Recent expansion of metabolic versatility in Diplonema papillatum, the model species of a highly speciose group of marine eukaryotes.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

Evolution: Mitochondrial Ribosomes Across Species.

The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor.

Estimating transcriptome complexities across eukaryotes.

BACKGROUND: Genomic complexity is a growing field of evolution, with case studies for comparative evolutionary analyses in model and emerging non-model systems. Understanding complexity and the functional components of the genome is an untapped wealth of knowledge ripe for exploration. With the "remarkable lack of correspondence" between genome size and complexity, there needs to be a way to quantify complexity across organisms. In this study, we use a set of complexity metrics that allow for evaluating changes in complexity using TranD. RESULTS: We ascertain if complexity is increasing or decreasing across transcriptomes and at what structural level, as complexity varies. In this study, we define three metrics - TpG, EpT, and EpG- to quantify the transcriptome's complexity that encapsulates the dynamics of alternative splicing. Here we compare complexity metrics across 1) whole genome annotations, 2) a filtered subset of orthologs, and 3) novel genes to elucidate the impacts of orthologs and novel genes in transcript model analysis. Effective Exon Number (EEN) issued to compare the distribution of exon sizes within transcripts against random expectations of uniform exon placement. EEN accounts for differences in exon size, which is important because novel gene differences in complexity for orthologs and whole-transcriptome analyses are biased towards low-complexity genes with few exons and few alternative transcripts. CONCLUSIONS: With our metric analyses, we are able to quantify changes in complexity across diverse lineages with greater precision and accuracy than previous cross-species comparisons under ortholog conditioning. These analyses represent a step toward whole-transcriptome analysis in the emerging field of non-model evolutionary genomics, with key insights for evolutionary inference of complexity changes on deep timescales across the tree of life. We suggest a means to quantify biases generated in ortholog calling and correct complexity analysis for lineage-specific effects. With these metrics, we directly assay the quantitative properties of newly formed lineage-specific genes as they lower complexity.

Direct tests of cytochrome c and c1 functions in the electron transport chain of malaria parasites.

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.

The role of lipid rafts in vesicle formation.

The formation of membrane vesicles is a common feature in all eukaryotes. Lipid rafts are the best-studied example of membrane domains for both eukaryotes and prokaryotes, and their existence also is suggested in Archaea membranes. Lipid rafts are involved in the formation of transport vesicles, endocytic vesicles, exocytic vesicles, synaptic vesicles and extracellular vesicles, as well as enveloped viruses. Two mechanisms of how rafts are involved in vesicle formation have been proposed: first, that raft proteins and/or lipids located in lipid rafts associate with coat proteins that form a budding vesicle, and second, vesicle budding is triggered by enzymatic generation of cone-shaped ceramides and inverted cone-shaped lyso-phospholipids. In both cases, induction of curvature is also facilitated by the relaxation of tension in the raft domain. In this Review, we discuss the role of raft-derived vesicles in several intracellular trafficking pathways. We also highlight their role in different pathways of endocytosis, and in the formation of intraluminal vesicles (ILVs) through budding inwards from the multivesicular body (MVB) membrane, because rafts inside MVB membranes are likely to be involved in loading RNA into ILVs. Finally, we discuss the association of glycoproteins with rafts via the glycocalyx.

De novo cholesterol biosynthesis in bacteria.

Eukaryotes produce highly modified sterols, including cholesterol, essential to eukaryotic physiology. Although few bacterial species are known to produce sterols, de novo production of cholesterol or other complex sterols in bacteria has not been reported. Here, we show that the marine myxobacterium Enhygromyxa salina produces cholesterol and provide evidence for further downstream modifications. Through bioinformatic analysis we identify a putative cholesterol biosynthesis pathway in E. salina largely homologous to the eukaryotic pathway. However, experimental evidence indicates that complete demethylation at C-4 occurs through unique bacterial proteins, distinguishing bacterial and eukaryotic cholesterol biosynthesis. Additionally, proteins from the cyanobacterium Calothrix sp. NIES-4105 are also capable of fully demethylating sterols at the C-4 position, suggesting complex sterol biosynthesis may be found in other bacterial phyla. Our results reveal an unappreciated complexity in bacterial sterol production that rivals eukaryotes and highlight the complicated evolutionary relationship between sterol biosynthesis in the bacterial and eukaryotic domains.

Distribution patterns and community assembly processes of eukaryotic microorganisms along an altitudinal gradient in the middle reaches of the Yarlung Zangbo River.

Eukaryotic microorganisms play an important role in the biogeochemical cycles of rivers. Dynamic hydrological processes in rivers are thought to influence the assembly processes of eukaryotic microbes, as well as affecting local geomorphology. These processes have not been extensively studied for eukaryotic river microbes in extreme environments on the Tibetan Plateau. This study used 18S rDNA gene amplification sequencing, a neutral community model, and a null model to analyze the spatial and temporal dynamics and assembly processes of eukaryotic microbial communities in the middle reaches of the Yarlung Zangbo River. We conducted analyses across wet and dry seasons, as well as varying altitudinal gradients. Our results showed that the diversity, structure, and taxonomic composition of eukaryotic microbial communities varied more with altitude than season, and the diversity of the communities first increased, then decreased, with increasing elevation. Distance-decay analysis showed that the correlation between eukaryotic microbial communities and environmental distance was stronger than the correlation between the microbial communities and geographical distance. Deterministic processes (homogeneous selection) dominated the construction of eukaryotic microbial communities, and water temperature, pH, and total phosphorus were the primary environmental factors that influenced the construction of eukaryotic microbial communities. These results expand our understanding of the characteristics of eukaryotic microbial communities in rivers on the Tibetan Plateau and provide clues to understanding the mechanisms that maintain eukaryotic microbial diversity in extreme environments.

Cell-type memory in a single-cell eukaryote requires the continuous presence of a specific transcription regulator.

A fundamental question in biology is how a eukaryotic cell type can be stably maintained through many rounds of DNA replication and cell division. In this paper, we investigate this question in a fungal species, Candida albicans, where two different cells types (white and opaque) arise from the same genome. Once formed, each cell type is stable for thousands of generations. Here, we investigate the mechanisms underlying opaque cell "memory." Using an auxin-mediated degradation system, we rapidly removed Wor1, the primary transcription activator of the opaque state and, using a variety of methods, determined how long cells can maintain the opaque state. Within approximately 1 h of Wor1 destruction, opaque cells irreversibly lose their memory and switch to the white cell state. This observation rules out several competing models for cell memory and demonstrates that the continuous presence of Wor1 is needed to maintain the opaque cell state-even across a single cell division cycle. We also provide evidence for a threshold concentration of Wor1 in opaque cells, below which opaque cells irreversibly switch to white cells. Finally, we provide a detailed description of the gene expression changes that occur during this switch in cell types.

ExpressAnalyst: A unified platform for RNA-sequencing analysis in non-model species.

The increasing application of RNA sequencing to study non-model species demands easy-to-use and efficient bioinformatics tools to help researchers quickly uncover biological and functional insights. We developed ExpressAnalyst ( www.expressanalyst.ca ), a web-based platform for processing, analyzing, and interpreting RNA-sequencing data from any eukaryotic species. ExpressAnalyst contains a series of modules that cover from processing and annotation of FASTQ files to statistical and functional analysis of count tables or gene lists. All modules are integrated with EcoOmicsDB, an ortholog database that enables comprehensive analysis for species without a reference transcriptome. By coupling ultra-fast read mapping algorithms with high-resolution ortholog databases through a user-friendly web interface, ExpressAnalyst allows researchers to obtain global expression profiles and gene-level insights from raw RNA-sequencing reads within 24 h. Here, we present ExpressAnalyst and demonstrate its utility with a case study of RNA-sequencing data from multiple non-model salamander species, including two that do not have a reference transcriptome.

Lagging Strand Initiation Processes in DNA Replication of Eukaryotes-Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes.

In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed.

Respiratory eukaryotic virome expansion and bacteriophage deficiency characterize childhood asthma.

Asthma development and exacerbation is linked to respiratory virus infections. There is limited information regarding the presence of viruses during non-exacerbation/infection periods. We investigated the nasopharyngeal/nasal virome during a period of asymptomatic state, in a subset of 21 healthy and 35 asthmatic preschool children from the Predicta cohort. Using metagenomics, we described the virome ecology and the cross-species interactions within the microbiome. The virome was dominated by eukaryotic viruses, while prokaryotic viruses (bacteriophages) were independently observed with low abundance. Rhinovirus B species consistently dominated the virome in asthma. Anelloviridae were the most abundant and rich family in both health and asthma. However, their richness and alpha diversity were increased in asthma, along with the co-occurrence of different Anellovirus genera. Bacteriophages were richer and more diverse in healthy individuals. Unsupervised clustering identified three virome profiles that were correlated to asthma severity and control and were independent of treatment, suggesting a link between the respiratory virome and asthma. Finally, we observed different cross-species ecological associations in the healthy versus the asthmatic virus-bacterial interactome, and an expanded interactome of eukaryotic viruses in asthma. Upper respiratory virome "dysbiosis" appears to be a novel feature of pre-school asthma during asymptomatic/non-infectious states and merits further investigation.

Qualitative and quantitative analysis of the effects of drinking water disinfection processes on eukaryotic microorganisms: A meta-analysis.

The risk of eukaryotic microorganisms in drinking water treatment has not received sufficient attention. As the final step in ensuring drinking water quality, the effectiveness of disinfection in inactivating eukaryotic microorganisms remains to be qualitatively and quantitatively demonstrated. In this study, we conducted a meta-analysis using a mixed effects model and bootstrapping analysis to assess the effects of the disinfection process on eukaryotic microorganisms. The results revealed significant reduction of eukaryotic microorganisms in drinking water associated with the disinfection process. The estimated logarithmic reduction rates for chlorination, ozone, and UV disinfection were 1.74, 1.82 and 2.15 log, respectively, for all eukaryotic microorganisms. Analysis of relative abundance variation of eukaryotic microorganisms also indicated certain phyla and classes exhibited tolerance and competitive advantage during disinfection. This study provides qualitatively and quantitatively analysis on the influence of drinking water disinfection processes on eukaryotic microorganisms, and highlights the persistent risk of eukaryotic microbial contamination in drinking water even after disinfection, calling for further optimization of current conventional disinfection methods.

Macroevolutionary patterns in intragenomic rDNA variability among planktonic foraminifera.

Ribosomal intragenomic variability in prokaryotes and eukaryotes is a genomic feature commonly studied for its inflationary impact on molecular diversity assessments. However, the evolutionary mechanisms and distribution of this phenomenon within a microbial group are rarely explored. Here, we investigate the intragenomic variability in 33 species of planktonic foraminifera, calcifying marine protists, by inspecting 2,403 partial SSU sequences obtained from single-cell clone libraries. Our analyses show that polymorphisms are common among planktonic foraminifera species, but the number of polymorphic sites significantly differs among clades. With our molecular simulations, we could assess that most of these mutations are located in paired regions that do not affect the secondary structure of the SSU fragment. Finally, by mapping the number of polymorphic sites on the phylogeny of the clades, we were able to discuss the evolution and potential sources of intragenomic variability in planktonic foraminifera, linking this trait to the distinctive nuclear and genomic dynamics of this microbial group.

Comparative transcriptome and antioxidant biomarker response reveal molecular mechanisms to cope with zinc ion exposure in the unicellular eukaryote Paramecium.

The development of industry has resulted in excessive environmental zinc exposure which has caused various health problems in a wide range of organisms including humans. The mechanisms by which aquatic microorganisms respond to environmental zinc stress are still poorly understood. Paramecium, a well-known ciliated protozoan and a popular cell model in heavy metal stress response studies, was chosen as the test unicellular eukaryotic organism in the present research. In this work, Paramecium cf. multimicronucleatum cells were exposed in different levels of zinc ion (0.1 and 1.0 mg/L) for different periods of exposure (1 and 4 days), and then analyzed population growth, transcriptomic profiles and physiological changes in antioxidant enzymes to explore the toxicity and detoxification mechanisms during the zinc stress response. Results demonstrated that long-term zinc exposure could have restrained population growth in ciliates, however, the response mechanism to zinc exposure in ciliates is likely to show a dosage-dependent and time-dependent manner. The differentially expressed genes (DEGs) were identified the characters by high-throughput sequencing, which remarkably enriched in the phagosome, indicating that the phagosome pathway might mediate the uptake of zinc, while the pathways of ABC transporters and Na+/K+-transporting ATPase contributed to the efflux transport of excessive zinc ions and the maintenance of osmotic balance, respectively. The accumulation of zinc ions triggered a series of adverse effects, including damage to DNA and proteins, disturbance of mitochondrial function, and oxidative stress. In addition, we found that gene expression changed significantly for metal ion binding, energy metabolism, and oxidation-reduction processes. RT-qPCR of ten genes involved in important biological functions further validated the results of the transcriptome analysis. We also continuously monitored changes in activity of four antioxidant enzymes (SOD, CAT, POD and GSH-PX), all of which peaked on day 4 in cells subjected to zinc stress. Collectively, our results indicate that excessive environmental zinc exposure initially causes damage to cellular structure and function and then initiates detoxification mechanisms to maintain homeostasis in P. cf. multimicronucleatum cells.

Maturases and Group II Introns in the Mitochondrial Genomes of the Deepest Jakobid Branch.

Ophirinina is a recently described suborder of jakobid protists (Excavata) with only one described species to date, Ophirina amphinema. Despite the acquisition and analysis of massive transcriptomic and mitogenomic sequence data from O. amphinema, its phylogenetic position among excavates remained inconclusive, branching as sister group either to all Jakobida or to all Discoba. From a morphological perspective, it has not only several typical jakobid features but also unusual traits for this group, including the morphology of mitochondrial cristae (sac-shaped to flattened-curved cristae) and the presence of two flagellar vanes. In this study, we have isolated, morphologically characterized, and sequenced genome and transcriptome data of two new Ophirinina species: Ophirina chinija sp. nov. and Agogonia voluta gen. et sp. nov. Ophirina chinija differs from O. amphinema in having rounded cell ends, subapically emerging flagella and a posterior cell protrusion. The much more distantly related A. voluta has several unique ultrastructural characteristics, including sac-shaped mitochondrial cristae and a complex "B" fiber. Phylogenomic analyses with a large conserved-marker dataset supported the monophyly of Ophirina and Agogonia within the Ophirinina and, more importantly, resolved the conflicting position of ophirinids as the sister clade to all other jakobids. The characterization of the mitochondrial genomes showed that Agogonia differs from all known gene-rich jakobid mitogenomes by the presence of two group II introns and their corresponding maturase protein genes. A phylogenetic analysis of the diversity of known maturases confirmed that the Agogonia proteins are highly divergent from each other and define distant families among the prokaryotic and eukaryotic maturases. This opens the intriguing possibility that, compared to other jakobids, Ophirinina may have retained additional mitochondrial elements that may help to understand the early diversification of eukaryotes and the evolution of mitochondria.

Recognition of γ-Subunit by ß-Subunit in Translation Initiation Factor 2. Stabilization of the GTP-Bound State of I/F 2 in Archaea and Eukaryotes.

Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) functions as a heterotrimeric complex. It consists of three subunits (α, ß, γ). α- and ß-subunits are bound to γ-subunit by hydrogen bonds and van der Waals interactions, but do not contact each other. Although main functions of the factor are performed by the γ-subunit, reliable formation of αγ and ßγ complexes is necessary for its proper functioning. In this work, we introduced mutations in the recognition part of the ßγ interface and showed that hydrophobic effect plays a crucial role in the recognition of subunits both in eukaryotes and archaea. Shape and properties of the groove on the surface of γ-subunit facilitates transition of the disordered recognition part of the ß-subunit into an α-helix containing approximately the same number of residues in archaea and eukaryotes. In addition, based on the newly obtained data, it was concluded that in archaea and eukaryotes, transition of the γ-subunit to the active state leads to additional contact between the region of switch 1 and C-terminal part of the ß-subunit, which stabilizes helical conformation of the switch.

Evolution: Was the nuclear-to-cytoplasmic ratio a key factor in the origin of animal multicellularity?

The ichthyosporean Sphaeroforma arctica, a protist closely related to animals, displays coenocytic development followed by cellularization and cell release. A new study reveals that the nuclear-to-cytoplasmic ratio drives cellularization in these fascinating organisms.

Large-scale invasion of unicellular eukaryotic genomes by integrating DNA viruses.

Eukaryotic genomes contain a variety of endogenous viral elements (EVEs), which are mostly derived from RNA and ssDNA viruses that are no longer functional and are considered to be "genomic fossils." Genomic surveys of EVEs, however, are strongly biased toward animals and plants, whereas protists, which represent the majority of eukaryotic diversity, remain poorly represented. Here, we show that protist genomes harbor tens to thousands of diverse, ~14 to 40 kbp long dsDNA viruses. These EVEs, composed of virophages, Polinton-like viruses, and related entities, have remained hitherto hidden owing to poor sequence conservation between virus groups and their repetitive nature that precluded accurate short-read assembly. We show that long-read sequencing technology is ideal for resolving virus insertions. Many protist EVEs appear intact, and most encode integrases, which suggests that they have actively colonized hosts across the tree of eukaryotes. We also found evidence for gene expression in host transcriptomes and that closely related virophage and Polinton-like virus genomes are abundant in viral metagenomes, indicating that many EVEs are probably functional viruses.