Ryoma Kamikawa

Complete genome of a nonphotosynthetic cyanobacterium in a diatom reveals recent adaptations to an intracellular lifestyle

Significance Members of the diatom family Rhopalodiaceae possess a cyanobacterial endosymbiont called a “spheroid body.” The spheroid body evolved much more recently than did mitochondria or plastids and is predicted to fix nitrogen. Here we present what is, to our knowledge, the first completely sequenced spheroid body genome from a rhopalodiacean diatom. Comparative analyses revealed that the endosymbiont is metabolically reduced, confirming its status as an obligate endosymbiont. The genome possesses genes for nitrogen fixation, and, to our surprise, no essential genes for photosynthesis. Thus, the spheroid body is, to our knowledge, the first known example of a nonphotosynthetic cyanobacterium, free-living or symbiotic. Rhopalodiacean diatoms have the potential to provide unique insight into the evolution of bacterial endosymbionts and their hosts. The evolution of mitochondria and plastids from bacterial endosymbionts were key events in the origin and diversification of eukaryotic cells. Although the ancient nature of these organelles makes it difficult to understand the earliest events that led to their establishment, the study of eukaryotic cells with recently evolved obligate endosymbiotic bacteria has the potential to provide important insight into the transformation of endosymbionts into organelles. Diatoms belonging to the family Rhopalodiaceae and their endosymbionts of cyanobacterial origin (i.e., “spheroid bodies”) are emerging as a useful model system in this regard. The spheroid bodies, which appear to enable rhopalodiacean diatoms to use gaseous nitrogen, became established after the divergence of extant diatom families. Here we report what is, to our knowledge, the first complete genome sequence of a spheroid body, that of the rhopalodiacean diatom Epithemia turgida. The E. turgida spheroid body (EtSB) genome was found to possess a gene set for nitrogen fixation, as anticipated, but is reduced in size and gene repertoire compared with the genomes of their closest known free-living relatives. The presence of numerous pseudogenes in the EtSB genome suggests that genome reduction is ongoing. Most strikingly, our genomic data convincingly show that the EtSB has lost photosynthetic ability and is metabolically dependent on its host cell, unprecedented characteristics among cyanobacteria, and cyanobacterial symbionts. The diatom–spheroid body endosymbiosis is thus a unique system for investigating the processes underlying the integration of a bacterial endosymbiont into eukaryotic cells.

Split Introns in the Genome of Giardia intestinalis Are Excised by Spliceosome-Mediated trans-Splicing

Spliceosomal introns are hallmarks of most eukaryotic genomes and are excised from premature mRNAs by a spliceosome that is among the largest, and most complex, molecular machine in cells. The divergent unicellular eukaryote Giardia intestinalis, the causative agent of giardiasis, also possesses spliceosomes, but only four canonical (cis-spliced) introns have been identified in its genome to date. We demonstrate that this organism has a novel form of spliceosome-mediated trans-splicing of split introns that is essential for generating mature mRNAs for at least two important genes: one encoding a heat shock protein 90 (HSP90), which controls the conformation of a suite of cellular proteins, and the other encoding a dynein molecular motor protein, involved in the motility of eukaryotic flagella. These split introns have properties that distinguish them from other trans-splicing systems known within eukaryotes, suggesting that Giardia independently evolved a unique system to splice split introns.

Development of a quantification assay for the cysts of the toxic dinoflagellate Alexandrium tamarense using real-time polymerase chain reaction

The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.

Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning‐related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.

Direct phylogenetic evidence for lateral transfer of elongation factor-like gene

Genes encoding elongation factor-like (EFL) proteins, which show high similarity to elongation factor-1α (EF-1α), have been found in phylogenetically distantly related eukaryotes. The sporadic distribution of “EFL-containing” lineages within “EF-1α-containing” lineages indirectly, but strongly, suggests lateral gene transfer as the principal driving force in EFL evolution. However, one of the most critical aspects in the above hypothesis, the donor lineages in any putative cases of lateral EFL gene transfer, remained unclear. In this study, we provide direct evidence for lateral transfer of an EFL gene through the analyses of 10 diatom EFL genes. All diatom EFL homologues tightly clustered in phylogenetic analyses, suggesting acquisition of the exogenous EFL gene early in diatom evolution. Our survey additionally identified Thalassiosira pseudonana as a eukaryote bearing EF-1α and EFL genes and secondary EFL gene loss in Phaeodactylum tricornutum, the complete genome of which encodes only the EF-1α gene. Most importantly, the EFL phylogeny recovered a robust grouping of homologues from diatoms, the cercozoan Bigelowiella natans, and the foraminifer Planoglabratella opecularis, with the diatoms nested within the Bigelowiella plus Planoglabratella (Rhizaria) grouping. The particular relationships recovered are further consistent with two characteristic sequence motifs. The best explanation of our data analyses is an EFL gene transfer from a foraminifer to a diatom, the first case in which the donor–recipient relationship was clarified. Finally, based on a reverse transcriptase quantitative PCR assay and the genome information of Thalassiosira and Phaeodactylum, we propose the loss of elongation factor function in Thalassiosira EF-1α.

Analysis of the mitochondrial genome, transcripts, and electron transport activity in the dinoflagellate Alexandrium catenella (Gonyaulacales, Dinophyceae)

The mitochondrial (mt) genomes of dinoflagellates are not completely sequenced due to frequent recombination events resulting in a shortage of information about the dinoflagellate mt genome. To obtain a large amount of information, we characterized 14 polymerase chain reaction (PCR) fragments of more than 27 kb of the mt genome of the toxic dinoflagellate Alexandrium catenella Whedon et Kofoid (Balech) using the cob and cox1 genes, the only identified functional mt genes of A. catenella excluding rRNA fragments. The mt PCR clones encode multiple copies of cytochrome b (cob) and cytochrome c oxidase subunit 1 (cox1) bearing several types of 5′ or 3′ sequences, and two rRNA fragments showing sequence similarity with a large subunit (LSU) rRNA D fragment and LSU RNA2 of apicomplexa. Each mt PCR clone showed different gene arrangements and intergenic sequences suggesting multiple contexts in the mt genome of A. catenella and frequent homologous recombinations. Reverse transcription PCR analysis suggested some types of the multiple copies of cob and cox1 genes are likely non‐transcriptional. Further, A. catenella mt mRNAs lacked in‐frame termination codons and a canonical initiation codon, excluding an ‘atg’ codon in cob mRNA. However, we successfully detected the activity of the electron transport proteins suggesting mt translation requires no canonical initiation and termination codons.

The Origin and Diversification of Mitochondria

Mitochondria are best known for their role in the generation of ATP by aerobic respiration. Yet, research in the past half century has shown that they perform a much larger suite of functions and that these functions can vary substantially among diverse eukaryotic lineages. Despite this diversity, all mitochondria derive from a common ancestral organelle that originated from the integration of an endosymbiotic alphaproteobacterium into a host cell related to Asgard Archaea. The transition from endosymbiotic bacterium to permanent organelle entailed a massive number of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metabolism and reproduction, genome reduction, endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated. Although many insights into this transition have been gained, controversy persists regarding the nature of the original endosymbiont, its initial interactions with the host and the timing of its integration relative to the origin of other features of eukaryote cells. Since the establishment of the organelle, proteins have been gained, lost, transferred and retargeted as mitochondria have specialized into the spectrum of functional types seen across the eukaryotic tree of life.

Proposal of a Twin Aarginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Development of a novel molecular marker on the mitochondrial genome of a toxic dinoflagellate, Alexandrium spp., and its application in single-cell PCR

Although the molecular data currently used for identifying dinoflagellates are generally limited to nuclear ribosomal RNA genes, some dinoflagellates cannot be identified by their gene sequence or morphotype, suggesting that additional effective molecular makers are required. We report here a novel species-specific marker on the mitochondrial (mt) genome of dinoflagellates belonging to six Alexandrium spp., namely, A. tamarense, A. catenella, A. tamiyavanichii, A. affine, A. hiranoi, and A. pseudogonyaulax. This new mt marker was able to clearly differentiate these six species. PCR analysis using a primer set for the A. tamarense-specific sequence confirmed that this sequence is conserved in A. tamarense strains but not in other dinoflagellate species. We also sequenced the mt genome containing the developed molecular marker using a single cell from a field sample, which suggests that this marker is a powerful tool for identifying unculturable dinoflagellates. The sequenced molecular region was also used to identify Alexandrium-like cells isolated from environmental seawater as A. tamarense and A. affine.

Plastid genome-based phylogeny pinpointed the origin of the green-colored plastid in the dinoflagellate Lepidodinium chlorophorum.

Unlike many other photosynthetic dinoflagellates, whose plastids contain a characteristic carotenoid peridinin, members of the genus Lepidodinium are the only known dinoflagellate species possessing green alga-derived plastids. However, the precise origin of Lepidodinium plastids has hitherto remained uncertain. In this study, we completely sequenced the plastid genome of Lepidodinium chlorophorum NIES-1868. Our phylogenetic analyses of 52 plastid-encoded proteins unite L. chlorophorum exclusively with a pedinophyte, Pedinomonas minor, indicating that the green-colored plastids in Lepidodinium spp. were derived from an endosymbiotic pedinophyte or a green alga closely related to pedinophytes. Our genome comparison incorporating the origin of the Lepidodinium plastids strongly suggests that the endosymbiont plastid genome acquired by the ancestral Lepidodinium species has lost genes encoding proteins involved in metabolism and biosynthesis, protein/metabolite transport, and plastid division during the endosymbiosis. We further discuss the commonalities and idiosyncrasies in genome evolution between the L. chlorophorum plastid and other plastids acquired through endosymbiosis of eukaryotic photoautotrophs.