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Abstract Background Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. Results We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. Conclusions In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

Abstract 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.

Abstract Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. Results We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. Conclusions The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.