BACKGROUND: Diplomonads are anaerobic flagellates classified within Metamonada. They contain both host-associated commensals and parasites that reside in the intestinal tracts of animals, including humans (e.g., Giardia intestinalis), as well as free-living representatives that inhabit freshwater and marine anoxic sediments (e.g., Hexamita inflata). The evolutionary trajectories within this group are particularly unusual as the free-living taxa appear to be nested within a clade of host-associated species, suggesting a reversal from host-dependence to a secondarily free-living lifestyle. This is thought to be an exceedingly rare event as parasites often lose genes for metabolic pathways that are essential to a free-living life strategy, as they become increasingly reliant on their host for nutrients and metabolites. To revert to a free-living lifestyle would require the reconstruction of numerous metabolic pathways. All previous studies of diplomonad evolution suffered from either low taxon sampling, low gene sampling, or both, especially among free-living diplomonads, which has weakened the phylogenetic resolution and hindered evolutionary insights into this fascinating transition. RESULTS: We sequenced transcriptomes from 1 host-associated and 13 free-living diplomonad isolates; expanding the genome scale data sampling for diplomonads by roughly threefold. Phylogenomic analyses clearly show that free-living diplomonads form several branches nested within endobiotic species. Moreover, the phylogenetic distribution of genes related to an endobiotic lifestyle suggest their acquisition at the root of diplomonads, while traces of these genes have been identified in free-living diplomonads as well. Based on these results, we propose an evolutionary scenario of ancestral and derived lifestyle transitions across diplomonads. CONCLUSIONS: Free-living taxa form several clades nested within endobiotic taxa in our phylogenomic analyses, implying multiple transitions between free-living and endobiotic lifestyles. The evolutionary history of numerous virulence factors corroborates the inference of an endobiotic ancestry of diplomonads, suggesting that there have been several reversals to a free-living lifestyle. Regaining host independence may have been facilitated by a subset of laterally transferred genes. We conclude that the extant diversity of diplomonads has evolved from a non-specialized endobiont, with some taxa becoming highly specialized parasites, others becoming free-living, and some becoming capable of both free-living and endobiotic lifestyles.

• Keywords
• Diplomonads, Parasitic ancestry signals, Phylogenetics, Phylogenomics, Transcriptomics,
• MeSH
• Biological Evolution MeSH
• Diplomonadida * genetics   MeSH
• Phylogeny * MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

• Keywords
• Cell cycle, Cytoskeleton, Flagellum, Giardia, Mitochondrial division, Mitochondrial inheritance, Mitosomes, Protist, mitochondrial evolution,
• MeSH
• Databases, Genetic MeSH
• Giardia lamblia * genetics   MeSH
• Mitochondrial Dynamics MeSH
• Mitochondria genetics   MeSH
• Organelles MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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).

• Keywords
• Evolution, HCF101, Ind1, Iron-sulfur cluster, Mitochondrion, Plastid,
• MeSH
• Cryptosporidium * MeSH
• Phylogeny MeSH
• Cryptosporidiosis * MeSH
• Iron-Sulfur Proteins * genetics   MeSH
• Sulfur MeSH
• Iron MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Iron-Sulfur Proteins * MeSH
• Sulfur MeSH
• Iron MeSH

The oxymonad Monocercomonoides exilis was recently reported to be the first eukaryote that has completely lost the mitochondrial compartment. It was proposed that an important prerequisite for such a radical evolutionary step was the acquisition of the SUF Fe-S cluster assembly pathway from prokaryotes, making the mitochondrial ISC pathway dispensable. We have investigated genomic and transcriptomic data from six oxymonad species and their relatives, composing the group Preaxostyla (Metamonada, Excavata), for the presence and absence of enzymes involved in Fe-S cluster biosynthesis. None possesses enzymes of mitochondrial ISC pathway and all apparently possess the SUF pathway, composed of SufB, C, D, S, and U proteins, altogether suggesting that the transition from ISC to SUF preceded their last common ancestor. Interestingly, we observed that SufDSU were fused in all three oxymonad genomes, and in the genome of Paratrimastix pyriformis. The donor of the SUF genes is not clear from phylogenetic analyses, but the enzyme composition of the pathway and the presence of SufDSU fusion suggests Firmicutes, Thermotogae, Spirochaetes, Proteobacteria, or Chloroflexi as donors. The inventory of the downstream CIA pathway enzymes is consistent with that of closely related species that retain ISC, indicating that the switch from ISC to SUF did not markedly affect the downstream process of maturation of cytosolic and nuclear Fe-S proteins.

• MeSH
• Phylogeny MeSH
• Genome, Protozoan * MeSH
• Evolution, Molecular * MeSH
• Oxymonadida genetics   metabolism   MeSH
• Iron-Sulfur Proteins genetics   MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists, organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

• Keywords
• Evolution, Excavata, Fe–S cluster, Mitochondria,
• MeSH
• Eukaryota cytology   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Iron MeSH

Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.

• MeSH
• Biotinylation MeSH
• Escherichia coli enzymology   MeSH
• Cell Fractionation MeSH
• Giardia lamblia chemistry   cytology   metabolism   MeSH
• Giardiasis parasitology   MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Carbon-Nitrogen Ligases metabolism   MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Organelles chemistry   metabolism   MeSH
• Escherichia coli Proteins metabolism   MeSH
• Protozoan Proteins analysis   isolation & purification   metabolism   MeSH
• Repressor Proteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• birA protein, E coli MeSH Browser
• Carbon-Nitrogen Ligases MeSH
• Escherichia coli Proteins MeSH
• Protozoan Proteins MeSH
• Repressor Proteins MeSH

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• Ferredoxins metabolism   MeSH
• Frataxin MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases chemistry   genetics   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Nuclear Matrix-Associated Proteins chemistry   genetics   metabolism   MeSH
• Iron-Binding Proteins metabolism   MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Trypanosoma brucei brucei enzymology   genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• cysteine desulfurase MeSH Browser
• Ferredoxins MeSH
• Nuclear Localization Signals MeSH
• Carbon-Sulfur Lyases MeSH
• Mitochondrial Proteins MeSH
• Nuclear Matrix-Associated Proteins MeSH
• Iron-Binding Proteins MeSH
• Protozoan Proteins MeSH

Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

• MeSH
• Energy Metabolism MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Organelles metabolism   ultrastructure   MeSH
• Oxidation-Reduction MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Protozoan Proteins chemistry   metabolism   MeSH
• Gene Expression Regulation MeSH
• Cluster Analysis MeSH
• Sulfur metabolism   MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Iron metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH
• Protozoan Proteins MeSH
• Sulfur MeSH
• Iron MeSH

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

• MeSH
• Amoeba genetics   metabolism   MeSH
• Cytosol metabolism   MeSH
• Gene Duplication * MeSH
• Entamoeba histolytica metabolism   MeSH
• Nitrogen Fixation genetics   MeSH
• Mitochondria metabolism   MeSH
• Molecular Sequence Data MeSH
• Protein Sorting Signals MeSH
• Iron-Sulfur Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Sorting Signals MeSH
• Iron-Sulfur Proteins MeSH

Gene duplication is an important evolutionary mechanism and no eukaryote has more duplicated gene families than the parasitic protist Trichomonas vaginalis. Iron is an essential nutrient for Trichomonas and plays a pivotal role in the establishment of infection, proliferation, and virulence. To gain insight into the role of iron in T. vaginalis gene expression and genome evolution, we screened iron-regulated genes using an oligonucleotide microarray for T. vaginalis and by comparative EST (expressed sequence tag) sequencing of cDNA libraries derived from trichomonads cultivated under iron-rich (+Fe) and iron-restricted (-Fe) conditions. Among 19,000 ESTs from both libraries, we identified 336 iron-regulated genes, of which 165 were upregulated under +Fe conditions and 171 under -Fe conditions. The microarray analysis revealed that 195 of 4,950 unique genes were differentially expressed. Of these, 117 genes were upregulated under +Fe conditions and 78 were upregulated under -Fe conditions. The results of both methods were congruent concerning the regulatory trends and the representation of gene categories. Under +Fe conditions, the expression of proteins involved in carbohydrate metabolism, particularly in the energy metabolism of hydrogenosomes, and in methionine catabolism was increased. The iron-sulfur cluster assembly machinery and certain cysteine proteases are of particular importance among the proteins upregulated under -Fe conditions. A unique feature of the T. vaginalis genome is the retention during evolution of multiple paralogous copies for a majority of all genes. Although the origins and reasons for this gene expansion remain unclear, the retention of multiple gene copies could provide an opportunity to evolve differential expression during growth in variable environmental conditions. For genes whose expression was affected by iron, we found that iron influenced the expression of only some of the paralogous copies, whereas the expression of the other paralogs was iron independent. This finding indicates a very stringent regulation of the differentially expressed paralogous genes in response to changes in the availability of exogenous nutrients and provides insight into the evolutionary rationale underlying massive paralog retention in the Trichomonas genome.

• MeSH
• Cysteine Proteases genetics   metabolism   MeSH
• Gene Duplication MeSH
• Expressed Sequence Tags MeSH
• Genome, Protozoan MeSH
• Gene Dosage MeSH
• Gene Library MeSH
• Glycolysis genetics   MeSH
• Evolution, Molecular MeSH
• Iron-Sulfur Proteins genetics   metabolism   MeSH
• Genes, Protozoan * MeSH
• Gene Expression Regulation * MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Transcriptome * MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cysteine Proteases MeSH
• Iron-Sulfur Proteins MeSH
• Iron MeSH