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![Unrooted ML tree of nonperiplasmic-like [FeFe]-hydrogenase sequences. Phylogenetic analyses were performed on 238 sequences and 346 sites, using RAxML and IQ-TREE. Bootstrap and ultrafast bootstrap values are indicated as in figure 4. Eukaryotes are shaded blue, and alphaproteobacteria magenta. [FeFe]-hydrogenases with C-terminal CysJ or flavodoxin (FLD) domains are indicated.](publication/303867345/figure/fig3/AS:560792336846848@1510714720917/Unrooted-ML-tree-of-nonperiplasmic-like-FeFe-hydrogenase-sequences-Phylogenetic.png)
Unrooted ML tree of nonperiplasmic-like [FeFe]-hydrogenase sequences. Phylogenetic analyses were performed on 238 sequences and 346 sites, using RAxML and IQ-TREE. Bootstrap and ultrafast bootstrap values are indicated as in figure 4. Eukaryotes are shaded blue, and alphaproteobacteria magenta. [FeFe]-hydrogenases with C-terminal CysJ or flavodoxin (FLD) domains are indicated.
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![FIG. 3. Immunogold localization of IscS and [FeFe]-hydrogenase in S....](publication/303867345/figure/fig1/AS:560792338087936@1510714720708/Immunogold-localization-of-IscS-and-FeFe-hydrogenase-in-S-incarcerata-cells-A-A_Q320.jpg)
![FIG. 5. Unrooted ML tree of nonperiplasmic-like [FeFe]-hydrogenase...](publication/303867345/figure/fig3/AS:560792336846848@1510714720917/Unrooted-ML-tree-of-nonperiplasmic-like-FeFe-hydrogenase-sequences-Phylogenetic_Q320.jpg)
Mitochondrion-related organelles (MROs) have arisen independently in a wide range of anaerobic protist lineages. Only a few
of these organelles and their functions have been investigated in detail, and most of what is known about MROs comes from
studies of parasitic organisms such as the parabasalid Trichomonas vaginalis. Here, we describe the MRO...
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Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric act...
Citations
... In addition to the "simple" hydrogenases, which are present in all species of Preaxostyla, [FeFe] hydrogenases with N-terminal homology to the NuoG subunit of NADH-quinone oxidoreductase and a C-terminal homology to NADPH-dependent sulfite reductase (CysJ), were identified in the MRO-containing T. marina and P. pyriformis (Figs 3 and D in S4 File). Similar "fused" hydrogenases have been previously reported in other eukaryotic anaerobes, including T. vaginalis [49], the breviate Pygsuia biforma [50], the jakobid Stygiella incarcerata [51], and the amoebozoan Pelomyxa schiedti [52]. Although they do not belong to the group of A3 trimeric hydrogenases [53] known to be capable of NADH oxidation via electron confurcation [54], they were hypothesized to catalyze NAD(P)H-dependent formation of H 2 [49]. ...
... The general design of the search followed the previously described methodology [7]. Briefly, a custom mitochondrial protein sequence database was compiled using the MitoMiner v4.0 database [22,23] as the core and supplemented with MROs proteins from sixteen different organisms [5,50,51,63,[111][112][113][114][115][116]. Redundant homologues (90% similarity threshold) were removed from the database using CD-HIT. ...
... HMM searches using HMMER 3.1b2 and profile HMMs previously employed by Karnkowska et al. [7] were performed to specifically identify proteins involved in mitochondrial protein import and translocation, as these were shown to be often divergent [51]. Mitochondrial targeting signals were detected using TargetP v1.1 [117] and MitoFates v1.1 [118]. ...
Author summary
Mitochondria are nearly ubiquitous components of eukaryotic cells that constitute bodies of animals, fungi, plants, algae, and a broad diversity of single-celled eukaryotes, a.k.a. protists. Many groups of protists have substantially reduced the complexity of their mitochondria because they live in oxygen-poor environments, so they are unable to utilize the most salient feature of mitochondria–their ATP-producing oxidative phosphorylation metabolism. However, for a long time, scientists thought that it is impossible to completely lose a mitochondrion because this organelle provides other essential services to the cell, e.g. synthesis of protein cofactors called iron-sulfur clusters. Detailed investigation of the chinchilla symbiont M. exilis documented the first case of an organism without mitochondrion, and it also provided a scenario explaining how this unique evolutionary experiment might have happened. In this work, we expand on this discovery by exploring genomes of multiple relatives of M. exilis. We show that the loss of the mitochondrion is not limited to a single species but possibly extends to its entire group, the oxymonads. We also compare the predicted metabolic capabilities of oxymonads to their closest known mitochondrion-containing relatives and map out various changes that occurred during the transition to amitochondriality.
... To maintain the redox balance and to maximize ATP production, the ameba reoxidizes NADH and reduced ferredoxin using long hydrogenases, which are characterized by an N-terminal NuoG domain, a [FeFe] hydrogenase domain, and a C-terminal CysJ domain, the latter containing an NADH binding pocket. Similar hydrogenases have been identified in Trichomonas vaginalis (Parabasalia) [60], Stygiella incarcerata [61], and Pygsuia biforma [62]. Phylogenetic analysis places them in a clade of bacterial NADH-dependent [FeFe] hydrogenases of group A6 (Fig. S2) It is possible that the long hydrogenases are electronconfurcating hydrogenases involved in the simultaneous reoxidation of reduced ferredoxin and NADH. ...
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 H 2 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.
... The multiple independent transitions from aerobically respiring mitochondria to MROs in the eukaryote tree in many cases involved the complete loss of the mitochondrial genome itself, the loss of aerobic metabolism genes encoded in the nucleus, and the gain of genes encoding novel biochemical pathways by lateral gene transfer (LGT) from bacteria, archaea or other anaerobic protists . For example, the 'hydrogenosomal' metabolism enzymes (Leger et al., 2016;Stairs et al., 2011Stairs et al., , 2021, the ability to synthesize rhodoquinone (Stairs et al., 2018) and oxygen defense enzymes (Jiménez-González et al., 2019) all appear to have been acquired by LGT in multiple distinct lineages from bacteria or other protists (Gawryluk & Stairs, 2021;Roger et al., 2017). The most striking examples of the remodeling of mitochondrial functions in anaerobic protists involve the wholesale replacement of the highly-conserved essential mitochondrial ISC system by laterally acquired prokaryotic Fe/S systems. ...
... We investigated the possibility that the lack of ISC system components in BRC members may be complemented by the presence of an alternative Fe/S cluster system such as the minimal Fe/S (MIS) or sulfur mobilization (SUF) systems found in a number of other anaerobic protists (Anwar et al., 2014;Stairs et al., 2014;Vacek et al., 2018;Žárský et al., 2021;Garcia et al. 2022). In all of the BRC genomes, we were able to identify a simple archaeal-type SUF-like minimal system (SMS) consisting of a gene encoding an SmsCB fusion protein that was most similar to the simple archaeal-type SmsCB systems found in the breviate Pygsuia biforma (Stairs et al., 2014), the anaerobic jakobid Stygiella incarcerata (Leger et al., 2016) and gut commensal opilinatan Stramenopiles (Tsaousis et al., 2012;Yubuki et al., 2020). To investigate the origin of this protein, we performed phylogenetic analysis separately for the SmsC and SmsB domains ( Supplementary Figures 1 & 2) before concatenating the domains together for the final tree ( Figure 3). ...
... The SmsCB fusion protein identified in each BRC member was added to previously constructed alignments (Leger et al., 2016) of SmsC and SmsBD. This dataset was aligned using mafft einsi with default settings and was trimmed by hand. ...
Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered 'mitochondrion-related organelles' (MROs) with reduced functions relative to aerobic mitochondria, however the degree of reduction varies markedly over the metamonad tree. To further investigate metamonad MRO diversity, we generated high quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses place these organisms in a clade sister to the Fornicata - a group of metamonads that includes parasitic and free-living diplomonads and Carpediemonas-like organisms. Extensive bioinformatic analyses of the manually curated gene models showed that these organisms have extremely reduced MROs in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster (ISC) assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system (SMS) Fe/S cluster pathway through lateral gene transfer (LGT). One of the isolates, named 'RC', appears to have undergone even more drastic mitochondrial reduction losing almost all other detectable MRO-related functions. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade is unprecedented and demonstrates that mitochondrial functions, under some conditions, can be almost completely lost even in free-living organisms.
... [FeFe] hydrogenases with N-terminal homology to the NuoG subunit of NADH-quinone oxidoreductase and a C-terminal homology to NADPH-dependent sulfite reductase (CysJ), were identified in T. marina and P. pyriformis (Fig. 5). Similar "fused" hydrogenases ( Fig. 5) have been previously reported in other eukaryotic anaerobes, including T. vaginalis (Tachezy and Doležal 2007), the breviate Pygsuia biforma (Stairs et al. 2014), and the jakobid Stygiella incarcerata (Leger et al. 2016). Although they do not belong to the group of A3 trimeric hydrogenases (Greening et al. 2016) The second reaction of the extended glycolysis, which yields ATP, acetate, and CoA, can be catalyzed either by a two-enzyme system consisting of acetate:succinate CoA-transferase (ASCT) and succinyl CoA synthetase (SCS) like in T. vaginalis, or by a single enzyme acetyl-CoA synthetase (ACS) like in G. intestinalis. ...
... The general design of the search followed the previously described methodology ). Briefly, a custom mitochondrial protein sequence database was established using the MitoMiner v4.0 database (Smith et al. 2012;Smith and Robinson 2018 Mi-ichi et al. 2009;Barberà et al. 2010;Alcock et al. 2012;Stairs et al. 2014;Noguchi et al. 2015;Nývltová et al. 2015;Leger et al. 2016;Leger et al. 2017;Pyrihová et al. 2018). Redundant homologues (90% similarity threshold) were removed from the database using CD-HIT (Li and Godzik 2006). ...
... Reciprocal BLAST analysis was performed for each predicted proteome with an e-value threshold of 0.001. HMM searches were used to identify proteins involved in protein import and translocation, as these were shown to be often divergent (Leger et al. 2016). Searches were done in HMMER 3.1b2 (Eddy 2011) using profile HMMs previously employed in Karnkowska et al. (2016). ...
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 whole group of Oxymonadida. This observation moves this unique loss to 100 MYA when oxymonad lineage diversified.
Author summary
Mitochondria are nearly ubiquitous components of eukaryotic cells that constitute bodies of animals, fungi, plants, algae, and a broad diversity of single-celled eukaryotes, aka protists. Many groups of protists have substantially reduced the complexity of their mitochondria because they live in oxygen-poor environments, so they are unable to utilize the most salient feature of mitochondria – their ATP-producing oxidative phosphorylation metabolism. However, for a long time, scientists thought that it is impossible to completely lose a mitochondrion because this organelle provides other essential services to the cell, e.g. synthesis of protein cofactors called iron-sulfur clusters. Detailed investigation of chinchilla symbiont M. exilis documented the first case of an organism without mitochondrion, and it also provided a scenario explaining how this unique evolutionary experiment might have happened. In this work, we expand on this discovery by exploring genomes of multiple relatives of M. exilis . We show that the loss of the mitochondrion is not limited to a single species but possibly extends to its entire group, the oxymonads. We also compare the predicted metabolic capabilities of oxymonads to their closest known mitochondrion-containing relatives and map out various changes that occurred during the transition to amitochondriality.
... Nevertheless, numerous eukaryotes, especially single-celled forms (protists), thrive in a variety of hypoxic and anoxic habitats, such as deep seas, marine sediments, and sewage (Hackstein 2018;Hu 2014;Zhu et al. 2021). These protists possess reduced mitochondrion-related organelles (MROs) as an adaptation to low-oxygen conditions (Leger et al. 2016;Müller et al. 2012). Among obligate and facultative anaerobic protists, ciliates contain numerous freeliving and endosymbiotic species that have been classified in most ciliate classes ( Fig. 1) Rotterová et al. 2020). ...
Unlabelled:
Adaptations of ciliates to hypoxic environments have arisen independently several times. Studies on mitochondrion-related organelle (MRO) metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes. To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis, mass-culture and single-cell transcriptomes of two anaerobic species, Metopus laminarius (class Armophorea) and Plagiopyla cf. narasimhamurtii (class Plagiopylea), were sequenced and their MRO metabolic maps were compared. In addition, we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes (i.e., Armophorea, Litostomatea, Muranotrichea, Oligohymenophorea, Parablepharismea and Plagiopylea). We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates. The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates, even among closely related species. Notably, our findings indicate the existence of group-specific functional relics of electron transport chains (ETCs). Detailed group-specific ETC functional patterns are as follows: full oxidative phosphorylation in Oligohymenophorea and Muranotrichea; only electron-transfer machinery in Armophorea; either of these functional types in Parablepharismea; and ETC functional absence in Litostomatea and Plagiopylea. These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times. Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates.
Supplementary information:
The online version contains supplementary material available at 10.1007/s42995-022-00147-w.
... Moreover, GCS and SHMT were predicted to be localized in the MRO of other free-living Metamonada, such as Barthelona sp., Anaeramoeba flamelloides, and A. Ignava 29,76 and a number of other free-living, but also endobiotic, anaerobic or microaerophilic protists from the lineages of Archamoebae, breviates, heteroloboseans, ciliates, gregarines, Brevimastigomonas motovehiculus, and Blastocystis sp. [77][78][79][80][81][82][83][84][85][86] Our data provide a potential reason for maintaining GCS and SHMT in these organelles, namely the production of 1C-charged folate species to supply cytosolic 1C metabolism, and particularly the reaction in the methionine cycle catalyzed by methionine synthase. In species without methionine synthase, typically parasites, 87 this MRO function is not required. ...
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.
... The glycine cleavage system (GCS) is at least partially retained in many MROs [42]. It consists of four enzymes (H-, L-, T-, and P-protein) and methylates tetrahydrofolate (THF) while decomposing glycine into CO 2 and ammonia. ...
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.
... The reduction of pyruvate to Acetyl-CoA in hydrogenosomes carried out by the enzyme Pyruvate: Ferredoxin Oxidoreductase (PFO) (Marvin-Sikkema et al. 1993;Stairs et al. 2015). The PFO enzyme reduces ferredoxin and catalyzes pyruvate decarboxylation process to form Acetyl-CoA and CO 2 (Atteia et al. 2013;Leger et al. 2016;Zimorski et al. 2019;Rotterova et al. 2020). ...
Agustina S, Wiryawan KG, Suharti S, Meryandini A. 2021. The enrichment process and morphological identification of anaerobic fungi isolated from buffalo rumen. Biodiversitas 23: 469-477. Anaerobic fungi are one of the microbes that have an important role in rumen fiber degradation because they can produce cellulase enzymes and penetrate feed particles. Nevertheless, few studies were performed to test the potential of anaerobic fungi in Indonesia. Therefore, the present study was carried out to evaluate the impact of the enrichment process on pH value, the zoospores population, NH3 (ammonia) concentration, and VFA (Volatile Fatty Acid) proportion. In addition, this research was also performed to isolate anaerobic fungi from buffalo rumen and identify their morphological characteristics. The enrichment stage of anaerobic fungi was carried out using the Hungate method. Results showed that the population of fungi zoospores, pH value, ammonia concentration, the proportion of acetate, and total VFA were significantly affected by the incubation time (P<0.01). In addition, Caecomyces, Neocallimastix, and Piromyces were rumen anaerobic fungi isolated from buffalo rumen with different morphological characteristics. It can be concluded that the incubation time increased the zoospore population, the concentration of NH3, acetate proportion, and total VFA but decreased media's pH value.
... Moreover, GCS and SHMT were predicted to be localized in the MRO of other free-living Metamonada, such as Barthelona sp., Anaeramoeba flamelloides, and A. Ignava 29,76 and a number of other free-living, but also endobiotic, anaerobic or microaerophilic protists from the lineages of Archamoebae, breviates, heteroloboseans, ciliates, gregarines, Brevimastigomonas motovehiculus, and Blastocystis sp. [77][78][79][80][81][82][83][84][85][86] Our data provide a potential reason for maintaining GCS and SHMT in these organelles, namely the production of 1C-charged folate species to supply cytosolic 1C metabolism, and particularly the reaction in the methionine cycle catalyzed by methionine synthase. In species without methionine synthase, typically parasites, 87 this MRO function is not required. ...
... Briefly, a custom mitochondrial protein sequence database was established using the MitoMiner v4.0 database [34]. The experimentally confirmed proteins (at least one GFP-tagging experiment or three different mass spectroscopy experiments) coming from H. sapiens, M. musculus, R. norvegicus, D. rerio, S. cerevisiae and S. pombe were used and supplemented by the published MROs' protein sets from sixteen species [35][36][37][38][39][40][41][42][43][44]. Redundant homologues (90 % similarity threshold) were removed from the database using cd-hit [25]. ...
... Reciprocal blast analysis was performed for each set of data with an e-value threshold of 0.001. Hidden Markov Model (HMM) searches were used to identify proteins involved in protein import and translocation, as these were shown to be often divergent [42]. Searches were done in HMMER 3.1b2 [45] using HMMs profiles used in Karnkowska et al. 2016 [5]. ...
Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.
