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

The notion that mitochondria cannot be lost was shattered with the report of an oxymonad Monocercomonoides exilis, the first eukaryote arguably without any mitochondrion. Yet, questions remain about whether this extends beyond the single species and how this transition took place. The Oxymonadida is a group of gut endobionts taxonomically housed in the Preaxostyla which also contains free-living flagellates of the genera Trimastix and Paratrimastix. The latter two taxa harbour conspicuous mitochondrion-related organelles (MROs). Here we report high-quality genome and transcriptome assemblies of two Preaxostyla representatives, the free-living Paratrimastix pyriformis and the oxymonad Blattamonas nauphoetae. We performed thorough comparisons among all available genomic and transcriptomic data of Preaxostyla to further decipher the evolutionary changes towards amitochondriality, endobiosis, and unstacked Golgi. Our results provide insights into the metabolic and endomembrane evolution, but most strikingly the data confirm the complete loss of mitochondria for all three oxymonad species investigated (M. exilis, B. nauphoetae, and Streblomastix strix), suggesting the amitochondriate status is common to a large part if not the whole group of Oxymonadida. This observation moves this unique loss to 100 MYA when oxymonad lineage diversified.

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

Pelomyxa is a genus of anaerobic amoebae that live in consortia with multiple prokaryotic endosymbionts. Although the symbionts represent a large fraction of the cellular biomass, their metabolic roles have not been investigated. Using single-cell genomics and transcriptomics, we have characterized the prokaryotic community associated with P. schiedti, which is composed of two bacteria, Candidatus Syntrophus pelomyxae (class Deltaproteobacteria) and Candidatus Vesiculincola pelomyxae (class Clostridia), and a methanogen, Candidatus Methanoregula pelomyxae. Fluorescence in situ hybridization and electron microscopy showed that Ca. Vesiculincola pelomyxae is localized inside vesicles, whereas the other endosymbionts occur freely in the cytosol, with Ca. Methanoregula pelomyxae enriched around the nucleus. Genome and transcriptome-based reconstructions of the metabolism suggests that the cellulolytic activity of P. schiedti produces simple sugars that fuel its own metabolism and the metabolism of a Ca. Vesiculincola pelomyxae, while Ca. Syntrophus pelomyxae energy metabolism relies on degradation of butyrate and isovalerate from the environment. Both species of bacteria and the ameba use hydrogenases to transfer the electrons from reduced equivalents to hydrogen, a process that requires a low hydrogen partial pressure. This is achieved by the third endosymbiont, Ca. Methanoregula pelomyxae, which consumes H2 and formate for methanogenesis. While the bacterial symbionts can be successfully eliminated by vancomycin treatment without affecting the viability of the amoebae, treatment with 2-bromoethanesulfonate, a specific inhibitor of methanogenesis, killed the amoebae, indicating the essentiality of the methanogenesis for this consortium.

Reduced mitochondria provide an essential function for the cytosolic methionine cycle.

The loss of mitochondria in oxymonad protists has been associated with the redirection of the essential Fe-S cluster assembly to the cytosol. Yet as our knowledge of diverse free-living protists broadens, the list of functions of their mitochondrial-related organelles (MROs) expands. We revealed another such function in the closest oxymonad relative, Paratrimastix pyriformis, after we solved the proteome of its MRO with high accuracy, using localization of organelle proteins by isotope tagging (LOPIT). The newly assigned enzymes connect to the glycine cleavage system (GCS) and produce folate derivatives with one-carbon units and formate. These are likely to be used by the cytosolic methionine cycle involved in S-adenosyl methionine recycling. The data provide consistency with the presence of the GCS in MROs of free-living species and its absence in most endobionts, which typically lose the methionine cycle and, in the case of oxymonads, the mitochondria.

A new lineage of non-photosynthetic green algae with extreme organellar genomes.

BACKGROUND: The plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them. RESULTS: We discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes. CONCLUSIONS: With its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.

Revealing the metabolic capacity of Streblomastix strix and its bacterial symbionts using single-cell metagenomics.

Lower termites harbor in their hindgut complex microbial communities that are involved in the digestion of cellulose. Among these are protists, which are usually associated with specific bacterial symbionts found on their surface or inside their cells. While these form the foundations of a classic system in symbiosis research, we still know little about the functional basis for most of these relationships. Here, we describe the complex functional relationship between one protist, the oxymonad Streblomastix strix, and its ectosymbiotic bacterial community using single-cell genomics. We generated partial assemblies of the host S. strix genome and Candidatus Ordinivivax streblomastigis, as well as a complex metagenome assembly of at least 8 other Bacteroidetes bacteria confirmed by ribosomal (r)RNA fluorescence in situ hybridization (FISH) to be associated with S. strix. Our data suggest that S. strix is probably not involved in the cellulose digestion, but the bacterial community on its surface secretes a complex array of glycosyl hydrolases, providing them with the ability to degrade cellulose to monomers and fueling the metabolism of S. strix In addition, some of the bacteria can fix nitrogen and can theoretically provide S. strix with essential amino acids and cofactors, which the protist cannot synthesize. On the contrary, most of the bacterial symbionts lack the essential glycolytic enzyme enolase, which may be overcome by the exchange of intermediates with S. strix This study demonstrates the value of the combined single-cell (meta)genomic and FISH approach for studies of complicated symbiotic systems.

The Oxymonad Genome Displays Canonical Eukaryotic Complexity in the Absence of a Mitochondrion.

The discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less "reduced" than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe-S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.

Fe-S Cluster Assembly in Oxymonads and Related Protists.

The oxymonad Monocercomonoides exilis was recently reported to be the first eukaryote that has completely lost the mitochondrial compartment. It was proposed that an important prerequisite for such a radical evolutionary step was the acquisition of the SUF Fe-S cluster assembly pathway from prokaryotes, making the mitochondrial ISC pathway dispensable. We have investigated genomic and transcriptomic data from six oxymonad species and their relatives, composing the group Preaxostyla (Metamonada, Excavata), for the presence and absence of enzymes involved in Fe-S cluster biosynthesis. None possesses enzymes of mitochondrial ISC pathway and all apparently possess the SUF pathway, composed of SufB, C, D, S, and U proteins, altogether suggesting that the transition from ISC to SUF preceded their last common ancestor. Interestingly, we observed that SufDSU were fused in all three oxymonad genomes, and in the genome of Paratrimastix pyriformis. The donor of the SUF genes is not clear from phylogenetic analyses, but the enzyme composition of the pathway and the presence of SufDSU fusion suggests Firmicutes, Thermotogae, Spirochaetes, Proteobacteria, or Chloroflexi as donors. The inventory of the downstream CIA pathway enzymes is consistent with that of closely related species that retain ISC, indicating that the switch from ISC to SUF did not markedly affect the downstream process of maturation of cytosolic and nuclear Fe-S proteins.

Molecular and Morphological Diversity of the Oxymonad Genera Monocercomonoides and Blattamonas gen. nov.

Oxymonads are a group of flagellates living as gut symbionts of insects or vertebrates. They have several unique features, one of them being the absence of mitochondria. Diversity of this group is seriously understudied, which is particularly true for small species from the family Polymastigidae. We isolated 34 strains of oxymonads with Polymastigidae-like morphology from 24 host species and unused cesspits and sequenced the SSU rRNA gene. Our strains formed two clades in the phylogenetic tree with Streblomastix strix branching between them. This topology was also supported by a three-gene phylogenetic analysis. Despite considerable genetic differences between the clades, light and electron microscopy revealed only subtle differences. The larger clade is considered genus Monocercomonoides and the isolates belonging here were classified into three new species (including the first potentially free-living species), two previously described species, and three unclassified lineages. The smaller clade, here described as Blattamonas gen. nov., consists of three newly described species. Concomitantly with the description of Blattamonas, we elevate the Monocercomonoides subgenus Brachymonas to the genus level. Our study shows that, despite their conserved morphology, the molecular diversity of Polymastigidae-like oxymonads is broad and represents a substantial part of the diversity of oxymonads.

A Eukaryote without a Mitochondrial Organelle.

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.