Biology subjects

Hampl, V.

Publications and source records attributed to Hampl, V..

8 recordsLinked to original sources

bioRxiv2024

Striated fibre assemblins localise in the feeding groove of the 'typical excavate' Paratrimastix pyriformis

The spatial arrangement of microtubules in the cytoskeleton in the cells of protists has been used for decades for taxonomy and phylogenetic inference at various levels. In contrast, the protein composition of non-microtubular structures is mostly unknown. Exceptions are system I fibers in algae, which are built of striated fiber assemblins (SFAs). Interestingly, SFAs are also components of a range of other, dissimilar structures, playing a role in the cortex of ciliates, cell division in apicomplexans, and adhesion of the parasite Giardia to the intestine. In a broad bioinformatic survey, we show the existence of three ancestral eukaryotic paralogs of SFA, and note that they are present in all "typical excavates": small heterotrophic flagellates bearing a ventral feeding groove. In one representative, Paratrimastix pyriformis, we detected two SFA paralogs using specific antibodies and expansion microscopy. We show that they co-localize selectively with several microtubules and structures attached to the basal body of the posterior flagellum, namely the right microtubular root, B-fiber, C-fiber, and composite fiber. We demonstrate that one of the paralogs self-assembles in vitro into striated filaments which, under negative staining and cryo-electron microscopy, resemble system I fibers as seen in previous studies. Given the facts that all three SFA paralogs appear to be ancestral to most eukaryotic lineages, as is probably the morphology of "typical excavates" with a ventral groove, we speculate that these proteins played roles in the support and development of the feeding apparatus of the last eukaryotic common ancestor. SIGNIFICANCE STATEMENTWe identified three paralogues of intermediate filament-like proteins from the family of striated fiber assemblins in the Last Eukaryotic Common Ancestor (LECA). Using expansion microscopy, we demonstrated that two of these proteins form a complex cytoskeleton that supports the feeding groove of Paratrimastix pyriformis. It is widely accepted that this flagellated protist retains the morphological and feeding characteristics of ancestral eukaryotes. Therefore, our results suggest that the striated fiber assemblin proteins, which form diverse structures in various extant eukaryotes, were initially components of the feeding apparatus in LECA.

microbiology↗

bioRxiv2024

Comprehensive analysis of the microbial consortium in the culture of flagellate Monocercomonoides exilis

Monocercomonoides exilis is the only known amitochondriate eukaryote, making it an excellent model for studying the implications of mitochondrial reduction from a cellular and evolutionary point of view. Although M. exilis is an endobiotic heterotroph, it can grow in vitro, albeit with an uncharacterized and complex prokaryotic community. All attempts to grow M. exilis axenically have been unsuccessful. Here, we use metagenomic sequencing at different time points during culture growth to describe the composition and dynamics of this community. We assembled genomes of 24 from at least the 30 different bacterial species within. Based on DNA read abundances, M. exilis represents less than 1.5%, and the representation of dominant bacterial members changes over time. Genome-scale metabolic reconstruction, differential expression analysis and measurements of metabolites in the media showed that the community depends on organic carbon oxidation, fermentation, and hydrogen production without methanogenesis. This is consistent with the rapid decline of amino acids, nucleotides, glyceraldehyde, lactate, fatty acids, and alcohols in the media. The community depends on recycling the external supply of amino acids since it has a limited capacity to fix nitrogen gas and lacks ammonia oxidizers to close the nitrogen cycle. With the senescence of the culture, we observe changes in the expression of several metabolic pathways in M. exilis, particularly those adapting to starvation. We do not reveal any clear metabolic link to explain the dependence of M. exilis on prokaryotes.

microbiology↗

bioRxiv2023

Characterisation of the SUF FeS cluster machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first eukaryotic organism described as a complete amitochondriate, yet it shares common features with heterotrophic anaerobic/microaerophilic protists, some of which bear divergent mitochondrion-related organelles or MROs. It has been postulated that the retention of these organelles stems from their involvement in the assembly of essential cytosolic and nuclear FeS proteins, whose maturation requires the evolutionarily conserved mitochondrial ISC and cytosolic CIA machineries. The amitochondriate M. exilis lacks genes encoding the ISC machinery yet contains a bacteria-derived SUF system (MeSuf), composed of the cysteine desulphurase SufS fused to SufD and SufU, as well as the FeS scaffolding components MeSufB and MeSufC. Here, we show that expression of the M. exilis SUF genes, either individually or in tandem, can restore the maturation of the FeS protein IscR in the Escherichia coli double mutants of {Delta}sufS {Delta}iscS and {Delta}sufB {Delta}iscUA. In vivo and in vitro studies indicate that purified MeSufB, MeSufC and MeSufDSU proteins interact suggesting that they act as a complex in the protist. MeSufBC can undergo conformational changes in the presence of ATP and assemble FeS clusters under anaerobic conditions in presence and absence of ATP in vitro. Altogether, these results indicate that the dynamically interacting MeSufDSUBC proteins may function as an FeS cluster assembly complex in M. exilis thereby being capable of replacing the organelle-enclosed ISC system of canonical eukaryotes.

biochemistry↗

bioRxiv2022

Euglenozoan kleptoplasty illuminates the early evolution of photoendosymbiosis

Kleptoplasts 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 247 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 stages in the endosymbiotic origin of euglenophyte plastids also involved kleptoplasty.

evolutionary biology↗

bioRxiv2022

Genomics of Preaxostyla Flagellates Illuminates Evolutionary Transitions and the Path Towards Mitochondrial Loss

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 summaryMitochondria 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.

microbiology↗

bioRxiv2022

Reduced mitochondria provide an essential function for the cytosolic methionine cycle

It has been long hypothesised that mitochondrial reduction is intrinsically related to the remodelling of Fe-S clusters assembly. Yet as our knowledge of divergent free-living protists broadens, so does the spectrum of variability within the range of mitochondrial-related organelles (MROs) fundamental functions. We resolved to high precision the MRO proteome of Paratrimastix pyriformis using Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and demonstrate its role in the synthesis of folate derivates bearing one-carbon (1C) units, its link to the glycine cleavage system (GCS) and their only conceivable role as suppliers for the cytosolic methionine cycle, involved in recycling of S-adenosine methionine. This observation provides congruity to the presence of GCS in MROs of free-living anaerobes and its absence in endobionts, which typically lose the methionine cycle and, in the case of oxymonads, also mitochondria.

cell biology↗

bioRxiv2021

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) - hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5I-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome. Author summaryA major part of the microbial diversity cannot be cultured in isolation, and so it escapes from traditional ways of investigation. In this paper, we demonstrate the successful approach for generating good-quality genome and transcriptome drafts from a peculiar amoeba Pelomyxa schiedti using single-cell methods. P. schiedti is a member of Archamoebae clade harboring microaerobic protists including a free-living Mastigamoeba balamuthi and a human parasite Entamoeba histolytica. Mitochondria and peroxisomes represent two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform to anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, all of which encompass different enzymatic capacity. Anaerobic peroxisomes have been first noticed in M. balamuthi, but their function remained unclear for now. Data obtained in this study were used for revealing the presence and for the detailed functional annotations of anaerobic derivates of mitochondria and peroxisomes in P. schiedti, which were corroborated by transmission electron microscopy.

evolutionary biology↗

bioRxiv2019

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Euglena gracilis is a well-studied biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids. Here we describe its plastid proteome obtained by high-resolution proteomics. We identified 1,345 candidate plastid proteins and assigned functional annotations to 774 of them. More than 120 proteins are affiliated neither to the host lineage nor the plastid ancestor and may represent horizontal acquisitions from various algal and prokaryotic groups. Reconstruction of plastid metabolism confirms both the presence of previously studied/predicted enzymes/pathways and also provides direct evidence for unusual features of its metabolism including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism and the presence of two sets of the SUF pathway for FeS cluster assembly. Most significantly, one of these was acquired by lateral gene transfer (LGT) from the chlamydiae. Plastidial paralogs of membrane trafficking-associated proteins likely mediating a poorly understood fusion of transport vesicles with the outermost plastid membrane were identified, as well as derlin-related proteins that potentially act as protein translocases of the middle membrane, supporting an extremely simplified TIC complex. The proposed innovations may be also linked to specific features of the transit peptide-like regions described here. Hence the Euglena plastid is demonstrated to be a product of several genomes and to combine novel and conserved metabolism and transport processes.

evolutionary biology↗