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Research Article

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

Takuro Nakayama, Ryoma Kamikawa, Goro Tanifuji, Yuichiro Kashiyama, Naohiko Ohkouchi, John M. Archibald, and Yuji Inagaki
  1. aCenter for Computational Sciences, University of Tsukuba, Tsukuba 305-8577, Japan;
  2. bDepartment of Biochemistry and Molecular Biology, Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Dalhousie University, Halifax, NS, Canada B3H 4R2;
  3. cNational Institute for Environmental Studies, Tsukuba 305-8506, Japan;
  4. dGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan;
  5. eGraduate School of Global Environmental Studies, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan;
  6. fJapan Agency for Marine-Earth Science and Technology, Yokosuka 237-0061, Japan;
  7. gDepartment of Environmental and Biological Chemistry, Fukui University of Technology, Fukui 910-8505, Japan; and
  8. hPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Chiyoda 153-8902, Japan

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PNAS August 5, 2014 111 (31) 11407-11412; first published July 21, 2014; https://doi.org/10.1073/pnas.1405222111
Takuro Nakayama
aCenter for Computational Sciences, University of Tsukuba, Tsukuba 305-8577, Japan;
bDepartment of Biochemistry and Molecular Biology, Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Dalhousie University, Halifax, NS, Canada B3H 4R2;
cNational Institute for Environmental Studies, Tsukuba 305-8506, Japan;
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  • For correspondence: ntakuro@ccs.tsukuba.ac.jp
Ryoma Kamikawa
dGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan;
eGraduate School of Global Environmental Studies, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan;
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Goro Tanifuji
bDepartment of Biochemistry and Molecular Biology, Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Dalhousie University, Halifax, NS, Canada B3H 4R2;
dGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan;
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Yuichiro Kashiyama
fJapan Agency for Marine-Earth Science and Technology, Yokosuka 237-0061, Japan;
gDepartment of Environmental and Biological Chemistry, Fukui University of Technology, Fukui 910-8505, Japan; and
hPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Chiyoda 153-8902, Japan
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Naohiko Ohkouchi
fJapan Agency for Marine-Earth Science and Technology, Yokosuka 237-0061, Japan;
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John M. Archibald
bDepartment of Biochemistry and Molecular Biology, Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity, Dalhousie University, Halifax, NS, Canada B3H 4R2;
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Yuji Inagaki
aCenter for Computational Sciences, University of Tsukuba, Tsukuba 305-8577, Japan;
dGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan;
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  1. Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 24, 2014 (received for review March 21, 2014)

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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.

Abstract

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.

  • photosynthesis
  • organelle evolution
  • pseudogenization

The establishment of two bacterium-derived organelles, mitochondria and plastids, triggered drastic changes in the metabolic capacity, genome architecture, and lifestyle of eukaryotic cells. Mitochondria, energy-producing organelles ubiquitously found in eukaryotic cells, can be traced back to a single endosymbiosis between the ancestral (i.e., amitochondrial) eukaryote and an α-proteobacterium. The plastids of photosynthetic eukaryotes are the result of an endosymbiosis between a heterotrophic eukaryote and a cyanobacterium, an event that took place after the mitochondrial endosymbiosis (1⇓⇓–4). Determining the evolutionary events that led to the origins of these organelles is key to understanding the evolution of eukaryotic cells and their genomes. However, these two endosymbioses occurred more than 1 billion years ago (2⇓⇓⇓–6), and present-day organisms provide limited information with which to explore the early stages of the transformation of an endosymbiotic bacterium into a fully integrated eukaryotic organelle.

Diatom species belonging to the family Rhopalodiaceae are unique among eukaryotes in possessing cyanobacterium-derived intracellular structures termed “spheroid bodies” (7), in addition to plastids and mitochondria. Phylogenetic analyses have shown that the spheroid bodies of rhopalodiacean diatoms originated from a single nitrogen-fixing cyanobacterium closely related to Cyanothece spp., which is distinct from the cyanobacterium that gave rise to the ancestral plastid (8, 9). Significantly, the spheroid bodies are considered obligate endosymbionts, as these structures are inseparable from the host (i.e., diatom) cells, and passed to daughter cells during host cell division (8, 10). The establishment of this diatom–cyanobacterium endosymbiosis can be traced back to the middle Miocene epoch ∼12 Ma (9), which is much more recent than the birth of mitochondria or plastids. Nitrogen fixation is predicted to be an important cellular function carried out by the spheroid bodies, as rhopalodiacean diatom cells can grow in media containing no nitrogen source (7). Indeed, previous studies experimentally confirmed nitrogenase activity in Rhopalodia gibba (8, 11), and genes involved in nitrogen fixation were found in partial genomic data from the spheroid body of R. gibba (RgSB) (12). Interestingly, despite its cyanobacterial ancestry, the spheroid body lacks chlorophyll autofluorescence (13), and photosynthetic genes in RgSB were predicted to be nonfunctional (12), suggesting that the symbiotic cyanobacteria are no longer capable of carrying out photosynthesis and are metabolically dependent on their host cells. Nonetheless, spheroid bodies still retain thylakoid membranes (7), the function of which is unclear, and there remains a possibility of retention of photosynthetic electron transport-related metabolism.

Present knowledge supports the idea that spheroid bodies depend on, and are controlled by, their host cells. The endosymbiotic relationship between rhopalodiacean diatoms and their spheroid bodies, as well as that between the testate amoeba Paulinella chromatophora and its cyanobacterium-derived photosynthetic organelle (14⇓–16), thus provides a unique opportunity to investigate how an endosymbiotic bacterium integrates with a eukaryotic cell at the biochemical and genetic level (9, 12, 17, 18). Toward the goal of understanding how, and the extent to which, spheroid bodies are integrated into their diatom hosts, we determined the complete genome sequence of the spheroid body in the rhopalodiacean diatom Epithemia turgida. The spheroid body genome was found to be considerably reduced compared with its most closely related free-living cyanobacteria, and, remarkably, has already lost key metabolic capacities, including photosynthesis.

Results and Discussion

The Spheroid Body Genome Is Reduced in Size and Gene Content.

The spheroid bodies in a clonal culture of E. turgida were isolated by discontinuous density Percoll gradient centrifugation. DNA extracted from the spheroid body-rich fraction was subjected to whole-genome amplification followed by 454 and Illumina sequencing. Sequence assembly was carried out by a combination of Velvet (19) and GS de novo assembler software, followed by PCR-based contig gap filling (Sanger sequencing). We obtained a single circular chromosome of 2.79 Mbp with a G+C content of 33.4% (Fig. 1). The genome has 39 tRNA genes (which can translate all 61 aa codons), two identical rRNA gene operons, and 1,720 protein-coding genes (Table 1). We also detected 225 pseudogenes, and no transposable elements were identified. The E. turgida spheroid body (EtSB) genome shares two major syntenic gene clusters with other cyanobacterial genomes—one for nitrogen fixation genes (nif genes) and the other for genes encoding ribosomal proteins. In addition to the EtSB genome, we assembled a 5.7-Kbp fragment with a low G+C content (Dataset S1). Most of the protein-coding genes on this fragment show no specific affinity to homologs of Cyanothece spp., which bear a close affinity to the spheroid bodies in cyanobacterial phylogeny (as detailed later). It is at present unclear whether this 5.7-Kbp fragment is an extrachromosomal element (e.g., plasmid) of EtSB origin; we did not analyze it further.

Fig. 1.
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Fig. 1.

Map of the circular chromosome of the spheroid body of the diatom E. turgida. Outer two circles (dark and light blue) show positions of protein-coding genes on plus and minus strands. The red bars on the third circle indicate pseudogenes. tRNA genes (yellow bars) and rRNA genes (green bars) are displayed on the fourth circle. The innermost circle shows G+C content (window size: 5,000 bp).

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Table 1.

Genome overview of the spheroid body of E. turgida and two closely related cyanobacteria

The spheroid bodies of rhopalodiacean diatoms have been shown to be specifically related to members of the cyanobacterial genus Cyanothece (9) (Fig. S1). Among cyanobacteria most closely related to spheroid bodies, complete genome data are available for three free-living Cyanothece species, strains PCC 8801 (National Center for Biotechnology Information BioProject ID PRJNA59027), PCC 8802 (PRJNA59143), and ATCC 51142 (PRJNA20319) (20, 21), as well as an uncultured oceanic species living symbiotically with a haptophyte alga (UCYN-A) (22, 23). The EtSB genome was found to be smaller in size and lower in G+C content than the free-living Cyanothece strains, which possess genomes of 4.68–5.36 Mbp in size and 37.9–39.8% G+C content (Table 1). Consistent with the observed differences in genome size, we found a marked difference in the number of ORFs between the EtSB genome and its free-living relatives. Whereas only 1,720 ORFs were identified in the EtSB genome, the genomes of the three free-living Cyanothece strains possess 4,367–5,304 ORFs. Moreover, 54.5% of the ORFs on the EtSB genome could be assigned to functional categories in Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO), whereas only 32.6–38.0% of the ORFs in the free-living Cyanothece strains could be given a functional designation. Overall, these data suggest that the EtSB genome has undergone significant reductive evolution, as seen in other obligate bacterial symbionts (15, 24⇓–26). Furthermore, the presence of numerous pseudogenes implies that genome reduction is still ongoing in the genome.

The partial nature of the R. gibba spheroid body genomic data obtained by Kneip et al. (12) precluded whole genome-scale comparisons between the spheroid body and free-living cyanobacteria, or between the spheroid body and other symbiotic cyanobacteria. Our KO-based analysis of the ORFs in the complete EtSB genome revealed that the spheroid body has already lost a large part of the metabolic functions presumed to have been present in its free-living ancestors. The numbers of KO IDs assigned to each predicted protein set for Cyanothece spp. ATCC 51142, PCC 8801, and PCC 8802 are 1,309, 1,325, and 1,318, respectively. In sharp contrast, only 849 KO IDs could be assigned to EtSB proteins, and most of these (842 KO IDs) were shared with at least one of the Cyanothece reference genomes (Fig. 2A). Large differences in the number of KO IDs between the EtSB and its free-living relatives were observed in several KEGG functional categories, most notably “energy metabolism,” “metabolism of cofactors and vitamins,” “carbohydrate metabolism,” “membrane transport,” and “signal transduction” (Fig. 2B). For instance, the EtSB genome possesses only half of the IDs present in sequenced Cyanothece genomes in the category of energy metabolism, mostly the result of a reduction in energy production abilities (e.g., photosynthesis, as detailed later). To the extent that the KO ID repertoire represents the metabolism of an organism, the EtSB genome has significantly reduced its metabolic capacity from its cyanobacterial progenitor, which is likely to have had a similar set of genes to strain PCC 8801, PCC 8802, or ATCC 51142.

Fig. 2.
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Fig. 2.

KO-based comparison between free-living Cyanothece strains and cyanobacterial symbionts. (A) Venn diagram of KO ID repertoires of the spheroid body of Epithemia turgida and Cyanothece spp. strain PCC 8801, PCC 8802, and ATCC 51142. The numbers in parentheses indicate the total number of unique KO IDs in each genome. (B) Comparison of KO ID numbers between the EtSB, PCC 8801, PCC 8802, and ATCC 51142. Each bar indicates numbers of unique KO IDs in each functional category. (C) Venn diagram of KO ID repertoires of the EtSB, UCYN-A, R. intracellularis and ‘N. azollae.’ (D) Comparison of KO ID numbers between the EtSB, UCYN-A, R. intracellularis, and ‘N. azollae' in functional categories.

In addition to the EtSB, two complete genome sequences are available for cyanobacterial nitrogen-fixing symbionts: ‘Nostoc azollae,’ which is a vertically inherited but extracellular symbiont of the small heterosporous water fern Azolla filiculoides (27), and the uncultured cyanobacterium UCYN-A, which is thought to have an extra- and/or intracellular symbiotic relationship with haptophyte algae (22, 23, 28). Additionally, there is a near-complete genome sequence for another cyanobacterial symbiont, Richelia intracellularis, which has a mutually dependent relationship with a diatom distantly related to rhopalodiacean species (29). Whereas the genomes of ‘N. azollae’ and R. intracellularis are larger than the EtSB genome (5.49 Mbp and 3.24 Mbp, respectively; Table S1), UCYN-A has a severely reduced genome of 1.44 Mbp, nearly half the size of the EtSB genome. As in EtSB, the UCYN-A, R. intracellularis, and ‘N. azollae’ genomes have smaller KO ID repertoires (683, 965, and 1,084, respectively; Fig. 2C and Table S1) than the free-living Cyanothece spp., which have >1,300 KO IDs (Fig. 2A). In the functional categories of energy metabolism and metabolism of cofactors and vitamins, UCYN-A and EtSB exhibit similar reductive trends compared with ‘N. azollae’ and R. intracellularis (Fig. 2D). Nevertheless, UCYN-A has fewer KO IDs than does EtSB in other functional categories, such as “carbohydrate metabolism,” “amino acid metabolism,” and “nucleotide metabolism,” suggesting that the genome of this uncultured cyanobacterial symbiont has experienced more severe reduction in terms of genome size and metabolic capacity than that of EtSB, R. intracellularis, and ‘N. azollae’ (Fig. 2 C and D and Table S1).

Spheroid Bodies Are Nonphotosynthetic Cyanobacterial Obligate Endosymbionts.

To our knowledge, the complete EtSB genome provides us with the first opportunity to predict the entire set of metabolic pathways carried out in the spheroid body (see Fig. 4). Pioneering studies detected no chlorophyll autofluorescence (13), and inactivated photosynthetic genes were found in a partially sequenced spheroid body genome (12). However, given the prominent thylakoid membranes of spheroid bodies (7), it was difficult to exclude the existence of facultative photosynthetic capacity of the endosymbiont based solely on the partial genome sequence of RgSB or microscopic observations. Analysis of the complete genome sequence of EtSB presented herein allows us to conclude that the cyanobacterial endosymbionts of rhopalodiacean diatoms truly lack photosynthetic ability. In the EtSB genome, genes for photosynthetic components such as photosystems I and II and phycobilisomes (the main peripheral antennae of cyanobacteria) were absent or clearly nonfunctional (Fig. 3). We also found that genes for the entire chlorophyll synthesis pathway, which converts protoporphyrin IX to chlorophyll a, appear to be inactivated by pseudogenization (Fig. S2). All things considered, the complete spheroid body genome sequence provides concrete and comprehensive evidence for the loss of photosynthesis. To our knowledge, the EtSB is the only known example of a nonphotosynthetic cyanobacterium, free-living or symbiotic.

Fig. 3.
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Fig. 3.

Schematic representation of the predicted electron flow in the thylakoid membrane of the spheroid body. Dashed red arrows indicate deduced electron flow. Complexes predicted to be inactivated by gene loss and/or pseudogenization are shown by dashed lines. Yellow- and blue-colored complexes are specific to respiration and photosynthesis, respectively, whereas those in green are involved in respiration and photosynthesis. COX, terminal cytochrome c oxidase; cyt b6f, cytochrome b6f complex; FNR, ferredoxin-NADP+ reductase; Fd, ferredoxin; Hup, uptake hydrogenase; NDH, type 1 NADPH dehydrogenase; PC, plastocyanin; PBS, phycobilisome; PQ, plastoquinone; PS I, photosystem I; PS II, photosystem II; SDH, succinate dehydrogenase.

Fig. 4.
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Fig. 4.

Overview of the deduced metabolism of the spheroid body in E. turgida. Single arrows indicate metabolic reactions and pathways involving multiple steps are represented by double arrows. Amino acids and nucleotides are highlighted by orange. Substances of particular interest are in bold. Question marks highlight pathways that are uncertain even among related Cyanothece strains. Transporter types: single oval, P-type ATPase transporter; oval coupled with small oval, ABC-transporters; single rounded rectangle, channels or secondary transporters. Ac-[acp], acetyl-[acyl-carrier protein]; Ac-CoA, acetyl-CoA; 1,3BPG, 1,3-bisphosphoglycerate; AICAR, 5-aminoimidazole-4-carboxamide ribotide; AIR, 5-aminoimidazole ribotide; APS, adenosine 5′-phosphosulfate; COX, terminal cytochrome c oxygenase; cyt b6f, cytochrome b6f complex; DHAP, dihydroxyacetone phosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; Fruc, fructose; GAP, glyceraldehyde-3-phosphate; Gluc, glucose; Mal-[acp], malonyl-[acyl-carrier protein]; Mal-CoA, malonyl CoA; NDH, type 1 NADPH dehydrogenase; PC, plastocyanin; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; PRPP, 5-phosphoribosyl 1-pyrophosphate.

Despite the absence of a photosynthetic apparatus, a cytochrome b6f complex, plastocyanin, and ATP synthase are most likely functional on the spheroid body thylakoid membranes, as intact ORFs for such proteins reside in the EtSB genome (Fig. 3). In cyanobacteria, the aforementioned set of proteins is involved in respiration together with NADH dehydrogenase and cytochrome c oxidase, in addition to photosynthesis (30). Thus, we predict that the EtSB still synthesizes ATP by an electrochemical proton gradient. The uptake hydrogenase (Hup, encoded by hupSL) (31), which can recycle dihydrogens produced through nitrogen fixation as electron donors, may be involved in the process described earlier as an additional supplier of electrons (Fig. 3). Therefore, although the spheroid body lacks photosynthetic capacity, its thylakoid membranes may be retained as the site of an intact respiratory chain. Furthermore, it is possible that the activity of cytochrome c oxidase in the spheroid body serves to protect extremely oxygen-sensitive nitrogenases by reducing oxygen molecules to water.

To gain insight into the energy-metabolic dependence of the spheroid body on its diatom host, we searched the EtSB genome for genes encoding enzymes involved in the Calvin and tricarboxylic acid (TCA) cycles. The Calvin cycle appears to be incomplete (Fig. S3). In particular, ORFs for the three subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL, rbcS, and rbcX) were found to be disrupted by stop codons, suggesting that the endosymbiont cannot fix carbon dioxide through the Calvin cycle. As the EtSB still possesses apparently functional genes for enzymes to catabolize carbohydrates (e.g., enzymes involved in the pentose phosphate pathway and glycolysis, except phosphofructokinase), the endosymbiont likely energetically depends on sugars that are “extracellularly” supplied from the host. This idea is also supported by the presence of a gene encoding an ABC-transporter for sugars in the EtSB genome (Fig. 4), despite the fact that the total repertoire of transporter genes is highly reduced compared with its free-living relatives (Fig. 2B, “membrane transport”). Additionally, the TCA cycle was found to be incomplete in the EtSB (Fig. 4 and Fig. S4); only intact ORFs for citrate synthase, aconitase, isocitrate dehydrogenase, and fumarase were identified. The TCA cycle in the spheroid body is thus unlikely to function in ATP synthesis, but the products from the incomplete cycle are likely used as substrates for the biosynthesis of certain amino acids (oxaloacetate and 2-oxoglutarate are required for biosynthesis of five and four amino acids, respectively; Fig. 4). The incomplete nature of the TCA cycle and the Calvin cycle deduced from the genome data strongly support the obligate endosymbiosis between spheroid bodies and rhopalodiacean diatoms. It is conceivable that some or all of the missing enzymes in the EtSB are imported from the host cell. Regardless, the obligate nature of the endosymbiosis would not be changed, as the host cell would still be required to complete the two pathways in the endosymbiont.

Amino acid biosynthetic pathways are often partially or entirely discarded in bacterial endosymbionts (15, 22, 25, 26, 32). In sharp contrast, the EtSB genome retains seemingly functional genes for the biosynthesis of all protein amino acids (Fig. 4). Likewise, the biosynthesis of purines, pyrimidines, and most cofactors [e.g., FMN, FAD, NAD(P)+, CoA, heme, and thiamine] is likely to be operating in the EtSB.

The key characteristics of the EtSB described earlier—(i) absence of photosynthesis, (ii) incomplete TCA cycle, and (iii) intact biosynthetic pathways for protein amino acids, nucleotides, and cofactors—places the spheroid bodies of rhopalodiacean diatoms on a distinct evolutionary trajectory from that of UCYN-A, which discarded the entire TCA cycle, and several biosynthetic pathways for amino acids and purine nucleotides, and indeed any endosymbiotic bacteria known to date.

Is the Spheroid Body Becoming a Nitrogen Fixing Organelle?

Nitrogenase activity has been experimentally confirmed in R. gibba cells (8, 11). To validate the nitrogen-fixing capacity of E. turgida experimentally, E. turgida cells were cultured in the presence of 15N2 gas, followed by analysis of the isotopic composition of chlorophyll a. Incorporation of isotopic nitrogen in chlorophyll a was detected (Table S2), indicating that this diatom species can indeed use gaseous nitrogen. A nearly complete set of nif genes for nitrogen fixation was identified in the EtSB genome, which is homologous to the gene set found in a 51-Kbp genome fragment of the RgSB (12). The composition and order of the nif genes were almost identical between the spheroid body genomes of E. turgida and R. gibba, with the exception of the nifU gene encoding a scaffold protein for [Fe2S2] cluster assembly. The partial genome of the RgSB harbors an ORF for NifU, which is highly truncated compared with other homologs (12), whereas the orthologous ORF in the EtSB genome is disrupted by multiple in-frame stop codons. We predict that the EtSB uses a NifU-like protein (ETSB_1030) located in a region distinct from the nif gene cluster, instead of the authentic NifU. It is presently not known how the products of nitrogen fixation are transported into the cytosol of the diatom host. Although a gene encoding a putative ammonium transporter persists in the EtSB genome (Fig. 4), its precise function is unclear.

It is also currently unclear how the spheroid body, which lacks a complete TCA cycle and photosynthetic ability (Fig. 3 and Fig. S4), generates sufficient ATP to support an energetically expensive nitrogenase activity (33). If glycolysis and/or the pentose phosphate pathway are the principal electron donors in the spheroid body, sugars or sugar phosphates need to be effectively imported from the host to the endosymbiont. It is also possible that the host supplies ATP to the spheroid bodies, an interesting hypothesis but one that cannot be confirmed or refuted based on the data presently in hand. Characterization of the complete set of spheroid body transporters will be necessary to elucidate the trafficking of metabolites between host and endosymbiont.

Reductive genome evolution has been demonstrated in EtSB, UCYN-A, ‘N. azollae,’ and R. intracellularis (22, 27, 29), but only the EtSB has discarded photosynthetic ability; photosynthesis genes (e.g., those for photosynthetic complexes) persist in the genomes of UCYN-A, ‘N. azollae,’ and R. intracellularis (22, 27, 29). For instance, UCYN-A retains complete gene sets for photosystem I and chlorophyll a synthesis, despite its small genome size (22). Altogether, with its lack of photosynthesis, the spheroid bodies of rhopalodiacean diatoms are highly integrated into their diatom host cells, and indeed distinct from all other cyanobacterial symbionts examined to date. However, based on the EtSB genome data currently in hand, it is not possible to determine whether the spheroid body is a bona fide organelle in the strictest sense. The evolution of protein import machinery, which enables proteins to move from host to endosymbiont, has been proposed as the critical event in the conversion of endosymbionts into organelles (34, 35). To address this issue, it will be important to survey the host diatom nuclear genome for genes possibly encoding proteins transported to, and functioning in, the spheroid body.

Conclusion

We have sequenced the genome of the E. turgida spheroid body, providing important insights into the unique evolutionary status of this nitrogen-fixing intracellular structure. The data strongly suggest that the spheroid bodies of rhopalodiacean diatoms have secondarily lost an important property of cyanobacteria (i.e., photosynthesis), and are unique among prokaryotic symbionts examined to date. However, there is still much to learn about how the diatom cell controls its spheroid bodies. Investigation of this host–symbiont system should provide clues to understanding the general evolutionary processes underlying the conversion of a free-living prokaryote into a fully integrated, host-controlled subcellular entity.

Materials and Methods

Culturing E. turgida Cells and Isolation of the Spheroid Bodies.

E. turgida, isolated from Lake Yunoko, Tochigi, Japan, was grown in CSi-N medium as described by Nakayama et al. (9). Harvested diatom cells were resuspended in PBS solution and mildly broken by vortexing with glass beads. The homogenate containing spheroid bodies was layered onto a discontinuous gradient of 90%, 70%, 60%, and 50% Percoll, and then centrifuged at 12,000 × g for 20 min. Intact spheroid bodies were taken from the boundary between the 60% and 70% Percoll fractions. Although the spheroid body-rich fraction also contained contaminating bacteria present in the E. turgida culture, DNA-containing organelles from the diatom cells (i.e., nuclei, mitochondria and plastids) were not detected under the light microscope. DNA was extracted from the spheroid body-rich fraction using the DNeasy Plant Mini Kit (Qiagen), followed by whole-genome amplification by using the REPLI-g mini kit (Qiagen).

Genome Sequencing and Assembly.

Approximately 10 μg of amplified DNA was sequenced on a Roche 454 GS FLX sequencer with Titanium reagents and an Illumina HiSeq 2000 at Hokkaido System Science. 454 and Illumina sequencing generated ∼195,000 reads (61 Mbp in total) and ∼58 million reads (5.8 Gbp in total), respectively. Illumina reads were assembled into contigs by using Velvet (19), and contigs were combined and assembled together with reads from 454 sequencing using the GS de novo assembler software (Roche Diagnostics). Ten large contigs (>5 Kbp) with high A+T content (>65%) were found in the final contig pool and selected as candidate spheroid body genome sequences. A contig containing the spheroid body rRNA gene operon [identical to a partial sequence already obtained by Nakayama et al. (9)] possessed a relatively low A+T content (∼5.2 Kbp, A+T of 49%) and was identified manually. Gaps between the remaining spheroid body-derived contigs were closed by PCR based on information from mate pairs of paired-end Illumina reads in the flanking regions of contigs. A single circular chromosome of 2.79 Mbp with coverage depth of ∼145× was obtained.

Genome Annotation.

The initial ORF prediction was performed by using GeneMarkS (36). Four short ORFs for genes rpl36, rpmG, petL, and ndhL were identified by tBLASTn searches. tRNA genes were detected by tRNAscan-SE (37), and rRNA genes were predicted based on nucleotide similarity. Putative pseudogenes on the spheroid body genome were identified by tBLASTn by using all proteins encoded in the genome of Cyanothece sp. PCC 8801 as queries with a threshold of 1.0 × 10−10. Coding regions interrupted by stop codons and/or disrupted by frame shifts, as well as severely truncated ORFs, were tagged as putative pseudogenes. A total of 176 ORFs initially predicted by GeneMarkS were also found to correspond to pseudogenes. The genome map was generated by using the DNAplotter (38). The fully annotated genome sequence is available in DNA Data Base in Japan/GenBank/European Molecular Biology Laboratory under accession number AP012549.

KO-Based Analyses.

KO ID assignment was performed for all 1,720 ORFs predicted in the spheroid body genome. The initial assignments were performed by using the KEGG Automatic Annotation Server (39) and then manually refined. A complete list of the KO assignments is provided in Dataset S2. All KO IDs assigned for genes from three Cyanothece strains (PCC 8801, PCC 8802, and ATCC 51142) and two cyanobacterial symbionts (‘N. azollae’ 0708 and UCYN-A) were retrieved from the KEGG database by using the KEGG API (40). KO IDs for protein sequences of R. intracellularis HH01 were assigned by using the KEGG Automatic Annotation Server. KO IDs for pseudogenes in the spheroid body genome were predicted from the KO IDs for their intact homologs in PCC 8801. Metabolic pathways in the EtSB were deduced by using KEGG mapper (41).

Phylogenetic Tree Construction.

Orthologous proteins for putative spheroid body proteins were searched against genome data from eight cyanobacterial strains by using BLASTp with each spheroid body protein as a query. Only top hits from each cyanobacterial strain with E-values of 0.0 were considered as orthologous proteins. A total of 241 orthologous proteins shared by all eight cyanobacterial strains and the EtSB were determined in this manner. The dataset is available upon request. The orthologous proteins were individually aligned by using MUSCLE version 3.6 (42); all positions containing gap(s) were removed. The alignments were concatenated into a single dataset (136,692 sites). Maximum likelihood phylogenetic analysis was performed by using RAxML 8.0.0 (43) under the LG + Γ + F model. Searches for the best trees were conducted starting from 10 random trees, and bootstrap values were obtained with nonparametric bootstrapping by using 100 replicates.

Nitrogen Isotope Tracing Analysis.

E. turgida cells and two diatom strains without spheroid bodies (Cyclotella meneghiniana NIES-805 and Fragilaria capucina NIES-391) were cultured in nitrogen-deficient CSi-N medium as described earlier for 4 d. The 15N2 gas was added to the gaseous phase of each flask on the first and second day of culturing. All three cultures were then collected through a glass filter (GF/F; Whatman). Extraction and isotopic analysis of chlorophyll a from the filters were done by using the procedure reported by Tyler et al. (44).

Acknowledgments

We thank Dr. Fumie Kasai (National Institute for Environmental Studies) for all her help and cooperation. This work was supported by Japan Society for the Promotion of Sciences Grants 22870037 (to T.N.), 24870004 (to R.K.), and 23117006 (to Y.I.); Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Scientific Research on Innovative Areas 3308; and a grant from the Institute for Fermentation (Osaka, Japan) (R.K.). T.N. was a JSPS Postdoctoral Fellow for Research Abroad. J.M.A. is a Senior Fellow of the Canadian Institute for Advanced Research, Program in Integrated Microbial Biodiversity.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: ntakuro{at}ccs.tsukuba.ac.jp.
  • Author contributions: T.N. and Y.I. designed research; T.N., R.K., G.T., Y.K., and N.O. performed research; T.N., R.K., G.T., Y.K., N.O., J.M.A., and Y.I. analyzed data; and T.N., R.K., G.T., Y.K., N.O., J.M.A., and Y.I. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AP012549).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405222111/-/DCSupplemental.

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Genome sequence of a spheroid body
Takuro Nakayama, Ryoma Kamikawa, Goro Tanifuji, Yuichiro Kashiyama, Naohiko Ohkouchi, John M. Archibald, Yuji Inagaki
Proceedings of the National Academy of Sciences Aug 2014, 111 (31) 11407-11412; DOI: 10.1073/pnas.1405222111

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Genome sequence of a spheroid body
Takuro Nakayama, Ryoma Kamikawa, Goro Tanifuji, Yuichiro Kashiyama, Naohiko Ohkouchi, John M. Archibald, Yuji Inagaki
Proceedings of the National Academy of Sciences Aug 2014, 111 (31) 11407-11412; DOI: 10.1073/pnas.1405222111
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