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.

• Keywords
• CAZymes, Ecological distribution, Feeding strategy, Gene-family evolution, Genome, Geographical distribution, Lateral gene transfer, Paradiplonema papillatum, Proteome, Protists, Transcriptome,
• MeSH
• Euglenozoa genetics   MeSH
• Eukaryota * genetics   MeSH
• Phylogeny MeSH
• Kinetoplastida * genetics   MeSH
• Humans MeSH
• Multigene Family MeSH
• Meiotic Prophase I MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. RESULTS: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. CONCLUSIONS: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

• Keywords
• Evolution, Mitochondrial ribosome, Phylogeny, Single-cell amplified genome,
• MeSH
• Euglenida * genetics   MeSH
• Euglenozoa genetics   MeSH
• Europium MeSH
• Phylogeny MeSH
• Genome, Mitochondrial * genetics   MeSH
• Genomics MeSH
• DNA, Mitochondrial MeSH
• RNA, Transfer MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Europium MeSH
• DNA, Mitochondrial MeSH
• RNA, Transfer MeSH

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

• MeSH
• Chromosomes genetics   MeSH
• RNA Editing genetics   MeSH
• Euglenozoa genetics   MeSH
• Phylogeny MeSH
• Genome, Mitochondrial * MeSH
• Genes * MeSH
• Conserved Sequence MeSH
• DNA, Mitochondrial genetics   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

• Keywords
• DNA content, kinetoplast DNA, mitochondrial DNA, protist,
• MeSH
• Euglenozoa genetics   MeSH
• Phylogeny MeSH
• Kinetoplastida genetics   MeSH
• DNA, Mitochondrial genetics   isolation & purification   ultrastructure   MeSH
• Mitochondria genetics   MeSH
• Trans-Splicing genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH

Diplonemids represent a hyperdiverse and abundant yet poorly studied group of marine protists. Here we describe two new members of the genus Diplonema (Diplonemea, Euglenozoa), Diplonema japonicum sp. nov. and Diplonema aggregatum sp. nov., based on life cycle, morphology, and 18S rRNA gene sequences. Along with euglenozoan apomorphies, they contain several unique features. Their life cycle is complex, consisting of a trophic stage that is, following the depletion of nutrients, transformed into a sessile stage and subsequently into a swimming stage. The latter two stages are characterized by the presence of tubular extrusomes and the emergence of a paraflagellar rod, the supportive structure of the flagellum, which is prominently lacking in the trophic stage. These two stages also differ dramatically in motility and flagellar size. Both diplonemid species host endosymbiotic bacteria that are closely related to each other and constitute a novel branch within Holosporales, for which a new genus, "Candidatus Cytomitobacter" gen. nov., has been established. Remarkably, the number of endosymbionts in the cytoplasm varies significantly, as does their localization within the cell, where they seem to penetrate the mitochondrion, a rare occurrence.IMPORTANCE We describe the morphology, behavior, and life cycle of two new Diplonema species that established a relationship with two Holospora-like bacteria in the first report of an endosymbiosis in diplonemids. Both endosymbionts reside in the cytoplasm and the mitochondrion, which establishes an extremely rare case. Within their life cycle, the diplonemids undergo transformation from a trophic to a sessile and eventually a highly motile swimming stage. These stages differ in several features, such as the presence or absence of tubular extrusomes and a paraflagellar rod, along with the length of the flagella. These morphological and behavioral interstage differences possibly reflect distinct functions in dispersion and invasion of the host and/or prey and may provide novel insight into the virtually unknown function of diplonemids in the oceanic ecosystem.

• Keywords
• Holosporales, diplonemid, endosymbionts, life cycle, ultrastructure,
• MeSH
• Bacteria genetics   MeSH
• Phylogeny MeSH
• Meiotic Prophase I physiology   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• Life Cycle Stages physiology   MeSH
• Symbiosis physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Ribosomal, 18S MeSH

The instructions to make proteins and structural RNAs are laid down in gene sequences. Yet, in certain instances, these primary instructions need to be modified considerably during gene expression, most often at the transcript level. Here we review a case of massive post-transcriptional revisions via trans-splicing and RNA editing, a phenomenon occurring in mitochondria of a recently recognized protist group, the diplonemids. As of now, the various post-transcriptional steps have been cataloged in detail, but how these processes function is still unknown. Since genetic manipulation techniques such as gene replacement and RNA interference have not yet been established for these organisms, alternative strategies have to be deployed. Here, we discuss the experimental and bioinformatics approaches that promise to unravel the molecular machineries of trans-splicing and RNA editing in Diplonema mitochondria.

• Keywords
• Cryptic genes, RNA editing, diplonemids, gene fragmentation, multipartite mtDNA, trans-splicing,
• MeSH
• RNA Editing MeSH
• Euglenozoa genetics   MeSH
• Mitochondrial Proteins genetics   MeSH
• Mitochondria genetics   MeSH
• RNA Processing, Post-Transcriptional MeSH
• Protozoan Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• RNA, Transfer metabolism   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Transfer MeSH

Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date.

• Keywords
• euglenozoa, mitochondria, mitochondrial genome,
• Publication type
• Journal Article MeSH
• Review MeSH

In this study, we describe the mitochondrial genome of the excavate flagellate Euglena gracilis. Its gene complement is reduced as compared with the well-studied sister groups Diplonemea and Kinetoplastea. We have identified seven protein-coding genes: Three subunits of respiratory complex I (nad1, nad4, and nad5), one subunit of complex III (cob), and three subunits of complex IV (cox1, cox2, and a highly divergent cox3). Moreover, fragments of ribosomal RNA genes have also been identified. Genes encoding subunits of complex V, ribosomal proteins and tRNAs were missing, and are likely located in the nuclear genome. Although mitochondrial genomes of diplonemids and kinetoplastids possess the most complex RNA processing machineries known, including trans-splicing and editing of the uridine insertion/deletion type, respectively, our transcriptomic data suggest their total absence in E. gracilis. This finding supports a scenario in which the complex mitochondrial processing machineries of both sister groups evolved relatively late in evolution from a streamlined genome and transcriptome of their common predecessor.

• Keywords
• Euglena gracilis, RNA editing, mitochondrial genome, transcription,
• MeSH
• RNA Editing MeSH
• Electron Transport Chain Complex Proteins genetics   MeSH
• Euglena gracilis genetics   MeSH
• Genome, Mitochondrial * MeSH
• Evolution, Molecular * MeSH
• RNA, Ribosomal genetics   MeSH
• RNA Splicing MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Electron Transport Chain Complex Proteins MeSH
• RNA, Ribosomal MeSH

Phylum Euglenozoa comprises three groups of eukaryotic microbes (kinetoplastids, diplonemids, and euglenids), the mitochondrial (mt) genomes of which exhibit radically different modes of organization and expression. Gene fragmentation is a striking feature of both euglenid and diplonemid mtDNAs. To rationalize the emergence of these highly divergent mtDNA types and the existence of insertion/deletion RNA editing (in kinetoplastids) and trans-splicing (in diplonemids), we propose that in the mitochondrion of the common evolutionary ancestor of Euglenozoa, small expressed gene fragments promoted a rampant neutral evolutionary pathway. Interactions between small antisense transcripts of these gene fragments and full-length transcripts, assisted by RNA-processing enzymes, permitted the emergence of RNA editing and/or trans-splicing activities, allowing the system to tolerate indel mutations and further gene fragmentation, respectively, and leading to accumulation of additional mutations. In this way, dramatically different mitochondrial genome structures and RNA-processing machineries were able to evolve. The paradigm of constructive neutral evolution acting on the widely different mitochondrial genetic systems in Euglenozoa posits the accretion of initially neutral molecular interactions by genetic drift, leading inevitably to the observed 'irremediable complexity'.

• MeSH
• RNA Editing MeSH
• Euglenozoa genetics   MeSH
• Genome, Mitochondrial * MeSH
• Models, Genetic MeSH
• Evolution, Molecular * MeSH
• DNA Replication MeSH
• Publication type
• Journal Article MeSH
• Review MeSH