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.

Department of Biochemistry Faculty of Natural Sciences Comenius University Bratislava Slovakia

Department of Parasitology Faculty of Science Charles University BIOCEV Vestec Czech Republic

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Zobrazit více v PubMed

Müller M, Mentel M, van Hellemond JJ, Henze K, Woehle C, Gould SB, et al. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol Mol Biol Rev. 2012;76:444–495. PubMed PMC

Roger AJ, Muñoz-Gómez SA, Kamikawa R. The origin and diversification of mitochondria. Curr Biol. 2017;27:R1177–R1192. PubMed

Gawryluk RMR, Stairs CW. Diversity of electron transport chains in anaerobic protists. Biochim Biophys Acta Bioenerg. 2021;1862:148334. PubMed

Zaremba-Niedzwiedzka K, Caceres EF, Saw JH, Di B, Juzokaite L, Vancaester E, et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature. 2017;541:353–358. PubMed

Sagan L. On the origin of mitosing cells. J Theor Biol. 1967;14:255–274. PubMed

Martijn J, Vosseberg J, Guy L, Offre P, Ettema TJG. Deep mitochondrial origin outside the sampled alphaproteobacteria. Nature. 2018;557:101–105. PubMed

Gawryluk RMR, Kamikawa R, Stairs CW, Silberman JD, Brown MW, Roger AJ. The earliest stages of mitochondrial adaptation to low oxygen revealed in a novel rhizarian. Curr Biol. 2016;26:2729–2738. PubMed

Leger MM, Kolisko M, Kamikawa R, Stairs CW, Kume K, Čepička I, et al. Organelles that illuminate the origins of Trichomonas hydrogenosomes and Giardia mitosomes. Nat Ecol Evol. 2017;1:0092. PubMed PMC

Karnkowska A, Vacek V, Zubáčová Z, Treitli SC, Petrželková R, Eme L, et al. A eukaryote without a mitochondrial organelle. Curr Biol. 2016;26:1274–1284. PubMed

Panigrahi AK, Ogata Y, Zíková A, Anupama A, Dalley RA, Acestor N, et al. A comprehensive analysis of Trypanosoma brucei mitochondrial proteome. Proteomics. 2009;9:434–450. PubMed PMC

Lee CP, Taylor NL, Harvey MA. Recent advances in the composition and heterogeneity of the Arabidopsis mitochondrial proteome. Front Plant Sci. 2013;4:4. PubMed PMC

Gawryluk RMR, Chisholm KA, Pinto DM, Gray MW. Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants. J Proteome. 2014;109:400–416. PubMed

Žárský V, Tachezy J. Evolutionary loss of peroxisomes - not limited to parasites. Biol Direct. 2015;10:74. PubMed PMC

Schlüter A, Fourcade S, Ripp R, Mandel JL, Poch O, Pujol A. The evolutionary origin of peroxisomes: an ER-peroxisome connection. Mol Biol Evol. 2006;23:838–845. PubMed

Gabaldón T. Peroxisome diversity and evolution. Philos Trans R Soc B Biol Sci. 2010;365:765–773. PubMed PMC

Gabaldón T, Ginger ML, Michels PAM. Peroxisomes in parasitic protists. Mol Biochem Parasitol. 2016;209:35–45. PubMed

Le T, Žárský V, Nývltová E, Rada P, Harant K, Vancová M, et al. Anaerobic peroxisomes in Mastigamoeba balamuthi. Proc Natl Acad Sci U S A. 2020;117:2065–2075. PubMed PMC

Verner Z, Žárský V, Le T, Narayanasamy RK, Rada P, Rozbeský D, et al. Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol. PLoS Pathog. 2021;17:e1010041. PubMed PMC

Pánek T, Zadrobílková E, Walker G, Brown MW, Gentekaki E, Hroudová M, et al. First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group. Mol Phylogenet Evol. 2016;98:41–51. PubMed

Kang S, Tice AK, Spiegel FW, Silberman JD, Pánek T, Čepička I, et al. Between a pod and a hard test: the deep evolution of Amoebae. Mol Biol Evol. 2017;34:2258–2270. PubMed PMC

Pearce XG, Annesley SJ, Fisher PR. The Dictyostelium model for mitochondrial biology and disease. Int J Dev Biol. 2019;63:497–508. PubMed

Leger MM, Gawryluk RMR, Gray MW, Roger AJ. Evidence for a hydrogenosomal-type anaerobic ATP generation pathway in Acanthamoeba castellanii. PLoS One. 2013;8:e69532. PubMed PMC

Mi-ichi F, Yousuf MA, Nakada-Tsukui K, Nozaki T. Mitosomes in Entamoeba histolytica contain a sulfate activation pathway. Proc Natl Acad Sci. 2009;106:21731–21736. PubMed PMC

Gill EE, Diaz-Triviño S, Barberà MJ, Silberman JD, Stechmann A, Gaston D, et al. Novel mitochondrion-related organelles in the anaerobic amoeba Mastigamoeba balamuthi. Mol Microbiol. 2007;66:1306–1320. PubMed

Nývltová E, Stairs CW, Hrdý I, Rídl J, Mach J, Pačes J, et al. Lateral gene transfer and gene duplication played a key role in the evolution of Mastigamoeba balamuthi hydrogenosomes. Mol Biol Evol. 2015;32:1039–1055. PubMed PMC

Nývltová E, Šuták R, Harant K, Šedinová M, Hrdy I, Paces J, et al. NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi. Proc Natl Acad Sci U S A. 2013;110:7371–7376. PubMed PMC

Zadrobílková E, Walker G, Čepička I. Morphological and molecular evidence support a close relationship between the free-living Archamoebae Mastigella and Pelomyxa. Protist. 2015;166:14–41. PubMed

Seravin L, Goodkov A. Cytoplasmic microbody-like granules of the amoeba Pelomyxa palustris. Tsitologiya. 1987;29:600–603.

Žárský V, Klimeš V, Pačes J, Vlček Č, Hradilová M, Beneš V, et al. The Mastigamoeba balamuthi genome and the nature of the free-living ancestor of Entamoeba. Mol Biol Evol. 2021;38:2240–2259. PubMed PMC

Wang D, Hancock J. IntronDB: a database for eukaryotic intron features. Bioinformatics. 2019;35:4400–4401. PubMed

Bolender N, Sickmann A, Wagner R, Meisinger C, Pfanner N. Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep. 2008;9:42–49. PubMed PMC

Makiuchi T, Mi-Ichi F, Nakada-Tsukui K, Nozaki T. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport. Sci Rep. 2013;3:1129. PubMed PMC

Dolezal P, Dagley MJ, Kono M, Wolynec P, Likić VA, Foo JH, et al. The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica. PLoS Pathog. 2010;6:e1000812. PubMed PMC

Schneider A. Evolution of mitochondrial protein import - lessons from trypanosomes. Biol Chem. 2020;401:663–676. PubMed

Makki A, Rada P, Žárský V, Kereïche S, Kováčik L, Novotný M, et al. Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis. PLoS Biol. 2019;17:e3000098. PubMed PMC

Garg S, Stölting J, Zimorski V, Rada P, Tachezy J, Martin WF, et al. Conservation of transit peptide-independent protein import into the mitochondrial and hydrogenosomal matrix. Genome Biol Evol. 2015;7:2716–2726. PubMed PMC

Hao H-X, Khalimonchuk O, Schraders M, Dephoure N, Bayley J-P, Kunst H, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science. 2009;325:1139–1142. PubMed PMC

Stairs CW, Eme L, Muñoz-Gómez SA, Cohen A, Dellaire G, Shepherd JN, et al. Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis. Elife. 2018;7:e34292. PubMed PMC

Castro-Guerrero NA, Jasso-Chávez R, Moreno-Sánchez R. Physiological role of rhodoquinone in Euglena gracilis mitochondria. Biochim Biophys Acta. 2005;1710:113–121. PubMed

Coustou V, Besteiro S, Rivière L, Biran M, Biteau N, Franconi JM, et al. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei. J Biol Chem. 2005;280:16559–16570. PubMed

Jitrapakdee S, St Maurice M, Rayment I, Cleland WW, Wallace JC, Attwood PV. Structure, mechanism and regulation of pyruvate carboxylase. Biochem J. 2008;413:369–387. PubMed PMC

Leger MM, Eme L, Hug LA, Roger AJ. Novel hydrogenosomes in the microaerophilic jakobid Stygiella incarcerata. Mol Biol Evol. 2016;33:2318–2336. PubMed PMC

Kikuchi G. The glycine cleavage system: composition, reaction mechanism, and physiological significance. Mol Cell Biochem. 1973;1:169–187. PubMed

Dale RA. Catabolism of threonine in mammals by coupling of L-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase. Biochim Biophys Acta. 1978;544:496–503. PubMed

Schertl P, Braun H-P. Respiratory electron transfer pathways in plant mitochondria. Front Plant Sci. 2014;5:163. PubMed PMC

Thomson JM, Gaucher EA, Burgan MF, De Kee DW, Li T, Aris JP, et al. Resurrecting ancestral alcohol dehydrogenases from yeast. Nat Genet. 2005;37:630–635. PubMed PMC

Hügler M, Wirsen CO, Fuchs G, Taylor CD, Sievert SM. Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the ε subdivision of proteobacteria. J Bacteriol. 2005;187:3020–3027. PubMed PMC

Jackson JB, Peake SJ, White SA. Structure and mechanism of proton-translocating transhydrogenase. FEBS Lett. 1999;464:1–8. PubMed

Yu Y, Samuelson J. Primary structure of an Entamoeba histolytica nicotinamide nucleotide transhydrogenase. Mol Biochem Parasitol. 1994;68:323–328. PubMed

Yousuf MA, Mi-ichi F, Nakada-Tsukui K, Nozaki T. Localization and targeting of an unusual pyridine nucleotide transhydrogenase in Entamoeba histolytica. Eukaryot Cell. 2010;9:926–933. PubMed PMC

Kuchenreuther JM, Britt RD, Swartz JR. New insights into [FeFe] hydrogenase activation and maturase function. PLoS One. 2012;7:e45850. PubMed PMC

Tachezy J, Doležal P. Iron–sulfur proteins and iron–sulfur cluster assembly in organisms with hydrogenosomes and mitosomes. In: Martin WF, Müller M, editors. Origin of mitochondria and hydrogenosomes. Berlin: Springer Berlin Heidelberg; 2007. pp. 105–133.

Stairs CW, Eme L, Brown MW, Mutsaers C, Susko E, Dellaire G, et al. A SUF Fe-S cluster biogenesis system in the mitochondrion-related organelles of the anaerobic protist Pygsuia. Curr Biol. 2014;24:1176–1186. PubMed

Thomé R, Gust A, Toci R, Mendel R, Bittner F, Magalon A, et al. A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli. J Biol Chem. 2012;287:4671–4678. PubMed PMC

Mansy SS, Wu G, Surerus KK, Cowan JA. Iron-sulfur cluster biosynthesis. Thermatoga maritima IscU is a structured iron-sulfur cluster assembly protein. J Biol Chem. 2002;277:21397–21404. PubMed

Kaiser JT, Clausen T, Bourenkow GP, Bartunik HD, Steinbacher S, Huber R. Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly. J Mol Biol. 2000;297:451–464. PubMed

Mi-ichi F, Makiuchi T, Furukawa A, Sato D, Nozaki T. Sulfate activation in mitosomes plays an important role in the proliferation of Entamoeba histolytica. PLoS Negl Trop Dis. 2011;5:e1263. PubMed PMC

Mi-ichi F, Nozawa A, Yoshida H, Tozawa Y, Nozaki T. Evidence that the Entamoeba histolytica mitochondrial carrier family links mitosomal and cytosolic pathways through exchange of 3′-phosphoadenosine 5′-phosphosulfate and ATP. Eukaryot Cell. 2015;14:1144–1150. PubMed PMC

Liu S, He L, Yao K. The antioxidative function of alpha-ketoglutarate and its applications. Biomed Res Int. 2018;2018:3408467. PubMed PMC

Sonneborn TM. Methods in the general biology and genetics of Paramecium aurelia. J Exp Zool. 1950;113:87–147.

Trager W. The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa. Biol Bull. 1934;66:182–190.

Picelli S, Faridani OR, Björklund AK, Winberg G, Sagasser S, Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014;9:171–181. PubMed

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–477. PubMed PMC

Dick GJ, Andersson AF, Baker BJ, Simmons SL, Thomas BC, Yelton AP, et al. Community-wide analysis of microbial genome sequence signatures. Genome Biol. 2009;10:R85–R85. PubMed PMC

Treitli SC, Kolisko M, Husník F, Keeling PJ, Hampl V. Revealing the metabolic capacity of Streblomastix strix and its bacterial symbionts using single-cell metagenomics. Proc Natl Acad Sci U S A. 2019;116:19675–19684. PubMed PMC

Zhu B-H, Xiao J, Xue W, Xu G-C, Sun M-Y, Li J-T. P_RNA_scaffolder: a fast and accurate genome scaffolder using paired-end RNA-sequencing reads. BMC Genomics. 2018;19:175. PubMed PMC

Stanke M, Schöffmann O, Morgenstern B, Waack S. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics. 2006;7:62. PubMed PMC

Haas BJ, Salzberg SL, Zhu W, Pertea M, Allen JE, Orvis J, et al. Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol. 2008;9:R7–R7. PubMed PMC

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–652. PubMed PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–359. PubMed PMC

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–915. PubMed PMC

Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–3212. PubMed

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. PubMed

Eddy SR. A new generation of homology search tools based on probabilistic inference. Genome Inf. 2009;23:205–211. PubMed

Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236–1240. PubMed PMC

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–1649. PubMed PMC

Almagro Armenteros JJ, Salvatore M, Emanuelsson O, Winther O, von Heijne G, Elofsson A, et al. Detecting sequence signals in targeting peptides using deep learning. Life Sci Alliance. 2019;2:e201900429. PubMed PMC

Horton P, Nakai K. Better prediction of protein cellular localization sites with the k nearest neighbors classifier. Proc Int Conf Intell Syst Mol Biol. 1997;5:147–152. PubMed

Blum T, Briesemeister S, Kohlbacher O. MultiLoc2: integrating phylogeny and gene ontology terms improves subcellular protein localization prediction. BMC Bioinformatics. 2009;10:274. PubMed PMC

Kume K, Amagasa T, Hashimoto T, Kitagawa H. NommPred: prediction of mitochondrial and mitochondrion-related organelle proteins of nonmodel organisms. Evol Bioinformatics Online. 2018;14:1176934318819835. PubMed PMC

Reumann S. Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiol. 2004;135:783–800. PubMed PMC

Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–580. PubMed

Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, Eisenhaber F. Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J Mol Biol. 2003;328:581–592. PubMed

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–780. PubMed PMC

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser. 1999;41:95–98.

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. PubMed PMC

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–1973. PubMed PMC

Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268–274. PubMed PMC

Wang H-C, Minh BQ, Susko E, Roger AJ. Modeling site heterogeneity with posterior mean site frequency profiles accelerates accurate phylogenomic estimation. Syst Biol. 2018;67:216–235. PubMed

Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol. 2017;35:518–522. PubMed PMC

Gietz RD, Woods RA. Yeast transformation by the LiAc/SS carrier DNA/PEG method. In: Xiao W, editor. Yeast Protocols. Totowa, NJ: Humana Press; 2006. pp. 107–120. PubMed

Malínská K, Malínský J, Opekarová M, Tanner W. Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol Biol Cell. 2003;14:4427–4436. PubMed PMC

Single-cell genome and transcriptome sequencing of Pelomyxa schiedti. NCBI. 2020. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA672820.

Two decades taken at speed: genomics, cell biology, ecology, and evolution of protists

Genomics of Preaxostyla Flagellates Illuminates the Path Towards the Loss of Mitochondria

Hydrogenotrophic methanogenesis is the key process in the obligately syntrophic consortium of the anaerobic ameba Pelomyxa schiedti

Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans

Independent and sensory human mitochondrial functions reflecting symbiotic evolution

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol