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

BACKGROUND: Apicomplexa is a diverse phylum comprising unicellular endobiotic animal parasites and contains some of the most well-studied microbial eukaryotes including the devastating human pathogens Plasmodium falciparum and Cryptosporidium hominis. In contrast, data on the invertebrate-infecting gregarines remains sparse and their evolutionary relationship to other apicomplexans remains obscure. Most apicomplexans retain a highly modified plastid, while their mitochondria remain metabolically conserved. Cryptosporidium spp. inhabit an anaerobic host-gut environment and represent the known exception, having completely lost their plastid while retaining an extremely reduced mitochondrion that has lost its genome. Recent advances in single-cell sequencing have enabled the first broad genome-scale explorations of gregarines, providing evidence of differential plastid retention throughout the group. However, little is known about the retention and metabolic capacity of gregarine mitochondria. RESULTS: Here, we sequenced transcriptomes from five species of gregarines isolated from cockroaches. We combined these data with those from other apicomplexans, performed detailed phylogenomic analyses, and characterized their mitochondrial metabolism. Our results support the placement of Cryptosporidium as the earliest diverging lineage of apicomplexans, which impacts our interpretation of evolutionary events within the phylum. By mapping in silico predictions of core mitochondrial pathways onto our phylogeny, we identified convergently reduced mitochondria. These data show that the electron transport chain has been independently lost three times across the phylum, twice within gregarines. CONCLUSIONS: Apicomplexan lineages show variable functional restructuring of mitochondrial metabolism that appears to have been driven by adaptations to parasitism and anaerobiosis. Our findings indicate that apicomplexans are rife with convergent adaptations, with shared features including morphology, energy metabolism, and intracellularity.

• Klíčová slova
• Anaerobic metabolism, Apicomplexa, Eugregarines, Evolution, Mitochondria, Mitosome, Parasitism, Phylogenomics,
• MeSH
• analýza jednotlivých buněk MeSH
• Apicomplexa * genetika   MeSH
• fylogeneze MeSH
• lidé MeSH
• mitochondrie * genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

• MeSH
• Archaea * MeSH
• Bacteria genetika   MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• geny rRNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.

• MeSH
• fylogeneze MeSH
• membránové proteiny metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• mitochondriální importní komplex MeSH
• mitochondrie metabolismus   MeSH
• organely MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny mitochondriální membrány metabolismus   MeSH
• transportní proteiny genetika   metabolismus   fyziologie   MeSH
• Trichomonas vaginalis metabolismus   patogenita   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• membránové transportní proteiny MeSH
• mitochondriální importní komplex MeSH
• transportní proteiny mitochondriální membrány MeSH
• transportní proteiny MeSH

BACKGROUND: Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree. RESULTS: We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain. CONCLUSIONS: At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

• Klíčová slova
• Hydrophobicity, Mitochondrial evolution, Protein transport, TAT translocase,
• MeSH
• Escherichia coli genetika   MeSH
• Eukaryota genetika   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• molekulární evoluce * MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové transportní proteiny MeSH
• proteiny z Escherichia coli MeSH

Modern syntheses of eukaryote diversity assign almost all taxa to one of three groups: Amorphea, Diaphoretickes and Excavata (comprising Discoba and Metamonada). The most glaring exception is Malawimonadidae, a group of small heterotrophic flagellates that resemble Excavata by morphology, but branch with Amorphea in most phylogenomic analyses. However, just one malawimonad, Malawimonas jakobiformis, has been studied with both morphological and molecular-phylogenetic approaches, raising the spectre of interpretation errors and phylogenetic artefacts from low taxon sampling. We report a morphological and phylogenomic study of a new deep-branching malawimonad, Gefionella okellyi n. gen. n. sp. Electron microscopy revealed all canonical features of 'typical excavates', including flagellar vanes (as an opposed pair, unlike M. jakobiformis but like many metamonads) and a composite fibre. Initial phylogenomic analyses grouped malawimonads with the Amorphea-related orphan lineage Collodictyon, separate from a Metamonada+Discoba clade. However, support for this topology weakened when more sophisticated evolutionary models were used, and/or fast-evolving sites and long-branching taxa (FS/LB) were excluded. Analyses of '-FS/LB' datasets instead suggested a relationship between malawimonads and metamonads. The 'malawimonad+metamonad signal' in morphological and molecular data argues against a strict Metamonada+Discoba clade (i.e. the predominant concept of Excavata). A Metamonad+Discoba clade should therefore not be assumed when inferring deep-level evolutionary history in eukaryotes.

• Klíčová slova
• Excavata, Metamonada, eukaryote evolution, protist, transcriptomics, ultrastructure,
• Publikační typ
• časopisecké články MeSH