Abstract 2017

ユーグレノゾア基部から分岐する新奇原生生物のミトコンドリアゲノム （第50回日本原生生物学会及び第1回日本共生学会）

ユーグレノゾアは主にユーグレナ類、ディプロネマ類、キネトプラスト類から構成され、系統毎に特徴的なミトコンドリア（Mt）ゲノム構造の進化が起こったと考えられるが、その過程は未解明である。従ってユーグレノゾアのMtゲノム構造多様化過程の解明には、その祖先的Mtゲノム構造の推測が必要である。新奇原生生物SRT308株は、153遺伝子連結系統解析によってユーグレノゾア基部から分岐することが示された。そのため本種はユーグレノゾア祖先的Mtゲノムの特徴を残していることが示唆された。そこでSRT308 Mtゲノムの解読を行ったところ、61キロ塩基長の環状分子であること、そのタンパク質遺伝子組成はユーグレノゾア主要生物種のMtゲノムのタンパク質遺伝子組成の和集合に極めて近いことが判明した。従って、SRT308を含むユーグレノゾアの共通祖先は、少なくとも19個のタンパク質遺伝子を持つ環状Mtゲノムをもっていたと推測できる。

Mitochondrial genome of a protist branched at the base of Euglenozoa. (MGE)

Euglenozoa is a large protist assemblage comprising three major subgroups, namly euglenids, diplonemids and kinetoplastea. Members of this assemblage are known for a large diversity in mitochondrial (mt) genome structure. The mt genome of Euglena gracilis, a representative of euglenids, are likely composed of multiple lineage chromosomes, which harbor only 6 protein-coding genes and fragmented ribosomal RNA genes. No RNA editing has been detected in the transcripts from Euglena mt genome. Diplonemid mitochondrial genomes were found to comprise multiple minicircular chromosomes, each of which carries a partial gene fragment, and initial transcripts from the mt minicircles receive both trans-splicing and RNA editing to yield mature mRNAs. Mitochondria of members of Kinetoplastea contain a complex network of two types of circular chromosomes, maxicircles and minicircles, and mt gene transcripts are edited intensively prior to translation. To understand how the complex genome structures and RNA editing mechanisms observed among the extant euglenozoans emerged, it is important to infer the mt genome structure of the ancestral euglenozoan. The aforementioned issue can be addressed by analyzing the mt genome of a novel protist strain SRT308, which was placed robustly at the base of the Euglenozoan clade in our phylogenomic analysis. We here report a 61 Kb-fragment of SRT308 mt genome. In this mt genome fragment, we identified genes encoding COX1, COX2, COX3 and subunits of NADH dehydrogenase, succinate dehydrogenase and ATP synthase. The partial SRT308 mt genome suggests that this protist (and the ancestral euglenozoan as well) possesses the mt genome larger in both size and gene content than the euglenozoan mt genomes studied to date.

新奇ヘテロロボサ生物SRT213における縮退的ミトコンドリア機能の推測（日本進化学会第19回大会）

パラオ共和国のマングローブ底泥嫌気サンプルから真核微生物SRT213株が単離された。SRT213は典型的なミトコンドリアの代わりに嫌気・微好気環境生物に特異な縮退的ミトコンドリア（mitochondrion-related organelle；MRO）をもつことが電子顕微鏡観察よって示唆された。また、SRT213はSSU rDNA系統解析で嫌気性ヘテロロボサ生物Creneis carolina (Pánek et al., 2014)と強い近縁性を示したため、MROをもつ新奇嫌気性ヘテロロボサ生物であると考えられる。これまでにMROに関する研究が行われたヘテロロボサ生物はPsalteriomonas lanterna（de Graaf et al., 2009）とSawyeria marylandensis（Barberà et al., 2010）のみであり、この生物群におけるミトコンドリア縮退進化の全体像を俯瞰するに十分とは言えない。本研究では、ヘテロロボサ生物におけるMRO縮退進化の理解を目指して、トランスクリプトームデータを基盤にSRT213のMRO機能を推測した。その結果、水素発生型ATP合成系関連遺伝子群を検出し、それらの一部のMROへの局在が示唆されたことから、SRT213は水素発生型MROを保持しているものと考えられた。さらに、これまでに解析された嫌気性ヘテロロボサ生物2種では検出されていないコハク酸脱水素酵素、F0 F1 ATP合成酵素群、TCAサイクル関連遺伝子が検出された。以上の結果をもとにSRT213を含む嫌気的ヘテロロボサ生物におけるMRO機能の進化を議論したい。

A heterolobosean strain SRT213 and the putative function of its mitochondrion-related organelle. (15th ICOP)

An amoeboflagellate, strain SRT213, was isolated from mangrove sediments sampled in the Republic of Palau in 2011, and has been maintained in the laboratory under the micro-aerobic condition with prey bacteria. A preliminary electron microscopic observation identified no typical mitochondrion, but double membrane-bound organelles that resembled superficially to mitochondrion-related organelles (MROs) found in diverse anaerobic/micro-aerophilic eukaryotes. Our small subunit ribosomal DNA phylogeny recovered a strong affinity between SRT213 and a heterolobosean Creneis carolina (Panek et al, 2014), suggesting that this amoeboflagellate is a new member of Heterolobosea. Prior to this study, MRO function was investigated only in two heterolobosean species, Psalteriomonas lanterna (de Graaf et al., 2009) and Sawyeria marylandensis (Barbera et al., 2010). In this study, we generated the transcriptomic data from SRT213 to deepen our understanding of the function of MROs in heteroloboseans. Homology searches against the SRT213 data successfully identified the transcripts encoding enzymes involved in anaerobic ATP generation and hydrogen production, suggesting that the MRO in this species belongs to “class 3/4.” In addition, we identified the transcripts for putative MRO proteins that were not detected in the data from the two previously studied heteroloboseans, such as subunits of succinate dehydrogenase (complex II), those of F0F1 ATP synthase (complex V), and the enzymes comprising the TCA cycle. In this presentation, we will discuss the differences and commonalities in MRO function between SRT213 and other anaerobic/microaerophilic eukaryotes.

Molecular tinkering in the evolution of the membrane attachment mechanisms of the Rheb GTPase. (15th ICOP)

Rheb is a highly conserved Ras-like GTPase involved in the cell growth and division regulation. A standard Rheb protein consists of a GTPase domain and a hypervariable tail with a C-terminal motif governing prenylation of a conserved cysteine residue. We conducted a detailed analysis of Rheb sequences based on a dense sampling of the eukaryote phylogeny, including minor lineages of key evolutionary importance. The analysis revealed that the canonical and apparently ancestral Rheb protein structure has been modified in multiple lineages in a way that affects the mode of membrane attachment of the protein. First, Rheb in Cryptista (including Palpitomonas bilix as the basal lineage) exhibits an N-terminal  extension  comprising  the  conserved  phosphoinositide-binding  PX  (Phox)  domain. This protein structure is the first defined candidate synapomorphy of the Cryptista clade. Rheb proteins in Euglenozoa and its sister lineage represented by the novel undescribed protist SRT308 share at the N-terminus an unrelated phosphoinositide-binding domain,  FYVE.  The  C-terminal  prenylation  motif  is  retained  in  the  Rheb  protein  of SRT308, but it was lost before euglenozoan radiation. In Euglenoidea, a second Rheb paralog emerged, lacking the FYVE domain but characterized by an N-terminal amphipathic helix that we demonstrated is myristoylated. All three lineages of the SAR clade (Stramenopiles, Alveolata, Rhizaria) exhibit a novel Rheb form with a long C-terminal extension including four transmembrane segments. This is the only Rheb variant in alveolates and rhizarians, whereas the canonical Rheb is widespread in stramenopiles, indicating Rheb duplication and modification of one paralog in the SAR stem followed by the loss of the standard form from some SAR lineages. Finally, Rheb proteins in several unrelated groups (ancyromonads, labyrinthulids) possess an N-terminal extension of varying length with multiple cysteine residues that might be palmitoylated. Rheb-membrane interaction is thus unexpectedly evolutionarily dynamic and represents an intriguing case of molecular tinkering. 

Morphology, ultrastructure and phylogeny of a new species of Glissandra(Protista incertae sedis) (15th ICOP)

Glissandra is an understudied genus of free-living marine biflagellates of which taxonomic position remains uncertain. These flagellates are characterized morphologically by possessing a short ventral groove and two long flagella inserted laterally from the groove. Both flagella tightly hold to substrate and only the tip of the anterior flagellum waves when gliding. Two species, G. innuerende Patterson and Simpson, 1996 and G. similis Lee, 2007, have been described so far. However, neither ultrastructural nor molecular study has been performed due to the absence of the laboratory culture. We recently established a culture of a new species of Glissandra from a seaweed sample of the Republic of Palau. In this study, the cultured Glissandra cell was subjected to light and electron microscopic observations, as well as a molecular phylogenetic analysis. Light microscopic observation indicated that the flagellate had the characteristics of Glissandra, such as a short ventral groove and two long flagella that attach to substrate. However, we noticed two differences between the new Glissandra species and the two species previously described. Firstly, the former cell is smaller in size than G. innuerende or G. similis. Secondly, the two flagella were inserted longitudinally in the new species, not laterally as observed in the two previously described species. In addition, the new species appeared to possess an oval depression, which is apart from the groove, at the ventral side of the cell. The transmission electron microscopic observation revealed that the cell membrane is lined by a thin theca, which is similar to that of apusozoans, and the rim of the oval depression is supported by a microtubular band. An 18S rRNA gene phylogeny recovered no strong affinity between the new species and any known eukaryotes/assemblages (including Apusozoa), suggesting that Glissandra represents a previously overlooked branch of the tree of eukaryotes.

The chlorophyll catabolism in a phycophagic cercozoan Paracercomonas sp. strain KMO002: exploring a biochemical/molecular biological approach. (15th ICOP)

Phycophagic protists ingest cells of microalgae that contain enormous amounts of chlorophylls under illuminated environments, none the less for the significant phototoxicity of these pigments. Recent studies have revealed that many lineages of protists are capable of detoxification catabolism of chlorophylls and produces 132,173-cyclopheophorbide enols (CPE) as its results. Despite accumulated evidences of CPE production in diverse lineages across supergroups of eukarya, little study has been successfully addressed to identify enzymes and/or genes responsible for on this metabolic process. We isolated a strain of phagotrophic amoeboflagellate (strain KMO002) by micropipetting from a freshwater sample collected in a small reservoir in Japan, and co-cultured with cyanobacterium Synechococcus elongates PCC 7942. The molecular phylogenetic analysis of 18S rDNA placed KMO002 in a clade of Cercomonadida, Cercozoa, that exclusively consists of genus Paracercomonas. KMO-002 was observed under microscope to actively prey on cells of PCC 7942 as well as other unicellular cyanobacteria and demonstrated to produce CPEs. Interestingly, when it was fed on Acaryochloris sp. that dominantly produces chlorophyll d instead of chlorophyll a, a species of CPE derived in chlorophyll d. Because such chlorophyll d-producing cyanobacteria are unlikely to be a natural prey in the environment, the results indicate a relatively loose substrate specificity of the enxyme(s) responsible for the CPE catabolism in the Paracercomonas. Here, we will also discuss on our transcriptomic analyses on this two-membered culture.

An obligate kleptoplastic phototrophy of a euglenoid Rapaza viridis. (15th ICOP)

Rapaza viridis Yamaguchi et al. 2012 was originally described as a mixotrophic alga that preys exclusively on a specific strain Tetraselmis sp. We were primarily interested in R. viridis for their CPE catabolism, the process catabolizing chlorophylls into non-phototoxic cyclopheophorbide enols (CPEs). Unexpectedly, however, R. viridis exhibited only trace amounts of CPE productions along with “phycophagy” on Tetraselmis cells, and no substantial generation of degradation products of chlorophylls were evident in any stage after predation. In addition, unlike typical phycophagic euglenoids, degradation of chloroplasts was not observed within cells of R. viridis. In a quantitative experiment, total chlorophylls were not significantly changed after the predation and only slightly reduced in the later stage; furthermore, total chlorophylls per cells of R. viridis during the stationary phase were unchanged through time, and so was chlorophyll a/b ratio. These evidences indicated that chlorophylls of ingested Tetraselmis had not substantially been altered in cells of R. viridis, hence implying retention of photosynthetic machinery of Tetraselmis intact. In optical and electron microscopic observations, interestingly, the ingested chloroplasts were shown to become subdivided into many pieces 8-14 hours after predation, which was followed by cell divisions of R. viridis where the subdivided chloroplasts were distributed to the daughter cells. Along with the division event, serial losses of typical features of the chloroplast of Tetraselmis were apparent, such as dispersant and eventual loss of intraplastidial eyespot globules, reduction of starch grains, and diminishment of Tetraselmis-type pyrenoids, thus the Tetraselmis-type chloroplast being indistinguishable from the Rapaza-type unlike the original description. We infer absence of proper chloroplast ofR. viridis and kleptoplastic origin of all chloroplasts retained therein. Furthermore, we measured photosynthetic activity of R.viridis and compared with that of Tetraselmis; the results indicated significant level of photosynthetic capacity of kleptochloroplasts, if so, was sustained in cells of R.viridis.

153 genes phylogenetic analysis indicated a newly single-celled eukaryote, strain SRT308, as a deep-branching Euglenozoan. (GEM)

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