We used culture-based and culture-independent approaches to discover diversity and ecology of anaerobic jakobids (Excavata: Jakobida), an overlooked, deep-branching lineage of free-living nanoflagellates related to Euglenozoa. Jakobids are among a few lineages of nanoflagellates frequently detected in anoxic habitats by PCR-based studies, however only two strains of a single jakobid species have been isolated from those habitats. We recovered 712 environmental sequences and cultured 21 new isolates of anaerobic jakobids that collectively represent at least ten different species in total, from which four are uncultured. Two cultured species have never been detected by environmental, PCR-based methods. Surprisingly, culture-based and culture-independent approaches were able to reveal a relatively high proportion of overall species diversity of anaerobic jakobids-60 or 80%, respectively. Our phylogenetic analyses based on SSU rDNA and six protein-coding genes showed that anaerobic jakobids constitute a clade of morphologically similar, but genetically and ecologically diverse protists-Stygiellidae fam. nov. Our investigation combines culture-based and environmental molecular-based approaches to capture a wider extent of species diversity and shows Stygiellidae as a group that ordinarily inhabits anoxic, sulfide- and ammonium-rich marine habitats worldwide.

• Klíčová slova
• anaerobic protists, cryptic species, environmental clones, marine communities, species diversity,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. RESULTS: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids. CONCLUSIONS: As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

• Klíčová slova
• Andalucia godoyi, Jakobids, Mitochondrial evolution, Mitochondrial genome, Mitochondrial proteome, Mitochondrion, Protist,
• MeSH
• buněčné jádro genetika   MeSH
• Eukaryota genetika   MeSH
• genom mitochondriální * MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• proteom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom * MeSH

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

• MeSH
• biologické modely MeSH
• Eukaryota klasifikace   genetika   metabolismus   MeSH
• fylogeneze MeSH
• gramnegativní bakterie klasifikace   genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• mitochondriální proteiny klasifikace   genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• molekulární modely MeSH
• Naegleria klasifikace   genetika   metabolismus   MeSH
• peroxizomy metabolismus   MeSH
• protozoální proteiny klasifikace   genetika   metabolismus   MeSH
• sekreční systém typu II klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH
• sekreční systém typu II MeSH

Circumscribing major eukaryote groups and resolving higher order relationships between them are among the most challenging tasks facing molecular evolutionists. Recently, evidence suggesting a new supergroup (the Excavata) comprising a wide array of flagellates has been collected. This group consists of diplomonads, retortamonads, Carpediemonas, heteroloboseans, Trimastix, jakobids, and Malawimonas, all of which possess a particular type of ventral feeding groove that is proposed to be homologous. Euglenozoans, parabasalids, and oxymonads have also been associated with Excavata as their relationships to one or more core excavate taxa were demonstrated. However, the main barrier to the general acceptance of Excavata is that its existence is founded primarily on cytoskeletal similarities, without consistent support from molecular phylogenetics. In gene trees, Excavata are typically not recovered together. In this paper, we present an analysis of the phylogenetic position of oxymonads (genus Monocercomonoides) based on concatenation of eight protein sequences (alpha-tubulin, beta-tubulin, gamma-tubulin, EF-1alpha, EF-2, cytosolic (cyt) HSP70, HSP90, and ubiquitin) and 18S rRNA. We demonstrate that the genes are in conflict regarding the position of oxymonads. Concatenation of alpha- and beta-tubulin placed oxymonads in the plant-chromist part of the tree, while the concatenation of other genes recovered a well-supported group of Metamonada (oxymonads, diplomonads, and parabasalids) that branched weakly with euglenozoans--connecting all four excavates included in the analyses and thus providing conditional support for the existence of Excavata.

• MeSH
• elongační faktor 1 genetika   MeSH
• elongační faktor 2 genetika   MeSH
• eukaryotické buňky * MeSH
• flagella MeSH
• fylogeneze * MeSH
• geny rRNA MeSH
• klasifikace MeSH
• molekulární evoluce * MeSH
• polymerázová řetězová reakce MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• tubulin genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elongační faktor 1 MeSH
• elongační faktor 2 MeSH
• tubulin MeSH

Telomeres, ubiquitous and essential structures of eukaryotic chromosomes, are known to come in a variety of forms, but knowledge about their actual diversity and evolution across the whole phylogenetic breadth of the eukaryotic life remains fragmentary. To fill this gap, we employed a complex experimental approach to probe telomeric minisatellites in various phylogenetically diverse groups of algae. Our most remarkable results include the following findings: 1) algae of the streptophyte class Klebsormidiophyceae possess the Chlamydomonas-type telomeric repeat (TTTTAGGG) or, in at least one species, a novel TTTTAGG repeat, indicating an evolutionary transition from the Arabidopsis-type repeat (TTTAGGG) ancestral for Chloroplastida; 2) the Arabidopsis-type repeat is also present in telomeres of Xanthophyceae, in contrast to the presence of the human-type repeat (TTAGGG) in other ochrophytes studied, and of the photosynthetic alveolate Chromera velia, consistent with its phylogenetic position close to apicomplexans and dinoflagellates; 3) glaucophytes and haptophytes exhibit the human-type repeat in their telomeres; and 4) ulvophytes and rhodophytes have unusual telomere structures recalcitrant to standard analysis. To obtain additional details on the distribution of different telomere types in eukaryotes, we performed in silico analyses of genomic data from major eukaryotic lineages, utilizing also genome assemblies from our on-going genome projects for representatives of three hitherto unsampled lineages (jakobids, malawimonads, and goniomonads). These analyses confirm the human-type repeat as the most common and possibly ancestral in eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages, some of them several times independently.

• MeSH
• DNA řas genetika   MeSH
• Eukaryota klasifikace   genetika   metabolismus   MeSH
• fylogeneze * MeSH
• genetická variace * MeSH
• genom MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• molekulární sekvence - údaje MeSH
• sekvence nukleotidů MeSH
• tandemové repetitivní sekvence MeSH
• telomery genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řas MeSH

BACKGROUND: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. RESULTS: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. CONCLUSIONS: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

• Klíčová slova
• Anaerobiosis, Cardiolipin, ERMES, Endoplasmic reticulum, Hydrogenosomes, Structure, Trichomonas vaginalis,
• MeSH
• anaerobióza MeSH
• endoplazmatické retikulum * metabolismus   MeSH
• fosfolipidy metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfolipidy MeSH
• membránové proteiny * MeSH

BACKGROUND: The cilium (flagellum) is a complex cellular structure inherited from the last eukaryotic common ancestor (LECA). A large number of ciliary proteins have been characterized in a few model organisms, but their evolutionary history often remains unexplored. One such protein is the small GTPase RABL2, recently implicated in the assembly of the sperm tail in mammals. RESULTS: Using the wealth of currently available genome and transcriptome sequences, including data from our on-going sequencing projects, we systematically analyzed the phylogenetic distribution and evolutionary history of RABL2 orthologs. Our dense taxonomic sampling revealed the presence of RABL2 genes in nearly all major eukaryotic lineages, including small "obscure" taxa such as breviates, ancyromonads, malawimonads, jakobids, picozoans, or palpitomonads. The phyletic pattern of RABL2 genes indicates that it was present already in the LECA. However, some organisms lack RABL2 as a result of secondary loss and our present sampling predicts well over 30 such independent events during the eukaryote evolution. The distribution of RABL2 genes correlates with the presence/absence of cilia: not a single well-established cilium-lacking species has retained a RABL2 ortholog. However, several ciliated taxa, most notably nematodes, some arthropods and platyhelminths, diplomonads, and ciliated subgroups of apicomplexans and embryophytes, lack RABL2 as well, suggesting some simplification in their cilium-associated functions. On the other hand, several algae currently unknown to form cilia, e.g., the "prasinophytes" of the genus Prasinoderma or the ochrophytes Pelagococcus subviridis and Pinguiococcus pyrenoidosus, turned out to encode not only RABL2, but also homologs of some hallmark ciliary proteins, suggesting the existence of a cryptic flagellated stage in their life cycles. We additionally obtained insights into the evolution of the RABL2 gene architecture, which seems to have ancestrally consisted of eight exons subsequently modified not only by lineage-specific intron loss and gain, but also by recurrent loss of the terminal exon encoding a poorly conserved C-terminal extension. CONCLUSIONS: Our comparative analysis supports the notion that RABL2 is an ancestral component of the eukaryotic cilium and underscores the still underappreciated magnitude of recurrent gene loss, or reductive evolution in general, in the history of eukaryotic genomes and cells.

• MeSH
• cilie metabolismus   MeSH
• Eukaryota klasifikace   genetika   MeSH
• fylogeneze MeSH
• molekulární evoluce * MeSH
• monomerní proteiny vázající GTP genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• monomerní proteiny vázající GTP MeSH

Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

• Klíčová slova
• Min proteins, MinCDE, mitochondria, mitochondrial division, mitochondrial fission,
• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• Arabidopsis genetika   MeSH
• Bacteria cytologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné dělení MeSH
• cytoskeletální proteiny genetika   MeSH
• databáze genetické MeSH
• Dictyostelium metabolismus   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• mitochondriální dynamika * MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• plastidy metabolismus   MeSH
• pravděpodobnostní funkce MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• bakteriální proteiny MeSH
• cytoskeletální proteiny MeSH
• DNA bakterií MeSH
• FtsZ protein, Bacteria MeSH Prohlížeč
• MinC protein, Bacteria MeSH Prohlížeč
• MinD protein, E coli MeSH Prohlížeč
• MinE protein, E coli MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny z Escherichia coli MeSH

The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

• Klíčová slova
• Diphoda, LECA, Opimoda, eukaryote phylogeny, phylogenomics,
• MeSH
• Bacteria klasifikace   genetika   metabolismus   MeSH
• bakteriální geny MeSH
• bakteriální proteiny fyziologie   MeSH
• datové soubory jako téma MeSH
• Eukaryota * MeSH
• fylogeneze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH

The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.

• Klíčová slova
• BLOC, BORC, comparative genomics, lysosome-related organelle, molecular evolution,
• MeSH
• biologická evoluce MeSH
• endozomy metabolismus   MeSH
• Eukaryota metabolismus   genetika   MeSH
• fylogeneze MeSH
• lyzozomy * metabolismus   MeSH
• molekulární evoluce * MeSH
• organely metabolismus   MeSH
• transport proteinů * MeSH
• Publikační typ
• časopisecké články MeSH