Archamoebae comprises free-living or endobiotic amoebiform protists that inhabit anaerobic or microaerophilic environments and possess mitochondrion-related organelles (MROs) adapted to function anaerobically. We compared in silico reconstructed MRO proteomes of eight species (six genera) and found that the common ancestor of Archamoebae possessed very few typical components of the protein translocation machinery, electron transport chain and tricarboxylic acid cycle. On the other hand, it contained a sulphate activation pathway and bacterial iron-sulphur (Fe-S) assembly system of MIS-type. The metabolic capacity of the MROs, however, varies markedly within this clade. The glycine cleavage system is widely conserved among Archamoebae, except in Entamoeba, probably owing to its role in catabolic function or one-carbon metabolism. MRO-based pyruvate metabolism was dispensed within subgroups Entamoebidae and Rhizomastixidae, whereas sulphate activation could have been lost in isolated cases of Rhizomastix libera, Mastigamoeba abducta and Endolimax sp. The MIS (Fe-S) assembly system was duplicated in the common ancestor of Mastigamoebidae and Pelomyxidae, and one of the copies took over Fe-S assembly in their MRO. In Entamoebidae and Rhizomastixidae, we hypothesize that Fe-S cluster assembly in both compartments may be facilitated by dual localization of the single system. We could not find evidence for changes in metabolic functions of the MRO in response to changes in habitat; it appears that such environmental drivers do not strongly affect MRO reduction in this group of eukaryotes.

• Klíčová slova
• anaerobiosis, comparative genomics, mitochondrion-related organelles, reductive evolution,
• MeSH
• anaerobióza MeSH
• Eukaryota * MeSH
• mitochondrie * genetika   MeSH
• sírany MeSH
• železo MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sírany MeSH
• železo MeSH

Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti, which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria) and Candidatus Vesiculincola pelomyxae (class Clostridia), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca. Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca. Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca. Vesiculincola pelomyxae, while Ca. Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca. Methanoregula pelomyxae, which consumes H2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.

• MeSH
• Amoeba * MeSH
• anaerobióza MeSH
• Bacteria genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• methan metabolismus   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methan MeSH
• vodík MeSH

BACKGROUND: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. RESULTS: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. CONCLUSIONS: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

• Klíčová slova
• Anaerobic peroxisome, Anaerobiosis, FeS cluster assembly, Hydrogenosome, Mitochondrion-related organelle, Pelomyxa, Single-cell genomics,
• MeSH
• Amoeba * genetika   metabolismus   MeSH
• anaerobióza MeSH
• Archamoebae * genetika   metabolismus   MeSH
• genomika MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• peroxizomy metabolismus   MeSH
• Saccharomyces cerevisiae MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

• Klíčová slova
• Amoebozoa, phylogenomics, phylotranscriptomics, reductive evolution, transcriptomes,
• MeSH
• Amoeba genetika   metabolismus   MeSH
• Amoebozoa genetika   MeSH
• bezobratlí genetika   MeSH
• biologická evoluce MeSH
• Eukaryota genetika   MeSH
• fylogeneze MeSH
• genová knihovna MeSH
• houby genetika   MeSH
• molekulární evoluce MeSH
• sekvenční analýza DNA metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH