Defining eukaryotes to dissect eukaryogenesis.

The origin of eukaryotes is among the most contentious debates in evolutionary biology, attracting multiple seemingly incompatible theories seeking to explain the sequence in which eukaryotic characteristics were acquired. Much of the controversy arises from differing views on the defining characteristics of eukaryotes. We argue that eukaryotes should be defined phylogenetically, and that doing so clarifies where competing hypotheses of eukaryogenesis agree and how we may test among aspects of disagreement. Some hypotheses make predictions about the phylogenetic origins of eukaryotic genes and are distinguishable on that basis. However, other hypotheses differ only in the order of key evolutionary steps, like mitochondrial endosymbiosis and nuclear assembly, which cannot currently be distinguished phylogenetically. Stages within eukaryogenesis may be made identifiable through the absolute dating of gene duplicates that map to eukaryotic traits, such as in genes of host or mitochondrial origin that duplicated and diverged functionally prior to emergence of the last eukaryotic common ancestor. In this way, it may finally be possible to distinguish heat from light in the debate over eukaryogenesis.

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.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

A New Model Trypanosomatid, Novymonas esmeraldas: Genomic Perception of Its "Candidatus Pandoraea novymonadis" Endosymbiont.

The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.

A mitochondrial cytidine deaminase is responsible for C to U editing of tRNATrp to decode the UGA codon in Trypanosoma brucei.

In kinetoplastid protists, all mitochondrial tRNAs are encoded in the nucleus and imported from the cytoplasm to maintain organellar translation. This also applies to the tryptophanyl tRNA (tRNATrp) encoded by a single-copy nuclear gene, with a CCA anticodon to read UGG codon used in the cytosolic translation. Yet, in the mitochondrion it is unable to decode the UGA codon specifying tryptophan. Following mitochondrial import of tRNATrp, this problem is solved at the RNA level by a single C34 to U34 editing event that creates the UCA anticodon, recognizing UGA. To identify the enzyme responsible for this critical editing activity, we scrutinized the genome of Trypanosoma brucei for putative cytidine deaminases as the most likely candidates. Using RNAi silencing and poisoned primer extension, we have identified a novel deaminase enzyme, named here TbmCDAT for mitochondrial Cytidine Deaminase Acting on tRNA, which is responsible for this organelle-specific activity in T. brucei. The ablation of TbmCDAT led to the downregulation of mitochondrial protein synthesis, supporting its role in decoding the UGA tryptophan codon.

A Uniquely Complex Mitochondrial Proteome from Euglena gracilis.

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

Author Correction: Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites.

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

Causes and Effects of Loss of Classical Nonhomologous End Joining Pathway in Parasitic Eukaryotes.

We report frequent losses of components of the classical nonhomologous end joining pathway (C-NHEJ), one of the main eukaryotic tools for end joining repair of DNA double-strand breaks, in several lineages of parasitic protists. Moreover, we have identified a single lineage among trypanosomatid flagellates that has lost Ku70 and Ku80, the core C-NHEJ components, and accumulated numerous insertions in many protein-coding genes. We propose a correlation between these two phenomena and discuss the possible impact of the C-NHEJ loss on genome evolution and transition to the parasitic lifestyle.IMPORTANCE Parasites tend to evolve small and compact genomes, generally endowed with a high mutation rate, compared with those of their free-living relatives. However, the mechanisms by which they achieve these features, independently in unrelated lineages, remain largely unknown. We argue that the loss of the classical nonhomologous end joining pathway components may be one of the crucial steps responsible for characteristic features of parasite genomes.

Transcriptome, proteome and draft genome of Euglena gracilis.

BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.

Farming, slaving and enslavement: histories of endosymbioses during kinetoplastid evolution.

Parasitic trypanosomatids diverged from free-living kinetoplastid ancestors several hundred million years ago. These parasites are relatively well known, due in part to several unusual cell biological and molecular traits and in part to the significance of a few - pathogenic Leishmania and Trypanosoma species - as aetiological agents of serious neglected tropical diseases. However, the majority of trypanosomatid biodiversity is represented by osmotrophic monoxenous parasites of insects. In two lineages, novymonads and strigomonads, osmotrophic lifestyles are supported by cytoplasmic endosymbionts, providing hosts with macromolecular precursors and vitamins. Here we discuss the two independent origins of endosymbiosis within trypanosomatids and subsequently different evolutionary trajectories that see entrainment vs tolerance of symbiont cell divisions cycles within those of the host. With the potential to inform on the transition to obligate parasitism in the trypanosomatids, interest in the biology and ecology of free-living, phagotrophic kinetoplastids is beginning to enjoy a renaissance. Thus, we take the opportunity to additionally consider the wider relevance of endosymbiosis during kinetoplastid evolution, including the indulged lifestyle and reductive evolution of basal kinetoplastid Perkinsela.

Not in your usual Top 10: protists that infect plants and algae.

Fungi, nematodes and oomycetes belong to the most prominent eukaryotic plant pathogenic organisms. Unicellular organisms from other eukaryotic lineages, commonly addressed as protists, also infect plants. This review provides an introduction to plant pathogenic protists, including algae infecting oomycetes, and their current state of research.

Genome of Ca. Pandoraea novymonadis, an Endosymbiotic Bacterium of the Trypanosomatid Novymonas esmeraldas.

We have sequenced, annotated, and analyzed the genome of Ca. Pandoraea novymonadis, a recently described bacterial endosymbiont of the trypanosomatid Novymonas esmeraldas. When compared with genomes of its free-living relatives, it has all the hallmarks of the endosymbionts' genomes, such as significantly reduced size, extensive gene loss, low GC content, numerous gene rearrangements, and low codon usage bias. In addition, Ca. P. novymonadis lacks mobile elements, has a strikingly low number of pseudogenes, and almost all genes are single copied. This suggests that it already passed the intensive period of host adaptation, which still can be observed in the genome of Polynucleobacter necessarius, a certainly recent endosymbiont. Phylogenetically, Ca. P. novymonadis is more related to P. necessarius, an intracytoplasmic bacterium of free-living ciliates, than to Ca. Kinetoplastibacterium spp., the only other known endosymbionts of trypanosomatid flagellates. As judged by the extent of the overall genome reduction and the loss of particular metabolic abilities correlating with the increasing dependence of the symbiont on its host, Ca. P. novymonadis occupies an intermediate position P. necessarius and Ca. Kinetoplastibacterium spp. We conclude that the relationships between Ca. P. novymonadis and N. esmeraldas are well-established, although not as fine-tuned as in the case of Strigomonadinae and their endosymbionts.