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Monocercomonoides
Monocercomonoides melolanthae
Scientific classification
Domain:
Eukaryota
(unranked):
Excavata
Phylum:
Metamonada
Class:
Preaxostyla
Order:
Oxymonadida
Family:
Polymastigidae
Genus:
Monocercomonoides

Travis, 1932
Species

Monocercomonoides is a genus of flagellate Excavata belonging to the order Oxymonadida. It was established by Bernard V. Travis and was first described as those with "polymastiginid flagellates having three anterior flagella and a trailing one originating at a single basal granule located in front of the anteriorly positioned nucleus, and a more or less well-defined axostyle".[14] It is the first eukaryotic genus to be found to completely lack mitochondria, and all hallmark proteins responsible for mitochondrial function. The genus also lacks any other mitochondrion-related organelles such as hydrogenosomes or mitosomes.[15] Data suggests that the absence of mitochondria is not an ancestral feature, but rather due to secondary loss. Monocercomonoides sp. was found to obtain energy through an enzymatic action of nutrients absorbed from the environment.[15] The genus has replaced the Iron–sulfur cluster assembly pathway with a cytosolic sulfur mobilization system, likely acquired by horizontal gene transfer from a eubacterium to a common ancestor of oxymonads.[16] These organisms are significant because they undermine assumptions that eukaryotes must have mitochondria to function properly. The genome of Monocercomonoides exilis has approximately 82 million base pairs (82 Mbp), with 18 152 predicted protein-coding genes.[17]

Habitat and ecology

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Most Monocercomonoides species are obligate animal symbionts that live in the digestive tracts of insects, amphibians, reptiles, and mammals.[18] Monocercomonoides are common in insect orders Orthoptera and Coleoptera. The species Monocercomonoides qadrii are found in the rectum of the larva of the dung-beetle (Oryctes rhinoceros).[19] M. caviae, M. wenrichi, M. quadrifunilis, and M. exilis are found in the caecum of guinea pigs, and M. caprae has been found in the rumen of goats.[20] Interestingly, some Monocercomonoides species were isolated from cesspits,[21] suggesting that they might be able to survive outside of the host in certain environmental conditions. The organism uses enzymes found in its cytoplasm to break down food and furnish energy since there is no mitochondria or oxygen presence.[22] It has been noted that Monocercomonoides ingest bacteria or wood and feed by pinocytosis, however, limited studies have been done on feeding style.

Morphology

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Monocercomonoides are small free-swimming, single-cell organisms ranging from 5-12μm in length, and 4.5-14.5μm in width.[19] The body may be ovoidal, pyriform, spherical or subspherical; however, they lack holdfasts and have small axostyles.[23] The axostyle is a single, contractible appendage made of microtubules that originates from the posterior end of the preaxostyle, and is situated near the posterior pair of the basal bodies (known as blepharoplast in older cytological literature).[23] The cytoskeleton is based around four basal bodies, an anterior pair and a posterior pair.[24] The preaxostyle runs between the two pairs of basal bodies and is composed of a broad, curved sheet of microtubules.[24] The inner face of the microtubule sheet adheres to a paracrystalline fibre (about 50 nm thick) which is a common characteristic of all oxymonads.[24] Monocercomonoides sp. has four flagella that originate in two pairs and arise from each basal body found in the anterior end.[23] Three of the four flagella are roughly equal in length (9.5-18μm) and the fourth trailing flagellum is slightly longer, measuring between 9.0 and 24.5μm.[19] The flagella have a beating action and are used for rapid movement. The proximal part of the long flagellum may adhere to the pellicle, which causes it to trail posteriorly.[23] The trailing flagellum is always directed backwards and is attached to the body for a considerable distance (6-9μm) by an accessory filament called a funis.[19] There are one to four filaments (rib-like strictures) extending backwards beneath the body surface.[19] In some parasites, the flagella end in acronemes. The nucleus is generally situated near the anterior end of the body and contains a central endosome surrounded by chromatin granules, some species have pelta-like structures below the nucleus.[23] The cytoplasm is granular with or without vacuoles.[23] Electron microscopic imaging of Monocercomonoides has found that the intracellular morphology lacks any Golgi apparatus, mitochondria, or potential homologs of the two; Golgi-associated proteins have been detected, but mitochondrial ones have not.[15]

Metabolic processes

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Monocercomonoides sp. strain PA203 (later described as M. exilis[21]) is the first eukaryote discovered to lack any trace of mitochondria. In all other eukaryotes that seemingly lack mitochondria, there are genes in the nucleus which mitochondria require; no such genes are present in Monocercomonoides.[15] It also lacks any genes ordinarily found in mitochondrial DNA, and genes used to make the energy-extracting enzymes present in mitochondria.[specify] Monocercomonoides are able to get some energy from glucose using anaerobic metabolic pathways that operate in the cytoplasm;[clarification needed] however, most of its energy is obtained using enzymes that break down the amino acid arginine.[24]

Glycolytic pathway

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Each molecule of glucose catabolized in Monocercomonoides yields less ATP compared to mitochondrial eukaryotes that use the tricarboxylic acid cycle and electron transport chain.[25] To aid in energy conservation, Monocercomonoides has adapted alternative glycolytic enzymes. Four alternative glycolytic enzymes include pyrophosphate-fructose-6-phosphate phosphotransferase (PFP), fructose-bisphosphate aldolase class II (FBA class II), 2,3-bisphosphoglycerate independent phosphoglycerate mutase (iPGM), and pyruvate phosphate dikinase (PPDK).[25] Glucose-6-phosphate isomerase (GPI) is predicted to be in Monocercomonoides  since it is universally distributed among Eukaryotes, Bacteria, and some Archaea and essential in catabolic glycolysis, but has not yet been found.[25] Most of the glycolytic enzymes are the standard eukaryotic versions, making Monocercomonoides' metabolic pathway a mosaic similar to that of other anaerobes.[25]

The addition of PPDK to the conversion of phosphoenolpyruvate to pyruvate (typically catalyzed solely by pyruvate kinase) has a strong effect on ATP conservation.[25] Both PFP and PPDK rely on inorganic phosphate (PPi) as the phosphate donor;  therefore rather than hydrolyzing ATP, the ATP yield is increased by using a by-product of the cell's anabolic processes as an energy source.[25] These reactions are able to allow for greater ATP conservation and regulation of glycolysis due to the PPDK's reversible nature and use of inorganic phosphate where pyruvate kinase only catalyzes the forward reaction.[25]

Arginine deiminase pathway

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In addition to the adjusted glycolysis, Monocercomonoides contain enzymes needed in the arginine deiminase (degradation) pathway.[15] The arginine deiminase pathway may be used for ATP production, as in Giardia intestinalis and Trichomonas vaginalis.[15] In G. intestinalis (an anaerobic unicellular eukaryote) this pathway produces eight times more ATP than sugar metabolism, and a similar output is expected in Monocercomonoides, but has yet to be confirmed.[15] All 3 enzymes of the arginine deiminase pathway are localized in the cytosol of Monocercomonoides exilis which may reflect an ancestral state in Metamonada.[26]

Iron-sulfur cluster

[edit]

Iron-sulfur clusters are important protein components that are synthesized by mitochondria.[16] The main function of these small inorganic prosthetic groups is mediating electron transport, which makes them a key part of photosynthesis, respiration, DNA replication/repair, and regulation of gene expression.[16] In eukaryotic cells, the common pathway for Fe-S cluster synthesis is ISC (iron-sulfur cluster). In the cytosol, a cytosolic iron-sulfur cluster assembly (CIA) forms Fe-S cluster-containing proteins that are responsible for the maturation of nuclear Fe-S proteins. CIA is unique to eukaryotes and does not have prokaryotic homologs.[16] The mitochondrial ISC pathway is believed to be necessary for the function of CIA since it synthesizes and transports uncharacterized sulfur-containing precursor to the cytosol, and is a major reason for retention of mitochondrial-related organelles in anaerobic eukaryotes.[16] The genus Monocercomonoides contains the CIA pathways but completely lacks the ISC pathway, along with any mitochondrial proteins.[16] The genus contains a reduced version of the SUF (sulfur utilization factor) pathway, having only three proteins - SufB, SufC, and SufU.[16] The SUF pathway is a known pathway of prokaryotes, and it is believed that the genes used to build Monocercomonoides' SUF system had to have come from prokaryotes.[16] However, Monocercomonoides' SUF proteins were found to not be related to plastid homologues, or any other microbial eukaryotes.[16] It was proposed that the pathway was acquired from a eubacterium by horizontal gene transfer (HGT) in the common ancestor of Monocercomonoides and Paratrismastrix (a sister taxon of oxymonads).[16] The genetic acquisition has not been demonstrated despite the assumption that it must have occurred.

Mitochondrial acquisition and loss

[edit]

Monocercomonoides contain no detectable sign that mitochondria were ever part of the organism.[15] However, since it is widely accepted that all eukaryotes have a common ancestor that evolved mitochondria, it is believed that mitochondria must have once been present in the ancestors to oxymonads and then secondarily lost. The amitchondrial genus demonstrates that mitochondria are not absolutely essential for life of a eukaryotic cell.

Genomic structure

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The lack of mitochondria or any mitochondria-related organelles in Monocercomonoides exilis is confirmed by its genome sequence. A complete genome sequence analysis of Monocercomonoides exilis strain PA203 from Chinchilla lanigera was conducted.[15] The estimated size of the genome is ~75Mb and the number of predicted protein-coding genes is 16,629.[15] A more recent re-sequencing of the genome using Oxford nanopore showed that the genome is ~82 Mbp in size.[17] Homology searches reveal a lack of genes that encode mitochondrial import machinery, metabolite transport proteins, and iron-sulfur clusters.[15][17] Additionally, an absence of targeted important genes and genes coding for mitochondrial membrane proteins were revealed when a search for specific N-terminal and C-terminal sequences was conducted.[15][17] Genes that are typically encoded on mitochondrial genomes (mtDNA) were not found among the assembled scaffold, suggesting Monocercomonoides lacks mtDNA.[15] 18S RNA genes were sequenced and found to be 2,927 nt long, and is among the longest known.[15][21] Some expansions were specific to Monocercomonoides, but many were similar to those in other oxymonad genera but substantially longer.[15] Comparisons of genes coding for 𝛼-tubulin, 𝛽-tubulin, 𝛾-tubulin, EF-1𝛼, EF-2, cytHSP70, ubiquitin, 18S rRNA, and HSP90 allow the placement of oxymonads near diplomonads and trichomonads, with Monocercomonoides and Streblomastix making up the oxymonad branch.[15]

References

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Monocercomonoides

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Monocercomonoides is a of anaerobic, unicellular protists in the order Oxymonadida, part of the supergroup , primarily inhabiting the guts of and vertebrates as symbionts. These microorganisms are characterized by their small size, typically 5–12 μm in length, and possession of four flagella, with a distinctive preaxostyle structure aiding in attachment and locomotion. Over 40 have been described in the genus based on morphological and molecular criteria, though recent phylogenetic analyses have refined its diversity into distinct clades. The genus gained significant attention in 2016 with the genomic characterization of Monocercomonoides sp. PA203, revealing it as the first known to completely lack mitochondria or any mitochondrion-related organelles (MROs). This secondary loss of mitochondrial functions is compensated by an expanded cytosolic glycolytic pathway for ATP production and a bacterial-type , acquired via lateral transfer, for iron-sulfur cluster assembly—replacing the canonical mitochondrial ISC machinery. Recent studies (as of 2024) have further characterized the SUF machinery, confirming its role in facilitating complete mitochondrial loss. Subsequent studies on species like M. exilis have confirmed haploid genome sizes ranging from 60 to 161 Mbp across strains, underscoring the genus's genomic compactness and uniformity in . Monocercomonoides species contribute to host gut as bacterivorous symbionts in and guts, aiding cycling in these low-oxygen environments. Their discovery challenges long-held assumptions about eukaryotic cellular architecture, demonstrating that mitochondria are not indispensable for eukaryotic life, and highlights the evolutionary plasticity within the Metamonada .

Taxonomy and Classification

Phylogenetic Position

Monocercomonoides belongs to the supergroup , phylum Metamonada, class Preaxostyla, and order Oxymonadida, within the family Polymastigidae. This placement positions it among other anaerobic, amitochondriate protists adapted to low-oxygen environments, such as and guts. The is characterized by its endobiotic lifestyle and morphological simplicity, including a reduced flagellar apparatus with four flagella emerging from a preaxostyle, distinguishing it from more complex excavates. Phylogenetic analyses using small subunit ribosomal RNA () and multiple protein-coding genes consistently place Monocercomonoides within Metamonada, closely related to diplomonads like and parabasalids like . Early multi-gene studies, incorporating eight proteins and , supported a Metamonada clade and weak Excavata monophyly, rejecting the primitive amitochondriate hypothesis. More recent phylogenomic datasets, including hundreds of conserved eukaryotic genes, confirm Monocercomonoides as part of a derived preaxostylid lineage, branching basal to free-living trimastigids like Trimastix. Recent phylogenomic studies (Stairs et al., 2023) have further supported the derived position within Preaxostyla and identified additional amitochondriate lineages. These relationships highlight convergent adaptations to anaerobiosis among metamonads, with shared traits like hydrogenosomes or mitosomes in relatives, though Monocercomonoides has completely lost such organelles. Historically, oxymonads including Monocercomonoides were grouped with trichomonads and diplomonads under informal amitochondriate assemblages in the late , based on shared ultrastructural features like the axostyle and lack of mitochondria. Molecular data from the 1990s and 2000s reclassified them firmly within , with key 2010s studies using genome-scale phylogenomics affirming their preaxostylid position and secondary mitochondrial loss around 100 million years ago. Unique traits supporting this placement include the anaerobic reliant on cytosolic pathways and simplified flagellar insertion, which align with evolutionary trends toward endobiotic simplification.

Species Diversity

The genus Monocercomonoides encompasses over 40 described , primarily distinguished through observations of gut-inhabiting flagellates in various hosts, though many classifications remain tentative due to limited molecular data. The model for the genus, particularly through the sequenced strain PA203, is Monocercomonoides exilis (described in 1911 from intestinal tracts), which has facilitated genomic studies revealing its unique amitochondriate nature. This typically exhibits a pear-shaped body 5–10 μm in length with four anterior flagella of unequal lengths, serving as a benchmark for morphological comparisons across the genus. The is M. melolonthae (Leidy, 1849). Other notable species include M. blattae from cockroaches and M. caviae from guinea pigs, alongside variants from reptilian and invertebrate hosts such as insects. Species distinctions often rely on subtle morphological traits, such as variations in overall cell dimensions, combined with genetic markers like SSU rRNA gene sequences that reveal interspecies divergences of up to 10–15%. For instance, recent molecular analyses have delineated three new Monocercomonoides species and multiple lineages within the genus, highlighting greater diversity than morphological surveys alone suggest. Taxonomic challenges persist due to cryptic diversity, where morphologically similar isolates harbor significant genetic differences, necessitating molecular barcoding for accurate identification. Current estimates indicate approximately 10–15 putative species based on emerging phylogenetic data, though historical descriptions may include synonyms, underscoring the need for integrated morphological and genomic approaches to resolve the genus's full diversity.

Habitat and Ecology

Distribution and Hosts

Monocercomonoides species inhabit the guts of insects and vertebrates. In insects, they are found in species such as dung beetles (Oryctes rhinoceros) and cockroaches, while in vertebrates, they primarily occur in rodents such as chinchillas (Chinchilla laniger) and other caviomorphs, as well as lagomorphs like rabbits. They are also reported from reptiles, including lizards and snakes. These protists exhibit an endoparasitic or commensal lifestyle within the anaerobic environment of the host's digestive tract, with no free-living forms known. The genus has a cosmopolitan distribution, with isolates documented from Europe (e.g., for M. exilis) and (e.g., for species in pocket gophers and other ). Transmission occurs via the fecal-oral route in host populations, facilitated by the passage of trophozoites in feces. varies, but surveys of rodent colonies indicate detection rates around 0.04% for Monocercomonoides sp. via wet mount examination, though higher incidences may occur in wild or specific captive settings. Distribution is influenced by host-specific factors, including the anaerobic conditions of the and diets rich in material, which are prevalent in herbivorous vertebrates like chinchillas and rabbits, as well as in detritivorous . These environmental niches support the protists' anaerobic metabolism and symbiotic associations within the gut .

Ecological Interactions

Monocercomonoides species primarily engage in commensal interactions within the anaerobic guts of herbivorous vertebrates and detritivorous , such as chinchillas and , where they inhabit the digestive tract without causing apparent harm to the host. These flagellates contribute indirectly to host by preying on prokaryotic members of the , thereby facilitating nutrient through bacterivory. This predatory behavior helps maintain microbial balance in the oxygen-depleted environment, potentially supporting the overall efficiency of fiber degradation processes mediated by . Their flagellar enables effective navigation and positioning within the viscous gut lumen to access prey. In polymicrobial gut communities, Monocercomonoides co-occurs with diverse bacterial taxa, forming interdependent consortia that mimic natural herbivore microbiomes. Metagenomic analyses of Monocercomonoides exilis cultures, derived from guts, reveal associations with (e.g., , Bacteroides thetaiotaomicron, Parabacteroides sp.) and Fusobacterium varium, which dominate the community and perform fermentation of organic substrates. Although methanogens are not prominently featured in these axenic culture proxies, broader gut surveys indicate potential syntrophic links with hydrogenotrophic in similar anaerobic niches, enhancing community stability through metabolic exchanges like scavenging. These partnerships underscore Monocercomonoides' role as a bacterivorous regulator rather than a direct mutualist. Monocercomonoides exhibits no documented pathogenicity toward hosts, positioning it as a benign symbiont that may bolster host via contributions to cycling. By phagocytosing , it recycles essential elements such as carbon, , and iron, supporting an incomplete and a complete within the . This activity indirectly aids host in fiber-rich diets by preventing bacterial overgrowth and promoting turnover of byproducts. In response to environmental stressors, such as limitation, Monocercomonoides demonstrates resilience through its embedded position in polymicrobial networks, where bacterial partners provide critical metabolites like polyamines, enabling survival in fluctuating gut conditions; however, direct perturbation studies remain limited.

Morphology and Ultrastructure

General Cell Features

Monocercomonoides cells are small flagellates, typically ranging from 5 to 15 μm in length, with a body shape that varies from ovoid to pear-shaped (pyriform). These dimensions and forms are observed across , contributing to their compact architecture suited for navigation in host gut environments. The cells are uninucleate, featuring a single spherical to ovoid nucleus positioned anteriorly, which often appears centrally located under light microscopy and contains a conspicuous . The cytoplasm is vacuolated, containing conspicuous and often hosting prokaryotic endosymbiotic , while housing storage granules that accumulate preferentially in association with the axostyle, supporting energy reserves in anaerobic conditions. Detailed genomic and microscopic studies have confirmed the complete absence of mitochondria or any mitochondrion-related organelles (MROs), with no remnants detected. Under light microscopy, Monocercomonoides cells appear colorless and non-pigmented, displaying a granular texture due to cytoplasmic inclusions and exhibiting moderate zig-zag . Although primarily free-swimming, certain possess a short ventral funis or striated that enables temporary attachment to gut surfaces. Cells bear four flagella emerging from anterior basal bodies, facilitating propulsion in viscous habitats.

Flagellar and Cytoskeletal Elements

Monocercomonoides features four anterior flagella arranged in two pairs emerging from basal bodies, consisting of two free flagella and two recurrent ones, with the recurrent flagella folding back along the cell surface to enable . The recurrent flagella are housed within a ventral channel supported by associated fibres, allowing the cell to move efficiently across surfaces in the host gut. Electron microscopy has revealed the basal body organization as two pairs in a V-shaped configuration, with posterior basal bodies (1 and 2) giving rise to the recurrent flagella and anterior ones (3 and 4) to the free flagella. Transition zones at the basal bodies exhibit a standard 9+2 axonemal structure with dynein arms, facilitating the undulating beat required for propulsion, while lacking any posterior flagella. The cytoskeleton includes a distinctive preaxostyle, a broad curved sheet of microtubules (homologous to root 2) originating between the basal body pairs and unique to preaxostylids, which provides ventral support and transitions into the axostyle—a slender rod of parallel microtubules extending longitudinally through the cell and protruding posteriorly for added stability. Additional elements, such as the pelta (a microtubular array associated with the anterior root) and striated fibres like the H fibre, reinforce the flagellar apparatus and channel walls. These flagellar and cytoskeletal components underpin toward gut nutrients and adherence to the host , primarily through the action of recurrent flagella and the supportive rigidity of the axostyle, adapting the to its anaerobic, endosymbiotic niche.

Metabolism

Glycolytic Pathway

Monocercomonoides employs a eukaryotic glycolytic pathway, known as the Embden-Meyerhof-Parnas (EMP) pathway, to catabolize glucose to pyruvate in the , with all requisite enzymes encoded by nuclear genes. This process generates a net yield of 2 ATP molecules per glucose through at the steps catalyzed by and . Key enzymes include , which phosphorylates glucose using ATP, and aldolase, which cleaves fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and . Adapted to its anaerobic lifestyle, the pathway incorporates alternative enzymes that enhance efficiency in oxygen-deprived environments, such as pyrophosphate-dependent phosphotransferase (PFP) in place of , class II , and cofactor-independent (iPGM). Additionally, pyruvate phosphate dikinase (PPDK) operates alongside , utilizing (PPi) generated earlier in the pathway to produce phosphoenolpyruvate, thereby conserving energy by avoiding PPi hydrolysis and supporting higher glycolytic flux. These modifications enable Monocercomonoides to maintain robust ATP production in the low-oxygen gut habitat of its hosts. Under anaerobic conditions, pyruvate is further metabolized to for additional ATP generation without mitochondrial involvement. Pyruvate: (PFO) decarboxylates pyruvate to , reducing in the process, followed by (ACS), which converts to while producing ATP from AMP and PPi. This extension yields up to 2 additional ATP per glucose when both pyruvates are directed to . NADH produced during the glyceraldehyde-3-phosphate dehydrogenase step is reoxidized in the primarily through ferredoxin-linked mechanisms, including the reduction of by PFO and its subsequent reoxidation by [FeFe]-hydrogenase to evolve hydrogen gas, preventing NADH accumulation and sustaining . Absent mitochondria or remnant organelles, Monocercomonoides lacks and relies entirely on these cytosolic fermentative routes for balance and energy homeostasis. Enzyme localization is exclusively cytosolic, with regulation favoring elevated glycolytic rates in anoxic settings through reversible PPi-dependent steps that minimize energy loss. This primary energy pathway from carbohydrate catabolism is supplemented by the deiminase pathway.

Arginine Deiminase Pathway

The deiminase (ADI) pathway in Monocercomonoides serves as an alternative mechanism for ATP generation through the anaerobic breakdown of , compensating for the organism's limited glycolytic capacity in the absence of mitochondria. This pathway consists of three sequential enzymatic steps: first, deiminase (ADI, encoded by arcA) hydrolyzes to citrulline and ; second, catabolic ornithine transcarbamylase (OTC, encoded by arcB) phosphorolyses citrulline to ornithine and ; and third, carbamate (CK, encoded by arcC) transfers the phosphate from to ADP, yielding ATP and . Overall, this process nets one ATP molecule per consumed via . The enzymes of the ADI pathway are encoded by a clustered set of arc genes (arcA, arcB, and arcC), a genomic organization conserved across anaerobic eukaryotes and prokaryotes that utilize this metabolism. This regulation supports the organism's survival in low-oxygen, host-associated environments typical of its intestinal habitat. Byproducts of the pathway include ammonia, which contributes to intracellular pH regulation by buffering acidic conditions, and ornithine, which serves as a precursor for polyamine synthesis essential for cell growth and stability. The ADI pathway integrates with cytosolic glycolysis to meet overall energy demands.

Iron-Sulfur Cluster Assembly

Monocercomonoides species, exemplified by M. exilis, assemble iron-sulfur (Fe-S) clusters exclusively in the , compensating for the complete absence of a mitochondrial ISC system. This amitochondriate relies on a bacterial-derived SUF pathway for de novo Fe-S cluster synthesis, acquired through lateral gene transfer, which provides scaffolds and sulfur mobilization machinery. Complementing this, a minimal cytosolic iron-sulfur assembly (CIA) pathway handles cluster maturation and targeting to apo-proteins, ensuring functionality of essential Fe-S enzymes without any organellar involvement. The SUF system in M. exilis features core components including SufB and SufC, which form a dynamic scaffold complex capable of binding Fe-S clusters in an ATP-dependent manner, as well as a fused SufDSU protein (comprising SufD, SufS, and SufU domains) that mobilizes via a pyridoxal 5'-phosphate (PLP) cofactor. This machinery assembles [2Fe-2S] and [4Fe-4S] clusters, with SufBC forming oligomers ranging from dimers to octamers, structurally analogous to bacterial SufBC₂D complexes from lineages such as Firmicutes and Proteobacteria. The SUF pathway's bacterial-like scaffolds trace back to horizontal transfer from prokaryotic donors, likely adapting an ancestral endosymbiotic contribution to eukaryotic Fe-S biogenesis in the absence of mitochondria. Experimental validation includes reconstitution showing 1.82 iron and 2.32 atoms per mole of MeSufBC under ATP conditions, alongside complementation in where MeSufBC reduced iscR-lacZ expression by 2.5-fold, demonstrating functional cluster maturation. The CIA pathway in Monocercomonoides is streamlined, encoding Nbp35 (a P-loop NTPase scaffold), Cia1, Nar1 (an iron-only hydrogenase-like protein), and Cia2 (homologous to Cfd1, forming the early maturation scaffold with Nbp35), but lacking Dre2, Tah18, and MMS19—features common in anaerobic protists. This minimal CIA receives nascent clusters from the SUF system, facilitating their transfer and insertion into cytosolic and nuclear Fe-S proteins, such as those involved in radical SAM reactions and DNA metabolism. Proteomic and transcriptomic analyses across Preaxostyla species, including M. exilis, confirm the expression and conservation of these CIA components, with no evidence of mitochondrial remnants, underscoring the pathway's standalone cytosolic operation. Functional Fe-S enzymes, verified through genomic inventories and heterologous expression, operate effectively, supporting core metabolism without organelle dependency.

Organelle Evolution

Mitochondrial Acquisition

The acquisition of mitochondria by the ancestors of Monocercomonoides traces back to the endosymbiotic event in the last eukaryotic common ancestor (LECA), where an alphaproteobacterium was engulfed by an archaeal host approximately 1.5 to 2 billion years ago. This foundational endosymbiosis, as proposed by the endosymbiotic theory, integrated the bacterial genome into the eukaryotic lineage through massive gene transfer to the host nucleus, enabling the evolution of complex cellular functions across all eukaryotes, including metamonads. Phylogenomic evidence for this ancient transfer is evident in the nuclear genomes of , which retain genes of alphaproteobacterial origin now functioning outside any mitochondrial compartment. For instance, chaperones such as the mitochondrial-type 70-kDa () and chaperonin 60 (cpn60) are present in the nucleus of related like , indicating their transfer from the and adaptation for cytosolic roles. Similarly, mitochondrial carrier proteins (MCPs) and other transporters, identified through comparative analyses, populate nuclear genomes, supporting the shared ancestry of mitochondrial functions across eukaryotic lineages. In the preaxostylid ancestors of Monocercomonoides—a subgroup within Metamonada—specific post-endosymbiotic acquisitions further shaped organelle evolution, such as the lateral gene transfer of a bacterial SUF system for iron-sulfur cluster assembly, serving as a precursor to hydrogenosome-like organelles in related lineages. This system, retained in the nuclear genome, replaced mitochondrial pathways and facilitated adaptations in anaerobic environments. Comparative phylogenomics of Monocercomonoides reveals that numerous mitochondrial-derived genes persist in its nucleus, repurposed for cytosolic functions like and metabolite transport, underscoring the deep integration of endosymbiotic contributions despite the organism's amitochondriate state. These genes cluster phylogenetically with alphaproteobacterial homologs, confirming their origin from the LECA endosymbiosis rather than independent acquisitions.

Mitochondrial Loss and Remnants

In 2016, genomic and transcriptomic analyses of Monocercomonoides exilis demonstrated the complete absence of mitochondria, mitosomes, or hydrogenosomes, identifying it as the first known lacking any mitochondrial remnant or related . Subsequent genomic analyses of additional oxymonad species, including Blattamonas nauphoetae and Streblomastix strix, have confirmed this complete absence across the order Oxymonadida. This loss is reflected in extensive gene loss patterns, with the absence of approximately 50 proteins typically targeted to mitochondria, such as those involved in protein import (TOM/TIM complexes) and metabolite transport, while around 20 genes encoding formerly mitochondrial proteins are retained but repurposed for cytosolic functions. The evolutionary timeline places this mitochondrial loss after the Last Eukaryotic Common Ancestor (LECA), likely occurring in the stem lineage of Oxymonadida at least 100 million years ago, coinciding with the diversification of the oxymonad lineage and enabling adaptation to oxygen-poor environments without mitochondrial contributions to energy metabolism or iron-sulfur cluster assembly. Functional replacements have arisen through the relocation of essential processes to the , including the acquisition of a bacterial SUF system for iron-sulfur cluster assembly via lateral transfer, which supplanted the lost mitochondrial ISC pathway (as detailed in the Iron-Sulfur Cluster Assembly section). Additionally, localization studies confirm that certain retained proteins, originally destined for mitochondria, possess N-terminal targeting signals that direct them to the in the absence of an ; for instance, experiments showed these proteins can still be imported into hydrogenosome-like organelles of related protists, underscoring their evolutionary repurposing. This complete elimination highlights the dispensability of mitochondria under specific ecological pressures, reshaping understandings of eukaryotic evolution.

Genomics

Genome Organization

The nuclear genome of Monocercomonoides exilis was initially sequenced in 2019 using a combination of Illumina short reads and PacBio long reads, yielding an assembly of approximately 75 Mbp across 2,092 scaffolds. An improved high-quality assembly published in 2021, incorporating (ONT) long reads, expanded the size to 82.3 Mbp and reduced fragmentation to 101 contigs. This assembly includes 10 full-length sequences ranging from 0.86 to 2.54 Mbp, along with 65 contigs featuring telomeric repeats (TTAGGG) at one end, indicating a likely total of 40–50 linear in the haploid . The genome exhibits a high AT content of approximately 63% ( 37%), which is elevated compared to many free-living eukaryotes but typical for certain parasitic and endobiotic protists. Its organization is compact, with 18,152 predicted protein-coding s spanning about 41% of the assembly; these genes are frequently interrupted by introns, averaging 1.95 per gene (range 0–>10) and a of 119 , resulting in roughly 85% of genes being interrupted. Exons average 278 in , and intergenic regions are short, contributing to the streamlined architecture despite the presence of 35,345 introns overall. As an amitochondriate , M. exilis lacks any mitochondrial or genomes, with all genetic material confined to the nucleus; this includes genes typically associated with functions, which have been relocated or lost during . Repetitive elements comprise a substantial portion of the in the improved assembly (46%, or 37.8 Mbp), dominated by unclassified repeats (36%), alongside DNA transposons (4.5%), simple repeats (3.3%), and LTR retrotransposons (1.7%); however, tandemly arrayed units (approximately 50 copies of 18S-5.8S-28S rRNA genes) form distinct clusters without extensive proliferation of other repeats. Telomeric repeats are conserved as vertebrate-like TTAGGG motifs, anchoring ends.

Key Gene Features

The genome of Monocercomonoides exilis encodes 18,152 protein-coding genes, a number comparable to many other unicellular eukaryotes despite the organism's highly derived metabolic and organellar features. This gene repertoire includes notable adaptations for anaerobic lifestyle, such as multiple alternative enzymes in the pathway. For instance, M. exilis possesses both ATP-dependent and pyrophosphate-dependent phosphofructokinases (PFKs), enabling energy-efficient ATP conservation during under low-oxygen conditions, a feature likely acquired through from . Additionally, the genome contains genes for the deiminase (ADI) pathway, including deiminase, catabolic carbamoyltransferase, and carbamate kinase, organized in a bacterial-like cluster resembling the arc , which supports ATP production from . Canonical mitochondrial genes are entirely absent in M. exilis, including hallmark respiratory chain components such as subunit 1 (cox1) and subunit 9 (atp9), consistent with the complete loss of the . This absence extends to all mitochondrial-targeted proteins, with no evidence of remnants or import machinery. Furthermore, recent analyses of genomes, including M. exilis, reveal an incomplete and segregation apparatus, lacking key components like most (ORC) subunits (except Orc5) and the Ndc80 complex, alongside reduced checkpoint kinases (retaining only Chk1 and Chk2); these deficiencies suggest reliance on unconventional, possibly origin-independent replication mechanisms. The cytoskeletal gene content supports M. exilis's flagellar , with expansions in families—including at least one copy each of α-, β-, γ-, δ-, and ε-tubulins—facilitating the assembly of multiple flagella characteristic of oxymonads. genes are present but limited, reflecting a streamlined set adapted for anaerobic flagellar function without mitochondrial support. Bacterial-like are retained in the , such as the SUF operon for iron-sulfur cluster assembly, which replaced the lost mitochondrial ISC system. Horizontal gene transfers from have contributed to anaerobiosis adaptations, exemplified by genes that enable in hydrogenosome-like processes and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) for glycolytic flux.

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