The trypanosomatid flagellates possess in their single mitochondrion a highly complex kinetoplast (k)DNA, which is composed of interlocked circular molecules of two types. Dozens of maxicircles represent a classical mitochondrial genome, and thousands of minicircles encode guide (g)RNAs, which direct the processive and essential uridine insertion/deletion messenger RNA (mRNA) editing of maxicircle transcripts. While the details of kDNA structure and this type of RNA editing are well established, our knowledge mostly relies on a narrow foray of intensely studied human parasites of the genera Leishmania and Trypanosoma. Here, we analyzed kDNA, its expression, and RNA editing of two members of the poorly characterized genus Vickermania with very different cultivation histories. In both Vickermania species, the gRNA-containing heterogeneous large (HL)-circles are atypically large with multiple gRNAs each. Examination of Vickermania spadyakhi HL-circle loci revealed a massive redundancy of gRNAs relative to the editing needs. In comparison, the HL-circle repertoire of extensively cultivated Vickermania ingenoplastis is greatly reduced. It correlates with V. ingenoplastis-specific loss of productive editing of transcripts encoding subunits of respiratory chain complex I and corresponding lack of complex I activity. This loss in a parasite already lacking genes for subunits of complexes III and IV suggests an apparent requirement for its mitochondrial adenosine triphosphate (ATP) synthase to work in reverse to maintain membrane potential. In contrast, V. spadyakhi retains a functional complex I that allows ATP synthase to work in its standard direction.

• Keywords
• ATP synthase, RNA editing, Vickermania, kinetoplast DNA, trypanosomatids,
• MeSH
• RNA Editing * genetics   MeSH
• Genome, Mitochondrial MeSH
• Genome, Protozoan * MeSH
• DNA, Kinetoplast * genetics   MeSH
• Evolution, Molecular * MeSH
• Trypanosomatina * genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Kinetoplast * MeSH

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

• MeSH
• Cell Nucleus * genetics   metabolism   MeSH
• RNA Editing * MeSH
• Genetic Code MeSH
• Genome, Mitochondrial * MeSH
• RNA, Guide, Kinetoplastida genetics   metabolism   MeSH
• Codon genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Open Reading Frames genetics   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• RNA, Transfer * genetics   metabolism   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Guide, Kinetoplastida MeSH
• Codon MeSH
• RNA, Messenger MeSH
• Protozoan Proteins MeSH
• RNA, Transfer * MeSH
• Codon, Terminator MeSH

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

• Keywords
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetics   MeSH
• Genetic Code MeSH
• Parasites * genetics   MeSH
• Codon, Terminator MeSH
• Trypanosoma brucei brucei * genetics   MeSH
• Trypanosomatina * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Codon, Terminator MeSH

Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.

• Keywords
• constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
• MeSH
• Eukaryota * genetics   MeSH
• Phylogeny MeSH
• Genomics MeSH
• Evolution, Molecular * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Barium and strontium are often used as proxies of marine productivity in palaeoceanographic reconstructions of global climate. However, long-searched biological drivers for such correlations remain unknown. Here, we report that taxa within one of the most abundant groups of marine planktonic protists, diplonemids (Euglenozoa), are potent accumulators of intracellular barite (BaSO4), celestite (SrSO4), and strontiobarite (Ba,Sr)SO4. In culture, Namystinia karyoxenos accumulates Ba2+ and Sr2+ 42,000 and 10,000 times higher than the surrounding medium, forming barite and celestite representing 90% of the dry weight, the greatest concentration in biomass known to date. As heterotrophs, diplonemids are not restricted to the photic zone, and they are widespread in the oceans in astonishing abundance and diversity, as their distribution correlates with environmental particulate barite and celestite, prevailing in the mesopelagic zone. We found diplonemid predators, the filter-feeding zooplankton that produces fecal pellets containing the undigested celestite from diplonemids, facilitating its deposition on the seafloor. To the best of our knowledge, evidence for diplonemid biomineralization presents the strongest explanation for the occurrence of particulate barite and celestite in the marine environment. Both structures of the crystals and their variable chemical compositions found in diplonemids fit the properties of environmentally sampled particulate barite and celestite. Finally, we propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players in Ba2+/Sr2+ cycling in the ocean that has possibly contributed to sedimentary rock formation over long geological periods. IMPORTANCE We have identified that diplonemids, an abundant group of marine planktonic protists, accumulate conspicuous amounts of Sr2+ and Ba2+ in the form of intracellular barite and celestite crystals, in concentrations that greatly exceed those of the most efficient Ba/Sr-accumulating organisms known to date. We propose that diplonemids are potential players in Ba2+/Sr2+ cycling in the ocean and have possibly contributed to sedimentary rock formation over long geological periods. These organisms emerged during the Neoproterozoic era (590 to 900 million years ago), prior to known coccolithophore carbonate biomineralization (~200 million years ago). Based on reported data, the distribution of diplonemids in the oceans is correlated with the occurrence of particulate barite and celestite. Finally, diplonemids may provide new insights into the long-questioned biogenic origin of particulate barite and celestite and bring more understanding of the observed spatial-temporal correlation of the minerals with marine productivity used in reconstructions of past global climate.

• Keywords
• Euglenozoa, barite, biocrystallization, biogeochemical cycles, celestite,
• MeSH
• Barium MeSH
• Minerals MeSH
• Oceans and Seas MeSH
• Plankton MeSH
• Barium Sulfate * MeSH
• Strontium * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Oceans and Seas MeSH
• Names of Substances
• Barium MeSH
• Minerals MeSH
• Barium Sulfate * MeSH
• Strontium * MeSH

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite's replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

• MeSH
• Cell Cycle MeSH
• Phosphatidate Phosphatase genetics   MeSH
• Leishmania guyanensis * MeSH
• Leishmaniavirus MeSH
• Leishmaniasis, Cutaneous * MeSH
• Lipids MeSH
• Mice MeSH
• Parasites * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidate Phosphatase MeSH
• Lipids MeSH

Most trypanosomatid flagellates do not have catalase. In the evolution of this group, the gene encoding catalase has been independently acquired at least three times from three different bacterial groups. Here, we demonstrate that the catalase of Vickermania was obtained by horizontal gene transfer from Gammaproteobacteria, extending the list of known bacterial sources of this gene. Comparative biochemical analyses revealed that the enzymes of V. ingenoplastis, Leptomonas pyrrhocoris, and Blastocrithidia sp., representing the three independent catalase-bearing trypanosomatid lineages, have similar properties, except for the unique cyanide resistance in the catalase of the latter species.

• Keywords
• Blastocrithidia sp., Leptomonas pyrrhocoris, Vickermania ingenoplastis, cyanide resistance,
• Publication type
• Journal Article 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.

• Keywords
• Adaptation, Diplonema, Euglenozoa, Hypoxia, Metabolism, Mitochondrion, Multiomics,
• MeSH
• Euglenozoa genetics   MeSH
• Eukaryota MeSH
• Phylogeny MeSH
• Oxygen MeSH
• Meiotic Prophase I * MeSH
• Proteomics * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oxygen MeSH

The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.

• Keywords
• Leishmaniinae, Trypanosomatidae, bacterial endosymbiont, genomics, metabolism,
• MeSH
• Bacteria classification   genetics   metabolism   MeSH
• Phylogeny MeSH
• Genome, Bacterial * MeSH
• Genomics MeSH
• Symbiosis genetics   MeSH
• Trypanosoma classification   metabolism   microbiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.

• Keywords
• Angomonas, Trypanosomatidae, bacterial endosymbionts, genome,
• Publication type
• Journal Article MeSH