Comparative analysis of mitochondrion-related organelles in anaerobic amoebozoans

Abstract

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.

Keywords: anaerobiosis; comparative genomics; mitochondrion-related organelles; reductive evolution.

Fig. 1.

Archamoebae species investigated in this study. (a) SSU rRNA gene phylogeny with branches with full and high bootstrap support (BS) indicated as thicker black and grey lines, respectively (for the detailed tree see Fig. S1). Species lifestyle is marked by pictograms explained in the graphical key above. Species with new data produced within this study are shown in bold. (b) The completeness of transcriptome-derived protein datasets produced in this study was evaluated by BUSCO v5 using the odb10_eukaryota database and compared with the completeness of genome-derived protein datasets from Entamoeba histolytica, Mastigamoeba balamuthi and P. schiedti.

Fig. 2.

Evaluation of localization prediction tools. Localizations were predicted for proteins residing outside MROs (ribosomal and replisomal proteins) and previously identified MRO proteins [10, 28–30, 42]. Venn diagrams depict the overlaps between reference sequences truly and falsely identified as mitochondrial (i.e. positives) or non-mitochondrial (i.e. negatives) by the three best-scoring targeting predictors (Table S2).

Fig. 3.

Mitochondrial pathways in Archamoebae species. Newly studied species are shown in bold. Presence/absence of components (explained in the graphical key) was compared with those of A. castellanii and/or yeast mitochondria. (a) Protein import machinery. TOM/TIM, translocase of the outer/inner membrane; SAM, sorting and assembly machinery; Pam, presequence translocase-assisted motor; Hsp70, heat shock protein 70; Mge1, nucleotide exchange factor; Cpn60/10, chaperonin 60/10; MPPα/β, mitochondrial processing peptidase α/β subunit. (b) Electron transfer chain and tricarboxylic acid cycle. cI, complex I; cIII–cV, complex III–V; SdhA–D, succinate dehydrogenase subunit A–D; SdhAF, succinate dehydrogenase assembly factor; ETFDH, electron transferring flavoprotein dehydrogenase; ETFα/β, electron transferring flavoprotein subunit α/β; RquA, rhodoquinone methyltransferase; CS, citrate synthase; ACO, aconitase; IDH, isocitrate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase; SCS, succinyl-coenzyme A synthetase; FH, fumarase; MDH, malate dehydrogenase; AAT, aspartate aminotransferase. (c) Glycine cleavage system and folate metabolism. GCS-H/L/P/T/R, glycine cleavage system H/L/P/T/R protein; LplA, lipoamide protein ligase; SHMT, serine hydroxymethyltransferase; TDH, threonine dehydrogenase; AKL, α-amino-β-ketobutyrate coenzyme A ligase; FPGS, folylpolyglutamate synthase; FolD, tetrahydrofolate dehydrogenase/cyclohydrolase; MTHFD, methylenetetrahydrofolate dehydrogenase; MetH, B₁₂-dependent methionine synthase. (d) Pyruvate metabolism and ATP generation. PFO, pyruvate:ferredoxin oxidoreductase; PNO, pyruvate:NADP⁺ oxidoreductase; PFL, pyruvate:formate lyase; PFLA, pyruvate:formate lyase activating enzyme; PC, pyruvate carboxylase; d-LDH, d-lactate dehydrogenase; HydA, [FeFe]-hydrogenase group A; HydB, [FeFe]-hydrogenase group B; HydE/F/G, hydrogenase maturases; Fd, ferredoxin; ASCT, acetate:succinate coenzyme A-transferase; ACS, acetyl-coenzyme A synthetase; AK, adenylate kinase. (e) Iron–sulphur cluster assembly. ISC, iron–sulphur cluster; NIF, nitrogen fixation; NifS, cysteine desulfurase; NifU, scaffold protein; FdhD, formate dehydrogenase accessory sulfurtransferase; FdhF, formate dehydrogenase. (f) Sulphate activation pathway. AS, ATP sulfurylase; APSK, adenosine-5′-phosphosulphate kinase; IPP, inorganic pyrophosphatase; NaS transp, sodium sulphate transporter; PAPS transp, 3′-phosphoadenosine 5′-phosphosulfate transporter.

Fig. 4.

The evolutionary history of MRO-localized pathways in Archamoebae. Ancestral, gained and lost enzymes and metabolic pathways are shown by different shades of blue on a schematic phylogenetic tree. Lifestyles are shown for all included species by a different symbol as explained in the graphical legend. Species with new data produced within this study are in bold type. Main clades of Archamoebae are shown at respective branches.