Discovery of a new photosynthetic euglenoid in Poland: Euglena mazurica sp. nov. (Euglenales, Euglenaceae).

Herein we describe a new photosynthetic euglenoid species found in Poland - Euglena mazurica. A large population exists in a small, eutrophic body of water located in a pasture near Mikolajki town inside the Masurian Landscape Park (covering a part of the Masurian Lake District in Poland). The unique cell shape (corkscrew-like) discerns it well from other previously described euglenoid species with metabolic cells. The new species possesses two plate-like chloroplasts each with a pyrenoid accompanied by two paramylon caps placed on either side of it (diplopyrenoids). On the phylogenetic tree, the new species is situated within the Euglena clade. Though it is a sister branch to three clades - one representing the similar Euglena agilis, characterized by its fusiform cells and two chloroplasts with diplopyrenoids, the two species are clearly morphologically and molecularly distinct.

Physiological responses and molecular mechanisms of biofilm formation induced by extracellular metabolites of euglena in Pseudomonas aeruginosa LNR1 for diesel biodegradation based on transcriptomic and proteomic.

Diesel, as a toxic and complex pollutant, is one of the main components in oily wastewater, and poses serious threats to the aquatic environment and the health of organisms. Employing environmentally friendly biostimulants to enhance the metabolic functions of microorganisms is currently the optimal choice to improve the biodegradation of oil-containing wastewater efficiency. This study takes Pseudomonas aeruginosa LNR1 as the target, analyzing the physiological responses and molecular mechanisms of biofilm formation when enhanced by the extracellular metabolites of euglena (EME) for diesel degradation. The results show that EME not only induces auto-aggregation behavior of strain LNR1, forming aerobic suspended granule biofilm, but also promotes the secretion of signaling molecules in the quorum sensing (QS) system. Transcriptomic and proteomic analyses indicate that the stimulatory effect of EME on strain LNR1 mainly manifests in biofilm formation, substance transmembrane transport, signal transduction, and other biological processes, especially the QS system in signal transduction, which plays a significant regulatory role in biofilm formation, chemotaxis, and two-component system (TCS). This study collectively unveils the molecular mechanisms of biostimulant EME inducing strain LNR1 to enhance diesel degradation from different aspects, providing theoretical guidance for the practical application of EME in oily wastewater pollution control.

NADPH-to-NADH conversion by mitochondrial transhydrogenase is indispensable for sustaining anaerobic metabolism in Euglena gracilis.

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

On the power per mitochondrion and the number of associated active ATP synthases.

Recent experiments and thermodynamic arguments suggest that mitochondrial temperatures are higher than those of the cytoplasm. A "hot mitochondrion" calls for a closer examination of the energy balance that endows it with these claimed elevated temperatures. As a first step in this effort, we present here a semi-quantitative bookkeeping whereby, in one stroke, a formula is proposed that yields the rate of heat production in a typical mitochondrion and a formula for estimating the number of "active" ATP synthase molecules per mitochondrion. The number of active ATP synthase molecules is the equivalent number of ATP synthases operating at 100% capacity to maintain the rate of mitochondrial heat generation. Scaling laws are shown to determine the number of active ATP synthase molecules in a mitochondrion and mitochondrial rate of heat production, whereby both appear to scale with cell volume. Four heterotrophic protozoan cell types are considered in this study. The studied cells, selected to cover a wide range of sizes (volumes) fromca.100µm3to 1 millionµm3, are estimated to exhibit a power per mitochondrion ranging fromca.1 pW to 0.03 pW. In these cells, the corresponding number of active ATP synthases per mitochondrion ranges from 5000 to just about a hundred. The absolute total number of ATP synthase molecules per mitochondrion, regardless of their activity status, can be up to two orders of magnitudes higher.

Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

Oxygenic Phototrophs Need ζ-Carotene Isomerase (Z-ISO) for Carotene Synthesis: Functional Analysis in Arthrospira and Euglena.

For carotenogenesis, two biosynthetic pathways from phytoene to lycopene are known. Most bacteria and fungi require only phytoene desaturase (PDS, CrtI), whereas land plants require four enzymes: PDS (CrtP), ζ-carotene desaturase (ZDS, CrtQ), ζ-carotene isomerase (Z-ISO) and cis-carotene isomerase (CrtISO, CrtH). The gene encoding Z-ISO has been functionally identified in only two species, Arabidopsis thaliana and Zea mays, and has been little studied in other organisms. In this study, we found that the deduced amino acid sequences of Arthrospira Z-ISO and Euglena Z-ISO have 58% and 62% identity, respectively, with functional Z-ISO from Arabidopsis. We studied the function of Z-ISO genes from the cyanobacterium Arthrospira platensis and eukaryotic microalga Euglena gracilis. The Z-ISO genes of Arthrospira and Euglena were transformed into Escherichia coli strains that produced mainly 9,15,9'-tri-cis-ζ-carotene in darkness. In the resulting E. coli transformants cultured under darkness, 9,9'-di-cis-ζ-carotene was accumulated predominantly as Z-ISO in Arabidopsis. This indicates that the Z-ISO genes were involved in the isomerization of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene in darkness. This is the first functional analysis of Z-ISO as a ζ-carotene isomerase in cyanobacteria and eukaryotic microalgae. Green sulfur bacteria and Chloracidobacterium also use CrtP, CrtQ and CrtH for lycopene synthesis as cyanobacteria, but their genomes did not comprise Z-ISO genes. Consequently, Z-ISO is needed in oxygenic phototrophs, whereas it is not found in anoxygenic species.

The dialogue between protozoa and bacteria in a microfluidic device.

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Response of aquatic protists to electric field exposure.

PURPOSE: To test the effects of short-term exposure of aquatic organisms to electric field (EF) with negligible magnetic component. MATERIALS AND METHODS: We built a plate capacitor that served as a source of EF of strengths that can be found in nature near transmission lines. We exposed two cultured protist species Euglena viridis and Paramecium caudatum to EFs for 24 hours and monitored their abundance, morphology, intracellular superoxide anion (by dihydroethidium [DHE]), hydrogen peroxide by (H2DCF) and lipid peroxidation (MDA) contents, catalase (CAT) and superoxide dismutase (SOD) activity. RESULTS: We found that even short-term exposure to low strength EF causes changes in population abundance, morphology and oxidative stress response in both species. As the EF strength increased, abundance of both species decreased. However, at weaker EFs, fission rates were seemingly promoted. We noted a decrease in size in both organisms in directions perpendicular to their fission planes correlated with EF strength. DHE and H2DCF fluorescence intensity and SOD activity were higher in organisms exposed to the stronger EFs. CONCLUSIONS: We suggest that the electric component of the field, rather than the magnetic, is the main cause of all the noted effects. As a result, aquatic organisms should be given greater importance in studies assessing the effects of EMFs in spite of the attenuating effects of water to EF strengths.

C2 metabolism in Euglena.

Euglenoids are able to assimilate fatty acids and alcohols with various carbon-chain lengths, and ethanol is known to be one of the best carbon sources to support the growth of Euglena gracilis. Ethanol is first oxidized to acetate by the sequential reactions of alcohol dehydrogenase and acetaldehyde dehydrogenase in the mitochondria, and then converted to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is metabolized through the glyoxylate cycle which is a modified tricarboxylic acid (TCA) cycle in which isocitrate lyase (ICL) and malate synthase (MS) function to bypass the two decarboxylation steps of the TCA cycle, enabling the net synthesis of carbohydrates from C2 compounds. ICL and MS form a unique bifunctional enzyme localized in Euglena mitochondria, not in glyoxysome as in other eukaryotes. The unique glyoxylate and glycolate metabolism during photorespiration is also discussed in this chapter.

Wax Ester Fermentation and Its Application for Biofuel Production.

In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i.e. wax esters, have relatively high molecular weights, are insoluble in water, and accumulate in the cells, in contrast to the common fermentation end products such as lactic acid and ethanol.A unique metabolic pathway involved in the wax ester fermentation is the mitochondrial fatty acid synthetic system. In this system, fatty acid are synthesized by the reversal of ß-oxidation with an exception that trans-2-enoyl-CoA reductase functions instead of acyl-CoA dehydrogenase. Therefore, acetyl-CoA is directly used as a C2 donor in this fatty acid synthesis, and the conversion of acetyl-CoA to malonyl-CoA, which requires ATP, is not necessary. Consequently, the mitochondrial fatty acid synthetic system makes possible the net gain of ATP through the synthesis of wax esters from paramylon. In addition, acetyl-CoA is provided in the anaerobic cells from pyruvate by the action of a unique enzyme, oxygen sensitive pyruvate:NADP+ oxidoreductase, instead of the common pyruvate dehydrogenase multienzyme complex.Wax esters produced by anaerobic Euglena are promising biofuels because myristic acid (C14:0) in contrast to other algal produced fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), has a low freezing point making it suitable as a drop-in jet fuel. To improve wax ester production, the molecular mechanisms by which wax ester fermentation is regulated in response to aerobic and anaerobic conditions have been gradually elucidated by identifying individual genes related to the wax ester fermentation metabolic pathway and by comprehensive gene/protein expression analysis. In addition, expression of the cyanobacterial Calvin cycle fructose-1,6-bisphosphatase/sedohepturose-1,7-bisphosphatase, in Euglena provided photosynthesis resulting in increased paramylon accumulation enhancing wax ester production. This chapter will discuss the biochemistry of the wax ester fermentation, recent advances in our understanding of the regulation of the wax ester fermentation and genetic engineering approaches to increase production of wax esters for biofuels.

Euglenophycin is produced in at least six species of euglenoid algae and six of seven strains of Euglena sanguinea.

Euglena sanguinea is known to produce the alkaloid toxin euglenophycin and is known to cause fish kills and inhibit mammalian tissue and microalgal culture growth. An analysis of over 30 species of euglenoids for accumulation of euglenophycin identified six additional species producing the toxin; and six of the seven E. sanguinea strains produced the toxin. A phylogenetic assessment of these species confirmed most taxa were in the Euglenaceae, whereas synthesis capability apparently has been lost in the Phacus, Eutreptiella, and Discoplastis branches.

Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae.

Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered.

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement 'Euglena'.

BACKGROUND: Microalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark. RESULTS: In this study, together with fatty acid profiling, we applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions. CONCLUSIONS: This study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.

Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday.

Selection and characterization of Euglena anabaena var. minor as a new candidate Euglena species for industrial application.

Euglena gracilis is a microalgae used as a model organism. Recently, mass cultivation of this species has been achieved for industrial applications. The genus Euglena includes more than 200 species that share common useful features, but the potential industrial applications of other Euglena species have not been evaluated. Thus, we conducted a pilot screening study to identify other species that proliferate at a sufficiently rapid rate to be used for mass cultivation; we found that Euglena anabaena var. minor had a rapid growth rate. In addition, its cells accumulated more than 40% weight of carbohydrate, most of which is considered to be a euglenoid specific type of beta-1-3-glucan, paramylon. Carbohydrate is stored in E. anabaena var. minor cells during normal culture, whereas E. gracilis requires nitrogen limitation to facilitate paramylon accumulation. These results suggest the potential industrial application of E. anabaena var. minor.

Diurnal changes in the xanthophyll cycle pigments of freshwater algae correlate with the environmental hydrogen peroxide concentration rather than non-photochemical quenching.

BACKGROUND AND AIMS: In photosynthetic organisms exposure to high light induces the production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), which in part is prevented by non-photochemical quenching (NPQ). As one of the most stable and longest-lived ROS, H2O2 is involved in key signalling pathways in development and stress responses, although in excess it can induce damage. A ubiquitous response to high light is the induction of the xanthophyll cycle, but its role in algae is unclear as it is not always associated with NPQ induction. The aim of this study was to reveal how diurnal changes in the level of H2O2 are regulated in a freshwater algal community. METHODS: A natural freshwater community of algae in a temporary rainwater pool was studied, comprising photosynthetic Euglena species, benthic Navicula diatoms, Chlamydomonas and Chlorella species. Diurnal measurements were made of photosynthetic performance, concentrations of photosynthetic pigments and H2O2. The frequently studied model organisms Chlamydomonas and Chlorella species were isolated to study photosynthesis-related H2O2 responses to high light. KEY RESULTS: NPQ was shown to prevent H2O2 release in Chlamydomonas and Chlorella species under high light; in addition, dissolved organic carbon excited by UV-B radiation was probably responsible for a part of the H2O2 produced in the water column. Concentrations of H2O2 peaked at 2 µm at midday and algae rapidly scavenged H2O2 rather than releasing it. A vertical H2O2 gradient was observed that was lowest next to diatom-rich benthic algal mats. The diurnal changes in photosynthetic pigments included the violaxanthin and diadinoxanthin cycles; the former was induced prior to the latter, but neither was strictly correlated with NPQ. CONCLUSIONS: The diurnal cycling of H2O2 was apparently modulated by the organisms in this freshwater algal community. Although the community showed flexibility in its levels of NPQ, the diurnal changes in xanthophylls correlated with H2O2 concentrations. Alternative NPQ mechanisms in algae involving proteins of the light-harvesting complex type and antioxidant protection of the thylakoid membrane by de-epoxidized carotenoids are discussed.

Biosynthesis of silver nanoparticles using Euglena gracilis, Euglena intermedia and their extract.

Extracellular and intracellular biosynthesis of silver nanoparticles (AgNPs) by Euglena gracilis (EG) strain and Euglena intermedia (EI) strain are reported in this study. The obtained nanoparticles showed an absorption peak approximates 420 nm in the UV-visible spectrum, corresponding to the plasmon resonance of AgNPs. According to the result of inductively coupled plasma-atomic emission spectrometer, the intakes of silver ions by EI and EG are roughly equal. The transmission electron microscope (TEM) analysis of the successful in vivo and in vitro synthesised AgNPs indicated the sizes, ranging from 6 to 24 nm and 15 to 60 nm in diameter, respectively, and a spherical-shaped polydispersal of the particles. The successful formation of AgNPs has been confirmed by energy dispersive X-ray analysis connected to the TEM. The Fourier transform infrared spectroscopy measurements reveal the presence of bioactive functional groups such as amines are found to be the capping and stabilising agents of nanoparticles. To our knowledge, this is the first report where two kinds of Euglena microalga were used as the potential source for in vivo and in vitro biosynthesis of AgNPs.

Arsenic hypertolerance in the protist Euglena mutabilis is mediated by specific transporters and functional integrity maintenance mechanisms.

Arsenic is a toxic metalloid known to cause multiple and severe cellular damages, including lipid peroxidation, protein misfolding, mutagenesis and double and single-stranded DNA breaks. Thus, exposure to this compound is lethal for most organisms but some species such as the photosynthetic protist Euglena mutabilis are able to cope with very high concentrations of this metalloid. Our comparative transcriptomic approaches performed on both an arsenic hypertolerant protist, i.e. E. mutabilis, and a more sensitive one, i.e. E. gracilis, revealed multiple mechanisms involved in arsenic tolerance. Indeed, E. mutabilis prevents efficiently the accumulation of arsenic in the cell through the expression of several transporters. More surprisingly, this protist induced the expression of active DNA reparation and protein turnover mechanisms, which allow E. mutabilis to maintain functional integrity of the cell under challenging conditions. Our observations suggest that this protist has acquired specific functions regarding arsenic and has developed an original metabolism to cope with acid mine drainages-related stresses.

Euglenoid flagellates: a multifaceted biotechnology platform.

Euglenoid flagellates are mainly fresh water protists growing in highly diverse environments making them well-suited for a multiplicity of biotechnology applications. Phototrophic euglenids possesses complex chloroplasts of green algal origin bounded by three membranes. Euglena nuclear and plastid genome organization, gene structure and gene expression are distinctly different from other organisms. Our observations on the model organism Euglena gracilis indicate that transcription of both the plastid and nuclear genome is insensitive to environmental changes and that gene expression is regulated mainly at the post-transcriptional level. Euglena plastids have been proposed as a site for the production of proteins and value added metabolites of biotechnological interest. Euglena has been shown to be a suitable protist species to be used for production of several compounds that are used in the production of cosmeceuticals and nutraceuticals, such as α-tocopherol, wax esters, polyunsaturated fatty acids, biotin and tyrosine. The storage polysaccharide, paramylon, has immunostimulatory properties and has shown a promise for biomaterials production. Euglena biomass can be used as a nutritional supplement in aquaculture and in animal feed. Diverse applications of Euglena in environmental biotechnology include ecotoxicological risk assessment, heavy metal bioremediation, bioremediation of industrial wastewater and contaminated water.