Screening of a Thraustochytrid Strain Collection for Carotenoid and Squalene Production Characterized by Cluster Analysis, Comparison of 18S rRNA Gene Sequences, Growth Behavior, and Morphology.

Carotenoids and squalene are important terpenes that are applied in a wide range of products in foods and cosmetics. Thraustochytrids might be used as alternative production organisms to improve production processes, but the taxon is rarely studied. A screening of 62 strains of thraustochytrids sensu lato for their potential to produce carotenoids and squalene was performed. A phylogenetic tree was built based on 18S rRNA gene sequences for taxonomic classification, revealing eight different clades of thraustochytrids. Design of experiments (DoE) and growth models identified high amounts of glucose (up to 60 g/L) and yeast extract (up to 15 g/L) as important factors for most of the strains. Squalene and carotenoid production was studied by UHPLC-PDA-MS measurements. Cluster analysis of the carotenoid composition partially mirrored the phylogenetic results, indicating a possible use for chemotaxonomy. Strains in five clades produced carotenoids. Squalene was found in all analyzed strains. Carotenoid and squalene synthesis was dependent on the strain, medium composition and solidity. Strains related to Thraustochytrium aureum and Thraustochytriidae sp. are promising candidates for carotenoid synthesis. Strains closely related to Schizochytrium aggregatum might be suitable for squalene production. Thraustochytrium striatum might be a good compromise for the production of both molecule groups.

Providing a phylogenetic framework for trait-based analyses in brown algae: Phylogenomic tree inferred from 32 nuclear protein-coding sequences.

In the study of the evolution of biological complexity, a reliable phylogenetic framework is needed. Many attempts have been made to resolve phylogenetic relationships between higher groups (i.e., interordinal) of brown algae (Phaeophyceae) based on molecular evidence, but most of these relationships remain unclear. Analyses based on small multi-gene data (including chloroplast, mitochondrial and nuclear sequences) have yielded inconclusive and sometimes contradictory results. To address this problem, we have analyzed 32 nuclear protein-coding sequences in 39 Phaeophycean species belonging to eight orders. The resulting nuclear-based phylogenomic trees provide virtually full support for the phylogenetic relationships within the studied taxa, with few exceptions. The relationships largely confirm phylogenetic trees based on nuclear, chloroplast and mitochondrial sequences, except for the placement of the Sphacelariales with weak bootstrap support. Our study indicates that nuclear protein-coding sequences provide significant support to conclusively resolve phylogenetic relationships among Phaeophyceae, and may be a powerful approach to fully resolve interordinal relationships with increased taxon sampling.

Evidence for evolutionary relationship between archaeplastidal and cyanobacterial phytochromes based on their chromophore pockets.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. In plants, phytochromes play a central role in growth and differentiation during the entire life cycle. Phytochromes of plants and other groups of archaeplastida have a common evolutionary origin in prokaryotes, but the exact prokaryotic origin is as yet uncertain. Two possibilities are presently discussed: either, archaeplastidal phytochromes arose from the last eukaryotic common ancestor (LECA) or they arose from the cyanobacterial endosymbiont that gave rise to plastids. We first constructed standard phylogenetic trees based on N-terminal protein sequences of the chromophore module. As usual, variation of algorithms and parameters led to different trees. A relationship between cyanobacteria and archaeplastida was observed in 7 out of 36 trees. The lack of consistency between results obtained from variation of parameters of tree constructions reflects the uncertainty of archaeplastidal origin. To gain more information about a possible cyanobacterial and archaeplastidal relationship, we performed phylogenetic studies based on the amino acids that line the chromophore pockets. These amino acids are highly conserved and could provide more accurate information about long evolutionary time scales, but the reduction of traits could also lead to insignificant results. From 30 selected chromophore-binding amino acids, 6 were invariant. The subsequent studies were thus based on the information dependent on 24 or fewer amino acid positions. Again, multiple trees were constructed to get information about the robustness of relationships. The very low number of information-containing traits resulted in low bootstrap values and many indistinguishable leaves. However, the major groups fungi, bacteria, cyanobacteria, and plants remained united. Without exception, cyanobacteria and archaeplastida were always closely linked. In this respect, the results were more robust than those of the classic approach, based on long contiguous sequences. We therefore consider cyanobacteria as the most likely origin of archaeplastidal phytochromes.

Mediocremonas mediterraneus, a New Member within the Developea.

The stramenopiles are a large and diverse group of eukaryotes that possess various lifestyles required to thrive in a broad array of environments. The stramenopiles branch with the alveolates, rhizarians, and telonemids, forming the supergroup TSAR. Here, we present a new genus and species of aquatic nanoflagellated stramenopile: Mediocremonas mediterraneus, a free-swimming heterotrophic predator. M. mediterraneus cell bodies measure between 2.0-4.0 µm in length and 1.2-3.7 µm in width, possessing two flagella and an oval body morphology. The growth and grazing rate of M. mediterraneus in batch cultures ranges from 0.68 to 1.83 d-1 and 1.99 to 5.38 bacteria/h, respectively. M. mediterraneus was found to be 93.9% phylogenetically similar with Developayella elegans and 94.7% with Develorapax marinus, two members within the class Developea. The phylogenetic position of the Developea and the ability of M. mediterraneus to remain in culture make it a good candidate for further genomic studies that could help us to better understand phagotrophy in marine systems as well as the transition from heterotrophy to phototrophy within the stramenopiles.

First Genome of Labyrinthula sp., an Opportunistic Seagrass Pathogen, Reveals Novel Insight into Marine Protist Phylogeny, Ecology and CAZyme Cell-Wall Degradation.

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula spp. virulence and ecology. Given these uncertainties, we investigated the Labyrinthula genus from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthula SR_Ha_C's actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialized substrate affinity and the scarcity of characterized protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula spp. ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between the Labyrinthula genus and its host substrate.

Laminarin, a Major Polysaccharide in Stramenopiles.

During the processes of primary and secondary endosymbiosis, different microalgae evolved to synthesis different storage polysaccharides. In stramenopiles, the main storage polysaccharides are ß-1,3-glucan, or laminarin, in vacuoles. Currently, laminarin is gaining considerable attention due to its application in the food, cosmetic and pharmaceuticals industries, and also its importance in global biogeochemical cycles (especially in the ocean carbon cycle). In this review, the structures, composition, contents, and bioactivity of laminarin were summarized in different algae. It was shown that the general features of laminarin are species-dependence. Furthermore, the proposed biosynthesis and catabolism pathways of laminarin, functions of key genes, and diel regulation of laminarin were also depicted and comprehensively discussed for the first time. However, the complete pathways, functions of genes, and diel regulatory mechanisms of laminarin require more biomolecular studies. This review provides more useful information and identifies the knowledge gap regarding the future studies of laminarin and its applications.

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31 h that damped within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.

Comparative plastid genomics of Synurophyceae: inverted repeat dynamics and gene content variation.

BACKGROUND: The Synurophyceae is one of most important photosynthetic stramenopile algal lineages in freshwater ecosystems. They are characterized by siliceous scales covering the cell or colony surface and possess plastids of red-algal secondary or tertiary endosymbiotic origin. Despite their ecological and evolutionary significance, the relationships amongst extant Synurophyceae are unclear, as is their relationship to most other stramenopiles. RESULTS: Here we report a comparative analysis of plastid genomes sequenced from five representative synurophycean algae. Most of these plastid genomes are highly conserved with respect to genome structure and coding capacity, with the exception of gene re-arrangements and partial duplications at the boundary of the inverted repeat and single-copy regions. Several lineage-specific gene loss/gain events and intron insertions were detected (e.g., cemA, dnaB, syfB, and trnL). CONCLUSIONS: Unexpectedly, the cemA gene of Synurophyceae shows a strong relationship with sequences from members of the green-algal lineage, suggesting the occurrence of a lateral gene transfer event. Using a molecular clock approach based on silica fossil record data, we infer the timing of genome re-arrangement and gene gain/loss events in the plastid genomes of Synurophyceae.

Diversity and evolution of cytochromes P450 in stramenopiles.

MAIN CONCLUSION: Comparative genomic analysis of cytochromes P450 revealed high diversification and dynamic changes in stramenopiles, associated with transcriptional responsiveness to various environmental stimuli. Comparative genomic and molecular evolution approaches were used to characterize cytochromes P450 (P450) diversity in stramenopiles. Phylogenetic analysis pointed to a high diversity of P450 in stramenopiles and identified three major clans. The CYP51 and CYP97 clans were present in brown algae, diatoms and Nannochloropsis gaditana, whereas the CYP5014 clan mainly includes oomycetes. Gene gain and loss patterns revealed that six CYP families-CYP51, CYP97, CYP5160, CYP5021, CYP5022, and CYP5165-predated the split of brown algae and diatoms. After they diverged, diatoms gained more CYP families, especially in the cold-adapted species Fragilariopsis cylindrus, in which eight new CYP families were found. Selection analysis revealed that the expanded CYP51 family in the brown alga Cladosiphon okamuranus exhibited a more relaxed selection constraint compared with those of other brown algae and diatoms. Our RNA-seq data further evidenced that most of P450s in Saccharina japonica are highly expressed in large sporophytes, which could potentially promote the large kelp formation in this developmental stage. A survey of Ectocarpus siliculosus and diatom transcriptomes showed that many P450s are responsive to stress, nutrient limitation or light quality, suggesting pivotal roles in detoxification or metabolic processes under adverse environmental conditions. The information provided in this study will be helpful in designing functional experiments and interpreting P450 roles in this particular lineage.

Assessing the viral content of uncultured picoeukaryotes in the global-ocean by single cell genomics.

Viruses are the most abundant biological entities on Earth and have fundamental ecological roles in controlling microbial communities. Yet, although their diversity is being increasingly explored, little is known about the extent of viral interactions with their protist hosts as most studies are limited to a few cultivated species. Here, we exploit the potential of single-cell genomics to unveil viral associations in 65 individual cells of 11 essentially uncultured stramenopiles lineages sampled during the Tara Oceans expedition. We identified viral signals in 57% of the cells, covering nearly every lineage and with narrow host specificity signal. Only seven out of the 64 detected viruses displayed homologies to known viral sequences. A search for our viral sequences in global ocean metagenomes showed that they were preferentially found at the DCM and within the 0.2-3 µm size fraction. Some of the viral signals were widely distributed, while others geographically constrained. Among the viral signals we detected an endogenous mavirus virophage potentially integrated within the nuclear genome of two distant uncultured stramenopiles. Virophages have been previously reported as a cell's defence mechanism against other viruses, and may therefore play an important ecological role in regulating protist populations. Our results point to single-cell genomics as a powerful tool to investigate viral associations in uncultured protists, suggesting a wide distribution of these relationships, and providing new insights into the global viral diversity.

Dictyochophyceae Plastid Genomes Reveal Unusual Variability in Their Organization.

Dictyochophyceae (silicoflagellates) are unicellular freshwater and marine algae (Heterokontophyta, stramenopiles). Despite their abundance in global oceans and potential ecological significance, discovered in recent years, neither nuclear nor organellar genomes of representatives of this group were sequenced until now. Here, we present the first complete plastid genome sequences of Dictyochophyceae, obtained from four species: Dictyocha speculum, Rhizochromulina marina, Florenciella parvula and Pseudopedinella elastica. Despite their comparable size and genetic content, these four plastid genomes exhibit variability in their organization: plastid genomes of F. parvula and P. elastica possess conventional quadripartite structure with a pair of inverted repeats, R. marina instead possesses two direct repeats with the same orientation and D. speculum possesses no repeats at all. We also observed a number of unusual traits in the plastid genome of D. speculum, including expansion of the intergenic regions, presence of an intron in the otherwise non-intron-bearing psaA gene, and an additional copy of the large subunit of RuBisCO gene (rbcL), the last of which has never been observed in any plastid genome. We conclude that despite noticeable gene content similarities between the plastid genomes of Dictyochophyceae and their relatives (pelagophytes, diatoms), the number of distinctive features observed in this lineage strongly suggests that additional taxa require further investigation.

Microbiology and foodborne pathogens in honey.

Honey has been considered a relatively safe foodstuff due to its compositional properties, with infant botulism caused by Clostridium botulinum being the most prominent health risk associated with it. Our review is focused on the honey microflora along the food chain and evaluates the pathogenic potential of those microorganisms found in honey. This product may contain a great variety of bacteria and, particularly, fungi that eventually entered the food chain at an early stage (e.g., via pollen). For many of these microorganisms, opportunistic infections in humans have been recorded (e.g., infections by Staphylococcus spp., Citrobacter spp., Escherichia coli, Hafnia alvei, Aspergillus spp., Fusarium spp., Trichoderma spp., Chaetomium spp.), although direct infections via honey were not registered.

The First Isolation and Characterisation of the Protist Labyrinthula sp. in Southeastern Australia.

As a result of anthropogenic influences and global climate change, emerging infectious marine diseases are thought to be increasingly more common and more severe than in the past. The aim of our investigation was to confirm the presence of Labyrinthula, the aetiological agent of the seagrass wasting disease, in Southeastern Australia and provide the first isolation and characterisation of this protist, in Australia. Colonies and individual cells were positively identified as Labyrinthula using published descriptions, diagrams, and photographs. Their identity was then confirmed using DNA barcoding of a region of the 18S rRNA gene. Species level identification of isolates was not possible as the taxonomy of the Labyrinthula is still poorly resolved. Still, a diversity of Labyrinthula was isolated from small sections of the southeast coast of Australia. The isolates were grouped into three haplotypes that are biogeographically restricted. These haplotypes are closely related to previously identified saprotrophic clades. The study highlights the need for further investigation into the global distribution of Labyrinthula, including phylogenetic pathogenicity and analysis of host-parasite interactions in response to stressors. Given the results of our analyses, it is prudent to continue research into disease and epidemic agents to better prepare researchers for potential future outbreaks.

Transcription factors in microalgae: genome-wide prediction and comparative analysis.

BACKGROUND: Studying transcription factors, which are some of the key players in gene expression, is of outstanding interest for the investigation of the evolutionary history of organisms through lineage-specific features. In this study we performed the first genome-wide TF identification and comparison between haptophytes and other algal lineages. RESULTS: For TF identification and classification, we created a comprehensive pipeline using a combination of BLAST, HMMER and InterProScan software. The accuracy evaluation of the pipeline shows its applicability for every alga, plant and cyanobacterium, with very good PPV and sensitivity. This pipeline allowed us to identify and classified the transcription factor complement of the three haptophytes Tisochrysis lutea, Emiliania huxleyi and Pavlova sp.; the two stramenopiles Phaeodactylum tricornutum and Nannochloropsis gaditana; the chlorophyte Chlamydomonas reinhardtii and the rhodophyte Porphyridium purpureum. By using T. lutea and Porphyridium purpureum, this work extends the variety of species included in such comparative studies, allowing the detection and detailed study of lineage-specific features, such as the presence of TF families specific to the green lineage in Porphyridium purpureum, haptophytes and stramenopiles. Our comprehensive pipeline also allowed us to identify fungal and cyanobacterial TF families in the algal nuclear genomes. CONCLUSIONS: This study provides examples illustrating the complex evolutionary history of algae, some of which support the involvement of a green alga in haptophyte and stramenopile evolution.

Complex evolution of two types of cardiolipin synthase in the eukaryotic lineage stramenopiles.

The phospholipid cardiolipin is indispensable for eukaryotes to activate mitochondria, and it was previously reported that two phylogenetically distinct types of enzyme synthesizing cardiolipin, one with two phospholipase D domains (CLS_pld) and the other with a CDP-alcohol phosphatidyltransferase domain (CLS_cap), are patchily and complementarily distributed at higher taxonomic (e.g., supergroup) levels of eukaryotes. Stramenopiles, one of the major eukaryotic clades, were considered to exclusively possess CLS_cap. However, through our present surveys with genome or transcriptome data from a broad range of stramenopile taxa, species with both CLS_cap and CLS_pld and species with only CLS_pld or CLS_cap were discovered among this group. Because these homologues of CLS_cap and CLS_pld retrieved from stramenopiles were likely inherited from the last eukaryotic common ancestor, it is reasonable to assume that a common ancestor of all stramenopiles harbored both CLS_cap and CLS_pld. Furthermore, based on the robust organismal phylogeny of stramenopiles unveiled with large-scale phylogenetic analyses, the earliest diverging lineage of stramenopiles (including bicosoecids, placidids, etc.) was found to comprise species with both CLS_cap and CLS_pld along with species with only either CLS_cap or CLS_pld. These findings suggest that a common ancestor of the most basal stramenopile lineage retained these two vertically inherited enzymes and that differential losses of either CLS_cap or CLS_pld occurred in this lineage. On the other hand, in the other stramenopile lineage composed of Ochrophyta, Pseudofungi, and Labyrinthulomycetes (to the exclusion of the most basal lineage), only CLS_cap was found, and therefore a common ancestor of these three groups likely lost CLS_pld. Based on our findings, the evolution of CLS_cap/CLS_pld in stramenopiles appears to be more complex than previously thought.

Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles.

Small but Manifold - Hidden Diversity in "Spumella-like Flagellates".

Colourless, nonscaled chrysophytes comprise morphologically similar or even indistinguishable flagellates which are important bacterivors in water and soil crucial for ecosystem functioning. However, phylogenetic analyses indicate a multiple origin of such colourless, nonscaled flagellate lineages. These flagellates are often referred to as "Spumella-like flagellates" in ecological and biogeographic studies. Although this denomination reflects an assumed polyphyly, it obscures the phylogenetic and taxonomic diversity of this important flagellate group and, thus, hinders progress in lineage- and taxon-specific ecological surveys. The smallest representatives of colourless chrysophytes have been addressed in very few taxonomic studies although they are among the dominant flagellates in field communities. To overcome the blurred picture and set the field for further investigation in biogeography and ecology of the organisms in question, we studied a set of strains of specifically small, colourless, nonscaled chrysomonad flagellates by means of electron microscopy and molecular analyses. They were isolated by a filtration-acclimatisation approach focusing on flagellates of around 5 µm. We present the phylogenetic position of eight different lineages on both the ordinal and the generic level. Accordingly, we describe the new genera Apoikiospumella, Chromulinospumella, Segregatospumella, Cornospumella and Acrispumella Boenigk et Grossmann n. g. and different species within them.

Blue-light-regulated transcription factor, Aureochrome, in photosynthetic stramenopiles.

During the course of evolution through various endosymbiotic processes, diverse photosynthetic eukaryotes acquired blue light (BL) responses that do not use photosynthetic pathways. Photosynthetic stramenopiles, which have red algae-derived chloroplasts through secondary symbiosis, are principal primary producers in aquatic environments, and play important roles in ecosystems and aquaculture. Through secondary symbiosis, these taxa acquired BL responses, such as phototropism, chloroplast photo-relocation movement, and photomorphogenesis similar to those which green plants acquired through primary symbiosis. Photosynthetic stramenopile BL receptors were undefined until the discovery in 2007, of a new type of BL receptor, the aureochrome (AUREO), from the photosynthetic stramenopile alga, Vaucheria. AUREO has a bZIP domain and a LOV domain, and thus BL-responsive transcription factor. AUREO orthologs are only conserved in photosynthetic stramenopiles, such as brown algae, diatoms, and red tide algae. Here, a brief review is presented of the role of AUREOs as photoreceptors for these diverse BL responses and their biochemical properties in photosynthetic stramenopiles.

The diversity of fungal genome.

The genome size of an organism varies from species to species. The C-value paradox enigma is a very complex puzzle with regards to vast diversity in genome sizes in eukaryotes. Here we reported the detailed genomic information of 172 fungal species among different fungal genomes and found that fungal genomes are very diverse in nature. In fungi, the diversity of genomes varies from 8.97 Mb to 177.57 Mb. The average genome sizes of Ascomycota and Basidiomycota fungi are 36.91 and 46.48 Mb respectively. But higher genome size is observed in Oomycota (74.85 Mb) species, a lineage of fungus-like eukaryotic microorganisms. The average coding genes of Oomycota species are almost doubled than that of Acomycota and Basidiomycota fungus.