Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion.

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study.

Draft genome of Parageobacillus thermoglucosidasius, a member of hydrogenogenic carbon monoxide utilizers, isolated from a freshwater lake sediment.

Parageobacillus thermoglucosidasius is a facultatively anaerobic thermophile and possesses carbon monoxide dehydrogenase and hydrogenase for carbon monoxide (CO) oxidation and hydrogen production, respectively. In this study, we report a draft genome of P. thermoglucosidasius isolated from a freshwater sediment, expanding our knowledge on the distribution of CO utilizers.

Population-level prokaryotic community structures associated with ferromanganese nodules in the Clarion-Clipperton Zone (Pacific Ocean) revealed by 16S rRNA gene amplicon sequencing.

Although deep-sea ferromanganese nodules are a potential resource for exploitation, their formation mechanisms remain unclear. Several nodule-associated prokaryotic species have been identified by amplicon sequencing of 16S rRNA genes and are assumed to contribute to nodule formation. However, the recent development of amplicon sequence variant (ASV)-level monitoring revealed that closely related prokaryotic populations within an operational taxonomic unit often exhibit distinct ecological properties. Thus, conventional species-level monitoring might have overlooked nodule-specific populations when distinct populations of the same species were present in surrounding environments. Herein, we examined the prokaryotic community diversity of nodules and surrounding environments at the Clarion-Clipperton Zone in Japanese licensed areas by 16S rRNA gene amplicon sequencing with ASV-level resolution for three cruises from 2017 to 2019. Prokaryotic community composition and diversity were distinct by habitat type: nodule, nodule-surface mud, sediment, bottom water and water column. Most ASVs (~80%) were habitat-specific. We identified 178 nodule-associated ASVs and 41 ASVs associated with nodule-surface mud via linear discriminant effect size analysis. Moreover, several ASVs, such as members of SAR324 and Woeseia, were highly specific to nodules. These nodule-specific ASVs are promising targets for future investigation of the nodule formation process.

Transcriptome data sets of free-living diplomonads, Trepomonas sp. and Hexamita sp.

Most species belonging to the diplomonad genera, Trepomonas and Hexamita, are considered to have secondarily adapted to free-living lifestyles from the parasitic ancestor. Here, we report the annotated transcriptome data of Trepomonas sp. NIES-1444 and Hexamita sp. NIES-1440, the analysis of which will provide insights into the lifestyle transitions.

A practical assembly guideline for genomes with various levels of heterozygosity.

Although current long-read sequencing technologies have a long-read length that facilitates assembly for genome reconstruction, they have high sequence errors. While various assemblers with different perspectives have been developed, no systematic evaluation of assemblers with long reads for diploid genomes with varying heterozygosity has been performed. Here, we evaluated a series of processes, including the estimation of genome characteristics such as genome size and heterozygosity, de novo assembly, polishing, and removal of allelic contigs, using six genomes with various heterozygosity levels. We evaluated five long-read-only assemblers (Canu, Flye, miniasm, NextDenovo and Redbean) and five hybrid assemblers that combine short and long reads (HASLR, MaSuRCA, Platanus-allee, SPAdes and WENGAN) and proposed a concrete guideline for the construction of haplotype representation according to the degree of heterozygosity, followed by polishing and purging haplotigs, using stable and high-performance assemblers: Redbean, Flye and MaSuRCA.

Construction of multiple metagenome assembled genomes containing carbon monoxide dehydrogenases from anaerobic carbon monoxide enrichment cultures.

Despite its toxicity to many organisms, including most prokaryotes, carbon monoxide (CO) is utilized by some aerobic and anaerobic prokaryotes. Hydrogenogenic CO utilizers employ carbon monoxide dehydrogenase (CODH) and energy-converting hydrogenase (ECH) to oxidize CO and reduce protons to produce H2. Those prokaryotes constitute a rare biosphere and are difficult to detect even with PCR amplification and with metagenomic analyses. In this study, anaerobic CO-enrichment cultures followed by construction of metagenome assembled genomes (MAGs) detected high-quality MAGs from potential hydrogenogenic CO utilizers. Of 32 MAGs constructed, 5 were potential CO utilizer harboring CODH genes. Of the five MAGs, two were classified into the genus Thermolithobacter on the basis of 16S rRNA sequence identity, related to Carboxydocella tharmautotrophica 41, with an average nucleotide identity (ANI) of approximately 72%. Additionally, two were related to Geoglobus acetivorans with ANI values ranging from 75 to 77% to G. acetivorans SBH6, and one MAG was identified as Desulfotomaculum kuznetsovii with an ANI > 96% to D. kuznetsovii DSM 6115. The two Thermolithobacter MAGs identified in this study contained CODH-ECH gene clusters, and were therefore identified as potential hydrogenogenic CO utilizers. However, these MAGs harbored three CODH gene clusters that showed distinct physiological functions in addition to CODH-ECH gene clusters. In total, the five potential CO utilizer MAGs contained sixteen CODH genes. Among those CODHs, four sets did not cluster with any known CODH protein sequences (with an identity of > 90%), and the CODH database was expanded.

Light-driven Proton Pumps as a Potential Regulator for Carbon Fixation in Marine Diatoms.

Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.

Draft Genome Sequence of Thermolongibacillus altinsuensis Strain B1-1, a Novel Hydrogenogenic CO Oxidizer Isolated from Sediment from Lake Biwa in Japan.

A facultative anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterial strain, B1-1, was isolated from a sediment sample from Lake Biwa, a freshwater lake in Japan. B1-1, which is a novel strain of Thermolongibacillus altinsuensis, is capable of hydrogenogenic CO oxidation. Here, we report the draft genome sequence of B1-1 (2.92 Mbp, with a GC content of 41.3%).

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases.

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO2-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A-G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.

Rappemonads are haptophyte phytoplankton.

Rapidly accumulating genetic data from environmental sequencing approaches have revealed an extraordinary level of unsuspected diversity within marine phytoplankton,1-11 which is responsible for around 50% of global net primary production.12,13 However, the phenotypic identity of many of the organisms distinguished by environmental DNA sequences remains unclear. The rappemonads represent a plastid-bearing protistan lineage that to date has only been identified by environmental plastid 16S rRNA sequences.14-17 The phenotypic identity of this group, which does not confidently cluster in any known algal clades in 16S rRNA phylogenetic reconstructions,15 has remained unknown since the first report of environmental sequences over two decades ago. We show that rappemonads are closely related to a haptophyte microalga, Pavlomulina ranunculiformis gen. nov. et sp. nov., and belong to a new haptophyte class, the Rappephyceae. Organellar phylogenomic analyses provide strong evidence for the inclusion of this lineage within the Haptophyta as a sister group to the Prymnesiophyceae. Members of this new class have a cosmopolitan distribution in coastal and oceanic regions. The relative read abundance of Rappephyceae in a large environmental barcoding dataset was comparable to, or greater than, those of major haptophyte species, such as the bloom-forming Gephyrocapsa huxleyi and Prymnesium parvum, and this result indicates that they likely have a significant impact as primary producers. Detailed characterization of Pavlomulina allowed for reconstruction of the ancient evolutionary history of the Haptophyta, a group that is one of the most important components of extant marine phytoplankton communities.

Highly Reduced Plastid Genomes of the Non-photosynthetic Dictyochophyceans Pteridomonas spp. (Ochrophyta, SAR) Are Retained for tRNA-Glu-Based Organellar Heme Biosynthesis.

Organisms that have lost their photosynthetic capabilities are present in a variety of eukaryotic lineages, such as plants and disparate algal groups. Most of such non-photosynthetic eukaryotes still carry plastids, as these organelles retain essential biological functions. Most non-photosynthetic plastids possess genomes with varied protein-coding contents. Such remnant plastids are known to be present in the non-photosynthetic, bacteriovorous alga Pteridomonas danica (Dictyochophyceae, Ochrophyta), which, regardless of its obligatory heterotrophic lifestyle, has been reported to retain the typically plastid-encoded gene for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). The presence of rbcL without photosynthetic activity suggests that investigating the function of plastids in Pteridomonas spp. would likely bring unique insights into understanding the reductive evolution of plastids, their genomes, and plastid functions retained after the loss of photosynthesis. In this study, we demonstrate that two newly established strains of the non-photosynthetic genus Pteridomonas possess highly reduced plastid genomes lacking rbcL gene, in contrast to the previous report. Interestingly, we discovered that all plastid-encoded proteins in Pteridomonas spp. are involved only in housekeeping processes (e.g., transcription, translation and protein degradation), indicating that all metabolite synthesis pathways in their plastids are supported fully by nuclear genome-encoded proteins. Moreover, through an in-depth survey of the available transcriptomic data of another strain of the genus, we detected no candidate sequences for nuclear-encoded, plastid-directed Fe-S cluster assembly pathway proteins, suggesting complete loss of this pathway in the organelle, despite its widespread conservation in non-photosynthetic plastids. Instead, the transcriptome contains plastid-targeted components of heme biosynthesis, glycolysis, and pentose phosphate pathways. The retention of the plastid genomes in Pteridomonas spp. is not explained by the Suf-mediated constraint against loss of plastid genomes, previously proposed for Alveolates, as they lack Suf genes. Bearing all these findings in mind, we propose the hypothesis that plastid DNA is retained in Pteridomonas spp. for the purpose of providing glutamyl-tRNA, encoded by trnE gene, as a substrate for the heme biosynthesis pathway.

Putative genome features of relic green alga-derived nuclei in dinoflagellates and future perspectives as model organisms.

Nucleomorphs, relic endosymbiont nuclei, have been studied as a model to elucidate the evolutionary process of integrating a eukaryotic endosymbiont into a host cell organelle. Recently, we reported two new dinoflagellates possessing nucleomorphs, and proposed them as new models in this research field based on the following findings: genome integration processes are incomplete, and the origins of the endosymbiont lineages were pinpointed. Here, we focused on the nucleomorph genome features in the two green dinoflagellates and compared them with those of the known nucleomorph genomes of cryptophytes and chlorarachniophytes. All nucleomorph genomes showed similar trends suggesting convergent evolution. However, the number of nucleomorph genes that are unrelated to housekeeping machineries in the two green dinoflagellates are greater than the numbers in cryptophytes and chlorarachniophytes, providing additional evidence that their genome reduction has not progressed much compared with those of cryptophytes and chlorarachniophytes. Finally, potential future work is discussed.

A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems.

BACKGROUND: Plastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems. RESULTS: Here we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids. CONCLUSION: The plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

Fine Structure Observation of Feeding Behavior, Nephroselmis spp.-derived Chloroplast Enlargement, and Mitotic Processes in the Katablepharid Hatena arenicola.

The difficult-to-cultivate katablepharid Hatena arenicola ingests green algae, Nephroselmis spp., and temporarily retains a Nephroselmis-derived cell compartment (kleptochloroplast), including a chloroplast within a phagocytotic vacuole. H. arenicola has a unique life history; during cell division, the Nephroselmis-derived cell compartment is only inherited by one of two daughter cells. However, the detailed morphological transition of the Nephroselmis cell to a kleptochloroplast and the mitotic process of the host cell remain unclear. Herein, we observed feeding behavior, enlargement of the Nephroselmis-derived chloroplast, and mitotic processes in H. arenicola using light and electron microscopy. During feeding behavior, H. arenicola peeled off the cell coverings and flagella of the Nephroselmis cell, which selectively accumulated in a vacuole separate to one containing a Nephroselmis cell body. An obvious nucleolus, but no heterochromatin was observed in the Nephroselmis-derived nucleus during the chloroplast-enlarging process, while compressed heterochromatin was explicitly observed in the nuclei of free-living Nephroselmis cells. The cell membrane of an ingested Nephroselmis cell disintegrated during enlargement of the Nephroselmis-derived chloroplast. The process of mitosis in H. arenicola was very similar to that of other katablepharids and cryptophytes.

Substrate specificity of plastid phosphate transporters in a non-photosynthetic diatom and its implication in evolution of red alga-derived complex plastids.

The triose phosphate transporter (TPT) is one of the prerequisites to exchange metabolites between the cytosol and plastids. In this study, we demonstrated that the four plastid TPT homologues in the non-photosynthetic diatom Nitzschia sp. NIES-3581 were highly likely integrated into plastid envelope membranes similar to counterparts in the model photosynthetic diatom Phaeodactylum tricornutum, in terms of target membranes and C-terminal orientations. Three of the four Nitzschia TPT homologues are capable of transporting various metabolites into proteo-liposomes including triose phosphates (TPs) and phosphoenolpyruvate (PEP), the transport substrates sufficient to support the metabolic pathways retained in the non-photosynthetic diatom plastid. Phylogenetic analysis of TPTs and closely related transporter proteins indicated that diatoms and other algae with red alga-derived complex plastids possess only TPT homologues but lack homologues of the glucose 6-phosphate transporter (GPT), xylulose 5-phosphate transporter (XPT), and phosphoenolpyruvate transporter (PPT). Comparative sequence analysis suggests that many TPT homologues of red alga-derived complex plastids potentially have the ability to transport mainly TPs and PEP. TPTs transporting both TPs and PEP highly likely mediate a metabolic crosstalk between a red alga-derived complex plastid and the cytosol in photosynthetic and non-photosynthetic species, which explains the lack of PPTs in all the lineages with red alga-derived complex plastids. The PEP-transporting TPTs might have emerged in an early phase of endosymbiosis between a red alga and a eukaryote host, given the broad distribution of that type of transporters in all branches of red alga-derived complex plastid-bearing lineages, and have probably played a key role in the establishment and retention of a controllable, intracellular metabolic connection in those organisms.