Novel virulent and temperate cyanophages predicted to infect Microcoleus associated with anatoxin-producing benthic mats.

Cyanophages are crucial for regulating cyanobacterial populations, but their influence on anatoxin-producing Microcoleus mat dynamics remains unexplored. Here, we use metagenomics to explore phage presence in benthic mats from the Wolastoq|Saint John River (New Brunswick, Canada) and the Eel River (California, USA). We recovered multiple viral-like sequences associated with different putative bacterial hosts, including two cyanophage genomes with apparently different replication strategies. A temperate cyanophage was found integrated in the genomes of Microcoleus sp. 3 recovered from the Eel River and is phylogenetically related to Phormidium phages. We also recovered novel virulent cyanophage genomes from Wolastoq and Eel River mats that were dominated by anatoxin-producing Microcoleus species predicted to be the host. Despite the geographical distance, these genomes have similar sizes (circa 239 kbp) and share numerous orthologous genes with high sequence identity. A considerable reduction of the anatoxin-producing Microcoleus species in Wolastoq mats following the emergence of the virulent phage suggests that phage infections have an important role in limiting the abundance of this toxigenic cyanobacterium and releasing anatoxins into the surrounding water. Our results constitute the first report of cyanophages predicted to infect mat-forming Microcoleus species associated with anatoxin production.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

A personal cost of cheating can stabilize reproductive altruism during the early evolution of clonal multicellularity.

Understanding how cooperation evolved and is maintained remains an important and often controversial topic because cheaters that reap the benefits of cooperation without paying the costs can threaten the evolutionary stability of cooperative traits. Cooperation-and especially reproductive altruism-is particularly relevant to the evolution of multicellularity, as somatic cells give up their reproductive potential in order to contribute to the fitness of the newly emerged multicellular individual. Here, we investigated cheating in a simple multicellular species-the green alga Volvox carteri, in the context of the mechanisms that can stabilize reproductive altruism during the early evolution of clonal multicellularity. We found that the benefits cheater mutants can gain in terms of their own reproduction are pre-empted by a cost in survival due to increased sensitivity to stress. This personal cost of cheating reflects the antagonistic pleiotropic effects that the gene coding for reproductive altruism-regA-has at the cell level. Specifically, the expression of regA in somatic cells results in the suppression of their reproduction potential but also confers them with increased resistance to stress. Since regA evolved from a life-history trade-off gene, we suggest that co-opting trade-off genes into cooperative traits can provide a built-in safety system against cheaters in other clonal multicellular lineages.

High Sequence Divergence but Limited Architectural Rearrangements in Organelle Genomes of Cyanophora (Glaucophyta) Species.

Cyanophora is the glaucophyte model taxon. Following the sequencing of the nuclear genome of C. paradoxa, studies based on single organelle and nuclear molecular markers revealed previously unrecognized species diversity within this glaucophyte genus. Here, we present the complete plastid (ptDNA) and mitochondrial (mtDNA) genomes of C. kugrensii, C. sudae, and C. biloba. The respective sizes and coding capacities of both ptDNAs and mtDNAs are conserved among Cyanophora species with only minor differences due to specific gene duplications. Organelle phylogenomic analyses consistently recover the species C. kugrensii and C. paradoxa as a clade and C. sudae and C. biloba as a separate group. The phylogenetic affiliations of the four Cyanophora species are consistent with architectural similarities shared at the organelle genomic level. Genetic distance estimations from both organelle sequences are also consistent with phylogenetic and architecture evidence. Comparative analyses confirm that the Cyanophora mitochondrial genes accumulate substitutions at 3-fold higher rates than plastid counterparts, suggesting that mtDNA markers are more appropriate to investigate glaucophyte diversity and evolutionary events that occur at a population level. The study of complete organelle genomes is becoming the standard for species delimitation and is particularly relevant to study cryptic diversity in microbial groups.

Seasonality and distribution of cyanobacteria and microcystin toxin genes in an oligotrophic lake of Atlantic Canada.

Cyanotoxins are an emerging threat to freshwater resources worldwide. The most frequently reported cyanotoxins are the microcystins, which threaten the health of humans, wildlife, and ecosystems. Determining the potential for microcystin production is hindered by a lack of morphological features that correlate with microcystin production. However, amplicon-based methods permit the detection of microcystin biosynthesis genes and were employed to assess the toxin potential in Lake Utopia, NB, Canada, an oligotrophic lake that occasionally experiences cyanobacteria blooms. Samples collected at 2 week intervals from June 27th to September 27th, 2016, were screened by polymerase chain reaction (PCR) for the microcystin synthetase E gene (mcyE). The mcyE gene was present in some samples every sampling day, despite microcystin not being detected via ELISA, and was most frequently associated with the larger pore size fractions of the serially filtered samples. Further PCR surveys using primer sets to amplify genus-specific (e.g., Microcystis, Anabaena/Dolichospermum, and Planktothrix) mcyE fragments identified Microcystis as the only taxa in Lake Utopia with toxigenic potential. Sequencing of the 16S rRNA V3-V4 region revealed a community dominated by members of the order Synechococcales (from 38 to 96% relative abundance), but with significant presence of taxa from Cyanobacteriales including Microcystaceae and Nostocaceae. A persistent Microcystis population was detected in samples both testing positive and negative for the mcyE gene, highlighting the importance of identifying cyanotoxin production potential by gene presence and not species identity. To our knowledge, this study represents the first application of amplicon-based approaches to studying toxic cyanobacteria in an understudied region-Atlantic Canada.

The Plastid Genome of Polytoma uvella Is the Largest Known among Colorless Algae and Plants and Reflects Contrasting Evolutionary Paths to Nonphotosynthetic Lifestyles.

The loss of photosynthesis is frequently associated with parasitic or pathogenic lifestyles, but it also can occur in free-living, plastid-bearing lineages. A common consequence of becoming nonphotosynthetic is the reduction in size and gene content of the plastid genome. In exceptional circumstances, it can even result in the complete loss of the plastid DNA (ptDNA) and its associated gene expression system, as reported recently in several lineages, including the nonphotosynthetic green algal genus Polytomella Closely related to Polytomella is the polyphyletic genus Polytoma, the members of which lost photosynthesis independently of Polytomella Species from both genera are free-living organisms that contain nonphotosynthetic plastids, but unlike Polytomella, Polytoma members have retained a genome in their colorless plastid. Here, we present the plastid genome of Polytoma uvella: to our knowledge, the first report of ptDNA from a nonphotosynthetic chlamydomonadalean alga. The P. uvella ptDNA contains 25 protein-coding genes, most of which are related to gene expression and none are connected to photosynthesis. However, despite its reduced coding capacity, the P. uvella ptDNA is inflated with short repeats and is tens of kilobases larger than the ptDNAs of its closest known photosynthetic relatives, Chlamydomonas leiostraca and Chlamydomonas applanata In fact, at approximately 230 kb, the ptDNA of P. uvella represents the largest plastid genome currently reported from a nonphotosynthetic alga or plant. Overall, the P. uvella and Polytomella plastid genomes reveal two very different evolutionary paths following the loss of photosynthesis: expansion and complete deletion, respectively. We hypothesize that recombination-based DNA-repair mechanisms are at least partially responsible for the different evolutionary outcomes observed in such closely related nonphotosynthetic algae.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Marine algae and land plants share conserved phytochrome signaling systems.

Phytochrome photosensors control a vast gene network in streptophyte plants, acting as master regulators of diverse growth and developmental processes throughout the life cycle. In contrast with their absence in known chlorophyte algal genomes and most sequenced prasinophyte algal genomes, a phytochrome is found in Micromonas pusilla, a widely distributed marine picoprasinophyte (<2 µm cell diameter). Together with phytochromes identified from other prasinophyte lineages, we establish that prasinophyte and streptophyte phytochromes share core light-input and signaling-output domain architectures except for the loss of C-terminal response regulator receiver domains in the streptophyte phytochrome lineage. Phylogenetic reconstructions robustly support the presence of phytochrome in the common progenitor of green algae and land plants. These analyses reveal a monophyletic clade containing streptophyte, prasinophyte, cryptophyte, and glaucophyte phytochromes implying an origin in the eukaryotic ancestor of the Archaeplastida. Transcriptomic measurements reveal diurnal regulation of phytochrome and bilin chromophore biosynthetic genes in Micromonas. Expression of these genes precedes both light-mediated phytochrome redistribution from the cytoplasm to the nucleus and increased expression of photosynthesis-associated genes. Prasinophyte phytochromes perceive wavelengths of light transmitted farther through seawater than the red/far-red light sensed by land plant phytochromes. Prasinophyte phytochromes also retain light-regulated histidine kinase activity lost in the streptophyte phytochrome lineage. Our studies demonstrate that light-mediated nuclear translocation of phytochrome predates the emergence of land plants and likely represents a widespread signaling mechanism in unicellular algae.

Nucleotide substitution analyses of the glaucophyte Cyanophora suggest an ancestrally lower mutation rate in plastid vs mitochondrial DNA for the Archaeplastida.

A lot is known about the evolution and architecture of plastid, mitochondrial, and nuclear genomes, but surprisingly little is known about their relative rates of mutation. Most available relative-rate data come from seed plants, which, with few exceptions, have a mitochondrial mutation rate that is lower than those of the plastid and nucleus. But new findings from diverse plastid-bearing lineages have shown that for some eukaryotes the mitochondrial mutation rate is an order of magnitude greater than those of the plastid and nucleus. Here, we explore for the first time relative rates of mutation within the Glaucophyta-one of three main lineages that make up the Archaeplastida (or Plantae sensu lato). Nucleotide substitution analyses from distinct isolates of the unicellular glaucophyte Cyanophora paradoxa reveal 4-5-fold lower rates of mutation in the plastid and nucleus than the mitochondrion, which is similar to the mutational pattern observed in red algae and haptophytes, but opposite to that of seed plants. These data, together with data from previous reports, suggest that for much of the known photosynthetic eukaryotic diversity, plastid DNA mutations occur less frequently than those in mitochondrial DNA.

Molecular markers from different genomic compartments reveal cryptic diversity within glaucophyte species.

Glaucophytes are the least studied of the three major Archaeplastida (Plantae sensu lato) lineages. It has been largely recognized that comprehensive investigations of glaucophyte genetic and species diversity will shed light on the early evolution of photosynthetic eukaryotes. Here we used molecular phylogenetics and genetic distance estimations of diverse molecular markers to explore strain and species diversity within the glaucophyte genera Cyanophora and Glaucocystis. Single gene and concatenated maximum likelihood analyses of markers from three different genetic compartments consistently recovered similar intrageneric genetic groups. Distance analyses of plastid (psbA and rbcL) and mitochondrial (cob and cox1) genes, and the nuclear internal transcribed spacer (ITS) region, revealed substantial genetic divergence between some Cyanophora paradoxa and Glaucocystis nostochinearum strains. The genetic distances estimated between some glaucophyte strains currently considered the same species are similar or greater than divergence values calculated between different species in other unicellular algae, such as certain green algae and diatoms. The analyzed molecular markers are prospective candidates for future studies of species diversity in glaucophytes. Overall, our results unveil previously unrecognized cryptic diversity within Cyanophora and Glaucocystis species.

Community dynamics of microbial eukaryotes in intertidal mudflats in the hypertidal Bay of Fundy.

Protists (microbial eukaryotes) are a critically important but understudied group of microorganisms. They are ubiquitous, represent most of the genetic and functional diversity among eukaryotes, and play essential roles in nutrient and energy cycling. Yet, protists remain a black box in marine sedimentary ecosystems like the intertidal mudflats in the Bay of Fundy. The harsh conditions of the intertidal zone and high energy nature of tides in the Bay of Fundy provide an ideal system for gaining insights into the major food web players, diversity patterns and potential structuring influences of protist communities. Our 18S rDNA metabarcoding study quantified seasonal variations and vertical stratification of protist communities in Bay of Fundy mudflat sediments. Three 'SAR' lineages were consistently dominant (in terms of abundance, richness, and prevalence), drove overall community dynamics and formed the core microbiome in sediments. They are Cercozoa (specifically thecate, benthic gliding forms), Bacillariophyta (mainly cosmopolitan, typically planktonic diatoms), and Dinophyceae (dominated by a toxigenic, bloom-forming species). Consumers were the dominant trophic functional group and were comprised mostly of eukaryvorous and bacterivorous Cercozoa, and omnivorous Ciliophora, while phototrophs were dominated by Bacillariophyta. The codominance of Apicomplexa (invertebrate parasites) and Syndiniales (protist parasites) in parasite assemblages, coupled with broader diversity patterns, highlighted the combined marine and terrestrial influences on microbial communities inhabiting intertidal sediments. Our findings, the most comprehensive in a hypertidal benthic system, suggest that synergistic interactions of both local and regional processes (notably benthic-pelagic coupling) may drive heterogenous microbial distribution in high-energy coastal systems.

Genomic characterization of coexisting anatoxin-producing and non-toxigenic Microcoleus subspecies in benthic mats from the Wolastoq, New Brunswick, Canada.

The presence of toxigenic benthic cyanobacteria in riverine ecosystems is an increasing concern around the world. In 2018, the death of three dogs along the Wolastoq (also known as the Saint John River) in New Brunswick, Canada, was attributed to anatoxin exposure after they ingested benthic microbial mats found along the shore. Here, we shotgun sequenced the DNA of 15 non-axenic cyanobacterial isolates derived from four anatoxin-containing benthic mat samples associated with the dog deaths. Anatoxins were produced by some of the isolates, but not all. We retrieved near-complete Microcoleus metagenome-assembled genomes (MAGs) from the isolates that are closely related to anatoxin-producing Microcoleus from the Cardrona River (New Zealand), although the Microcoleus MAGs from the Wolastoq varied in the presence/absence of the anatoxin-a biosynthesis cluster. Sequence similarity at the genomic level suggests that toxigenic and non-toxigenic Microcoleus MAGs from the Wolastoq belong to the same species but are separate subspecies. The toxigenic and nontoxic Wolastoq Microcoleus subspecies coexisted in the mat samples in similar relative abundance. Overall genomic comparisons revealed that toxigenic Microcoleus MAGs are longer and code for more accessory genes than their non-toxigenic relatives, suggesting a differential responsiveness to changing environments, stress conditions and nutrient availability.

The two-component regulatory system CenK-CenR regulates expression of a previously uncharacterized protein required for salinity and oxidative stress tolerance in Sinorhizobium meliloti.

Genes of unknown function constitute a considerable fraction of most bacterial genomes. In a Tn5-based search for stress response genes in the nitrogen-fixing facultative endosymbiont Sinorhizobium (Ensifer) meliloti, we identified a previously uncharacterized gene required for growth on solid media with increased NaCl concentrations. The encoded protein carries a predicted thioredoxin fold and deletion of the gene also results in increased sensitivity to hydrogen peroxide and cumene hydroperoxide. We have designated the gene srlA (stress resistance locus A) based on these phenotypes. A deletion mutant yields phenotypic revertants on high salt medium and genome sequencing revealed that all revertants carry a mutation in genes homologous to either cenK or cenR. srlA promoter activity is abolished in these revertant host backgrounds and in a strain carrying a deletion in cenK. We also observed that the srlA promoter is autoregulated, displaying low activity in a wildtype (wt) host background and high activity in the srl deletion mutant background. The srlA promoter includes a conserved inverted repeat directly upstream of the predicted -35 subsequence. A mutational analysis demonstrated that the site is required for the high promoter activity in the srlA deletion background. Electromobility shift assays using purified wildtype CenR response regulator and a D55E phosphomimetic derivative suggest this protein acts as a likely Class II activator by binding promoter DNA. These results document the first identified CenK-CenR regulon member in S. meliloti and demonstrate this two-component regulatory system and gene srlA influences cellular growth and persistence under certain stress-inducing conditions.

The plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architectures after the loss of photosynthesis.

The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free-living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadaleans are surprisingly diverse in architecture, including highly expanded forms (Polytoma uvella and Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62-27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62-1c), each representing independent losses of photosynthesis within the Chlamydophyceae. The Hm. oviformis ptDNA is moderately sized (132 kb) with a reduced gene complement (but still encoding the ATPase subunits) and is in fact smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11-48b (198.3 kb). The Hg. fusiforme plastome, however, is the largest yet observed in nonphotosynthetic plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the nonphotosynthetic lineage of most recent origin among known colorless Chlamydophyceae. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free-living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic.

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.

Differential gene retention in plastids of common recent origin.

The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote-eukaryote (primary) endosymbiosis that occurred approximately 60 mya. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence with the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus-Prochlorococcus group, and the cyanobacterial provenance of all plastid genes suggests that these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/dS ratios <<1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution.

Winogradsky columns as a strategy to study typically rare microbial eukaryotes.

Winogradsky columns have been widely used to study soil microbial communities, but the vast majority of those investigations have focused on the ecology and diversity of bacteria. In contrast, microbial eukaryotes (ME) have been regularly overlooked in studies based on experimental soil columns. Despite the recognized ecological relevance of ME in soil communities, investigations focused on ME diversity and the abundance of certain groups of interest are still scarce. In the present study, we used DNA metabarcoding (high-throughput sequencing of the V4 region of the 18S rRNA locus) to survey the ME diversity and abundance in an experimental Winogradsky soil column. Consistent with previous surveys in natural soils, our survey identified members of Cercozoa (Rhizaria; 31.2%), Apicomplexa and Ciliophora (Alveolata; 12.5%) as the predominant ME groups, but at particular depths we also detected the abundant presence of ME lineages that are typically rare in natural environments, such as members of the Vampyrellida (Rhizaria) and Breviatea (Amorphea). Our survey demonstrates that experimental soil columns are an efficient enrichment-culture approach that can enhance investigations about the diversity and ecology of ME in soils.

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii.

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.

A single origin of the photosynthetic organelle in different Paulinella lineages.

BACKGROUND: Gaining the ability to photosynthesize was a key event in eukaryotic evolution because algae and plants form the base of the food chain on our planet. The eukaryotic machines of photosynthesis are plastids (e.g., chloroplast in plants) that evolved from cyanobacteria through primary endosymbiosis. Our knowledge of plastid evolution, however, remains limited because the primary endosymbiosis occurred more than a billion years ago. In this context, the thecate "green amoeba" Paulinella chromatophora is remarkable because it very recently (i.e., minimum age of approximately 60 million years ago) acquired a photosynthetic organelle (termed a "chromatophore"; i.e., plastid) via an independent primary endosymbiosis involving a Prochlorococcus or Synechococcus-like cyanobacterium. All data regarding P. chromatophora stem from a single isolate from Germany (strain M0880/a). Here we brought into culture a novel photosynthetic Paulinella strain (FK01) and generated molecular sequence data from these cells and from four different cell samples, all isolated from freshwater habitats in Japan. Our study had two aims. The first was to compare and contrast cell ultrastructure of the M0880/a and FK01 strains using scanning electron microscopy. The second was to assess the phylogenetic diversity of photosynthetic Paulinella to test the hypothesis they share a vertically inherited plastid that originated in their common ancestor. RESULTS: Comparative morphological analyses show that Paulinella FK01 cells are smaller than M0880/a and differ with respect to the number of scales per column. There are more distinctive, multiple fine pores on the external surface of FK01 than in M0880/a. Molecular phylogenetic analyses using multiple gene markers demonstrate these strains are genetically distinct and likely comprise separate species. The well-supported monophyly of the Paulinella chromatophora strains analyzed here using plastid-encoded 16S rRNA suggests strongly that they all share a common photosynthetic ancestor. The strain M0880/a is most closely related to Japanese isolates (Kanazawa-1, -2, and Kaga), whereas FK01 groups closely with a Kawaguchi isolate. CONCLUSION: Our results indicate that Paulinella chromatophora comprises at least two distinct evolutionary lineages and likely encompasses a broader taxonomic diversity than previously thought. The finding of a single plastid origin for both lineages shows these taxa to be valuable models for studying post-endosymbiotic cell and genome evolution.

Cyanobacterial contribution to algal nuclear genomes is primarily limited to plastid functions.

A single cyanobacterial primary endosymbiosis that occurred approximately 1.5 billion years ago is believed to have given rise to the plastid in the common ancestor of the Plantae or Archaeplastida--the eukaryotic supergroup comprising red, green (including land plants), and glaucophyte algae. Critical to plastid establishment was the transfer of endosymbiont genes to the host nucleus (i.e., endosymbiotic gene transfer [EGT]). It has been postulated that plastid-derived EGT played a significant role in plant nuclear-genome evolution, with 18% (or 4,500) of all nuclear genes in Arabidopsis thaliana having a cyanobacterial origin with about one-half of these recruited for nonplastid functions. Here, we determine whether the level of cyanobacterial gene recruitment proposed for Arabidopsis is of the same magnitude in the algal sisters of plants by analyzing expressed-sequence tag (EST) data from the glaucophyte alga Cyanophora paradoxa. Bioinformatic analysis of 3,576 Cyanophora nuclear genes shows that 10.8% of these with significant database hits are of cyanobacterial origin and one-ninth of these have nonplastid functions. Our data indicate that unlike plants, early-diverging algal groups appear to retain a smaller number of endosymbiont genes in their nucleus, with only a minor proportion of these recruited for nonplastid functions.