With the first oxymonad genome sequence in hand, we focused our attention on one of the most puzzling aspects of their biology—the elusive nature of their mitochondrion.

Using the 454 whole-genome shotgun sequencing methodology, we generated a draft genome sequence of the oxymonad Monocercomonoides sp. PA203, assembled into 2,095 scaffolds at ∼35× coverage (see Experimental Procedures ). The estimated size of the genome (∼75 Mb) and the number of predicted protein-coding genes (16,629) is intermediate between what is found in diplomonads and T. vaginalis ( Table 1 ). Almost 67% of predicted protein-coding genes contain introns (∼1.9 introns per gene on average; Table 1 ). The assembly contains genes encoding tRNAs for all 20 amino acids, and ∼50 ribosomal DNA units were identified on small contigs outside the main assembly (see Supplemental Experimental Procedures ). To estimate completeness of the genome sequence, we performed transcriptome mapping, in which 96.9% of transcripts mapped to the genome (see Supplemental Experimental Procedures ), and checked the representation of core eukaryotic genes. Using the Core Eukaryotic Genes Mapping Approach (CEGMA) [], we recovered 63.3% of core eukaryotic genes, a greater fraction than in the G. intestinalis genome (46.6%). However, when we excluded genes encoding mitochondrial proteins from the CEGMA dataset and used manually curated Monocercomonoides sp. gene models, the percentage of recovered genes increased to 90% ( Table S1 ). For another set of 163 conserved eukaryotic genes used for phylogenomic analyses, the percentage of recovered genes exceeded 95% ( Table S2 ). As the last measure of completeness, we identified 77 out of 78 conserved families of cytosolic eukaryotic ribosomal proteins [] ( Table S3 ), with the single exception of L41e, which is very short, difficult to detect, and has not been identified in other Metamonada genomes. Phylogenomic analysis ( Figure 1 ) confirmed the relationship of Monocercomonoides sp. to P. pyriformis and other Metamonada and demonstrated that the Monocercomonoides lineage forms a much shorter branch relative to parabasalids and diplomonads. All these measures suggest that the assembled Monocercomonoides sp. genome sequence is nearly complete and its encoded proteins are, on average, less divergent than those of G. intestinalis and T. vaginalis.

The tree displayed was inferred using PhyloBayes (CAT + Poisson substitution model). A maximum-likelihood (ML) tree inferred from the same supermatrix using RAxML (not shown) was very similar to the PhyloBayes tree, with the topological differences in the poorly resolved area comprising Chloroplastida, Cryptophyta, Glaucophyta, and Haptophyta, and in the position of Metamonada, in the ML tree placed sister (with strong bootstrap support) to Discoba. The branch support values shown are posterior probabilities (>0.95) from the PhyloBayes analysis and bootstrap values (>50%) from the ML analysis. Three branches are shown shortened to the indicated percentage of their actual length to fit them on the page. See also Table S2

Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale.

Absence of Mitochondrial Proteins

18 Jedelský P.L.

Doležal P.

Rada P.

Pyrih J.

Smíd O.

Hrdý I.

Sedinová M.

Marcinčiková M.

Voleman L.

Perry A.J.

et al. The minimal proteome in the reduced mitochondrion of the parasitic protist Giardia intestinalis. 19 Tsaousis A.D.

Kunji E.R.S.

Goldberg A.V.

Lucocq J.M.

Hirt R.P.

Embley T.M. A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi. All MROs, with the exception of the G. intestinalis mitosome [], are known to export or import ATP and other metabolites typically using transporters from the mitochondrial carrier family (MCF) or, in mitosomes of the microsporidian Encephalitozoon cuniculi [], by the bacterial-type (NTT-like) nucleotide transporters. We did not identify in the Monocercomonoides sp. genome any homologs of genes encoding known mitochondrial metabolite transport proteins ( Figure 2 A; Table S4 ).

20 Lill R. Function and biogenesis of iron-sulphur proteins. 21 Tsaousis A.D.

Gentekaki E.

Eme L.

Gaston D.

Roger A.J. Evolution of the cytosolic iron-sulfur cluster assembly machinery in Blastocystis species and other microbial eukaryotes. Fe-S clusters are essential biological cofactors associated with many different proteins and are therefore synthesized de novo in every organism across the tree of life []. In eukaryotes, this is done mostly by the mitochondrial ISC assembly system and the cytosolic iron-sulfur assembly (CIA) system []. Analyses of the Monocercomonoides sp. genome revealed the presence of a CIA system but a complete lack of components of the ISC system ( Figure 2 A; Table S3 Experimental Procedures ).

22 Tian H.-F.

Feng J.-M.

Wen J.-F. The evolution of cardiolipin biosynthesis and maturation pathways and its implications for the evolution of eukaryotes. 22 Tian H.-F.

Feng J.-M.

Wen J.-F. The evolution of cardiolipin biosynthesis and maturation pathways and its implications for the evolution of eukaryotes. 23 Wideman J.G.

Gawryluk R.M.R.

Gray M.W.

Dacks J.B. The ancient and widespread nature of the ER-mitochondria encounter structure. We could not identify either of two possible enzymes involved in the synthesis of cardiolipin, a phospholipid specific for energy-transducing membranes []. The majority of eukaryotes synthesize cardiolipins, and the process is localized to mitochondria, but a complete lack of cardiolipin has been experimentally shown for G. intestinalis, T. vaginalis, and E. cuniculi []. Furthermore, we could not identify any component of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES; Figure 2 A) [].

11 Zubáčová Z.

Novák L.

Bublíková J.

Vacek V.

Fousek J.

Rídl J.

Tachezy J.

Doležal P.

Vlček C.

Hampl V. The mitochondrion-like organelle of Trimastix pyriformis contains the complete glycine cleavage system. 24 Yarlett N.

Lindmark D.G.

Goldberg B.

Moharrami M.A.

Bacchi C.J. Subcellular localization of the enzymes of the arginine dihydrolase pathway in Trichomonas vaginalis and Tritrichomonas foetus. 25 Yousuf M.A.

Mi-ichi F.

Nakada-Tsukui K.

Nozaki T. Localization and targeting of an unusual pyridine nucleotide transhydrogenase in Entamoeba histolytica. We identified only two orthologs of the set of proteins predicted to localize to the mitochondrion-related compartment of the closely related P. pyriformis []: aspartate/ornithine carbamoyltransferase family protein and pyridine nucleotide transhydrogenase. Neither protein has an exclusively mitochondrial localization in eukaryotes [], and the Monocercomonoides sp. orthologs do not contain predicted mitochondrial targeting sequences.

26 Smith A.C.

Blackshaw J.A.

Robinson A.J. MitoMiner: a data warehouse for mitochondrial proteomics data. To complement the targeted homology-based searches, we also performed an extensive search for putative homologs of known mitochondrial proteins using a pipeline based on the Mitominer database [], which was enriched with identified mitochondrial proteins of diverse anaerobic eukaryotes with MROs ( Experimental Procedures ). The search recovered 76 Monocercomonoides sp. proteins as candidates for functions in a putative mitochondrion ( Figure 2 B; Table S5 ). Similarly to G. intestinalis, T. vaginalis, and E. histolytica, used as controls, the selected candidates were mainly proteins that are obviously not mitochondrial (e.g., histones) or for which the annotation is too general (e.g. “kinase domain-containing protein”), indicating that the specificity of the pipeline in organisms with divergent mitochondrion is low. However, unlike all other control organisms, in which the search always recovered at least a few mitochondrial hallmark proteins, the set of 76 Monocercomonoides sp. candidates did not contain any such proteins. Only 11 of the Monocercomonoides candidates fall in the GO category “metabolism,” but they do not assemble any obvious metabolic pathway. In summary, this approach ( Table S5 ) failed to reveal any credible set of mitochondrial protein in Monocercomonoides sp.

14 Dolezal P.

Likic V.

Tachezy J.

Lithgow T. Evolution of the molecular machines for protein import into mitochondria. 27 Lucattini R.

Likic V.A.

Lithgow T. Bacterial proteins predisposed for targeting to mitochondria. As an alternative to homology searches, we have also attempted to identify mitochondrial proteins by searching for several types of signature sequences. The matrix proteins of mitochondria and MROs are expected to contain conserved N-terminal targeting signals needed for the targeted import into MROs []. We performed in silico prediction of mitochondrial targeting signals in the predicted Monocercomonoides sp. proteome and identified 107 candidate proteins ( Figure 2 A; Experimental Procedures Table S6 A). The presence of a predicted targeting signal by itself does not prove the targeting, as such amino acid sequences can also appear at random []. Functional annotation revealed that a majority of proteins recovered by this search fall into the Kyoto Encyclopedia of Genes and Genomes (KEGG) category “genetic information processing.” Given the absence of a mitochondrial genome, or organellar translation machinery, it is unlikely that these proteins function in an MRO. Only eight candidates were assigned to the KEGG category “metabolism,” and they are part of several different metabolic pathways. Finally, only three proteins were predicted to have a mitochondrial targeting signal and homology to a Mitominer protein (hydrolase-like family protein MONOS_10795, cytosolic TCP-1/cpn60 chaperonin family protein MONOS_13132, and ribonuclease Z MONOS_6181). This also suggests that both pipelines failed to recover specific sets of mitochondrial proteins but instead detected only low-specificity “noise.”

28 Denic V. A portrait of the GET pathway as a surprisingly complicated young man. 29 Borgese N.

Brambillasca S.

Colombo S. How tails guide tail-anchored proteins to their destinations. The outer mitochondrial membranes accommodate two special classes of proteins, β-barrel and tail-anchored (TA) proteins, which are devoid of the N-terminal targeting signals and instead use specific C-terminal signals []. We have identified 32 candidates for TA proteins in the predicted proteome, several of which appeared to be ER-targeted proteins. None of these had the hallmark characteristics of proteins targeted to the mitochondrial outer membrane ( Figure 2 A; Experimental Procedures Table S6 B). We also failed to identify any credible candidates for β-barrel outer membrane proteins (BOMPs) ( Figure 2 A; Experimental Procedures ).

In summary, our comprehensive examination of the Monocercomonoides sp. genome based on homology searches and searches for specific N-terminal and C-terminal signals failed to recover proteins typically associated with MROs, including mitochondrial translocases, metabolite transporters and the ISC system for Fe-S cluster synthesis, ERMES, and enzymes responsible for cardiolipin synthesis.

30 Mowbrey K.

Dacks J.B. Evolution and diversity of the Golgi body. In order to verify that our inability to find any reliable mitochondrial proteins is not caused by possible unprecedented divergence of Monocercomonoides sp. proteins or a failure of our methods, we searched for hallmark proteins of another cellular system, so far not observed in Monocercomonoides sp.—the Golgi complex. In this case, using homology-based searches, we detected numerous Golgi-associated proteins, including components of the COPI, AP-1, AP-3, AP-4, COG, GARP, TRAPPI, and Retromer complexes and Rab GTPases regulating transport to and from the Golgi ( Table S3 ). This suggests the presence of Golgi-like compartments in oxymonads [], despite the absence of a cytologically discernible Golgi apparatus.

The specific absence of mitochondria-associated proteins in Monocercomonoides sp. implies the legitimate absence of a mitochondrial compartment. If so, then how does the Monocercomonoides cell function without this organelle?