コバヤシ ユウスケ小林 優介准教授Yusuke Kobayashi
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論文
- Identification of a plastid-targeted RecQ-like helicase in the red alga Cyanidioschyzon merolae.
Shoko Kawato; Minori Ebihara; Yui Mizuki; Takayuki Fujiwara; Shin-Ya Miyagishima; Yusuke Kobayashi, Plastids retain their own genomic DNA (plastid DNA, ptDNA) which must be faithfully maintained for proper organelle function. However, the molecular mechanisms responsible for ptDNA maintenance remain poorly understood, particularly in red algae. Here, we identified a plastid-targeted RecQ-like helicase (ptRECQ) in the unicellular red alga Cyanidioschyzon merolae. Biochemical assays demonstrated that ptRECQ possesses ATP-dependent helicase activity and preferentially unwinds splayed DNA substrates that mimic replication fork structures. ptrecQ mutant cells were hypersensitive to nalidixic acid, a ptDNA gyrase inhibitor that induces replication stress, indicating a role for ptRECQ in plastid genome maintenance under replication stress. Phylogenetic analyses showed that ptRECQ is broadly conserved in red algae, absent from green algae and land plants, and phylogenetically distinct from cyanobacterial RecQ proteins. These results suggest that red algae employ a ptDNA maintenance system that differs from that of the green lineage.
The Journal of general and applied microbiology, 2026年05月28日 - The closed nutrient recycling system in the Paramecium - Chlorella photosymbiosis contributes to survival under oligotrophic conditions
Kaoru Okada; Takayuki Fujiwara; Shunsuke Hirooka; Yusuke Kobayashi; Ryo Onuma; Shin-ya Miyagishima, Endosymbiotic relationships between a heterotrophic host and a unicellular algal endosymbiont are observed across many eukaryotic lineages. Although these relationships are prevalent in oligotrophic environments, how they function and provide an advantage under such conditions remains largely unknown. To address these issues, we examined the behavior of the ciliate Paramecium bursaria hosting Chlorella endosymbionts under nitrogen- and prey-depleted conditions. The Paramecium host survived for up to 5 weeks while maintaining the number of Chlorella endosymbionts, whereas aposymbiotic Paramecium and free-living Chlorella either died or bleached, respectively, under the same conditions. In the symbiotic state, the host continuously fed on the endosymbionts without excreting nitrogenous waste into the medium, while the remaining endosymbionts continued to proliferate using heterotrophic metabolites from the host and light energy. Thus, the cyclical farming of endosymbionts by the host maintains a high concentration of nutrients within the closed system, providing a selective advantage in oligotrophic environments., American Association for the Advancement of Science (AAAS)
Science Advances, 2025年10月31日, ［査読有り］ - HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii
Mari Takusagawa; Yusuke Kobayashi; Yoichiro Fukao; Kumi Hidaka; Masayuki Endo; Hiroshi Sugiyama; Takashi Hamaji; Yoshinobu Kato; Isamu Miyakawa; Osami Misumi; Toshiharu Shikanai; Yoshiki Nishimura, Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA–protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green algaChlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of theHBD1 gene by CRISPR-Cas9–mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeastΔabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids., Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences, 2021年05月18日, ［査読有り］ - Holliday Junction Resolvase MOC1 Maintains Plastid and Mitochondrial Genome Integrity in Algae and Bryophytes
Yusuke Kobayashi; Masaki Odahara; Yasuhiko Sekine; Takashi Hamaji; Sumire Fujiwara; Yoshiki Nishimura; Shin-ya Miyagishima, 筆頭著者, When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants., Oxford University Press (OUP)
Plant Physiology, 2020年12月, ［査読有り］ - Dynamic Motion of Chloroplast Nucleoids Captured by the Microfluidic System
Kamimura Y; Kobayashi Y; Nishimura Y
Cyotologia, 2020年09月, ［査読有り］ - Molecular structure and evolution of chloroplast nucleoids
Yusuke Kobayashi, 筆頭著者, The Japanese Society of Plant Morphology
PLANT MORPHOLOGY, 2020年, ［査読有り］, ［招待有り］ - Evolutionary Changes in DnaA-Dependent Chromosomal Replication in Cyanobacteria.
Ryudo Ohbayashi; Shunsuke Hirooka; Ryo Onuma; Yu Kanesaki; Yuu Hirose; Yusuke Kobayashi; Takayuki Fujiwara; Chikara Furusawa; Shin-Ya Miyagishima, Replication of the circular bacterial chromosome is initiated at a unique origin (oriC) in a DnaA-dependent manner in which replication proceeds bidirectionally from oriC to ter. The nucleotide compositions of most bacteria differ between the leading and lagging DNA strands. Thus, the chromosomal DNA sequence typically exhibits an asymmetric GC skew profile. Further, free-living bacteria without genomes encoding dnaA were unknown. Thus, a DnaA-oriC-dependent replication initiation mechanism may be essential for most bacteria. However, most cyanobacterial genomes exhibit irregular GC skew profiles. We previously found that the Synechococcus elongatus chromosome, which exhibits a regular GC skew profile, is replicated in a DnaA-oriC-dependent manner, whereas chromosomes of Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, which exhibit an irregular GC skew profile, are replicated from multiple origins in a DnaA-independent manner. Here we investigate the variation in the mechanisms of cyanobacterial chromosome replication. We found that the genomes of certain free-living species do not encode dnaA and such species, including Cyanobacterium aponinum PCC 10605 and Geminocystis sp. NIES-3708, replicate their chromosomes from multiple origins. Synechococcus sp. PCC 7002, which is phylogenetically closely related to dnaA-lacking free-living species as well as to dnaA-encoding but DnaA-oriC-independent Synechocystis sp. PCC 6803, possesses dnaA. In Synechococcus sp. PCC 7002, dnaA was not essential and its chromosomes were replicated from a unique origin in a DnaA-oriC independent manner. Our results also suggest that loss of DnaA-oriC-dependency independently occurred multiple times during cyanobacterial evolution and raises a possibility that the loss of dnaA or loss of DnaA-oriC dependency correlated with an increase in ploidy level.
Frontiers in microbiology, 2020年, ［査読有り］ - Responses of unicellular predators to cope with the phototoxicity of photosynthetic prey
Uzuka A†; Kobayashi Y†; Onuma R†; Hirooka S.; Kanesaki Y.; Yoshikawa H.; Fujiwara T.; Miyagishima SY; These authors contributed equally to this work, 筆頭著者
Nature communications, 2019年12月, ［査読有り］ - Chloroplast nucleoids as a transformable network revealed by live imaging with a microfluidic device
Kamimura Y; Tanaka H; Kobayashi Y; Shikanai T; Nishimura Y
Communications Biology, 2018年05月, ［査読有り］ - Identification of Holliday junction resolvases crucial for the chloroplast nucleoid morphology and segregation
Yusuke Kobayashi; Osami Misumi; Yoshiki Nishimura, 筆頭著者, The Japanese Society of Plant Morphology
PLANT MORPHOLOGY, 2018年, ［査読有り］ - Finding holliday junction resolvases: A crucial factor for chloroplast nucleoid segregation
Yusuke Kobayashi; Osami Misumi; Yoshiki Nishimura, 筆頭著者, Japan Mendel Society
Cytologia, 2017年12月01日, ［査読有り］, ［招待有り］ - Holliday junction resolvases mediate chloroplast nucleoid segregation
Yusuke Kobayashi; Osami Misumi; Masaki Odahara; Kota Ishibashi; Masafumi Hirono; Kumi Hidaka; Masayuki Endo; Hiroshi Sugiyama; Hiroshi Iwasaki; Tsuneyoshi Kuroiwa; Toshiharu Shikanai; Yoshiki Nishimura, 筆頭著者
SCIENCE, 2017年05月, ［査読有り］ - Dynamic Interplay between Nucleoid Segregation and Genome Integrity in Chlamydomonas Chloroplasts
Masaki Odahara; Yusuke Kobayashi; Toshiharu Shikanai; Yoshiki Nishimura
PLANT PHYSIOLOGY, 2016年12月, ［査読有り］ - C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants
Yusuke Kobayashi; Takuto Otani; Kota Ishibashi; Toshiharu Shikanai; Yoshiki Nishimura, 筆頭著者
GENOME BIOLOGY AND EVOLUTION, 2016年05月, ［査読有り］ - Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution
Yusuke Kobayashi; Mari Takusagawa; Naomi Harada; Yoichiro Fukao; Shohei Yamaoka; Takayuki Kohchi; Koichi Hori; Hiroyuki Ohta; Toshiharu Shikanai; Yoshiki Nishimura, 筆頭著者
GENOME BIOLOGY AND EVOLUTION, 2016年01月, ［査読有り］ - Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon merolae
Nobuko Sumiya; Takayuki Fujiwara; Yusuke Kobayashi; Osami Misumi; Shin-ya Miyagishima
PLOS ONE, 2014年10月, ［査読有り］ - Algae Sense Exact Temperatures: Small Heat Shock Proteins Are Expressed at the Survival Threshold Temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii
Yusuke Kobayashi; Naomi Harada; Yoshiki Nishimura; Takafumi Saito; Mami Nakamura; Takayuki Fujiwara; Tsuneyoshi Kuroiwa; Osami Misumi, 筆頭著者
GENOME BIOLOGY AND EVOLUTION, 2014年10月, ［査読有り］
MISC
- 葉緑体核様体の進化と構造のダイナミクス
小林 優介; 三角 修己; 西村 芳樹
化学と生物, 2018年09月, ［招待有り］ - 葉緑体ゲノムの分配はHollidayジャンクション解離酵素MOC1により保障される
小林 優介; 三角 修己; 西村 芳樹
ライフサイエンス 新着論文レビュー, 2017年05月, ［招待有り］ - 葉緑体核様体コア因子の多様性と進化
小林優介; 田草川真理; 田草川真理; 原田尚実; 深尾陽一朗; 深尾陽一朗; 山岡尚平; 河内孝之; 堀孝一; 太田啓之; 太田啓之; 鹿内利治; 西村芳樹
日本植物学会大会研究発表記録, 2015年
講演・口頭発表等
- 葉緑体母性遺伝を制御する天然変性タンパク質の解析
第１９回クラミドモナス研究会, 2026年03月 - 葉緑体核様体の「かたち」と運命 ：分配機構と母性遺伝における制御
第48回日本分子生物学会年会シンポジウム, 2025年12月03日, ［招待有り］ - クラミドモナスで蛍光タンパク質を使いやすく
中村陽世里; 高木杏子; 海老原美紀; 水木結唯; 小林優介
日本植物形態学会第37回大会, 2025年09月 - 単細胞藻類を用いた葉緑体DNAの複製・ 修復機構の解析
海老原美紀; 小林優介
日本植物形態学会第37回大会, 2025年09月 - クラミドモナスの葉緑体核様体ライブイメージング解析に最適な蛍光タンパク質の探索
高木杏子; 水木結唯; 海老原美紀; 池田彩乃; 小林優介
第66回日本植物生理学会年会, 2025年03月 - 葉緑体DNAの遺伝機構
日本遺伝学会第96回大会＠高知工科大学, 2024年09月, ［招待有り］ - Hollidayジャンクション切断酵素MOC1は色素体とミトコンドリアDNAの安定性に寄与する
Yusuke Kobayashi
2020年度日本分子生物学会ワークショップ, 2020年12月, ［招待有り］ - 葉緑体DNA遺伝に必須な Holliday ジャンクション切断機構の発見
小林 優介
第20回植物オルガネラワークショップ, 2018年03月, ［招待有り］ - Hollidayジャンクション解離酵素MOC1は葉緑体核様体の形態・分配を保障する
小林 優介
新光合成＆光合成若手の会ジョイント若手ワークショップ, 2017年08月, ［招待有り］
共同研究・競争的資金等の研究課題
