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.

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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.

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