Endosymbiotic Origin Research Articles

Genomes of nitrogen-fixing eukaryotes reveal a non-canonical model of organellogenesis.

Endosymbiont gene transfer and import of host-encoded proteins are considered hallmarks of organelles necessary for stable integration of two cells. However, newer endosymbiotic models have challenged the origin and timing of such genetic integration during organellogenesis. Epithemia diatoms contain diazoplasts, closely related to recently-described nitrogen-fixing organelles, that are also stably integrated and co-speciating with their host algae. We report genomic analyses of two species, freshwater E.clementina and marine E.pelagica, which are highly divergent but share a common endosymbiotic origin. We found minimal evidence of genetic integration: nonfunctional diazoplast-to-nuclear DNA transfers in the E.clementina genome and 6 host-encoded proteins of unknown function in the E.clementina diazoplast proteome, far fewer than in other recently-acquired organelles. Epithemia diazoplasts are a valuable counterpoint to existing organellogenesis models, demonstrating that endosymbionts can be stably integrated and inherited absent significant genetic integration. The minimal genetic integration makes diazoplasts valuable blueprints for bioengineering endosymbiotic compartments de novo.

Membrane association of active genes organizes the chloroplast nucleoid structure

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

Coordinating mitochondrial translation with assembly of the OXPHOS complexes.

The mitochondrial oxidative phosphorylation (OXPHOS) system produces the majority of energy required by cells. Given the mitochondrion's endosymbiotic origin, the OXPHOS machinery is still under dual genetic control where most OXPHOS subunits are encoded by the nuclear DNA and imported into mitochondria, while a small subset is encoded on the mitochondrion's own genome, the mitochondrial DNA (mtDNA). The nuclear and mtDNA encoded subunits must be expressed and assembled in a highly orchestrated fashion to form a functional OXPHOS system and meanwhile prevent the generation of any harmful assembly intermediates. While several mechanisms have evolved in eukaryotes to achieve such a coordinated expression, this review will focus on how the translation of mtDNA encoded OXPHOS subunits is tailored to OXPHOS assembly.

A molecular atlas of plastid and mitochondrial proteins reveals organellar remodeling during plant evolutionary transitions from algae to angiosperms.

Algae and plants carry 2 organelles of endosymbiotic origin that have been co-evolving in their host cells for more than a billion years. The biology of plastids and mitochondria can differ significantly across major lineages and organelle changes likely accompanied the adaptation to new ecological niches such as the terrestrial habitat. Based on organelle proteome data and the genomes of 168 phototrophic (Archaeplastida) versus a broad range of 518 non-phototrophic eukaryotes, we screened for changes in plastid and mitochondrial biology across 1 billion years of evolution. Taking into account 331,571 protein families (or orthogroups), we identify 31,625 protein families that are unique to primary plastid-bearing eukaryotes. The 1,906 and 825 protein families are predicted to operate in plastids and mitochondria, respectively. Tracing the evolutionary history of these protein families through evolutionary time uncovers the significant remodeling the organelles experienced from algae to land plants. The analyses of gained orthogroups identifies molecular changes of organelle biology that connect to the diversification of major lineages and facilitated major transitions from chlorophytes en route to the global greening and origin of angiosperms.

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor

Mitochondria are membrane bound organelles of endosymbiotic origin with limited protein coding capacity. The import of nuclear-encoded protein and nucleic acids is required and essential for maintaining organelle mass, number and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with 6 monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

A break in mitochondrial endosymbiosis as a basis for inflammatory diseases.

Mitochondria retain bacterial traits due to their endosymbiotic origin, but host cells do not recognize them as foreign because the organelles are sequestered. However, the regulated release of mitochondrial factors into the cytosol can trigger cell death, innate immunity and inflammation. This selective breakdown in the 2-billion-year-old endosymbiotic relationship enables mitochondria to act as intracellular signalling hubs. Mitochondrial signals include proteins, nucleic acids, phospholipids, metabolites and reactive oxygen species, which have many modes of release from mitochondria, and of decoding in the cytosol and nucleus. Because these mitochondrial signals probably contribute to the homeostatic role of inflammation, dysregulation of these processes may lead to autoimmune and inflammatory diseases. A potential reason for the increased incidence of these diseases may be changes in mitochondrial function and signalling in response to such recent phenomena as obesity, dietary changes and other environmental factors. Focusing on the mixed heritage of mitochondria therefore leads to predictions for future insights, research paths and therapeutic opportunities. Thus, whereas mitochondria can be considered 'the enemy within' the cell, evolution has used this strained relationship in intriguing ways, with increasing evidence pointing to the recent failure of endosymbiosis being critical for the pathogenesis of inflammatory diseases.

Components of iron–Sulfur cluster assembly machineries are robust phylogenetic markers to trace the origin of mitochondria and plastids

Establishing the origin of mitochondria and plastids is key to understand 2 founding events in the origin and early evolution of eukaryotes. Recent advances in the exploration of microbial diversity and in phylogenomics approaches have indicated a deep origin of mitochondria and plastids during the diversification of Alphaproteobacteria and Cyanobacteria, respectively. Here, we strongly support these placements by analyzing the machineries for assembly of iron–sulfur ([Fe–S]) clusters, an essential function in eukaryotic cells that is carried out in mitochondria by the ISC machinery and in plastids by the SUF machinery. We assessed the taxonomic distribution of ISC and SUF in representatives of major eukaryotic supergroups and analyzed the phylogenetic relationships with their prokaryotic homologues. Concatenation datasets of core ISC proteins show an early branching of mitochondria within Alphaproteobacteria, right after the emergence of Magnetococcales. Similar analyses with the SUF machinery place primary plastids as sister to Gloeomargarita within Cyanobacteria. Our results add to the growing evidence of an early emergence of primary organelles and show that the analysis of essential machineries of endosymbiotic origin provide a robust signal to resolve ancient and fundamental steps in eukaryotic evolution.

PlastiC: A pipeline for recovery and characterization of plastid genomes from metagenomic datasets

The use of culture independent molecular methods, often referred to as metagenomics, have revolutionized the ability to explore and characterize microbial communities from diverse environmental sources. Most metagenomic workflows have been developed for identification of prokaryotic and eukaryotic community constituents, but tools for identification of plastid genomes are lacking. The endosymbiotic origin of plastids also poses challenges where plastid metagenomic assembled genomes (MAGs) may be misidentified as low-quality bacterial MAGs. Current tools are limited to classification of contigs as plastid and do not provide further assessment or characterization of plastid MAGs. plastiC is a workflow that allows users to identify plastid genomes in metagenome assemblies, assess completeness, and predict taxonomic association from diverse environmental sources. plastiC is a Snakemake workflow available at https://github.com/Finn-Lab/plastiC. We demonstrate the utility of this workflow with the successful recover of algal plastid MAGs from publicly available lichen metagenomes.

Characterization of signal and transit peptides based on motif composition and taxon-specific patterns

Targeting peptides or presequences are N-terminal extensions of proteins that encode information about their cellular localization. They include signal peptides (SP), which target proteins to the endoplasmic reticulum, and transit peptides (TP) directing proteins to the organelles of endosymbiotic origin: chloroplasts and mitochondria. TPs were hypothesized to have evolved from antimicrobial peptides (AMPs), which are responsible for the host defence against microorganisms, including bacteria, fungi and viruses. In this study, we performed comprehensive bioinformatic analyses of amino acid motifs of targeting peptides and AMPs using a curated set of experimentally verified proteins. We identified motifs frequently occurring in each type of presequence showing specific patterns associated with their amino acid composition, and investigated their position within the presequence. We also compared motif patterns among different taxonomic groups and identified taxon-specific features, providing some evolutionary insights. Considering the functional relevance and many practical applications of targeting peptides and AMPs, we believe that our analyses will prove useful for their design, and better understanding of protein import mechanism and presequence evolution.

Apoptotic Factors Are Evolutionarily Conserved Since Mitochondrial Domestication.

The mechanisms initiating apoptotic programmed cell death in diverse eukaryotes are very similar. Basically, the mitochondrial permeability transition activates apoptotic proteases, DNases, and flavoproteins such as apoptosis-inducing factors (AIFs). According to the hypothesis of the endosymbiotic origin of apoptosis, these mechanisms evolved during mitochondrial domestication. Various phylogenetic analyses, including ours, have suggested that apoptotic factors were eubacterial protomitochondrial toxins used for killing protoeukaryotic hosts. Here, we tested whether the function of yeast Saccharomyces cerevisiae apoptotic proteases (metacaspases Mca1 and Nma111), DNase Nuc1, and flavoprotein Ndi1 can be substituted with orthologs from remotely related eukaryotes such as plants, protists, and eubacteria. We found that orthologs of remotely related eukaryotic and even eubacterial proteins can initiate apoptosis in yeast when triggered by chemical stresses. This observation suggests that apoptotic mechanisms have been maintained since mitochondrial domestication, which occurred approximately 1,800 Mya. Additionally, it supports the hypothesis that some of these apoptotic factors could be modified eubacterial toxins.

The apicoplast is important for the viability and persistence of Toxoplasma gondii bradyzoites

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.

Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis.

Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.

Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.

Mitochondria are eukaryotic organelles of endosymbiotic origin that contain their own genetic material, mitochondrial DNA (mtDNA), and dedicated systems for mtDNA maintenance and expression. MtDNA molecules encode a limited number of proteins that are nevertheless all essential subunits of the mitochondrial oxidative phosphorylation system. Here, we describe protocols to monitor DNA and RNA synthesis in intact, isolated mitochondria. These in organello synthesis protocols are valuable techniques for studying the mechanisms and regulation of mtDNA maintenance and expression.

J-Domain Proteins Orchestrate the Multifunctionality of Hsp70s in Mitochondria: Insights from Mechanistic and Evolutionary Analyses.

Mitochondrial J-domain protein (JDP) co-chaperones orchestrate the function of their Hsp70 chaperone partner(s) in critical organellar processes that are essential for cell function. These include folding, refolding, and import of mitochondrial proteins, maintenance of mitochondrial DNA, and biogenesis of iron-sulfur cluster(s) (FeS), prosthetic groups needed for function of mitochondrial and cytosolic proteins. Consistent with the organelle's endosymbiotic origin, mitochondrial Hsp70 and the JDPs' functioning in protein folding and FeS biogenesis clearly descended from bacteria, while the origin of the JDP involved in protein import is less evident. Regardless of their origin, all mitochondrial JDP/Hsp70 systems evolved unique features that allowed them to perform mitochondria-specific functions. Their modes of functional diversification and specialization illustrate the versatility of JDP/Hsp70 systems and inform our understanding of system functioning in other cellular compartments.

Chloroplast Envelope Membrane Subfractionation from Arabidopsis and Pea.

Due to their endosymbiotic origin, chloroplasts harbor several subcompartments and membrane systems. Each of these has a different protein and lipid composition that dynamically changes either naturally during plant development or induced by environmental stimuli. Here, we describe a protocol for chloroplast envelope membrane subfractionation via discontinuous sucrose gradients, which offers the possibility to separate the different plastid subcompartments for several downstream applications. It is a strong tool for protein sublocalization studies as well as for tracking dynamic movement patterns. Furthermore, it can be combined with in vitro import studies of radioactively labeled proteins, which allows sublocalization of putative envelope proteins independent of the availability of specific antisera.

Challenging the Importance of Plastid Genome Structure Conservation: New Insights From Euglenophytes.

Plastids, similar to mitochondria, are organelles of endosymbiotic origin, which retained their vestigial genomes (ptDNA). Their unique architecture, commonly referred to as the quadripartite (four-part) structure, is considered to be strictly conserved; however, the bulk of our knowledge on their variability and evolutionary transformations comes from studies of the primary plastids of green algae and land plants. To broaden our perspective, we obtained seven new ptDNA sequences from freshwater species of photosynthetic euglenids-a group that obtained secondary plastids, known to have dynamically evolving genome structure, via endosymbiosis with a green alga. Our analyses have demonstrated that the evolutionary history of euglenid plastid genome structure is exceptionally convoluted, with a patchy distribution of inverted ribosomal operon (rDNA) repeats, as well as several independent acquisitions of tandemly repeated rDNA copies. Moreover, we have shown that inverted repeats in euglenid ptDNA do not share their genome-stabilizing property documented in chlorophytes. We hypothesize that the degeneration of the quadripartite structure of euglenid plastid genomes is connected to the group II intron expansion. These findings challenge the current global paradigms of plastid genome architecture evolution and underscore the often-underestimated divergence between the functionality of shared traits in primary and complex plastid organelles.

The complete mitochondrial genome of Isochrysis galbana harbors a unique repeat structure and a specific trans-spliced cox1 gene.

The haptophyte Isochrysis galbana is considered as a promising source for food supplements due to its rich fucoxanthin and polyunsaturated fatty acids content. Here, the I. galbana mitochondrial genome (mitogenome) was sequenced using a combination of Illumina and PacBio sequencing platforms. This 39,258 bp circular mitogenome has a total of 46 genes, including 20 protein-coding genes, 24 tRNA genes and two rRNA genes. A large block of repeats (~12.7 kb) was segregated in one region of the mitogenome, accounting for almost one third of the total size. A trans-spliced gene cox1 was first identified in I. galbana mitogenome and was verified by RNA-seq and DNA-seq data. The massive expansion of tandem repeat size and cis- to trans-splicing shift could be explained by the high mitogenome rearrangement rates in haptophytes. Strict SNP calling based on deep transcriptome sequencing data suggested the lack of RNA editing in both organelles in this species, consistent with previous studies in other algal lineages. To gain insight into haptophyte mitogenome evolution, a comparative analysis of mitogenomes within haptophytes and among eight main algal lineages was performed. A core gene set of 15 energy and metabolism genes is present in haptophyte mitogenomes, consisting of 1 cob, 3 cox, 7 nad, 2 atp and 2 ribosomal genes. Gene content and order was poorly conserved in this lineage. Haptophyte mitogenomes have lost many functional genes found in many other eukaryotes including rps/rpl, sdh, tat, secY genes, which make it contain the smallest gene set among all algal taxa. All these implied the rapid-evolving and more recently evolved mitogenomes of haptophytes compared to other algal lineages. The phylogenetic tree constructed by cox1 genes of 204 algal mitogenomes yielded well-resolved internal relationships, providing new evidence for red-lineages that contained plastids of red algal secondary endosymbiotic origin. This newly assembled mitogenome will add to our knowledge of general trends in algal mitogenome evolution within haptophytes and among different algal taxa.

New insights on thioredoxins (Trxs) and glutaredoxins (Grxs) by in silico amino acid sequence, phylogenetic and comparative structural analyses in organisms of three domains of life

Thioredoxins (Trxs) and Glutaredoxins (Grxs) regulate several cellular processes by controlling the redox state of their target proteins. Trxs and Grxs belong to thioredoxin superfamily and possess characteristic Trx/Grx fold. Several phylogenetic, biochemical and structural studies have contributed to our overall understanding of Trxs and Grxs. However, comparative study of closely related Trxs and Grxs in organisms of all domains of life was missing. Here, we conducted in silico comparative structural analysis combined with amino acid sequence and phylogenetic analyses of 65 Trxs and 88 Grxs from 12 organisms of three domains of life to get insights into evolutionary and structural relationship of two proteins. Outcomes suggested that despite diversity in their amino acids composition in distantly related organisms, both Trxs and Grxs strictly conserved functionally and structurally important residues. Also, position of these residues was highly conserved in all studied Trxs and Grxs. Notably, if any substitution occurred during evolution, preference was given to amino acids having similar chemical properties. Trxs and Grxs were found more different in eukaryotes than prokaryotes due to altered helical conformation. The surface of Trxs was negatively charged, while Grxs surface was positively charged, however, the active site was constituted by uncharged amino acids in both proteins. Also, phylogenetic analysis of Trxs and Grxs in three domains of life supported endosymbiotic origins of chloroplast and mitochondria, and suggested their usefulness in molecular systematics. We also report previously unknown catalytic motifs of two proteins, and discuss in detail about effect of abovementioned parameters on overall structural and functional diversity of Trxs and Grxs.

The greening ashore.

More than half a billion years ago a streptophyte algal lineage began terraforming the terrestrial habitat and the Earth's atmosphere. This pioneering step enabled the subsequent evolution of all complex life on land, and the past decade has uncovered that many traits, both morphological and genetic, once thought to be unique to land plants, are conserved across some streptophyte algae. They provided the common ancestor of land plants with a repertoire of genes, of which many were adapted to overcome the new biotic and abiotic challenges. Exploring these molecular adaptations in non-tracheophyte species may help us to better prepare all green life, including our crops, for the challenges precipitated by the climate change of the Anthropocene because the challenges mostly differ by the speed with which they are now being met.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria