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.

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion.

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study.

A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix.

The diverse GTPases of the dynamin superfamily play various roles in the cell, as exemplified by the dynamin-related proteins (DRPs) Mgm1 and Opa1, which remodel the mitochondrial inner membrane in fungi and metazoans, respectively. Via an exhaustive search of genomic and metagenomic databases, we found previously unknown DRP types occurring in diverse eukaryotes and giant viruses (phylum Nucleocytoviricota). One novel DRP clade, termed MidX, combined hitherto uncharacterized proteins from giant viruses and six distantly related eukaryote taxa (Stramenopiles, Telonemia, Picozoa, Amoebozoa, Apusomonadida, and Choanoflagellata). MidX stood out because it was not only predicted to be mitochondria-targeted but also to assume a tertiary structure not observed in other DRPs before. To understand how MidX affects mitochondria, we exogenously expressed MidX from Hyperionvirus in the kinetoplastid Trypanosoma brucei, which lacks Mgm1 or Opa1 orthologs. MidX massively affected mitochondrial morphology from inside the matrix, where it closely associates with the inner membrane. This unprecedented mode of action contrasts to those of Mgm1 and Opa1, which mediate inner membrane remodeling in the intermembrane space. We speculate that MidX was acquired in Nucleocytoviricota evolution by horizontal gene transfer from eukaryotes and is used by giant viruses to remodel host mitochondria during infection. MidX's unique structure may be an adaptation for reshaping mitochondria from the inside. Finally, Mgm1 forms a sister group to MidX and not Opa1 in our phylogenetic analysis, throwing into question the long-presumed homology of these DRPs with similar roles in sister lineages.

PhyloFisher: A phylogenomic package for resolving eukaryotic relationships.

Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic "single-copy orthogroup" datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset.

Hydrocytium expands the phylogenetic, morphological, and genomic diversity of the poorly known green algal order Chaetopeltidales.

PREMISE: Chaetopeltidales is a small, understudied order of the green algal class Chlorophyceae, that is slowly expanding with the occasional discoveries of novel algae. Here we demonstrate that hitherto unrecognized chaetopeltidaleans also exist among previously described but neglected and misclassified species. METHODS: Strain SAG 40.91 of Characium acuminatum, shown by previous preliminary evidence to have affinities with the orders Oedogoniales, Chaetophorales, and Chaetopeltidales (together constituting the OCC clade), was investigated with light and electron microscopy to characterize its morphology and ultrastructure. Sequence assemblies of the organellar and nuclear genomes were obtained and utilized in bioinformatic and phylogenetic analyses to address the phylogenetic position of the alga and its salient genomic features. RESULTS: The characterization of strain SAG 40.91 and a critical literature review led us to reinstate the forgotten genus Hydrocytium A.Braun 1855, with SAG 40.91 representing its type species, Hydrocytium acuminatum. Independent molecular markers converged on placing H. acuminatum as a deeply diverged lineage of the order Chaetopeltidales, formalized as the new family Hydrocytiaceae. Both chloroplast and mitochondrial genomes shared characteristics with other members of Chaetopeltidales and were bloated by repetitive sequences. Notably, the mitochondrial cox2a gene was transferred into the nuclear genome in the H. acuminatum lineage, independently of the same event in Volvocales. The nuclear genome data from H. acuminatum and from another chaetopeltidalean that was reported by others revealed endogenized viral sequences corresponding to novel members of the phylum Nucleocytoviricota. CONCLUSIONS: The resurrected genus Hydrocytium expands the known diversity of chaetopeltidalean algae and provides the first glimpse into their virosphere.

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.

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria.

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba.

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

Redefining Chlorobotryaceae as one of the principal and most diverse lineages of eustigmatophyte algae.

Eustigmatophyceae is one of the ∼17 classes of the vast algal phylum Ochrophyta. Over the last decade, the eustigmatophytes emerged as an expansive group that has grown from the initially recognized handful of species to well over 200 genetically distinct entities (potential species). Yet the majority of eustigs, remain represented by unidentified strains, or even only metabarcode sequences obtained from environmental samples. Moreover, the formal classification of the group has not yet been harmonized with the recently uncovered diversity and phylogenetic relationships within the class. Here we make a major step towards resolving this issue by addressing the diversity, phylogeny and classification of one of the most prominent eustigmatophyte clades previously informally called the "Eustigmataceae group". We obtained 18S rDNA and rbcL gene sequences from four new strains from the "Eustigmataceae group", and from several additional eustig strains, and performed the most comprehensive phylogenetic analyses of Eustigmatophyceae to date. Our results of these analyses confirm the monophyly of the "Eustigmataceae group" and define its major subclades. We also sequenced plastid genomes of five "Eustigmataceae group" strains to not only improve our understanding of the plastid gene content evolution in eustigs, but also to obtain a robustly resolved eustigmatophyte phylogeny. With this new genomic data, we have solidified the view of the "Eustigmataceae group" as a well-defined family level clade. Crucially, we also have firmly established the genus Chlorobotrys as a member of the "Eustigmataceae group". This new molecular evidence, together with a critical analysis of the literature going back to the 19th century, provided the basis to radically redefine the historical concept of the family Chlorobotryaceae as the formal taxonomic rubric corresponding to the "Eustigmataceae group". With this change, the family names Eustigmataceae and Characiopsidaceae are reduced to synonymy with the Chlorobotryaceae, with the latter having taxonomic priority. We additionally studied in detail the morphology and ultrastructure of two Chlorobotryaceae members, which we describe as Neustupella aerophytica gen. et sp. nov. and Lietzensia polymorpha gen. et sp. nov. Finally, our analyses of partial genomic data from several Chlorobotryaceae representatives identified genes for hallmark flagellar proteins in all of these strains. The presence of the flagellar proteins strongly suggests that zoosporogenesis is a common trait of the family and also occurs in the members never observed to produce flagellated stages. Altogether, our work paints a rich picture of one of the most diverse principal lineages of eustigmatophyte algae.

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Biodegradable Nanohybrid Materials as Candidates for Self-Sanitizing Filters Aimed at Protection from SARS-CoV-2 in Public Areas.

The COVID-19 pandemic has raised the problem of efficient, low-cost materials enabling the effective protection of people from viruses transmitted through the air or via surfaces. Nanofibers can be a great candidate for efficient air filtration due to their structure, although they cannot protect from viruses. In this work, we prepared a wide range of nanofibrous biodegradable samples containing Ag (up to 0.6 at.%) and Cu (up to 20.4 at.%) exhibiting various wettability. By adjusting the magnetron current (0.3 A) and implanter voltage (5 kV), the deposition of TiO2 and Ag+ implantation into PCL/PEO nanofibers was optimized in order to achieve implantation of Ag+ without damaging the nanofibrous structure of the PCL/PEO. The optimal conditions to implant silver were achieved for the PCL-Ti0.3-Ag-5kV sample. The coating of PCL nanofibers by a Cu layer was successfully realized by magnetron sputtering. The antiviral activity evaluated by widely used methodology involving the cultivation of VeroE6 cells was the highest for PCL-Cu and PCL-COOH, where the VeroE6 viability was 73.1 and 68.1%, respectively, which is significantly higher compared to SARS-CoV-2 samples without self-sanitizing (42.8%). Interestingly, the samples with implanted silver and TiO2 exhibited no antiviral effect. This difference between Cu and Ag containing nanofibers might be related to the different concentrations of ions released from the samples: 80 µg/L/day for Cu2+ versus 15 µg/L/day for Ag+. The high antiviral activity of PCL-Cu opens up an exciting opportunity to prepare low-cost self-sanitizing surfaces for anti-SARS-CoV-2 protection and can be essential for air filtration application and facemasks. The rough cost estimation for the production of a biodegradable nanohybrid PCL-Cu facemask revealed ~$0.28/piece, and the business case for the production of these facemasks would be highly positive, with an Internal Rate of Return of 34%.

Olisthodiscus represents a new class of Ochrophyta.

The phylogenetic diversity of Ochrophyta, a diverse and ecologically important radiation of algae, is still incompletely understood even at the level of the principal lineages. One taxon that has eluded simple classification is the marine flagellate genus Olisthodiscus. We investigated Olisthodiscus luteus K-0444 and documented its morphological and genetic differences from the NIES-15 strain, which we described as Olisthodiscus tomasii sp. nov. Phylogenetic analyses of combined 18S and 28S rRNA sequences confirmed that Olisthodiscus constitutes a separate, deep, ochrophyte lineage, but its position could not be resolved. To overcome this problem, we sequenced the plastid genome of O. luteus K-0444 and used the new data in multigene phylogenetic analyses, which suggested that Olisthodiscus is a sister lineage of the class Pinguiophyceae within a broader clade additionally including Chrysophyceae, Synchromophyceae, and Eustigmatophyceae. Surprisingly, the Olisthodiscus plastid genome contained three genes, ycf80, cysT, and cysW, inherited from the rhodophyte ancestor of the ochrophyte plastid yet lost from all other ochrophyte groups studied so far. Combined with nuclear genes for CysA and Sbp proteins, Olisthodiscus is the only known ochrophyte possessing a plastidial sulfate transporter SulT. In addition, the finding of a cemA gene in the Olisthodiscus plastid genome and an updated phylogenetic analysis ruled out the previously proposed hypothesis invoking horizontal cemA transfer from a green algal plastid into Synurales. Altogether, Olisthodiscus clearly represents a novel phylogenetically distinct ochrophyte lineage, which we have proposed as a new class, Olisthodiscophyceae.

The draft nuclear genome sequence and predicted mitochondrial proteome of Andalucia godoyi, a protist with the most gene-rich and bacteria-like mitochondrial genome.

BACKGROUND: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. RESULTS: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids. CONCLUSIONS: As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

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.

Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid.

Euglena spp. are phototrophic flagellates with considerable ecological presence and impact. Euglena gracilis harbours secondary green plastids, but an incompletely characterised proteome precludes accurate understanding of both plastid function and evolutionary history. Using subcellular fractionation, an improved sequence database and MS we determined the composition, evolutionary relationships and hence predicted functions of the E. gracilis plastid proteome. We confidently identified 1345 distinct plastid protein groups and found that at least 100 proteins represent horizontal acquisitions from organisms other than green algae or prokaryotes. Metabolic reconstruction confirmed previously studied/predicted enzymes/pathways and provided evidence for multiple unusual features, including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism, and dual sets of the SUF pathway for FeS cluster assembly, one of which was acquired by lateral gene transfer from Chlamydiae. Plastid paralogues of trafficking-associated proteins potentially mediating fusion of transport vesicles with the outermost plastid membrane were identified, together with derlin-related proteins, potential translocases across the middle membrane, and an extremely simplified TIC complex. The Euglena plastid, as the product of many genomes, combines novel and conserved features of metabolism and transport.

Evolution of the genetic code in the mitochondria of Labyrinthulea (Stramenopiles).

Mitochondrial translation often exhibits departures from the standard genetic code, but the full spectrum of these changes has certainly not yet been described and the molecular mechanisms behind the changes in codon meaning are rarely studied. Here we report a detailed analysis of the mitochondrial genetic code in the stramenopile group Labyrinthulea (Labyrinthulomycetes) and their relatives. In the genus Aplanochytrium, UAG is not a termination codon but encodes tyrosine, in contrast to the unaffected meaning of the UAA codon. This change is evolutionarily independent of the reassignment of both UAG and UAA as tyrosine codons recently reported from two uncultivated labyrinthuleans (S2 and S4), which we show are not thraustochytrids as proposed before, but represent the clade LAB14 previously recognised in environmental 18S rRNA gene surveys. We provide rigorous evidence that the UUA codon in the mitochondria of all labyrinthuleans serves as a termination codon instead of encoding leucine, and propose that a sense-to-stop reassignment has also affected the AGG and AGA codons in the LAB14 clade. The distribution of the different forms of sense-to-stop and stop-to-sense reassignments correlates with specific modifications of the mitochondrial release factor mtRF2a in different subsets of labyrinthuleans, and with the unprecedented loss of mtRF1a in Aplanochytrium and perhaps also in the LAB14 clade, pointing towards a possible mechanistic basis of the code changes observed. Curiously, we show that labyrinthulean mitochondria also exhibit a sense-to-sense codon reassignment, manifested as AUA encoding methionine instead of isoleucine. Furthermore, we show that this change evolved independently in the uncultivated stramenopile lineage MAST8b, together with the reassignment of the AGR codons from arginine to serine. Altogether, our study has uncovered novel variants of the mitochondrial genetic code and previously unknown modifications of the mitochondrial translation machinery, further enriching our understanding of the rules governing the evolution of one of the central molecular process in the cell.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

Toward Modern Classification of Eustigmatophytes, Including the Description of Neomonodaceae Fam. Nov. and Three New Genera1.

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as 10 new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.), we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).

Analysis of epoxy functionalized layers synthesized by plasma polymerization of allyl glycidyl ether.

The deposition of epoxide groups by plasma polymerization opens new horizons for robust and quick immobilization of biomolecules on any type of substrate. However, as of now there are just very few papers dealing with the deposition of epoxy layers by plasma polymerization, probably due to the high reactivity of this group leading to a low functionalization efficiency. In this work we carried out an extensive experimental and theoretical investigation of plasma synthesis of epoxide groups from a low pressure allyl glycydyl ether (AGE) plasma. The influence of composite parameter W/F and the working pressure on the density of epoxide groups and the layer stability was thoroughly addressed. It was found that by increasing the working pressure it is possible to sufficiently raise the concentration of epoxide groups. The composite parameter W/F was shown to be a crucial parameter in affecting the density of epoxides. An optimal value of W/F of around 2.3 eV per molecule leading to the highest density of epoxides produced in the process at 15 Pa was revealed through FT-IR and XPS findings. This value correlates well with the ab initio calculations suggesting that the lowest bond dissociation energy belongs to the C-O bond of the epoxide ring. Therefore, in order to increase the density of epoxides deposited by plasma polymerization, a precursor molecule containing at least two epoxide rings is strongly advised to be employed.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.