Plastids of diatoms and related algae with complex plastids of red algal origin are surrounded by four membranes, which also define the periplastidic compartment (PPC), the space between the second and third membranes. Metabolic reactions as well as cell biological processes take place in the PPC; however, genome-wide predictions of the proteins targeted to this compartment were so far based on manual annotation work. Using published experimental protein localizations as reference data, we developed the first automatic prediction method for PPC proteins, which we included as a new feature in an updated version of the plastid protein predictor ASAFind. With our method, at least a subset of the PPC proteins can be predicted with high specificity, with an estimate of at least 81 proteins (0.7% of the predicted proteome) targeted to the PPC in the model diatom Phaeodactylum tricornutum. The proportion of PPC proteins varies, since 180 PPC proteins (1.3% of the predicted proteome) were predicted in the genome of the diatom Thalassiosira pseudonana. The new ASAFind version can also generate a newly designed graphical output that visualizes the contribution of each position in the sequence to the score and accepts the output of the recent versions of SignalP (5.0) and TargetP (2.0) as input data. Furthermore, we release a script to calculate custom scoring matrices that can be used for predictions in a simplified score cut-off mode. This allows for adjustments of the method to other groups of algae.

• Klíčová slova
• chloroplast, diatoms, evolution, gene transfer, genome annotation, mitochondria, organelle, periplastidic compartment, protein transport, secretory pathway, technical advance,
• MeSH
• bílkoviny řas * metabolismus   MeSH
• plastidy * metabolismus   MeSH
• proteom MeSH
• Rhodophyta metabolismus   MeSH
• rozsivky * metabolismus   genetika   MeSH
• software * MeSH
• výpočetní biologie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas * MeSH
• proteom MeSH

This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis, an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.

• MeSH
• chloroplasty ultrastruktura   MeSH
• cytoskelet * ultrastruktura   MeSH
• Euglena gracilis * ultrastruktura   MeSH
• flagella ultrastruktura   MeSH
• fluorescenční mikroskopie * metody   MeSH
• mitochondrie ultrastruktura   MeSH
• mitóza MeSH
• protilátky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH

BACKGROUND: Plastids are usually involved in photosynthesis, but the secondary loss of this function is a widespread phenomenon in various lineages of algae and plants. In addition to the loss of genes associated with photosynthesis, the plastid genomes of colorless algae are frequently reduced further. To understand the pathways of reductive evolution associated with the loss of photosynthesis, it is necessary to study a number of closely related strains. Prototheca, a chlorophyte genus of facultative pathogens, provides an excellent opportunity to study this process with its well-sampled array of diverse colorless strains. RESULTS: We have sequenced the plastid genomes of 13 Prototheca strains and reconstructed a comprehensive phylogeny that reveals evolutionary patterns within the genus and among its closest relatives. Our phylogenomic analysis revealed three independent losses of photosynthesis among the Prototheca strains and varied protein-coding gene content in their ptDNA. Despite this diversity, all Prototheca strains retain the same key plastid functions. These include processes related to gene expression, as well as crucial roles in fatty acid and cysteine biosynthesis, and membrane transport. CONCLUSIONS: The retention of vestigial genomes in colorless plastids is typically associated with the biosynthesis of secondary metabolites. In contrast, the remarkable conservation of plastid membrane transport system components in the nonphotosynthetic genera Prototheca and Helicosporidium provides an additional constraint against the loss of ptDNA in this lineage. Furthermore, these genes can potentially serve as targets for therapeutic intervention, indicating their importance beyond the evolutionary context.

• Klíčová slova
• Prototheca, Chlorophyta, Colorless plastids, Plastid genomes,
• MeSH
• fotosyntéza genetika   MeSH
• fylogeneze * MeSH
• genom plastidový * MeSH
• molekulární evoluce MeSH
• Prototheca * genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Complete plastid loss seems to be very rare among secondarily non-photosynthetic eukaryotes. Leukarachnion sp. PRA-24, an amoeboid colourless protist related to the photosynthetic algal class Synchromophyceae (Ochrophyta), is a candidate for such a case based on a previous investigation by transmission electron microscopy. Here, we characterize this organism in further detail and describe it as Leucomyxa plasmidifera gen. et sp. nov., additionally demonstrating it is the first known representative of a broader clade of non-photosynthetic ochrophytes. We recovered its complete plastid genome, exhibiting a reduced gene set similar to plastomes of other non-photosynthetic ochrophytes, yet being even more extreme in sequence divergence. Identification of components of the plastid protein import machinery in the L. plasmidifera transcriptome assembly corroborated that the organism possesses a cryptic plastid organelle. According to our bioinformatic reconstruction, the plastid contains a unique combination of biosynthetic pathways producing haem, a folate precursor and tocotrienols. As another twist to its organellar biology, L. plasmidifera turned out to contain an unusual long insertion in its mitogenome related to a newly discovered mitochondrial plasmid exhibiting unprecedented features in terms of its size and coding capacity. Combined, our work uncovered further striking outcomes of the evolutionary course of semiautonomous organelles in protists.

• Klíčová slova
• Leukarachnion, mitochondrial plasmids, non-photosynthetic plastid, plastid evolution, plastid genome, stramenopiles,
• MeSH
• fylogeneze * MeSH
• genom mitochondriální MeSH
• genom plastidový * MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• plastidy * genetika   metabolismus   MeSH
• plazmidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.

• Klíčová slova
• Automated Tree Sorting, Myzozoa, Post-Endosymbiotic Organelle Evolution, Protists, Shopping Bag Model,
• MeSH
• Dinoflagellata * genetika   metabolismus   MeSH
• fylogeneze MeSH
• plastidy genetika   MeSH
• proteom genetika   metabolismus   MeSH
• symbióza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

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.

• Klíčová slova
• DNA polymerase, endosymbiosis, last eukaryotic common ancestor, lateral gene transfer, mitochondria, plastids,
• MeSH
• DNA-dependentní DNA-polymerasy genetika   MeSH
• fylogeneze MeSH
• mitochondrie MeSH
• organely * genetika   MeSH
• plastidy genetika   MeSH
• sinice * genetika   MeSH
• symbióza MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH

Kleptoplasts (kP) are distinct among photosynthetic organelles in eukaryotes (i.e., plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light-dark cycles, and 13C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 276 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis. These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early evolutionary stages in the green alga-derived secondary plastids of euglenophytes also involved kleptoplasty.

• Klíčová slova
• endosymbiosis, euglenozoa, horizontal gene transfer, kleptoplasty, plastid evolution,
• MeSH
• Chlorophyta * genetika   metabolismus   MeSH
• Eukaryota genetika   MeSH
• fotosyntéza * genetika   MeSH
• fylogeneze MeSH
• plastidy genetika   metabolismus   MeSH
• symbióza genetika   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. RESULTS: We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. CONCLUSIONS: The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

• Klíčová slova
• Adaptation, Diplonema, Euglenozoa, Hypoxia, Metabolism, Mitochondrion, Multiomics,
• MeSH
• Euglenozoa genetika   MeSH
• Eukaryota MeSH
• fylogeneze MeSH
• kyslík MeSH
• profáze meiózy I * MeSH
• proteomika * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyslík MeSH

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

• MeSH
• biologická evoluce MeSH
• Chromatium genetika   metabolismus   MeSH
• Euglenida genetika   metabolismus   MeSH
• exony genetika   MeSH
• fylogeneze MeSH
• genom MeSH
• heterotrofní procesy MeSH
• introny genetika   MeSH
• mitochondrie genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea. To date, the cellular distribution of these proteins is considered to be highly conserved with only a few exceptions. RESULTS: We searched for the genes of all members of the Nbp35-like protein family and analyzed their targeting sequences. Nbp35 and Cfd1 were predicted to reside in the cytoplasm with some exceptions of Nbp35 localization to the mitochondria; Ind1was found in the mitochondria, and HCF101 was predicted to reside in plastids (chHCF101) of all photosynthetically active eukaryotes. Surprisingly, we found a second HCF101 paralog in all members of Cryptista, Haptista, and SAR that was predicted to predominantly target mitochondria (mHCF101), whereas Ind1 appeared to be absent in these organisms. We also identified a few exceptions, as apicomplexans possess mHCF101 predicted to localize in the cytosol and Nbp35 in the mitochondria. Our predictions were experimentally confirmed in selected representatives of Apicomplexa (Toxoplasma gondii), Stramenopila (Phaeodactylum tricornutum, Thalassiosira pseudonana), and Ciliophora (Tetrahymena thermophila) by tagging proteins with a transgenic reporter. Phylogenetic analysis suggested that chHCF101 and mHCF101 evolved from a common ancestral HCF101 independently of the Nbp35/Cfd1 and Ind1 proteins. Interestingly, phylogenetic analysis supports rather a lateral gene transfer of ancestral HCF101 from bacteria than its acquisition being associated with either α-proteobacterial or cyanobacterial endosymbionts. CONCLUSION: Our searches for Nbp35-like proteins across eukaryotic lineages revealed that SAR, Haptista, and Cryptista possess mitochondrial HCF101. Because plastid localization of HCF101 was only known thus far, the discovery of its mitochondrial paralog explains confusion regarding the presence of HCF101 in organisms that possibly lost secondary plastids (e.g., ciliates, Cryptosporidium) or possess reduced nonphotosynthetic plastids (apicomplexans).

• Klíčová slova
• Evolution, HCF101, Ind1, Iron-sulfur cluster, Mitochondrion, Plastid,
• MeSH
• Cryptosporidium * MeSH
• fylogeneze MeSH
• kryptosporidióza * MeSH
• proteiny obsahující železo a síru * genetika   MeSH
• síra MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny obsahující železo a síru * MeSH
• síra MeSH
• železo MeSH