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

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

• Klíčová slova
• Mastigamoeba, Archamoebae, chitinous cysts, evolution of parasitism, lateral gene transfer, pathway complexity,
• MeSH
• anaerobióza genetika   MeSH
• Archamoebae genetika   metabolismus   MeSH
• biologická adaptace genetika   MeSH
• biologická evoluce * MeSH
• délka genomu MeSH
• Entamoeba histolytica genetika   MeSH
• genom protozoální * MeSH
• paraziti genetika   MeSH
• přenos genů horizontální MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

• Klíčová slova
• Mastigamoeba balamuthi, anaerobiosis, mitochodria, peroxisome,
• MeSH
• anaerobióza MeSH
• Archamoebae genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• oxidace-redukce MeSH
• peroxiny genetika   metabolismus   MeSH
• peroxizomy genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peroxiny MeSH
• protozoální proteiny MeSH
• reaktivní formy kyslíku MeSH

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

• Klíčová slova
• Algae, amoebae, biodiversity, ciliate, ecology, flagellate, fungus, microbiology, parasite, plankton, protozoa, systematics, taxonomy,
• MeSH
• biodiverzita * MeSH
• Eukaryota klasifikace   MeSH
• fylogeneze * MeSH
• terminologie jako téma * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. 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