Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left.
The cytosolic iron/sulfur cluster assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages, such as animals and fungi. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. strain NandII. We identified transcripts encoding six of the components-Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18-and showed using immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation that the last three of them localized to the cytoplasm of the cell. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the last eukaryotic common ancestor (LECA). The Nbp35, Nar1 Cia1, and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic sulfur utilization factors (SUF) system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in nonmodel organisms and how it interfaces with various iron/sulfur cluster systems (i.e., the iron/sulfur cluster, nitrogen fixation, and/or SUF system) found in eukaryotic cells.
The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses.
Protist diversity is currently a much debated issue in eukaryotic microbiology. Recent evidence suggests that morphological and genetic diversity might be decoupled in some groups of protists, including ciliates, and that these organisms might be much more diverse than their morphology implies. We sought to assess the genetic and morphological diversity of Carchesium polypinum, a widely distributed peritrich ciliate. The mitochondrial marker cytochrome c oxidase subunit I and the nuclear small subunit ribosomal RNA were used to examine genetic diversity. For the morphological assessment, live microscopy and Protargol staining were used. The mitochondrial marker revealed six robust, deeply diverging, and strongly supported clades, while the nuclear gene was congruent for three of these clades. There were no major differences among individuals from the different clades in any of the morphological features examined. Thus, the underlying genetic diversity in C. polypinum is greater than what its morphology suggests, indicating that morphology and genetics are not congruent in this organism. Furthermore, because the clades identified by the mitochondrial marker are so genetically diverse and are confirmed by a conserved nuclear marker in at least three cases, we propose that C. polypinum be designated as a "cryptic species complex." Our results provide another example where species diversity can be underestimated in microbial eukaryotes when using only morphological criteria to estimate species richness.
Most recent studies of geographic distribution of microbial eukaryotes have focused on marine rather than freshwater protists. Here, we used the freshwater peritrich ciliate Carchesium polypinum to quantify the degree of genetic diversity of four closely related and previously described lineages and to determine whether patterns of genetic differentiation showed geographic partitioning. Using an expanded dataset of 100 isolates and employing the mitochondrial marker cytochrome oxidase c subunit I (cox-1), we enriched the 6 previously identified clades of Carchesium polypinum. We found a large degree of geographic overlap among the different clades (e.g. to the level of range of sampling), but also a spatially restricted clade (e.g. to the level of one river basin). Furthermore, we present evidence of a clear geographic separation in one of the lineages with Canadian and North Carolinian isolates grouping in two distinct clusters.
Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasites cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.
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