regions. Molecular analyses revealed the presence of a single independent lineage within the Chlamydomonadales. The genus Sanguina is described, with Sanguina nivaloides as its type. It is distinguishable from other red cysts forming alga by the number of cell wall layers, cell size, cell surface morphology and habitat preference. Sanguina nivaloides is a diverse species containing a total of 18 haplotypes according to nuclear ribosomal DNA internal transcribed spacer 2, with low nucleotide divergence (≤3.5%). Based on molecular data we demonstrate that it has a cosmopolitan distribution with an absence of geographical structuring, indicating an effective dispersal strategy with the cysts being transported all around the globe, including trans-equatorially. Additionally, Sanguina aurantia is described, with small spherical orange cysts often clustered by means of mucilaginous sheaths, and causing orange blooms in snow in subarctic and Arctic regions.
- MeSH
- Chlorophyta klasifikace genetika fyziologie MeSH
- ekosystém MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- mezerníky ribozomální DNA MeSH
- Rhodophyta MeSH
- sníh mikrobiologie MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Jižní Amerika MeSH
- Severní Amerika MeSH
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.
Mass spectrometry-based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes-phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)2 Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long-chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)2 Cer decreased. Molecular species having C18-LCB were associated with low cultivation temperature, and a higher proportion of C20-LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long-chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2-hydrocylo-lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.
The traditional green algal genus Chloromonas accommodates mesophilic, cold-tolerant and cold-adapted microorganisms. In this paper, we studied a new strain isolated from a wet hummock meadow in the High Arctic. We used morphological, ultrastructural and molecular data to assess the taxonomic position and phylogenetic relationships of the new isolate. The observed morphological features generally corresponded to the cold-tolerant Chloromonas characteristics. However, ellipsoidal or wide ellipsoidal vegetative cells, a massive parietal cup-shaped chloroplast with a number of continuously connected lobes, a thick cell wall, a prominent hemispherical papilla and the anterior position of an oblong or round eyespot distinguished the alga from all previously described Chloromonas species. Analyses of rbcL and 18S rRNA genes showed that the new strain formed an independent lineage within a clade containing mesophilic and psychrotolerant Chloromonas species. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker supported a separate species identity of the new isolate. Considering the morphological and molecular differences from its relatives, a new psychrotolerant species, Chloromonas svalbardensis, is proposed. Further, our results demonstrated the paraphyletic origin of Chloromonas within Chloromonadinia with genetically, morphologically and ecologically well-defined clades. We discuss a scenario of a possible Chloromonas split and revision.
- MeSH
- buněčná stěna ultrastruktura MeSH
- Chlorophyceae klasifikace cytologie genetika fyziologie MeSH
- chloroplasty ultrastruktura MeSH
- DNA rostlinná analýza MeSH
- fylogeneze * MeSH
- ribozomální DNA genetika MeSH
- ribulosa-1,5-bisfosfát-karboxylasa genetika MeSH
- RNA ribozomální 18S genetika MeSH
- rostlinné geny genetika MeSH
- sníh MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Arktida MeSH
- Norsko MeSH
Enantiomers of triacylglycerols (TAGs) containing any combination of very long chain fatty acids (VLCFAs) and/or very long chain polyunsaturated fatty acids (VLCPUFAs) with diolein, dilinolein and didocosahexaenoin were synthesized. Gradient non-aqueous reversed-phase high-performance liquid chromatography/high resolution atmospheric pressure chemical ionization-tandem mass spectrometry (NARP-HPLC/HRMS2-APCI) and chiral liquid chromatography were used for the separation and identification of molecular species of these TAGs. Further, NARP-LC and chiral LC were used to separate natural mixtures of TAGs obtained from four natural sources, i.e. ximenia oil (Ximenia americana), green alga (Botryococcus braunii), breweŕs yeast (Saccharomyces pastorianus) and a dinoflagellate (Amphidinium carterae). The ratio of regioisomers and enantiomers in individual samples was determined and a hypothesis has been confirmed on the biosynthetic pathway of natural TAGs, which is based on the preferential representation of VLCFAs and VLCPUFAs in the sn-1 position of the glycerol backbone.
- MeSH
- atmosférický tlak MeSH
- Chlorophyta chemie metabolismus MeSH
- chromatografie s reverzní fází MeSH
- Dinoflagellata chemie metabolismus MeSH
- kyseliny mastné neesterifikované chemie MeSH
- nenasycené mastné kyseliny chemie MeSH
- Saccharomyces chemie metabolismus MeSH
- stereoizomerie MeSH
- tandemová hmotnostní spektrometrie MeSH
- triglyceridy analýza chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
