Researchers Database

SAEKI Kazuhiko

FacultyFaculty Division of Natural Sciences Research Group of Biological Sciences
PositionProfessor
Last Updated :2022/10/05

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Profile and Settings

  • Name (Japanese)

    Saeki
  • Name (Kana)

    Kazuhiko

Research Interests

  • Nitrogen fixation, Symbiosis, Plant-Microbe interaction, Protein secretion

Research Areas

  • Life sciences, Functional biochemistry
  • Life sciences, Plants: molecular biology and physiology

Research Experience

  • Apr. 2012, Professor, School of Natural Sciences, Nara Women's University
  • Oct. 2004, Mar. 2012, Professor, Department of Biological Science, Nara Women's University
  • May 2000, Sep. 2004, Associate Professor, Department of Biology, Graduate School of Science, Osaka University
  • Apr. 1996, Apr. 2000, Lecturer, Department of Biology, Graduate School of Science, Osaka University
  • Jun. 1995, Mar. 1996, Lecturer, Department of Biology, Faculty of Science, Osaka University
  • Nov. 1989, May 1995, Instructor, Department of Biology, Faculty of Science, Osaka University
  • Sep. 1987, Oct. 1989, Instructor, Department of Biology, Faculty of Science, Osaka University
  • Aug. 1986, Aug. 1987, Visiting Research Associate, Department of Biochemistry, Michigan State University and Michigan Biotechnology Institute, USA

Education

  • 1986, Osaka University, Graduate School of Science, Course for Biological Chemistry
  • Mar. 1981, The University of Tokyo, Faculty of Science, Department of Biophysics and Biochemistry

Teaching Experience

  • 20 Sep. 2018
  • 20 Apr. 2018
  • 20 Oct. 2018
  • 20 Apr. 2018
  • 20 Oct. 2019
  • 20 Dec. 2018
  • 20 Apr. 2018
  • 20 Apr. 2018
  • 20 Apr. 2018
  • 20 Oct. 2018
  • 20 Apr. 2018
  • 20 Apr. 2018
  • 20 Apr. 2018
  • 20 Apr. 2012
  • 20 Jun. 2015
  • 20 Apr. 2015
  • 20 Apr. 2021
  • 20 Oct. 2018
  • 20 Apr. 2018
  • 20 Apr. 2019
  • 20 Apr. 2015
  • 20 Oct. 2018

Association Memberships

  • Japanese Society for Plant Physiologists
  • The Japanese Biochemical Society
  • American Society for Microbiology
  • 植物微生物研究会
  • International Society for Molecular-Plant Microbe Interaction
  • 日本ゲノム微生物学会

Ⅱ.研究活動実績

Published Papers

  • Refereed, Journal of Plant Research, Springer Science and Business Media LLC, Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis, Yuji Tokumoto; Kayo Hashimoto; Takashi Soyano; Seishiro Aoki; Wataru Iwasaki; Mai Fukuhara; Tomomi Nakagawa; Kazuhiko Saeki; Jun Yokoyama; Hironori Fujita; Masayoshi Kawaguchi, Jan. 2020, 133, 1, 109, 122, Scientific journal
  • Refereed, Microbes and environments, Lotus Accessions Possess Multiple Checkpoints Triggered by Different Type III Secretion System Effectors of the Wide-Host-Range Symbiont Bradyrhizobium elkanii USDA61., Shohei Kusakabe; Nahoko Higasitani; Takakazu Kaneko; Michiko Yasuda; Hiroki Miwa; Shin Okazaki; Kazuhiko Saeki; Atsushi Higashitani; Shusei Sato, Bradyrhizobium elkanii, a rhizobium with a relatively wide host range, possesses a functional type III secretion system (T3SS) that is involved in symbiotic incompatibility against Rj4-genotype soybean (Glycine max) and some accessions of mung bean (Vigna radiata). To expand our knowledge on the T3SS-mediated partner selection mechanism in the symbiotic legume-rhizobia association, we inoculated three Lotus experimental accessions with wild-type and T3SS-mutant strains of B. elkanii USDA61. Different responses were induced by T3SS in a host genotype-dependent manner. Lotus japonicus Gifu inhibited infection; L. burttii allowed infection, but inhibited nodule maturation at the post-infection stage; and L. burttii and L. japonicus MG-20 both displayed a nodule early senescence-like response. By conducting inoculation tests with mutants of previously reported and newly identified effector protein genes of B. elkanii USDA61, we identified NopF as the effector protein triggering the inhibition of infection, and NopM as the effector protein triggering the nodule early senescence-like response. Consistent with these results, the B. elkanii USDA61 gene for NopF introduced into the Lotus symbiont Mesorhizobium japonicum induced infection inhibition in L. japonicus Gifu, but did not induce any response in L. burttii or L. japonicus MG-20. These results suggest that Lotus accessions possess at least three checkpoints to eliminate unfavorable symbionts, including the post-infection stage, by recognizing different T3SS effector proteins at each checkpoint., 2020, 35, 1, Scientific journal, False
  • Refereed, Genome Announcements, American Society for Microbiology, Whole-Genome Sequence of the Nitrogen-Fixing Symbiotic Rhizobium Mesorhizobium loti Strain TONO, Yoshikazu Shimoda; Hideki Hirakawa; Shusei Sato; Kazuhiko Saeki; Makoto Hayashi, Mesorhizobium loti is the nitrogen-fixing microsymbiont for legumes of the genus Lotus . Here, we report the whole-genome sequence of a Mesorhizobium loti strain, TONO, which is used as a symbiont for the model legume Lotus japonicus . The whole-genome sequence of the strain TONO will be a solid platform for comparative genomics analyses and for the identification of genes responsible for the symbiotic properties of Mesorhizobium species., 27 Oct. 2016, 4, 5, Scientific journal
  • Refereed, Soil Science and Plant Nutrition, Informa UK Limited, Peribacteroid solution of soybean root nodules partly induces genomic loci for differentiation into bacteroids of free-living Bradyrhizobium japonicum cells, Naoko Ohkama-Ohtsu; Sachiko Ichida; Hiroko Yamaya; Takuji Ohwada; Manabu Itakura; Yoshino Hara; Hisayuki Mitsui; Takakazu Kaneko; Satoshi Tabata; Kouhei Tejima; Kazuhiko Saeki; Hirofumi Omori; Makoto Hayashi; Takaki Maekawa; Yoshikatsu Murooka; Shigeyuki Tajima; Kenshiro Simomura; Mika Nomura; Toshiki Uchiumi; Akihiro Suzuki; Yoshikazu Shimoda; Mikiko Abe; Kiwamu Minamisawa; Yasuhiro Arima; Tadashi Yokoyama, 04 May 2015, 61, 3, 461, 470, Scientific journal
  • Refereed, PLOS ONE, Public Library of Science (PLoS), Genome Analysis of a Novel Bradyrhizobium sp. DOA9 Carrying a Symbiotic Plasmid, Shin Okazaki; Rujirek Noisangiam; Takashi Okubo; Takakazu Kaneko; Kenshiro Oshima; Masahira Hattori; Kamonluck Teamtisong; Pongpan Songwattana; Panlada Tittabutr; Nantakorn Boonkerd; Kazuhiko Saeki; Shusei Sato; Toshiki Uchiumi; Kiwamu Minamisawa; Neung Teaumroong, 24 Feb. 2015, 10, 2, e0117392, e0117392, Scientific journal
  • Refereed, Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system, S. Okazaki; T. Kaneko; S. Sato; K. Saeki, 15 Oct. 2013, 110, 42, 17131, 17136, Scientific journal
  • Refereed, Plant and Cell Physiology, Oxford University Press (OUP), LjMATE1: A Citrate Transporter Responsible for Iron Supply to the Nodule Infection Zone of Lotus japonicus, Kojiro Takanashi; Kengo Yokosho; Kazuhiko Saeki; Akifumi Sugiyama; Shusei Sato; Satoshi Tabata; Jian Feng Ma; Kazufumi Yazaki, Symbiotic nitrogen fixation by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. While exchanged molecules imply nitrogen compounds, carbohydrates and also various minerals, knowledge of the molecular basis of plant transporters that mediate those metabolite exchanges is still limited. In this study, we have shown that a multidrug and toxic compound extrusion (MATE) protein, LjMATE1, is specifically induced during nodule formation, which nearly paralleled nodule maturation, in a model legume Lotus japonicus. Reporter gene experiments indicated that the expression of LjMATE1 was restricted to the infection zone of nodules. To characterize the transport function of LjMATE1, we conducted a biochemical analysis using a heterologous expression system, Xenopus oocytes, and found that LjMATE1 is a specific transporter for citrate. The physiological role of LjMATE1 was analyzed after generation of L. japonicus RNA interference (RNAi) lines. One RNAi knock-down line revealed limited growth under nitrogen-deficient conditions with inoculation of rhizobia compared with the controls (the wild type and an RNAi line in which LjMATE1 was not suppressed). It was noteworthy that Fe localization was clearly altered in nodule tissues of the knock-down line. These results strongly suggest that LjMATE1 is a nodule-specific transporter that assists the translocation of Fe from the root to nodules by providing citrate. © 2013 The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com., Apr. 2013, 54, 4, 585, 594, Scientific journal
  • Refereed, Microbes and environments, Commonalities and differences among symbiosis islands of three Mesorhizobium loti strains., Hiroko Kasai-Maita; Hideki Hirakawa; Yasukazu Nakamura; Takakazu Kaneko; Kumiko Miki; Jumpei Maruya; Shin Okazaki; Satoshi Tabata; Kazuhiko Saeki; Shusei Sato, To shed light on the breadth of the host range of Mesorhizobium loti strain NZP2037, we determined the sequence of the NZP2037 symbiosis island and compared it with those of strain MAFF303099 and R7A islands. The determined 533 kb sequence of NZP2037 symbiosis island, on which 504 genes were predicted, implied its integration into a phenylalanine-tRNA gene and subsequent genome rearrangement. Comparative analysis revealed that the core regions of the three symbiosis islands consisted of 165 genes. We also identified several NZP2037-specific genes with putative functions in nodulation-related events, suggesting that these genes contribute to broaden the host range of NZP2037., 2013, 28, 2, 275, 8, False
  • Refereed, Microbes and Environments, Japanese Society of Microbial Ecology, Involvement of a Novel Genistein-Inducible Multidrug Efflux Pump of Bradyrhizobium japonicum Early in the Interaction with Glycine max (L.) Merr, Keisuke Takeshima; Tatsuo Hidaka; Min Wei; Tadashi Yokoyama; Kiwamu Minamisawa; Hisayuki Mitsui; Manabu Itakura; Takakazu Kaneko; Satoshi Tabata; Kazuhiko Saeki; Hirofumi Oomori; Shigeyuki Tajima; Toshiki Uchiumi; Mikiko Abe; Yoshihiko Tokuji; Takuji Ohwada, The early molecular dialogue between soybean and the bacterium Bradyrhizobium japonicum is crucial for triggering their symbiotic interaction. Here we found a single large genomic locus that is widely separated from the symbiosis island and was conspicuously induced within minutes after the addition of genistein. This locus (named BjG30) contains genes for the multidrug efflux pump, TetR family transcriptional regulator, and polyhydroxybutyrate (PHB) metabolism. The induction of BjG30 by genistein was competitively inhibited by daidzein, although both genistein and daidzein are soybean-derived inducers of nodulation (nod) genes. Such a differential expression pattern is also observed in some legume-derived flavonoids, which structurally differ in the hydroxy/deoxy group at the 5-position. In addition, not only did the induction start far in advance of nodW and nodD1 after the addition of genistein, but the levels showed distinct concentration dependence, indicating that the induction pattern of BjG30 is completely different from that of nod genes. The deletion of genes encoding either the multidrug efflux pump or PHB metabolism, especially the former, resulted in defective nodulation performance and nitrogen-fixing capability. Taken together, these results indicate that BjG30, and especially its multidrug efflux pump, may play a key role in the early stage of symbiosis by balancing the dual functions of genistein as both a nod gene inducer and toxicant.https://www.jstage.jst.go.jp/article/jsme2/28/4/28_ME13057/_article/-char/ja/, 2013, 28, 4, 414, 421, Scientific journal
  • Refereed, CELLULAR AND MOLECULAR LIFE SCIENCES, SPRINGER BASEL AG, Rhizobial measures to evade host defense strategies and endogenous threats to persistent symbiotic nitrogen fixation: a focus on two legume-rhizobium model systems, Kazuhiko Saeki, The establishment and maintenance of rhizobium-legume symbioses require a sequence of highly regulated and coordinated events between the organisms. Although the interaction is mutually beneficial under nitrogen-limited conditions, it can resemble a pathogenic infection at some stages. Some host legumes mount defense reactions, including the production of reactive oxygen species (ROS) and defensin-like antimicrobial compounds. To subvert these host defenses, the infecting rhizobial cells can use measures to passively protect themselves and actively modulate host functions. This review first describes the establishment and maintenance of active nodules, as well as the external and endogenous attack and threat stages. Next, recent studies of ROS scavenging enzymes, the BacA protein originally found in Sinorhizobium meliloti, and the type III/IV secretion systems are discussed, with a focus on two legume-rhizobium model systems., Apr. 2011, 68, 8, 1327, 1339
  • Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, The bacA Gene Homolog, mlr7400, in Mesorhizobium loti MAFF303099 is Dispensable for Symbiosis with Lotus japonicus but Partially Capable of Supporting the Symbiotic Function of bacA in Sinorhizobium meliloti, Jumpei Maruya; Kazuhiko Saeki, Establishment of rhizobiumlegume symbiosis requires a series of mutual authentication, which might involve bacterial evasion of host defense. One such evasion-related genes is Sinorhizobium meliloti bacA that is essential for bacteroid formation. BacA is a transmembrane protein highly similar to Escherichia coli SbmA, a predicted transporter, and has homologs even in animal pathogens, such as Brucella abortus in which the homolog contributes to effective survival in host macrophages. Despite such a significance in hostmicrobe interactions, studies on rhizobial BacA have been mostly performed with the MedicagoSinorhizobium model system that forms indeterminate cylindrical nodules. Since Lotus japonicusMesorhizobium loti constitutes another model system that forms determinate globular nodules, we genetically analyzed the bacA homolog with the locus tag mlr7400 in M. loti MAFF303099. We found that the mlr7400-null mutant ML7400DK was able to establish quasi-healthy symbiosis with the Lotus plant with 5080 nitrogen-fixing capacity. This dispensability for symbiosis was in contrast to the indispensability of S. meliloti BacA for symbiosis. However, free-living phenotypes of ML7400DK paralleled those of known bacA mutants, i.e. ML7400DK showed decreased sensitivity to the antibiotics bleomycin and gentamicin as well as increased sensitivity to membrane-disturbing reagents such as SDS. Conservation of the free-living function between Mlr7400 protein and S. meliloti BacA was further confirmed by heterologous complementation experiments. Although simple introduction of mlr7400 into the S. meliloti bacA mutant did not increase the symbiotic capacity at all, a significant but marginal increase was obtained when mlr7400 was fused to the S. meliloti bacA promoter. These findings might indicate currently progressing evolutionary specialization among BacASbmA proteins., Sep. 2010, 51, 9, 1443, 1452, Scientific journal
  • Refereed, MOLECULAR PLANT-MICROBE INTERACTIONS, AMER PHYTOPATHOLOGICAL SOC, Temperature-Dependent Expression of Type III Secretion System Genes and Its Regulation in Bradyrhizobium japonicum, Min Wei; Keisuke Takeshima; Tadashi Yokoyama; Kiwamu Minamisawa; Hisayuki Mitsui; Manabu Itakura; Takakazu Kaneko; Satoshi Tabata; Kazuhiko Saeki; Hirofumi Omori; Shigeyuki Tajima; Toshiki Uchiumi; Mikiko Abe; Satoshi Ishii; Takuji Ohwada, The genome-wide expression profiles of Bradyrhizobium japonicum in response to soybean (Glycine max (L.) Merr.) seed extract (SSE) and genistein were monitored with time at a low temperature (15 degrees C). A comparison with the expression profiles of the B. japonicum genome previously captured at the common growth temperature (30 degrees C) revealed that the expression of SSE preferentially induced genomic loci, including a large gene cluster encoding the type III secretion system (T3SS), were considerably delayed at 15 degrees C, whereas most nodulation (nod) gene loci, including nodD1 and nodW, were rapidly and strongly induced by both SSE and genistein. Induction of the T3SS genes was progressively activated upon the elevation of temperature to 30 degrees C and positively responded to culture population density. In addition, genes nolA and nodD2 were dramatically induced by SSE, concomitantly with the expression of T3SS genes. However, the deletion mutation of nodD2 but not nolA led to elimination of the T3SS genes expression. These results indicate that the expression of the T3SS gene cluster is tightly regulated with integration of environmental cues such as temperature and that NodD2 may be involved in its efficient induction in B. japonicum., May 2010, 23, 5, 628, 637, Scientific journal
  • Refereed, MOLECULAR PLANT-MICROBE INTERACTIONS, AMER PHYTOPATHOLOGICAL SOC, Identification and Functional Analysis of Type III Effector Proteins in Mesorhizobium loti, Shin Okazaki; Saori Okabe; Miku Higashi; Yoshikazu Shimoda; Shusei Sato; Satoshi Tabata; Masatsugu Hashiguchi; Ryo Akashi; Michael Goettfert; Kazuhiko Saeki, Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, possesses a cluster of genes (tts) that encode a type III secretion system (T3SS). In the presence of heterologous nodD from Rhizobium leguminosarum and a flavonoid naringenin, we observed elevated expression of the tts genes and secretion of several proteins into the culture medium. Inoculation experiments with wild-type and T3SS mutant strains revealed that the presence of the T3SS affected nodulation at a species level within the Lotus genus either positively (L. corniculatus subsp. frondosus and L. filicaulis) or negatively (L. halophilus and two other species). By inoculating L. halophilus with mutants of various type III effector candidate genes, we identified open reading frame mlr6361 as a major determinant of the nodulation restriction observed for L. halophilus. The predicted gene product of mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl terminus. Homologues with similar repeat sequences are present in the hypersensitive-response and pathogenicity regions of several plant pathogens, including strains of Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas species. These results suggest that L. halophilus recognizes Mlr6361 as potentially pathogen derived and subsequently halts the infection process., Feb. 2010, 23, 2, 223, 234, Scientific journal
  • Refereed, JOURNAL OF BACTERIOLOGY, AMER SOC MICROBIOLOGY, Functional Differences of Two Distinct Catalases in Mesorhizobium loti MAFF303099 under Free-Living and Symbiotic Conditions, Masaki Hanyu; Hanae Fujimoto; Kouhei Tejima; Kazuhiko Saeki, Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the establishment and maintenance of symbiosis, as well as under free-living conditions, because legume hosts might assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated mutants of two potential catalase genes in Mesorhizobium loti MAFF303099 to investigate their physiological significance. Biochemical results indicated that genes with the locus tags mlr2101 and mlr6940 encoded a monofunctional catalase and a bifunctional catalase-peroxidase, respectively, that were named katE and katG. Under free-living conditions, the katG mutant demonstrated an extended generation time and elevated sensitivity to exogenous H2O2, whereas the katE mutant exhibited no generation time extension and only a slight increase in sensitivity to exogenous H2O2. However, the katE mutant showed a marked decrease in its survival rate during the stationary phase. With regard to symbiotic capacities with Lotus japonicus, the katG mutant was indistinguishable from the wild type; nevertheless, the mutants with disrupted katE formed nodules with decreased nitrogen fixation capacities (about 50 to 60%) compared to those formed by the wild type. These mutant phenotypes agreed with the expression profiles showing that transcription of katG, but not katE, was high during the exponential growth phase and that transcription levels of katE versus sigA were elevated during stationary phase and were approximately fourfold higher in bacteroids than mid-exponential-phase cells. Our results revealed functional separation of the two catalases, as well as the importance of KatE under conditions of strong growth limitation., Mar. 2009, 191, 5, 1463, 1471, Scientific journal
  • Refereed, ISME JOURNAL, NATURE PUBLISHING GROUP, Genomic comparison of Bradyrhizobium japonicum strains with different symbiotic nitrogen-fixing capabilities and other Bradyrhizobiaceae members, Manabu Itakura; Kazuhiko Saeki; Hirofumi Omori; Tadashi Yokoyama; Takakazu Kaneko; Satoshi Tabata; Takuji Ohwada; Shigeyuki Tajima; Toshiki Uchiumi; Keina Honnma; Konosuke Fujita; Hiroyoshi Iwata; Yuichi Saeki; Yoshino Hara; Seishi Ikeda; Shima Eda; Hisayuki Mitsui; Kiwamu Minamisawa, Comparative genomic hybridization (CGH) was performed with nine strains of Bradyrhizobium japonicum (a symbiotic nitrogen-fixing bacterium associated with soybean) and eight other members of the Bradyrhizobiaceae by DNA macroarray of B. japonicum USDA110. CGH clearly discriminated genomic variations in B. japonicum strains, but similar CGH patterns were observed in other members of the Bradyrhizobiaceae. The most variable regions were 14 genomic islands (4-97 kb) and low G + C regions on the USDA110 genome, some of which were missing in several strains of B. japonicum and other members of the Bradyrhizobiaceae. The CGH profiles of B. japonicum were classified into three genome types: 110, 122 and 6. Analysis of DNA sequences around the boundary regions showed that at least seven genomic islands were missing in genome type 122 as compared with type 110. Phylogenetic analysis for internal transcribed sequences revealed that strains belonging to genome types 110 and 122 formed separate clades. Thus genomic islands were horizontally inserted into the ancestor genome of type 110 after divergence of the type 110 and 122 strains. To search for functional relationships of variable genomic islands, we conducted linear models of the correlation between the existence of genomic regions and the parameters associated with symbiotic nitrogen fixation in soybean. Variable genomic regions including genomic islands were associated with the enhancement of symbiotic nitrogen fixation in B. japonicum USDA110., Mar. 2009, 3, 3, 326, 339, Scientific journal
  • Refereed, DNA RESEARCH, OXFORD UNIV PRESS, Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr, Min Wei; Tadashi Yokoyama; Kiwamu Minamisawa; Hisayuki Mitsui; Manabu Itakura; Takakazu Kaneko; Satoshi Tabata; Kazuhiko Saeki; Hirofumi Omori; Shigeyuki Tajima; Toshiki Uchium; Mlkiko Abe; Takuji Ohwada, Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 mu M), nevertheless SSE-supplemented medium contained 4.7 mu M genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. in addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis., Aug. 2008, 15, 4, 201, 214, Scientific journal
  • Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Requirement for Mesorhizobium loti ornithine transcarbamoylase for successful symbiosis with Lotus japonicus as revealed by an unexpected long-range genome deletion, Elina Mishima; Atsuko Hosokawa; Haruko Imaizumi-Anraku; Katsuharu Saito; Masayoshi Kawaguchi; Kazuhiko Saeki, With the original aim of surveying the role of exopolysaccharide (EPS) in LotusMesorhizobium symbiosis, we carried out Tn5 mutagenesis of Mesorhizobium loti and obtained 32 mutants with defects in EPS biosynthesis. One of the mutants, HIA22, formed pseudonodules and failed to fix nitrogen with Lotus japonicus. However, complementation analysis unexpectedly revealed that the potential gene with the locus tag, mll2073, interrupted by Tn5 was responsible for neither normal EPS synthesis nor symbiosis. Further analysis uncovered that HIA22 had a genome deletion of approximately 20 kbp, resulting in the loss of two separate genes responsible for EPS biosynthesis and symbiosis. One gene with the locus tag, mll5669, was needed to synthesize normal EPS that fluoresced on medium containing Calcofluor and encoded a homolog of O-antigen acetyl transferase in Salmonella typhimurium. A specific mutant of mll5669, EMB-B58, successfully fixed nitrogen when infected onto L. japonicus. Another gene, mlr5647, was needed to establish fully functional nodules and encoded ornithine carbamoyl transferase [ArgF (EC 2.1.3.3)], which participates in arginine biosynthesis. A specific mutant of mlr5647, EMB-Y2, showed arginine auxotrophy and formed infection threads, but the nodules formed by this strain had few infected cells filled with bacteroids. These mutant phenotypes were complemented by supplementation of arginine or citrulline to bacterial or plant medium. EMB-Y2 represented a novel class of rhizobial arginine auxotrophs with symbiotic deficiency, and its phenotypes indicated that sufficient supply of citrulline or its derivative is essential for successful infection or for a stage in the infection process in LotusMesorhizobium symbiosis., Mar. 2008, 49, 3, 301, 313, Scientific journal
  • Refereed, JOURNAL OF BACTERIOLOGY, AMER SOC MICROBIOLOGY, The Mesorhizobium loti purB gene is involved in infection thread formation and nodule development in Lotus japonicus, Shin Okazaki; Yoshiyuki Hattori; Kazuhiko Saeki, The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis., Nov. 2007, 189, 22, 8347, 8352, Scientific journal
  • Not Refereed, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme., Rhizobial networks for symbiosis with legumes, Kiwamu Minamisawa; Kazuhiko Saeki; Shusei Sato; Yoshikazu Shimoda, 01 Aug. 2006, 51, 1044, 1050
  • Refereed, Microbes and Environments, Japanese Society of Microbial Ecology, Global Gene Expression in Bradyrhizobium japonicum Cultured with Vanillin, Vanillate, 4-Hydroxybenzoate and Protocatechuate, Naofumi Ito; Manabu Itakura; Shima Eda; Kazuhiko Saeki; Hirofumi Oomori; Tadashi Yokoyama; Takakazu Kaneko; Satoshi Tabata; Takuji Ohwada; Shigeyuki Tajima; Toshiki Uchiumi; Eiji Masai; Masataka Tsuda; Hisayuki Mitsui; Kiwamu Minamisawa, Pathways for aerobic degradation of naturally occurring aromatics were estimated from the entire genome sequence of Bradyrhizobium japonicum strain USDA 110, a symbiotic nitrogen-fixing bacterium in soil. Many homologs for the genes encoding various oxygenases and enzymes for the β-ketoadipate pathway in the degradation of vanillin, vanillate, protocatechuate, and 4-hydroxybenzoate were scattered over nine loci of the genome. Using a macroarray developed for B. japonicum strain USDA 110, we compared gene expression profiles in cells grown in each of these aromatic compounds as a sole carbon source with those of succinate-fed cells. One set of oxygenase genes homologous to pcaGH, pobA, and vanAB and structurally accompanied by transcriptional regulator homologs was markedly upregulated in their expression by one or more of the four aromatics, whereas no marked change was observed in the expression levels of pcaBCDIJF genes for the β-ketoadipate pathway. In addition, cells fed vanillin and vanillate showed high levels of expression of genes for a glutathione-dependent pathway of formaldehyde oxidation, suggesting that the formaldehyde generated from vanillate's demethylation is oxidized via C1 metabolism in B. japonicum. The expression of the above genes was confirmed by quantitative reverse transcription PCR. The implications of these results are discussed in terms of degradation pathways, gene regulation, and the soil environment., 2006, 21, 4, 240, 250, Scientific journal
  • Refereed, Plant Physiology, American Society of Plant Biologists (ASPB), Characterization of the Lotus japonicus Symbiotic Mutant lot1 That Shows a Reduced Nodule Number and Distorted Trichomes, Yasuhiro Ooki; Mari Banba; Koji Yano; Jumpei Maruya; Shusei Sato; Satoshi Tabata; Kazuhiko Saeki; Makoto Hayashi; Masayoshi Kawaguchi; Katsura Izui; Shingo Hata, Apr. 2005, 137, 4, 1261, 1271, Scientific journal
  • Refereed, Journal of Bacteriology, American Society for Microbiology, Expression Islands Clustered on the Symbiosis Island of the Mesorhizobium loti Genome, Toshiki Uchiumi; Takuji Ohwada; Manabu Itakura; Hisayuki Mitsui; Noriyuki Nukui; Pramod Dawadi; Takakazu Kaneko; Satoshi Tabata; Tadashi Yokoyama; Kouhei Tejima; Kazuhiko Saeki; Hirofumi Omori; Makoto Hayashi; Takaki Maekawa; Rutchadaporn Sriprang; Yoshikatsu Murooka; Shigeyuki Tajima; Kenshiro Simomura; Mika Nomura; Akihiro Suzuki; Yoshikazu Shimoda; Kouki Sioya; Mikiko Abe; Kiwamu Minamisawa, ABSTRACT Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes. The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners. We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation. Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated. This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation. Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN. The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions. The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression. In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids. A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis., 15 Apr. 2004, 186, 8, 2439, 2448, Scientific journal
  • Refereed, JOURNAL OF ENDOTOXIN RESEARCH, MANEY PUBLISHING, Characteristic biological activities of lipopolysaccharides from Sinorhizobium and Mesorhizobium, Y Tsukushi; N Kido; K Saeki; T Sugiyama; N Koide; Mori, I; T Yoshida; T Yokochi, The biological actions of lipopolysaccharides (LPSs) from Sinorhizobium meliloti, Mesorhizobium loti and Escherichia coli were compared. In biological activities including lethality, production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO), adjuvant action and Limulus activity, LPS from S. meliloti exhibited stronger actions than LPS from M. loti, but had a weaker action than LPS from E. coli. On the other hand, M. loti LPS showed a higher activity to activate human complement than S. meliloti LPS. Further, there was a significant difference in polymyxin B binding between S. meliloti LPS and M. loti LPS, suggesting a difference in the lipid A structure. LPSs from S. meliloti and M. loti seem to exhibit characteristic biological actions that may be dependent on the difference in the lipid A structure., 2004, 10, 1, 25, 31, Scientific journal
  • Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis, Y Hattori; H Omori; M Hanyu; N Kaseda; E Mishima; T Kaneko; S Tabata; K Saeki, For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobium loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host-range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively. The genome of M. loti consists of a single chromosome and two plasmids. The chromosome (7,036,071 bp) was covered 99.68% by 445 clones with four gaps, although two clones were unstable in E. coli. The larger plasmid pMLa (351,911 bp) was completely covered by 23 clones, while the smaller pMLb (208,315 bp) was covered 98.85% by 12 clones with two gaps. We have also made ancillary plasmids to facilitate the construction of deletion mutants using derivatives of the library clones. As a pilot experiment to uncover regions which contain novel symbiotic genes, 13 deletion mutants were constructed to lack in total 180.5 kbp of the genome. All the mutants formed apparently normal nodules and supported symbiotic nitrogen fixation, however, one mutant that lacked a 5.3 kbp chromosomal region, 4,551,930-4,557,222, did not produce normal exopolysaccharides as judged by fluorescence on medium containing Calcofluor. The results supported the effectiveness of the approach to detect gene functions., Dec. 2002, 43, 12, 1542, 1557, Scientific journal
  • Refereed, JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE BV, A novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia, R Sriprang; M Hayashi; M Yamashita; H Ono; K Saeki; Y Murooka, A novel plant-bacterial remediation system for heavy metals (HM) was developed by expression of tetrameric human metallothionein (MTL4) in Mesorhizobium huakuii subsp. rengei B3, a strain which infects and forms nodules on a green manure, Astragalus sinicus. The MTL4 gene was fused to the nifH and nolB promoters, which generated nodule- specific expression of the MTL4 gene. The expression analysis of the MTL4 gene was demonstrated in free-living cells in the presence of Cd2+ and Cu2+, under the low oxygen condition. The MTL4 under the nifH and nolB promoters was expressed and increased the accumulation of Cd2+, but not Cu2+ in free-living cells. The expression of the integrated nifH-MTL4 gene in the chromosome of strain B3 was also expressed stably and accumulated Cd2+ in the bacterial cells. The MTL4 transcripts were detected by in situ hybridization in bacteroids of mature nodules of A. sinicus containing nifH-MTL4 and nolB-MTL4 fusion gene. Moreover the MTL4 protein was detected by immunostaining. By infection of the recombinant B3, A. sinicus established symbiosis with the recombinant B3 that was grown in Cd2+ and Cu2+-polluted soils. The symbionts increased Cd2+ accumulation in nodules 1.7-2.0-fold, whereas, no significantly increase in Cu2+ accumulation was noted. (C) 2002 Elsevier Science B.V. All rights reserved., Nov. 2002, 99, 3, 279, 293, Scientific journal
  • Refereed, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, TAYLOR & FRANCIS LTD, Primary structure and phylogenetic analysis of the coat protein of a Toyama isolate of tobacco necrosis virus, K Saeki; Y Takahashi; H Oh-Oka; T Umeoka; Y Oda; K Fukuyama, The amino acid sequence of the coat protein (CP) of a tobacco necrosis virus (TNV) strain, Toyama isolate, was determined by a combination of peptide and cDNA sequencing. The deduced sequence of 276 residues was compared with CPs of other TNV isolates and other plant virus isolates of Tombusviridae. It shelved the highest similarity to the TNV Nebraska isolate with 92% identity and moderate similarity to the TNV strain A with 51% identity, confirming the previous serological analysis. It also showed overall similarity with CPs of mostly genera Necrovirus and Sobemovirus, and partial similarity with CPs of genera Tombusvirus and Carmovirus. Among 13 CPs that showed overall similarity, there were 10 completely conserved residues. These included three residues that participate in Ca2+ ligation at the interfaces of virion subunits in TNV crystal structure, suggesting that similar metal binding occur in the viruses of genera Necrovirus and Sobemovirus., Mar. 2001, 65, 3, 719, 724, Scientific journal
  • Refereed, JOURNAL OF PLANT RESEARCH, BOTANICAL SOC JAPAN, The lotus symbiont, Mesorhizobium loti: Molecular genetic techniques and application, K Saeki; H Kouchi, Despite the recent attention given to the model legume Lotus japonicus, orderly information on its symbiotic partner Mesorhizobium loti is still lacking. To improve this situation, we review the history of the M. loti nomenclature and compare several strains commonly used, since classification of rhizobia has been confusing during the last decade. We also refer to the large symbiotic gene cluster, the 'symbiosis island', on M. loti chromosome. Then, molecular techniques for M. loti currently available and required to cope with the post-genome sequencing era will be summarized. Finally, we review current knowledge on the structure of M. loti Nod factors., Dec. 2000, 113, 1112, 457, 465, Scientific journal
  • Refereed, JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS LTD, Crystal structure of tobacco necrosis virus at 2.25 angstrom resolution, Y Oda; K Saeki; Y Takahashi; T Maeda; H Naitow; T Tsukihara; K Fukuyama, The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R = 25.3 % for diffraction data to 2.25 Angstrom resolution. A total of 180 subunits form a T = 3 virus shell with a diameter of about 280 Angstrom and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T = 3 shell. (C) 2000 Academic Press., Jun. 2000, 300, 1, 153, 169, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, OXFORD UNIV PRESS, Hyperproduction of recombinant ferredoxins in Escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster, M Nakamura; K Saeki; Y Takahashi, Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli, Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids, The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 X YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins., Jul. 1999, 126, 1, 10, 18, Scientific journal
  • Refereed, ARCHIVES OF MICROBIOLOGY, SPRINGER VERLAG, The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark, K Saeki; H Kumagai, The rnf genes in Rhodobacter capsulatus are essential for nitrogen fixation in the light. Because R. capsulatus grows readily on N-2 in the dark by anaerobic respiration with dimethylsulfoxide, the diazotrophic capacities of various strains in the dark were examined. No I nf mutants tested grew diazotrophically, and a nonpolar fdxN-null mutant showed decreased diazotrophic growth in the dark, suggesting that the Rnf and FdxN proteins form the primary electron donor pathway to nitrogenase in the dark as well as in the light. Nonphotosynthetic mutants lacking the component of cyclic electron transport grew diazotrophically and the levels of Rnf proteins were similar to those of the wild-type. These results indicate that I-nf gene products play an essential role in nitrogen fixation without any functional link to the cyclic electron transport system., May 1998, 169, 5, 464, 467, Scientific journal
  • Refereed, Biochemistry, American Chemical Society (ACS), Membrane Localization, Topology, and Mutual Stabilization of the rnfABC Gene Products in Rhodobacter capsulatus and Implications for a New Family of Energy-Coupling NADH Oxidoreductases†, Hirotaka Kumagai; Takeshi Fujiwara; Hiroshi Matsubara; Kazuhiko Saeki, May 1997, 36, 18, 5509, 5521, Scientific journal
  • Refereed, JOURNAL OF BIOLOGICAL CHEMISTRY, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Site-specific mutagenesis of Rhodobacter capsulatus ferredoxin I, FdxN, that functions in nitrogen fixation - Role of extra residues, K Saeki; K Tokuda; K Fukuyama; H Matsubara; K Nadanami; M Go; S Itoh, One of the two [4Fe-4S] type clusters of the Rhodobacter capsulatus ferredoxin I, FdxN, was modified through site-specific mutagenesis of the distinctive features of the second cluster-binding motif, Cys(38)-X(2)-Cys(41)-X(8)-Cys(50)-X(8)-Cys(50)-X(3)-Cys(59). First, various mutagenized products were tested to learn whether they could rescue the decreased capacity of an fdxN-null strain MSA1 to fix nitrogen: the phenotype of MSA1 was reassessed to Nif(s) (slow growth by nitrogen fixation) from our previous description of Nif(-) (Saeki, K., Suetsugu, Y., Tokuda, H., Miyatake, P., Young, D. A., Marrs, B. L. and Matsubara, H. (1991) J. Biol. Chem. 266, 12889-12895). Substitution of Cys(59) to Ser yielded an almost fully active product, while that of Cys(54) did not. Gradual deletions and deletion-substitution of the 8 residues between Cys(41) and Cys(50) also yielded active products. Second, three of the modified FdxN proteins were subjected to purification. Only the GA protein, whose 8 residues between positions 42 and 49 were replaced by the Gly-Ala sequence, was purified. The GA protein and the authentic FdxN showed similar optical properties. The two clusters in the former had E(m) values of -490 and -430 mV, while those in the latter had an identical value of -490 mV, when determined by EPR analysis. It was concluded that: 1) Cys(59) is not a ligand to [4Fe-4S] clusters but is important for structural integrity, 2) the residues between positions 42 and 49 may form a ''loop-out'' from a structure analogous to the Peptococcus aerogenes ferredoxin, and 3) the loop-out region does not have functional significance in nitrogen fixation but may be responsible for maintaining the highly negative redox potential of one of the two clusters., Dec. 1996, 271, 49, 31399, 31406, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, MOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCES OF CDNAS ENCODING SUBUNITS-I, SUBUNIT-II, AND SUBUNIT-IX OF EUGLENA-GRACILIS MITOCHONDRIAL COMPLEX-III, JY CUI; K MUKAI; K SAEKI; H MATSUBARA, cDNA clones encoding subunits I, II, and IX of Euglena gracilis mitochondrial complex III have been isolated from a lambdagt11 cDNA expression library by immunoscreening with an antiserum against the complex of the organism. Determination of the nucleotide sequences and amino-terminal amino acid sequences of purified subunits revealed that subunits II and IX, respectively, consist of 432 and 70 amino acids as their mature forms and possess potential presequences of 42 and 30 amino acids. The amino-terminal parts of the presequences had typical structural features of the mitochondrial targeting signal. Such features were also found at the amino-terminal region of the predicted subunit I protein, which comprises 494 residues. However, the amino terminus of the purified subunit I could not be detected, possibly because of a post-translational modification. Euglena subunits I and II both showed similarities to the members of the protein family which comprises complex III core proteins, mitochondrial processing peptidases (MPP) and processing enhancing proteins (PEP). Namely, the Euglena subunit I could be assigned to core 1 protein and the subunit II to core 2 protein in the family. In contrast, the subunit IX seemed to be peculiar to Euglena complex III. At 5'-untranslated regions, the three cloned cDNAs for subunits I, II, and IX had a common poly(T)CG structure which has also been reported for other Euglena cDNAs of nuclear genes., Jan. 1994, 115, 1, 98, 107, Scientific journal
  • Refereed, PLANT AND CELL PHYSIOLOGY, JAPANESE SOC PLANT PHYSIOLOGISTS, NUCLEOTIDE-SEQUENCE AND GENETIC-ANALYSIS OF THE REGION ESSENTIAL FOR FUNCTIONAL EXPRESSION OF THE GENE FOR FERREDOXIN-I, FDXN, IN RHODOBACTER-CAPSULATUS - SHARING OF ONE UPSTREAM ACTIVATOR SEQUENCE IN OPPOSITE DIRECTIONS BY 2 OPERONS RELATED TO NITROGEN-FIXATION, K SAEKI; K TOKUDA; T FUJIWARA; H MATSUBARA, Nucleotide sequencing of the region upstream of two ferredoxin genes, fdxC and fdxN, of Rhodobacter capsulatus revealed the existence of one open reading frame (ORF), ORFU1, in the same orientation as these genes and two other ORFs, ORFU2 and ORFU3, in the opposite orientation. Two potential -24/-12 promoters were found in front of ORFU1 and ORFU2, respectively, and there was a putative upstream activator sequence (UAS) or NifA-binding site between them. The ORFs corresponded to no known nif genes. However, analysis of their putative products showed that the product of ORFU1 (M(r) 47,912) and that of ORFU3 (M(r) 19,090) had a flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-like domain, respectively, and that the product of ORFU2 (M(r) 20,424) was a hydrophobic protein with six potential membrane-spanning portions. Results of interposon mutagenesis and complementation experiments indicated that ORFU2 but not ORFU1 is essential for nitrogen fixation and that additional gene(s) essential for nitrogen fixation must be present in the unsequenced region adjacent to ORFU3. Translational fusion analysis involving lacZYA and fdxN or ORFU3 provided evidence that the putative UAS is responsible for regulation of both ORFU1-fdxC-fdxN and ORFU2-ORFU3 operons in opposite orientations, and that the control of the latter is stricter than that of the former., Mar. 1993, 34, 2, 185, 199, Scientific journal
  • Refereed, Advances in Inorganic Chemistry, Elsevier, Structural and Functional Diversity of Ferredoxins and Related Proteins, Hiroshi Matsubara; Kazuhiko Saeki, 1992, 38, 223, 280, In book
  • Refereed, JOURNAL OF BIOLOGICAL CHEMISTRY, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, GENETIC-ANALYSIS OF FUNCTIONAL DIFFERENCES AMONG DISTINCT FERREDOXINS IN RHODOBACTER-CAPSULATUS, K SAEKI; Y SUETSUGU; K TOKUDA; Y MIYATAKE; DA YOUNG; BL MARRS; H MATSUBARA, Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S][4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN., Jul. 1991, 266, 20, 12889, 12895, Scientific journal
  • Refereed, PLANT AND CELL PHYSIOLOGY, JAPANESE SOC PLANT PHYSIOLOGISTS, FA/FB PROTEIN FROM THE SPINACH PHOTOSYSTEM-I COMPLEX - ISOLATION IN A NATIVE-STATE AND SOME PROPERTIES OF THE IRON-SULFUR CLUSTERS, H OHOKA; S ITOH; K SAEKI; Y TAKAHASHI; H MATSUBARA, The F(A)/F(B) protein of the photosystem I complex was isolated from spinach leaves in a native state by use of anaerobic systems. The protein contained 8.5 non-heme iron atoms and 8.0 acid-labile sulfur atoms per molecule, consistent with the current concept that it has two [4Fe-4S] clusters. Its absorption spectrum was very similar to those of bacterial-type ferredoxins. The ratio of the absorbance at 390 nm to that at 280 nm was 0.6, and the molar extinction coefficient at 390 nm was 32,000 M-1.cm-1. The oxidation-reduction properties of the iron-sulfur clusters were examined by redox potentiometry and EPR spectroscopy. The two clusters were distinguishable in terms of their oxidation-reduction midpoint potentials; their E(m) values were determined to be about -470 mV and -560 mV, respectively., Jan. 1991, 32, 1, 11, 17, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPAN BIOCHEMICAL SOC, 2 DISTINCT FERREDOXINS FROM RHODOBACTER-CAPSULATUS - COMPLETE AMINO-ACID-SEQUENCES AND MOLECULAR EVOLUTION, K SAEKI; Y SUETSUGU; Y YAO; T HORIO; BL MARRS; H MATSUBARA, Sep. 1990, 108, 3, 475, 482, Scientific journal
  • Refereed, NUCLEIC ACIDS RESEARCH, OXFORD UNIV PRESS UNITED KINGDOM, A PLANT-FERREDOXIN-LIKE GENE IS LOCATED UPSTREAM OF FERREDOXIN-I GENE (FDXN) OF RHODOBACTER-CAPSULATUS, K SAEKI; Y MIYATAKE; DA YOUNG; BL MARRS; H MATSUBARA, Feb. 1990, 18, 4, 1060, 1060
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, FERREDOXIN AND RUBREDOXIN FROM BUTYRIBACTERIUM-METHYLOTROPHICUM - COMPLETE PRIMARY STRUCTURES AND CONSTRUCTION OF PHYLOGENETIC TREES, K SAEKI; Y YAO; S WAKABAYASHI; GJ SHEN; JG ZEIKUS; H MATSUBARA, Oct. 1989, 106, 4, 656, 662, Scientific journal
  • Refereed, JOURNAL OF BACTERIOLOGY, AMER SOC MICROBIOLOGY, PURIFICATION AND PROPERTIES OF FERREDOXIN AND RUBREDOXIN FROM BUTYRIBACTERIUM-METHYLOTROPHICUM, K SAEKI; MK JAIN; GJ SHEN; RC PRINCE; JG ZEIKUS, Sep. 1989, 171, 9, 4736, 4741, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, PSEUDOMONAS-STUTZERI FERREDOXIN - CLOSE SIMILARITY TO AZOTOBACTER-VINELANDII AND PSEUDOMONAS-OVALIS FERREDOXINS, K SAEKI; S WAKABAYASHI; WG ZUMFT; H MATSUBARA, Aug. 1988, 104, 2, 242, 246, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, THE PROTEIN RESPONSIBLE FOR CENTER A/B IN SPINACH PHOTOSYSTEM-I - ISOLATION WITH IRON-SULFUR CLUSTER(S) AND COMPLETE SEQUENCE-ANALYSIS, H OHOKA; Y TAKAHASHI; K KURIYAMA; K SAEKI; H MATSUBARA, Jun. 1988, 103, 6, 962, 968, Scientific journal
  • Refereed, JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS LTD, PRELIMINARY CRYSTALLOGRAPHIC STUDY OF A RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE FROM CHROMATIUM-VINOSUM, H NAKAGAWA; M SUGIMOTO; Y KAI; S HARADA; K MIKI; N KASAI; K SAEKI; T KAKUNO; T HORIO, Oct. 1986, 191, 3, 577, 578
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, BARLEY LEAF PEROXIDASE - PURIFICATION AND CHARACTERIZATION, K SAEKI; O ISHIKAWA; T FUKUOKA; H NAKAGAWA; Y KAI; T KAKUNO; J YAMASHITA; N KASAI; T HORIO, Feb. 1986, 99, 2, 485, 494, Scientific journal
  • Refereed, JOURNAL OF BIOCHEMISTRY, JAPANESE BIOCHEMICAL SOC, A NOVEL FAD-PROTEIN THAT ALLOWS EFFECTIVE REDUCTION OF METHYL VIOLOGEN BY NADH (NADH-METHYL VIOLOGEN REDUCTASE) FROM PHOTOSYNTHETIC BACTERIUM, RHODOSPIRILLUM-RUBRUM - PURIFICATION AND CHARACTERIZATION, K SAEKI; T HARUNA; T KAKUNO; J YAMASHITA; T HORIO, Feb. 1986, 99, 2, 423, 435, Scientific journal
  • COG分類を考慮した遺伝子発現プロファイルデータのクラスタリング手法, 大古田,みのり; 石川,由羽; 高田,雅美; 佐伯,和彦; 城,和貴, Jun. 2015, 23
  • Compendium of Plant Genomes, Genome Sequence and Gene Functions in Mesorhizobium loti and Relatives, Kazuhiko Saeki, 27 Sep. 2014
  • Refereed, Microbes and Environments, Japanese Society of Microbial Ecology, Phenolic Acids Induce Nod Factor Production in Lotus japonicus-Mesorhizobium Symbiosis, Masayuki Shimamura; Takashi Kumaki; Shun Hashimoto; Kazuhiko Saeki; Shin-ichi Ayabe; Atsushi Higashitani; Tomoyoshi Akashi; Shusei Sato; Toshio Aoki, Mar. 2022, 37, 1, n/a, n/a, Scientific journal
  • Journal of Plant Research, Correction to: Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis (Journal of Plant Research, (2020), 133, 1, (109-122), 10.1007/s10265-019-01159-x), Yuji Tokumoto; Kayo Hashimoto; Takashi Soyano; Seishiro Aoki; Wataru Iwasaki; Mai Fukuhara; Tomomi Nakagawa; Kazuhiko Saeki; Jun Yokoyama; Hironori Fujita; Masayoshi Kawaguchi, A correction to this paper has been published: https://doi.org/10.1007/s10265-021-01295-3., Jul. 2021, 134, 4, 885, Scientific journal

MISC

  • Not Refereed, 第15回国際分子・植物・微生物相互作用学会(IS-MPMI) 2012.7.29-2012.8.2, A MATE-type transporter responsible for iron supply to nodule infection zone of Lotus japonicus., 高梨功次郎; 横正健剛; 高橋宏和; 佐伯和彦; 杉山暁史; 佐藤修正; 田畑哲之; 中園幹生; 馬建鋒; 矢崎一史, 2012, Summary international conference
  • Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Symbiotic significance of type III secretion system in Mesorhizobium loti, Saori Okabe; Shin Okazaki; Kouhei Tejima; Michael Gottfert; Kazuhiko Saeki, 2007, 48, S47, S47, Summary international conference
  • Not Refereed, 蛋白質・核酸・酵素, ミヤコグサで解き明かす菌根・根粒共生系の分子基盤, SAEKI Kazuhiko, Sep. 2006, 51, 9, 1044-1050
  • Not Refereed, 蛋白質・核酸・酵素, 共立出版, 根粒菌からみた共生システム, 南澤 究; 佐伯和彦; 佐藤修正; 下田宜司, 2006, 51, 9, 1044, 1050, Introduction scientific journal
  • Not Refereed, Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, SPRINGER, Characterization of a novel Lotus japonicus symbiotic mutant, Lot1, that shows reduced nodule numberand distorted trichomes, Y Ooki; M Banba; K Yano; J Maruya; S Sato; S Tabata; K Saeki; M Hayashi; M Kawaguchi; K Izui; S Hata, 2005, 41, 233, 233, Summary international conference
  • Not Refereed, 農業技術, 農業技術協会, 共生窒素固定研究の最新情報(10):根粒菌研究のためのリソース, SAEKI Kazuhiko, Apr. 2003, 58, 4, 183-188, 188
  • Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Analysis of gene expression of mesorhizobium loti by genomic macro-array, T Ohwada; K Minamisawa; P Dawady; H Mitsui; M Itakura; T Kaneko; S Tabata; T Yokoyama; K Tejima; K Saeki; H Ohmori; Y Murooka; M Hayashi; S Tajima; M Nomura; K Shimomura; M Abe; A Suzuki; Y Shimoda; T Uchiumi, 2003, 44, S210, S210, Summary international conference
  • Not Refereed, 日本微生物生態学会講演要旨集, 日本微生物生態学会, B-06 炭素飢餓条件下におけるミヤコグサ根粒菌の網羅的な遺伝子発現解析(生理・増殖2,口頭発表), 板倉 学; 大和田 琢二; 折笠 善丈; 南澤 究; 三井 久幸; 金子 貴一; 田畑 哲之; 横山 正; 手島 光平; 佐伯 和彦; 室岡 義勝; 林 誠; 田島 茂行; 野村 美加; 下村 憲司朗; 阿部 美紀子; 鈴木 章弘; 下田 宜司; 内海 俊樹, 15 Nov. 2002, 18, 74, 74
  • Not Refereed, 日本土壌肥料学会講演要旨集, 一般社団法人日本土壌肥料学会, 6-21 全ゲノム塩基配列情報に基づくミヤコグサ根粒菌の網羅的遺伝子発現解析(6.土壌生物), 大和田 琢二; 折笠 善丈; 南澤 究; 三井 久幸; 板倉 学; 田畑 哲之; 金子 貴一; 横山 正; 手島 光平; 佐伯 和彦; 大森 博文; 室岡 義勝; 林 誠; 田島 茂行; 野村 美加; 下村 憲司朗; 阿部 美紀子; 内海 俊樹; 鈴木 章弘; 下田 宜司, 25 Mar. 2002, 48
  • Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Genetic analysis of the Mesorhizobium loti mutant exo-22 that has defects in exopolysaccharide synthesis and symbiotic nitrogen fixation., E Mishima; H Imaizumi-Anraku; M Kawaguchi; K Saeki, 2002, 43, S72, S72, Summary international conference
  • Not Refereed, 化学と生物, 光合成細菌の多様なフェレドキシン, SAEKI Kazuhiko, Nov. 1993, 31, 11, 750-754
  • Not Refereed, PHOTOSYNTHESIS RESEARCH, KLUWER ACADEMIC PUBL, EVOLUTIONARY ASPECTS OF IRON-SULFUR PROTEINS IN PHOTOSYNTHETIC APPARATUS, Y FUJITA; Y TAKAHASHI; H OHOKA; K SAEKI; K FUKUYAMA; H MATSUBARA, Oct. 1992, 34, 1, 96, 96, Summary international conference
  • Not Refereed, ADVANCES IN INORGANIC CHEMISTRY, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, STRUCTURAL AND FUNCTIONAL DIVERSITY OF FERREDOXINS AND RELATED PROTEINS, H MATSUBARA; K SAEKI, 1992, 38, 223, 280, Book review
  • Not Refereed, FEBS LETTERS, ELSEVIER SCIENCE BV, TRANSCRIPTIONAL ANALYSIS OF 2 RHODOBACTER-CAPSULATUS FERREDOXINS BY TRANSLATIONAL FUSION TO ESCHERICHIA-COLI-LACZ, Y SUETSUGU; K SAEKI; H MATSUBARA, Plasmids which contained the translational fusion of Escherichia coli lacZ to Rhodobacter capsulatus ferredoxin genes, fdxN and fdxA, were constructed. Effects of growth conditions on the expression of each ferredoxin were analyzed by measuring the beta-galactosidase activity in R. capsulatus which harbored a corresponding plasmid. Transcription of fdxN::lacZ, the ferredoxin I fusion gene, was regulated at least 100-fold by either NH4+ or O2 but not by illumination, confirming that fdxN belongs to the nif-gene family. Transcription of fdxA::lacZ, the ferredoxin II fusion gene, however, was constant under all the conditions surveyed, suggesting that the protein has some constitutive function(s)., Nov. 1991, 292, 1-2, 13, 16
  • Not Refereed, Cell Science, 古細菌のフェレドキシン, SAEKI Kazuhiko, Apr. 1990, 6, 4, 293-296
  • Not Refereed, FASEB JOURNAL, FEDERATION AMER SOC EXP BIOL, A PROTEIN CARRYING FE-S CENTER A/B IN SPINACH CHLOROPLAST PHOTOSYSTEM-I (PSI), H MATSUBARA; H OHOKA; Y TAKAHASHI; K SAEKI, Mar. 1988, 2, 4, A768, A768, Summary international conference
  • Not Refereed, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, AMER CHEMICAL SOC, REGULATORY METABOLISM OF SINGLE CARBON-COMPOUNDS BY BUTYRIBACTERIUM-METHYLOTROPHICUM, MK JAIN; GJ SHEN; K SAEKI; JG ZEIKUS, Aug. 1987, 194, 34, MBTD, Summary international conference
  • PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, LjMATE1: A Citrate Transporter Responsible for Iron Supply to the Nodule Infection Zone of Lotus japonicas (vol 54, pg 585, 2013), Kojiro Takanashi; Kengo Yokosho; Kazuhiko Saeki; Akifumi Sugiyama; Shusei Sato; Satoshi Tabata; Jian Feng Ma; Kazufumi Yazaki, Oct. 2013, 54, 10, 1749, 1749, Others
  • 日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology, Rhizobial use of factors common among virulent bacteria, SAEKI Kazuhiko, 31 Mar. 2011, 37, 23, 23
  • Not Refereed, PLANT AND CELL PHYSIOLOGY, OXFORD UNIV PRESS, Global analysis of gene expression of Mesorhizobium loti MAFF303099, T Uchiumi; T Ohwada; Y Orikasa; K Minamisawa; H Mitsui; M Itakura; T Kaneko; S Tabata; T Yokoyama; K Tejima; K Saeki; H Omori; Y Murooka; M Hayashi; S Tajima; M Nomura; K Shimomura; M Abe; A Suzuki; Y Shimoda, 2002, 43, S73, S73, Summary international conference
  • Abstracts of the meeting, the Society of the Science of Soil and Manure, Japanese Society of Soil Science and Plant Nutrition, 8-10 Rhizobial determinants for nodulation and nitrogen fixation with bred soybean, Teamtisong Kamonluck; Teaumroong Neung; Okazaki Shin; Saeki Kazuhiko; Kaneko Takakazu; Tabata Satoshi; Minamisawa Kiwamu, 20 Aug. 2003, 49, 58, 58
  • 日本土壌肥料学会講演要旨集, 一般社団法人日本土壌肥料学会, 8-17 共生・微好気・炭素飢餓条件におけるミヤコグサ根粒菌の網羅的遺伝子発現解析(8.共生), 大和田 琢二; 南澤 究; 三井 久幸; 板倉 学; 貫井 憲之; 田畑 哲之; 金子 貴一; 横山 正; 手島 光平; 佐伯 和彦; 大森 博文; 室岡 義勝; 林 誠; 前川 隆紀; 田島 茂行; 野村 美加; 下村 憲司朗; 阿部 美紀子; 内海 俊樹; 鈴木 章弘; 下田 宣司; 塩谷 幸樹, 20 Aug. 2003, 49, 60, 60
  • Not Refereed, Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, SPRINGER, Genome-wide view of Mesorhizobium and Bradyrhizobium, expression island clustard on symbiosis island and genome diversity, T Uchiumi; T Ohwada; T Kaneko; T Yokoyama; K Saeki; M Hayashi; S Tajima; K Minamisawa, 2005, 41, 139, 139, Summary international conference
  • 日本土壌肥料学会講演要旨集, 一般社団法人日本土壌肥料学会, マクロアレイによるダイズ根粒菌株のゲノム比較(6. 土壌生物, 2004年度大会講演要旨集), 南澤 究; 板倉 学; 増田 幸子; 佐伯 和彦; 大森 博文; 横山 正; 金子 貴一; 田畑 哲之; 大和田 琢二; 田島 茂行; 内海 俊樹, 14 Sep. 2004, 50, 40, 40
  • 日本土壌肥料学会講演要旨集, 一般社団法人日本土壌肥料学会, 8-12 ダイズ根粒シンビオソーム溶液を処理した単生ダイズ根粒菌に誘導される新規遺伝子応答の網羅的解析(8. 共生, 2006年度秋田大会講演要旨), 横山 正; 大石 真子; 有馬 泰紘; 大和田 琢二; 南澤 究; 三井 久幸; 板倉 学; 佐伯 和彦; 田島 茂行; 内海 俊樹; 阿部 美紀子, 05 Sep. 2006, 52, 44, 44
  • 日本土壌肥料学会講演要旨集, 一般社団法人日本土壌肥料学会, 6-8 ダイズ根粒菌のゲノムの構造比較と共生窒素固定能(6. 土壌生物, 2006年度秋田大会講演要旨), 板倉 学; 佐伯 和彦; 大森 博文; 横山 正; 金子 貴一; 田畑 哲之; 大和田 琢二; 田島 茂行; 内海 俊樹; 藤田 耕之助; 本間 佳奈; 鮫島 玲子; 三井 久幸; 南澤 究, 05 Sep. 2006, 52, 32, 32
  • 日本微生物生態学会講演要旨集, 日本微生物生態学会, A-12 マクロアレイによるダイズ根粒菌株のゲノム比較 : Bradyrhizobium属細菌のゲノム進化と多様性(分類・系統解析,口頭発表), 板倉 学; 増田 幸子; 佐伯 和彦; 大森 博文; 横山 正; 金子 貴一; 田畑 哲之; 大和田 琢二; 田島 茂行; 内海 俊樹; 南澤 究, 2004, 20, 134, 134
  • 日本微生物生態学会講演要旨集, 日本微生物生態学会, B-08 マクロアレイによるダイズ根粒菌バクテロイドの網羅的遺伝子発現解析(遺伝子伝播,口頭発表), 原 圭乃; 板倉 学; 佐伯 和彦; 大森 博文; 横山 正; 金子 貴一; 田畑 哲之; 大和田 琢二; 田島 茂行; 内海 俊樹; 三井 久幸; 南澤 究, 2004, 20, 170, 170
  • 日本微生物生態学会講演要旨集, 日本微生物生態学会, 27-C-21 炭素源飢餓条件下で発現誘導されるミヤコグサ根粒菌の遺伝子(生理・増殖,一般講演), 板倉 学; 大和田 琢二; 南澤 究; 三井 久幸; 金子 貴一; 田畑 哲之; 横山 正; 手島 光平; 佐伯 和彦; 大森 博文; 室岡 義勝; 林 誠; 田島 茂行; 下村 憲司朗; 阿部 美紀子; 内海 俊樹; 鈴木 章弘; 下田 宜司, 2003, 19, 79, 79

Books etc

  • “Chapter 5: Genome sequence and gene functions in Mesorhizobium loti and relatives” In The Lotus japonicus Genome (Eds. Tabata S and Stougaard J), Springer-Verlag, Berlin, Germany, SAEKI Kazuhiko, 分担, Oct. 2014, 41-57, Not Refereed, 9783662442692
  • 光合成微生物の機能と応用, シーエムシー出版, SAEKI Kazuhiko, 分担, Dec. 2006, 170-176, Not Refereed
  • モデル植物の実験プロトコール(第3版)イネ・シロイヌナズナ・ミヤコグサ編, 秀潤社, SAEKI Kazuhiko, 分担, Apr. 2005, 315-317, Not Refereed
  • “Electron Transport to Nitrogenase: Diverse Routes for A Common Destination” In Genetics and Regulation of Nitrogen Fixing in Free-Living Bacteria (Eds. W. Klipp, B. Masepohl, J.R. Gallon & W.E. Newton) pp.257-290, Kluwer Academic Publishers, Dortrecht, Netherland (ISBN: 1-4020-2178-X), SAEKI Kazuhiko, 筆頭著者, 2004, Not Refereed
  • “Genome analysis of Mesorhizobium loti” In Biotechnology in Agriculture and Forestry Vol 52. Brassica and Legumes: From genome structure to plant breeding (Eds. T. Nagata and S. Tabata) pp.203-216, Springer-Verlag, Berlin, Germany (ISBN: 3-540-42728-7), SAEKI Kazuhiko, 分担, 2003, Not Refereed
  • “Electron Transport Pathway to Nitrogenase in Rhodobacter capsulatus : Rnf Complex and Its Relatives in Non-Diazotrophs” in Achievements and Objectives in Nitrogen Fixation beyond Horizon (F. Pedorosa, A. Kondorosi & G. Walker eds.), Kluwer Academic Publishers, Dortrecht, Netherland (ISBN: 0-7923-6233-0), SAEKI Kazuhiko, 筆頭著者, Mar. 2000, Not Refereed
  • 蛋白質・酵素の基礎実験法 改訂第2版, 南江堂, SAEKI Kazuhiko, 分担, Nov. 1994, 232-241, 355-361, Not Refereed, 9784524401239

Presentations

  • SAEKI Kazuhiko, 第26回植物細菌病談話会, 根粒菌菌体外分泌系による窒素固定共生の制御, Oct. 2014, 岡山空港温泉 レスパール藤ケ鳴, False
  • SAEKI Kazuhiko, 第55回日本植物生理学会年会, GFP派生物導入変異体を用いた共生成立過程における根粒菌体pHの測定にむけて, Mar. 2014, 富山大学・五福キャンパス, False
  • SAEKI Kazuhiko; Kazuhiko Saeki; Hiroko Kasai-Maita; Kazuna Kubota; Takakazu Kaneko; Hideki Hirakawa; Satoshi Tabata; Shusei Sato, 18th International Congress on Nitrogen Fixation, Core gene sets and evolution of symbiosis islands in rhizobial isolates from Lotus japonicas indigenous to Japan, Oct. 2013, Phoenix Seagaia Resort; World Convention Center Summit, Miyazaki, Japan, True
  • SAEKI Kazuhiko, 第54回日本植物生理学会年会・シンポジウム10・微生物エフェクター:植物と微生物の攻防と調和の鍵を握る分子, 根粒菌エフェクターによるマメ科植物との共生成立の制御, Mar. 2013, 岡山大学・津島キャンパス, False
  • 清水香織; 高市真一; 佐伯和彦, 第93回日本生化学会大会, β-カロテンを蓄積するゲノム改変光合成細菌株を用いたレチナール大量合成条件の探索, Poster presentation, Sep. 2020, 14 Sep. 2020, 16 Sep. 2020
  • 清水香織; 高市真一; 佐伯和彦, 第61回日本植物生理学会年会, β-カロテン生産改良株を用いたレチナール合成の試み, Poster presentation, Mar. 2020, 19 Mar. 2020, 21 Mar. 2020
  • 清水香織; 高市真一; 佐伯和彦, 第92回日本生化学会大会, Rhodobacter capsulatus におけるレチナール合成の試み, Poster presentation, 19 Sep. 2019, 18 Sep. 2019, 20 Sep. 2019
  • 三浦帆波; 西川佳那; 本田裕樹; 藤井浩; 佐伯和彦, 第92回日本生化学会大会, ミヤコグサ根粒菌カタラーゼKatEはプロトヘムとヘムdをもつ, Poster presentation, 18 Sep. 2019, 18 Sep. 2019, 20 Sep. 2019
  • 三浦帆波; 佐伯和彦, 第91回日本生化学会大会(京都国際会館), ミヤコグサ根粒菌5-アミノレブリン酸合成酵素は大腸菌内での根粒菌カタラーゼのホロ酵素発現を補助するか?, Poster presentation, 24 Sep. 2018, 24 Sep. 2018, 26 Sep. 2018
  • 日下部 翔平; 金子 貴一; 安田 美智子; 三輪 大樹; 岡崎 伸; 佐伯 和彦; 佐藤 修正, 植物微生物研究会・第28回研究交流会(鳥取大学農学部), Bradyrhizobium elkanii USDA61 株の3型分泌エフェクターはミヤコグサに複数の防御反応を誘導する, Poster presentation, Sep. 2018, 19 Sep. 2018, 21 Sep. 2018
  • 三浦 帆波; 白井 理恵; 浅野 美和; 藤井 浩; 佐伯 和彦, 植物微生物研究会・第28回研究交流会(鳥取大学農学部), 大腸菌を宿主とするミヤコグサ根粒菌のカタラーゼと5-アミノレブリン酸合成酵素の共発現, Poster presentation, Sep. 2018, 19 Sep. 2018, 21 Sep. 2018
  • 疋田 真穂; 佐伯 和彦, 植物微生物研究会・第28回研究交流会(鳥取大学農学部), 宮古島の土壌とミヤコグサから単離された根粒菌の多様性調査, Poster presentation, Sep. 2018, 18 Sep. 2018, 21 Sep. 2018
  • 清水香織; 高市真一; 佐伯和彦, 第59回日本植物生理学会(札幌コンベンションセンター), 光合成細菌のクロマトフォア上での動物光受容体の発現に向けて, Mar. 2018, 28 Mar. 2018, 30 Mar. 2018

Works

  • 科学技術総合研究委託費・委託業務・平成19年度「科学技術連携施策群の効果的・効率的な推進 食料・生物生産研究」、 「植物・微生物間共生におけるゲノム相互作用」のうち、「3-2.宿主防御システムに対抗し回避・抑制するための根粒菌マシナリー」, Sep. 2007, Mar. 2010

Research Projects

  • Grant-in-Aid for Scientific Research (B), 01 Apr. 2015, 31 Mar. 2019, 15H02960, Estimate of Quaternary paleoenvironment from molecular information of microfossils, TAKADA Masashi; Shimada Aiko, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Nara Women's University, 16640000, 12800000, 3840000, We examined possibilities that new methods could be introduced into Quaternary paleoenvironment reconstruction using DNA analysis of pollen fossils and oxygen isotope ratio of phytolith fossils. We examined PCR amplification conditions for chloroplast genome analysis using modern conifers as test samples. Although we could obtain primers suitable for analysis of phylogenetic comparison evolution above the genus level, we could not find the amplification condition suitable for species identification from total DNA samples. Further examination of amplification conditions is necessary for the DNA analysis of pollen fossils, which are anticipated to decrease and fragment the DNA content itself due to aging. Since differences in oxygen isotope ratio analysis results by different methods were recognized for phytoliths extracted from modern plants, it is necessary to establish a routine way to measure the oxygen isotope ratio of phytoliths with standard samples., url
  • Grant-in-Aid for Scientific Research (C), 01 Apr. 2015, 31 Mar. 2019, 15K07003, On the Mechanism of Functional Expression of Rhizobial Type III Secretion System, Saeki Kazuhiko, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Nara Women's University, 5070000, 3900000, 1170000, The Type III Secretion System (T3SS) was found as a toxin-injection system from pathogenic bacteria to mammalian cell. Genes for T3SS are identified not only in animal and plant pathogens but also in symbiotic bacteria such as rhizobia. Here, we have studied Mesorhizobial T3SS, which shows limited homology even to T3SSs in plant pathogens. We have investigated target genes of the T3SS transcriptional activator protein, TtsI, and found a few new binding sites. We also constructed reporter systems to access the secretion activity as well as transcriptional activation and utilized the systems to evaluate the necessity for secretion activity of the relatively small genes that situate around the main T3SS gene cluster. Simultaneously, we constructed new broad-host range vector-sets that are comprised of controllable promoter plasmid and temperature sensitive replication., url
  • 新学術領域研究(研究領域提案型), 01 Apr. 2012, 31 Mar. 2014, 24113512, 宿主からの防御・制裁への対抗に関連する根粒菌側因子が共生の進化に及ぼす効果, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 新学術領域研究(研究領域提案型), 奈良女子大学, 9880000, 7600000, 2280000, 根粒菌とマメ科植物が営む窒素固定共生は、相利共生の代表的な例である。しかし、宿主植物が自然免疫や病原応答類似の手段によって根粒菌に対して攻撃を加える場合や、逆に根粒菌が寄生的に振る舞う場合もある。これら共生生物間の攻撃と防御機構の進化を解明する土台を築くことが本研究の目標である。 本課題の第一の目的は、宿主の防御機構に対する根粒菌の対抗手段はどの様に進化しつつあるのかを解析することである。まず、ミヤコグサ根粒菌Mesorhizobium loti MAFF303099株の3型分泌エフェクターのうち、中近東原産のミヤコグサ近縁種Lotus halophilusとの共生に負の効果を持ち、植物病原菌にもホモローグの存在するMlr6361タンパク質について、負の効果の原因ドメインを明らかにした。また、ダイズ根粒菌Bradyrhizobium elkanii USDA61株が、Nodファクター受容体NFRに欠陥を持つダイズ変異体(rj1ダイズ:一般的なダイズ根粒菌の感染では根粒を形成しない)に対してさえ根粒を形成して窒素固定を行うこと、これは3型分泌系によりNFRを経ずに宿主の根粒形成遺伝子群を活性化しているためであることを示した(学会誌1)。さらに、活性酸素種除去酵素の一つであるスーパーオキシドジスムターゼ(SOD)を欠損するM. loti変異株は、宿主系統により根粒着生と窒素固定能が異なるという結果を得た(投稿準備中)。 本課題の第二の目的は、寄生的根粒菌系統に対する宿主からの制裁は存在して共進化における選択圧であるのかを解明するモデル実験系を確立することにある。このために、根粒形成や窒素固定能の異なる様々なM. loti変異株に特異標識を付加した派生株を作製した。標識菌株群と宿主を構成成分とするミニ生態系の構築実験を開始した。
  • Grant-in-Aid for Scientific Research (C), 2011, 2013, 23510235, Deduction of minimum sysmbiosis island gene sets for Lotus rhizobia, SAEKI Kazuhiko; SATO Shusei, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Nara Women's University, 5460000, 4200000, 1260000, In an attempt to deduce the rhizobial minimum symbiosis gene sets for mutualism between Mesorhizobium loti and Lotus species, we compared nucleotide sequences of symbiosis islands of three M. loti strains and found 155 genes are completely conserved among them. We made controllable promoters and new selectable markers to be used with M. loti strains. We also made a temperature sensitive derivative of the broad-host range vector pBBR1MCS2, by mutagenesis of its rep gene. In addition, we performed Next Generation Sequencing analysis of several M. loti strains and mapped the obtained reads on the genome sequence of the strain MAFF303099. The results supported the divergence of main chromosomes among M. loti strains with conserved core structure of symbiosis island., url
  • 挑戦的萌芽研究, 2008, 2010, 20657023, 光合成細菌を宿主とする膜タンパク質の高効率大量発現系の構築, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 挑戦的萌芽研究, 奈良女子大学, 3200000, 3200000, 本研究では、光合成細菌が暗所・微好気条件下でも多量の細胞内膜陥入構造(クロマトフォア)を形成し大量の光化学系関連の膜タンパク質を蓄積しうる点に着目して、膜含量自体を増大させた上で光化学系の替わりに外来の膜タンパク質を安定かつ多量に発現させることを目指した。Sec分泌系を用いて発現させた場合に引き起こされる基質輸送体や電子伝達系、ATP合成酵素などの機能発現との競合を避けるために、膜配置・分泌シグナルとして、枯草菌由来のMISTIC(Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs)タンパク質を用いることとし、MISTIC人工遺伝子を光合成細菌Rhodobacter capsulatusのコドン頻度に合わせて作製した。クロマトフォアを形成しない好気条件下で目的の膜タンパク質を発現せず、微好気条件でのみ多量に発現させるために、酸素分圧の低下により制御を受けるpufプロモーターを人工遺伝子の上流に配置した。また、下流にはρ因子非依存性の転写終結を惹起する人工配列を配置した。なお、産物のアミノ末端側にヘキサHis配列とカルボキシ末端側に血液凝固系のタンパク質分解酵素トロンビン認識配列などを付加して、発現させる膜タンパク質の精製を容易にした。人工遺伝子とのin-frame融合により、GFPならびにR.capsulatus自身のRnfの過剰発現を確認した。安定発現のため宿主にpuhBとdegPの変異を導入したが、効果は限定的であった。また、長時間の培養時に宿主株の示す溶菌傾向に変化は認められなかった。一方、膜画分から標的タンパク質を可溶化・精製の際に、カロチノイド色素が標的と類似の挙動を示した。今後、溶菌傾向と色素生成能を軽減させた株を作出する必要が有る。
  • Grant-in-Aid for Scientific Research on Priority Areas, 2005, 2009, 17018041, Comprehensive analysis of interactions between plants and microbes, TABATA Satoshi; KAWATUCHI Masayoshi; SAEKI Kazuhiko; MINAMISAWA Kiwamu, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas, Kazusa DNA Research Institute, 146900000, 146900000, Molecular mechanisms of symbiotic nitrogen-fixation between legumes and rhizobia have been investigated by molecular-genetics and genomics approaches. Genes that are essential or related to the establishment of symbiosis, nodule formation and its regulation, and nitrogen fixation were isolated, and their functions were identified., url
  • 特別研究員奨励費, 2004, 2005, 04F04176, ミヤコグサ根粒菌におけるアミノ酸代謝と宿主との物質交換の分子遺伝学的・生化学的解析, 佐伯 和彦; KUMAR Anvita, 日本学術振興会, 科学研究費助成事業 特別研究員奨励費, 奈良女子大学, 2400000, 2400000, 1)実験系の評価:材料とする菌株自体の特性を明確にするため、ミヤコグサまたは近縁のマメ科植物(西洋ミヤコグサやネビキミヤコグサ)の根粒から単離された国内外15株の根粒菌の16S rRNAならびにnodJ-nodS領域の部分塩基配列を新たに決定した。15菌株の宿主特異性を調べた結果は、16S rRNAを用いた結果より、nodJ-nodS領域を用いた結果より強くリンクしていた。これは宿主特異性に共生アイランドが主要な役割を果たしていることを示唆した。 2)変異株作製:MAFF303099株のアミノ酸輸送系遺伝子や菌体表層構造の変異性を蓄積するために、大腸菌アルカリ性フォスファターゼphoA遺伝子の成熟タンパク質コード領域を含むmini-TnphoAを用いたトランスポゾン挿入実験を行った。約4000の挿入株から、357株のフォスファターゼ発現株を得た。これらは膜貫通タンパク質のペリプラズム露出部位またはペリプラズムタンパク質にin-frame融合しているものと期待できる。しかし、挿入株総数と期待される膜タンパク質遺伝子の数を勘案すると、フォスファターゼ発現株の数は多すぎると考えられた。これらの栄養要求性や菌体表層構造ならびに根粒形成能・共生能の評価を行うとともに、TnphoAによって破壊されていないと想定されるアルギニン合成系遺伝子などの有無をPCRで調べた。その結果は、集積した変異株候補のうち少なくとも65%、最大で85%程度が、非根粒菌の混入に由来するものと判断された。但し、残る15から30%程度の変異株の中には、共生不全のものが含まれ、新規の遺伝子の発見に繋がることが期待される。しかしながら、フェローの在日中には、inverse PCRやクローニングによる挿入断片の回収が十分に達成できず、遺伝子の同定は今後の課題である。
  • 特定領域研究, 2004, 2004, 16013225, ミヤコグサ根粒菌ゲノムの多様性・可塑性の解析に基づく共生システム分子基盤の解明, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 特定領域研究, 奈良女子大学, 3400000, 3400000, ミヤコグサと共生する根粒菌の菌株間の差異に注目し、共生アイランドの構造上の相違と機能上の相違の相関、アイランド外のゲノム領域の共生における意義を調べるために、以下の実験を行った。1)ミヤコグサ根粒菌の菌株比較:4種の宿主を用いて、国内外でミヤコグサと近縁種の根粒から単離された15菌株について、共生アイランドに特異的な構造と宿主域を検定した。国内の6株はどれもTypeIIIタンパク質分泌装置、ニュージーランドや北米の9株はいずれもTypeIV装置の遺伝子群を持つと見積もられた。国内の6株はいずれもL.japonicusともL.corniculatusとも共生したが,ミヤコグサ近縁種Lotus pedunculatusには根粒を形成しても共生不能で、木本のマメ科植物ギンネムLeucaena leucocephalaには根粒形成しなかった。一方、ニュージーランドと北米で単離の9株はいずれもL.pedunculatusとL.leucocephalaに根粒を形成したが、両者に共生可能なものは4株で、残り5株は窒素固定不能であった。分泌系との関連は現在検証中である。2)共生アイランド水平移動モデル化に向けたリコンビナーゼ系の開発:インテグラーゼ・ファミリーに属する配列特異的なリコンビナーゼの標的配列(FRTやRS)をsingle-crossoverによりゲノムに組込む方法を確立した。3)ミヤコグサに早期老化根粒を形成するインゲン根粒菌の異種相補実験:インゲン根粒菌株Rhizobium etli CE3株は、M.lotiと同じNod因子を産生し、ミヤコグサと不完全な共生を行う。整列化コスミドクローンとその派生物を個々別々に導入した結果、R.etliとミヤコグサの共生を部分的に高めるクローンを13得てきた。アセチレン還元法によりニトロゲナーゼ活性測定を行った結果、コスミド非導入株では感染2週間後にほぼ活性を失うのに対し、これらクローン導入株ではより窒素固定活性が長期にわたり保持されていた。さらに、共生能の向上は根粒数の持続的増加による量的なものと根粒数の変わらぬ質的なものの2種に分別された。個別責任遺伝子の同定と効果の加算性を検討しつつある。
  • 特定領域研究, 2003, 2003, 15013232, ミヤコグサ根粒菌ゲノムの多様性・可塑性の解析に基づく共生システム分子基盤の解明, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 特定領域研究, 大阪大学, 4800000, 4800000, 本研究では、Mesorhizobium loti MAFF303099株ゲノムの情報を基盤に、ミヤコグサLotus japonicus根粒由来の菌株(国内単離6株)と西洋ミヤコグサLotus corniculatusなどミヤコグサ近縁種の根粒由来の菌株(M.loti標準株NZP2213株やR7A株などニュージーランドと北米単離の9株)更に,広宿主域菌Rhjzobium spp.NGR234株やミヤコグサと不完全な共生を行うRhizobium etli CE3株について、ゲノムの多様性・可塑性と宿主特異性を解析することにより、非根粒菌から根粒菌への変換に必要な因子及び宿主域を決定する因子の解明を目指している。(1)ゲノム構造の粗比較:16S rRNA配列およびnodJ-nodS領域の塩基配列に基づき系統解析を行った。(2)共生アイランド内の分泌系遺伝子の相違:MAFF303099とR7Aの2つの共生アイランドは、前者がTypeIII蛋白質分泌系遺伝子群を持つのに対し、後者はTypeIV蛋白質分泌系群を持つ点で著しく異なる。PCRとSouthern解析により、国内株はいずれもTypeIII、ニュージーランドや北米の株はTypeIV遺伝子群を持つと見積もられた。(3)生育条件・宿主域の比較:国内株は、ほぼ同一の抗菌剤感受性スペクトル示し,ミヤコグサ以外にも西洋ミヤコグサと有効な共生を行うが,Lotus pedunculatusには根粒を形成するが共生不能であった。以上の結果は,16S rRNA配列よりは,共生アイランド内の局所構造に基づく分類か,宿主域とよく相関していることを示した。(4)共生アイランド水平移動のモデル系構築準備:リコンビナーゼを用いてゲノム改変を行うため、広域宿主ベクターpBBR1MCS2のrep遺伝子にError-Prone PCRを行い、温度感受性を示す派生物1クローンを得た。
  • 基盤研究(C), 2003, 2003, 15638002, 全ゲノム塩基配列情報に基づく根粒菌の共生機構の解明, 南澤 究; 横山 正; 田島 茂行; 佐伯 和彦; 永田 裕二; 大和田 琢二, 日本学術振興会, 科学研究費助成事業 基盤研究(C), 東北大学, 3400000, 3400000, 本調査の目的は、根粒菌とマメ科植物の共生機構および生態を根粒菌ポストゲノム解析により明らかにする総合的な戦略を練り、食糧生産および環境保全に資する共生科学を創成することにある。8月12-13日に仙台において、ワークショップ「根粒菌のポストゲノム研究の展開」を開催し、数回の打合せ行い、以下のような項目について調査を行った。 1)モデルマメ科植物分子遺伝解析に対応したミヤコグサ根粒菌の戦略の検討:根粒菌の遺伝子が宿主植物側の共生受容系や防御応答系とどのような相互作用を通じて共生成立に至るのかという点を、モデルマメ科植物ミヤコグサの分子遺伝解析タイアップしながら進行させる必要がある。既に共同研究として論文発表した網羅的な遺伝子発現解析・破壊株の共生特性の解析以外に、CalcofluorやTn-phoA変異などの根粒菌の細胞膜構造、分泌タンパク、多糖生産の変異株が、相互作用機構解明に適したアプローチ方法であると結論された。 2)ダイズ根粒菌のアレイ解析・ライブラリー構築利用の基本戦略の策定:ミヤコグサ根粒菌と同様に整列化コスミッドライブラリーが、品種特異的共生因子の同定に有効であると判断された。また、ミヤコグサ根粒菌で使用したマクロアレイ系のダイズ根粒菌への適用は、クローンの選択・RNA抽出方法などの改善が必要であると考えられた。 3)食糧生産および環境保全への応用:根粒菌ゲノム情報から難分解物質分解系遺伝子、脱窒遺伝子群は、根粒菌接種に環境保全への付加価値をつけ、また機能性遺伝子導入根粒菌による共生環境修復への発展性が期待された。また、グリシンベタインによる浸透圧(塩)耐性、乾燥耐性は、応用研究に有効であると考えられた。野外利用はセルフクローニング系が有望であった。 4)クローン管理およびデータベース構築:データベース構築・公開は大阪大学で、クローン管理は東北大学と東京農工大学で共同で行うこととした。
  • Grant-in-Aid for Scientific Research (C), 2002, 2003, 14540594, Similarity and Diversity of Electron Transport Pathways to Nitrogense, Studies on Rhodobacter and Mesorhizobium, SAEKI Kazuhiko; FUKUYAMA Keiichi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Osaka University, 3400000, 3400000, For structural and biochemical analysis of proteins that participate electron transport to nitrogenase, the following studies were carried out on in Rhodobacter capsulatus rnf and nif gene products and Mesorhizobium loti fixABCX products. (1) Affinity chromatography procedures were optimized for successful purification of the proteins under strict anaerobic conditions with dithionite-containing buffer system, using (His)6-tagged model electron carrier protein. (2) Effective purification procedures were established for R. capsulatus NifF protein after expression in Escherichia coli. (3) The M loti fixX gene was over-expressed in E. coli under various conditions including co-expression of GroES-GroEL chaperones, however, the products formed inclusion body. Reconstitution conditions were investigated to make holo-FixX with Fe-S cluster from denatured inclusion bodies. (4) The M loti fixA, fixB, fixC and fixX genes were co-expressed in E. coli, however, most of the product were situated on membranes. (5) For effective exploitation of the genome sequence information of Lotus microsymbiont, Mesorhizobiurn loti MAFF303099, to discover gene functions, we have constructed an ordered and mutually overlapping cosmid library using an IncP broad host range vector. The library consisted of 480 clones to cover approximately 99.6% of the genome with average insert size and overlap of 26.9 and 11.1 kbp, respectively.
  • 特定領域研究, 1999, 2002, 11169223, タバコネクロシスウィルスの高分解能立体構造と粒子形成機構の解析, 福山 恵一; 大岡 宏造; 佐伯 和彦; 角田 佳充, 日本学術振興会, 科学研究費助成事業 特定領域研究, 大阪大学, 27000000, 27000000, タバコネクロシスウィルス(TNV)は一本鎖RNAをゲノムとして持つ球状ウィルスであり、アガサ・ナス・マメ・ユリ科などの植物に感染し、壊疽症状を起こす。TNVのサブユニットは276アミノ酸残基からなる一次構造上一種類のタンパクであるが、これが180個集合してT=3の正20面体対称を持つキャプシドを形成している。TNV粒子はN末端領域の立体構造が異なる3種類のサブユニットから構成されていることを既に明らかにしている。さらにサブユニットの境界にCa^<2+>イオンがあり、EDTA処理により粒子は解離し、またCa^<2+>の添加により元の粒子と区別できない粒子に再構成することも明らかにしている。本研究では、N末端が粒子形成にどのように影響するか、さらにその構造基盤を明らかにすべく解析を進めた。まずN末端を欠いたサブユニットををコードする遺伝子の発現系を構築し、大腸菌で大量発現させた。このタンパク質は不溶性画分に得られたので、これを尿素で可溶化した後、尿素を除くことでリフォールドさせた。このタンパク質はCa^<2+>存在下で天然の粒子より小さな粒子に再構成した。この粒子の形・サイズから正20面対称的に60個会合したと考えられる。この粒子の立体構造解析のため、結晶化を試みたところ、約0.2mmの大きさの単結晶を得た。この結晶は正方晶系に属し、単位格子中に4粒子含まれる。放射光を用いたX線回折実験で、2.7Å以上の分解能の回折点を観測できた。数百万の回折強度データを収集し、現在これに基づいて構造解析中である。
  • Grant-in-Aid for Scientific Research (B), 1999, 2001, 11440236, Studies on a Novel Energy-Coupling Enzyme Family Rnf That Functions at Very Low Redox Potential, SAEKI Kazuhiko; TAKAHASHI Yasuhiro; FUKUYAMA Keiichi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Osaka University, 7700000, 7700000, The rnf(rhodobacter nitrogen fixation) operon in Rhodobacter capsulatus is essential for nitrogen fixation under light. The genes encode a protein complex in chromatophore membrane that might function as an energy-consuming NADH-ferredoxin oxidoreductase. 1) We have partially purified the Rnf complex using Ni-NTA affinity column with hexahistidine-tagged RnfB expressed the cells of non-polar rnfB mutant strain KF11l containing the corresponding modified rnfB gene. However, the iron-sulfur center in RnfB protein was so unstable that complete purification was unsuccessful. 2) Analysis by phoA-fusion method revealed that RnfA and RnfE both span membrane six times with opposite Membrane topology of transmembrane subunits RnfA, RnfD and RnfE was analyzed by the. It was revealed that RnfA and RnfE span membrane 6 times with an opposite orientation and that RnfD protein spans membrane 8 times with its central hydrophilic region exposed to periplasm. 3) Downstream analysis of the known rnf genomic region revealed there is another gene (rnfI) that is essential for nitrogen fixation. RnfI protein shows similarity to the FADbinding subunit of periplasmic flavocytochrome c sulfide dehydrogenase.
  • Grant-in-Aid for Scientific Research (C), 1997, 1998, 09640771, A STUDY ON A NOVEL ENERGY-COUPLING PROTEIN COMPLEX THAT REDUCE FERREDOXIN BY NADH, SAEKI Kazuhiko, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Osaka University, 2700000, 2700000, The rnf(rbodobacter nitrogen fixation)operon in Rhodobacter capsutatus is essential for nitrogen fixation under light. We showed that products of the rnf operon constitute a protein complex in chromatophore membrane and proposed that the complex has a chimerie construct of two subcomplexes, one transmembrane subcomplex similar to Na^+-translocating NADH quinone oxidoreductase and the other peripheral subcomptex similar to H^+-translocating NADH quinone oxidoreductase. Its function should : be energy-consuming NADH-ferredoxin oxidoreductase. 1) We revealed that the complex is also required for nitrogen fixation under dark anaerobic conditions using dimethylsulfoxide as a terminal electron acceptor this agrees that the Rnf complex functions without direct link to photosynthetic reaction center. 2) Presence of at least one Fe-S center in Rnf complex was identified by EPR analysis The cluster showed a novel signal, g=1 .84, at 10K with extremely low redox potential. Since other strong signals from succinate-quinone oxidoreductase interfered detailed analysis, we are currently seeking conditions and mutants to overcome this difficulty. 3) Escheriehia coil and four other non-diazotrophic bacteria possess homologous of the rnf operon. Over expression of the E.coil homologue in E.ccli seemed lethal. This E.coli operon did not complemented the rnf-null mutants of R.capsulatus. 4) Chemical crosslinking studies showed that RnfC subunit of the complex interacts closely with a 20K Dalton peptide, which is possibly RnfB or RnfA peptide.
  • Grant-in-Aid for international Scientific Research, 1996, 1998, 08044203, Crystallographic and Genetic Study of Cytochrome bc1 Complex, FUKUYAMA Keiichi; OH-OKA Hirozo; TAKAHASHI Yasuhiro; SAEKI Kazuhiko; DALDAL Fevzi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for international Scientific Research, Osaka University, 9900000, 9900000, For efficient supply of crystallization materials, two fast and highly efficient methods were developed to facilitate large scale purification of the cytochrome (cyt) bc_1 complex from Rhodobacer capsulatus. The methods rely on the high affinity of hexa-histidine tag for Ni^<2+>2-nitrilotriacetic acid (NTA) agarose. Genes for two type of (His) _6-tagged bc_1 complex were engineered : one was fused at the C-terminus of cyt c^1 (ct-tagged), and another was at the C-terminus of Rieske subunit (Rieske-tagged). Each engineered gene was transferred into a bc^1complex-null mutant MT-RBCl. The resultant strains and the bc^1 complexes they produce were analyzed in detail. Both Rieske- and c1 ^1-tagged bc^1 complex supported photosynthetic growth of R.capsulatus. The enzymes in chromatophore membrane showed the steady-state activity of about 20% and 35%, and the flash-induced single turnover kinetics of about 70% and 90% of those of wild-type complex, respectively. The tagged bcl^1 complex can be readily purified with high yield by Ni^<2+>-NTA-agarose chromatography. Their crystallization are under study.
  • Grant-in-Aid for Scientific Research (C), 1995, 1996, 07640862, BIOCHEMICAL GENETIC ANALYSIS OF THE LINKAGEFACTOR OF PHOTOSYNTHETIS AND NITROGEN FIXATION IN PURPLE BACTERIA, SAEKI Kazuhiko, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Osaka University, 2100000, 2100000, Nitrogen fixation is one of the most important biological processes, because it is the main gateway of nitrogen atom in biosphere. Biological nitrogen fixation is catalyzed by nitrogen enzyme which reduce nitrogen gas to ammonium ion with hydrolysis of ATP.Despite the wealth of knowledge on structure, enzymology and synthesis of nitrogenase itself, little is known about how reducing power is supplied to the enzyme except in the genetically well studied bacterium Klebsiella pneumonia. We have studied the electron transport pathway to nitrogenase in the purple photosynthetic bacterium Rhodobacter capsulatus which is taxonomically close to rhizobia. First, we have site-specifically engineered R.capsulatus ferredoxin I that is the primary electron donor to nitrogenase. With series of engineered genes and a purified products, the unique structural feature of this group of ferredoxins were related to their extremely low redox potential. Second, we analyzed the R.capsulatus rnf genes that are essential for nitrogen fixation under illuminated conditions. RnfA protein was shown to span the chromatophore membrane with its odd-numbered hydrophilic regions exposed to periplasm, whereas RnfB and RnfC proteins were revealed to situate at the periphery of the chromatophore membranes. The contents in cellular fractions indicated that the three proteins stabilize each other, supporting a hypothesis that the Rnf products are subunits of a membrane complex. Finally, we detected homologs of rnf genes in Haemophilus influenzae, Vibrio alginolyticus and E.coli. Close comparisons revealed that RnfC has potential binding sites for NADH and FMN which are similar to those found in proton-translocating NADH-quinone oxidoreductases and that RnfA,RnfD and RnfE show similarity to subunits of sodium-translocating NADH-quinone oxidoreductases. We predict that the putative Rnf complex represents a novel family of energy-coupling NADH-oxidoreductases.
  • Grant-in-Aid for international Scientific Research, 1994, 1994, 06044086, Regulation Mechanism of the Electron Transport in Chloroplasts, WADA Keishiro; GOLBECK John h.; BUCHANAN Bob b.; OH-OKA Hirozo; SAEKI Kazuhiko; HOSHINA Satoshi; MATSUBARA Hiroshi; BLANKENSHIP Robert e., Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for international Scientific Research, Kanazawa University, Faculty of Science, 3000000, 3000000, Regulation mechanism of the electron transport in chloroplast and the related plastids was investigated by the group including three American scientists who was experts in each field. Experiments were planed by focussing on 1) regulation in cyclic electron transport system, 2) regulation of photon cupturation by two photosystems, 3) balance between NADP photoreduction and photophosphorylation, 4) partitioning of electrons from reduced ferredoxin to electron requiring system, such as NADP reduction, reductive assimilation of nitrogen, sulfur and carbon, ferredoxin-dependent thioredoxin system. Electron transport in photosystem I reaction center complex and Fe-S type reaction center was intensively investigated by spectrophotometrical and biochemical procedures and site-directed mutagenesis of genes encoding subunit proteins of the complex. It was investigated if the c-type cytochrome deficient mutant of photosynthetic bacterium (Rhodobacter capsulata) was compensated by the introduction of gene encoding human cytochrome c. At the moment no positive result was obtained. Some proteins components (ferredoxin and ferredoxin-NADP^+ oxido-reductase) of electron transport system in non-photosynthetic plastids were biochemically compared with those in chloroplast. There were isoenzymes or isoproteins differently expressed in chloroplast and non-photosynthetic plastids, which can differently function in the various electron transport systems in plastids.
  • 奨励研究(A), 1993, 1993, 05780457, 7鉄8硫黄フェレドキシンの分子機能の解明:3鉄クラスターは特有の生理機能を持つか, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 奨励研究(A), 大阪大学, 900000, 900000, 咋年度までに,光合成細菌Rhodobacter capsulatusの3種のフェレドキシン遺伝子fdxN,fdxA,fdxCに関する分子生物学的解析により,FdxNとCが窒素固定系で機能するのに対し,7鉄型のFdxAは恒常的に発現する必須蛋白質であること,また,この機能上の相違は発現量ではなく構造上の相違に起因することを明らかにした。また,FdxAは生理機能不明な必須蛋白質で,破壊株に変異遺伝子を導入する定法を適用できないため,予め変異fdxA^*遺伝子をプラスミドとして保持させた部分2倍体の野性型fdxAだけを選択的に破壊するシステムを構築し,構造-機能解析を行なう土台を作っている。これにより,変異fdxA^*と生理機能の相関をより精密に解析した。 1)[3Fe-4S]クラスター周辺のCys11とCys16間の配列を,Lys-Tyr-Thr-AspからGly-Alaに変換する改変遺伝子(fdxAGA)を従来作製していたが,さらにGly-AspならびGly-Asnとするもの(fdxAGDとfdxAGN)を作製して解析を加えた。この結果,fdxAGDのみを持つ変異株は,fdxAGAのみを持つ変異株同様,光合成により生育可能だが,呼吸では生育不能であった。一方,fdxAGNのみを持つ変異株は全く得られなかった。従って,Cys16隣接残基の側鎖へのアミノ基の導入が,生理機能の完全な喪失をもたらすことが明らかとなった。 2)既に,C末端側10残基がこのフェレドキシンの生理活性に必須であることを明らかにしていたので,C末端側より2,6ないしは8残基を欠失させる変異遺伝子(fdxA109,gdxA105およびfdxA103)を作製し,この領域の精密な解析を行なった。C末端6残基を欠失させたfdxA105のみを持つ株は,光合成により生育可能であるが,呼吸では生育不能であった。この株から比較的高頻度(10-3程度)に呼吸でも生育可能な2次変異株が生み出されることが見出した。現在,この復帰変異に関わる遺伝子のクローニングを試みている。
  • 奨励研究(A), 1992, 1992, 04780207, 7鉄8硫黄フェレドキシンの分子機能の解明:3鉄クラスターは特有の生理機能を持つか, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 奨励研究(A), 大阪大学, 900000, 900000
  • 重点領域研究, 1992, 1992, 04225218, 蛋白質部分による-硫黄クラスターの物性と制御機構, 福山 恵一; 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 重点領域研究, 大阪大学, 1300000, 1300000, フェレドキシン(Fd)は広く生物界に存在し、様々な代謝系で電子伝達体として機能する鉄-硫黄蛋白質である。本研究では光合成細菌Rhodoacter capsulatusが持つ幾つかのFdの一つ(FdI)をとりあげ、その変異Fdの生理機能を評価するとともに、大量発現系を構築し変異Fdの物理化学的性質を調べた。既にFdI遺伝子(fdxN)のクローニングは完了しており、またfdxN破壊株は窒素固定条件下で生育が著しく遅くなることを明らかにしている。平成4年度の研究成果は具体的に以下の通りである。 1。部位特異的変異を加えたfdxNを含むフラグメントを発現ベクターに挿入し、発現プラスミドを得た。ここで光化学系蛋白質をコードする遺伝子群のプロモーターを用いた。E.coliから接合により発現プラスミドをR.capsulatusのfdxN欠損株MSAIへ導入し、変異Fdの大量現系を構築した。 2。本Fdに特徴的なC41とC50の間の8残基を少なくするようなプラスミドをMSA1に導入したところ、どれも窒素固定条件下で野生株と同程度に生育した。このことからこのループアウトは、生理機能上重要でないといえる。またC54またはC59がセリンになるようなプラスミドを導入したところ、後者は野生株と同程度に生育したか、前者はMSA1と同程度にしか生育しなかった。 3。FdIの変異蛋白質を嫌気条件下で精製した。C41とC50の間の残基をGAに置換した変異Fdは、可視近紫外のCDスペクトルでは野生型FdIと似ていたが、紫外部でのCDスペクトルでは違いがみられた。変異FdIのEPRスペクトルは野生型FdIと異なり、典型的な2[4Fe-4S]型Fdのスペクトルに近かった。
  • 重点領域研究, 1991, 1991, 03241216, 蛋白質部分による鉄ー硫黄クラスタ-の物性と反応性の制御機構, 福山 恵一; 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 重点領域研究, 大阪大学, 1300000, 1300000, フェレドキシン(Fd)は鉄と硫黄からなるクラスタ-を持ち、光合成系や窒素固定系において電子伝達の役割を果している。このクラスタ-の機能や性質が、それを取り囲むペプチド構造によっていかに制御されているかを明らかにするため、光合成細菌Rhodofacter capsulatusのFd(FdIとFclII)をとりあげた。本研究では分子遺学的手法でこれらのFdの生理学的意義を明らかにすると同時に、化学的性質を調べるために大量発現系を構築した。 合成オリゴヌクレオチドを用いて、本細菌のゲノムのコスミドライブラリ-をスクリ-ニングすることにより、Fd遺伝子を含む断片を単離し、その塩基配列を決定した。FdI遺伝子(fdxN)の他に、2つのオ-プンリ-ディングフレ-ム(fdxCとURF1)が含まれていた。fdxNおよびFdxC遺伝子にカナマイシン耐性遺伝子を挿入することにより、これらの遺伝子を破壊したミュ-タントを得た。これらのミュ-タントは窒素固定能が著しく低下しているが、アンモニアなどの固定窒素源があると、光照射嫌気的条件下でも、暗所で好気的条件下でも、野生株と同じ生育をする。またfdxNまたはfdxCを持つプラスミドをこれらのミュ-タントに導入すると、窒素固定能が回復した。本実験から、窒素固定能にはfdxNおよびfdxC遺伝子の両方が必要であること、またこの系を用いてミュ-タント蛋白の生理活性を評価できることが示された。 本細菌のFdの性質を化学的に理解するため、本細菌の光化学中心をコ-ドする遺伝子群の持つ強力なプロモ-タ-領域を含むシャトルベクタ-pTSVシリ-ズを開発した。これらにfdxNなどを含むDNA断片をサブクロ-ンし、対応する蛋白を多量に発現することができた。現在種々のミュ-タント蛋白を発現させ、これらの性質を調べている。
  • 奨励研究(A), 1990, 1990, 02780236, フェレドキシンの遺伝子工学的改変による鉄硫黄クラスター保持機構の解析, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 奨励研究(A), 大阪大学, 800000, 800000
  • Grant-in-Aid for General Scientific Research (B), 1989, 1990, 01470148, Structure, Function, and Evolution of Eukaryotic and Prokaryotic Respiratory Electron Transfer Systems, MATSUBARA Hiroshi; SAEKI Kazuhiko; WAKABAYASHI Sadao; FUKUYAMA Keiichi, Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B), Osaka University, 6400000, 6400000, 1) Bovine heart mitochondrial complex III (bc_1 was prepared by an improved method. This preparation showed a ping-pong mechanism analyzed by a steady-state reaction kinetics, when it transfers electrons from QH_2 to cytochrome c. 2) This bc_1 complex was reproducibly crystallized in a large scale to reddish parallelpipe forms by treating the preparation with polyethyleneglycol containing sucrose within 1-2 weeks at room temperature. 3) The crystal diffracted X-ray at less than 8 A resolution, but it was rather fragile for mechanical shocks and unstable by exposing it to air. Therefore, some improvements in crystallization and manipulations to X-ray analysis. 4) Euglena cytochrome c was found to be stable if it was prepared in the reduced form, promisingit to be a good preparation for crystallization. 5) The base sequence of cDNA encoding Euglena cytochrome c_1 was completed. The comparison of various c_1 sequences revealed that His and Met were the ligands to the heme iron atom. Heme C was bound to only one cysteine through a thioether bond and Phe was in the position of Cys usually binding the heme together with the other. 6) The site directed mutations of putative heme ligands, His and Met, in yeast cytochrome c_1 showed that these two have important roles for the iron chelation and assembly of the bc_1 complex. 7) One of the two acidic regions supposed to interact with cytochrome c at around position 70 in yeast cytochrome c_1 was found to be not essential for the interaction by gene deletion experiments. 8) Sequence analyses of 13kDa(two different polypeptides), 2okDa, and 20kDa Sub-units revealed that there are several regions forming iron-sulfur clusters in mitochondrial Complex I. 9) A cyanobacterial b_6f complex was found to be able to transfer electrons to cytochrome aa_3 through cytochrome c_<553>.
  • 奨励研究(A), 1988, 1988, 63780246, 光合成細菌の嫌気呼吸にともなう電子伝達とその光制御の酵素化学的解析, 佐伯 和彦, 日本学術振興会, 科学研究費助成事業 奨励研究(A), 大阪大学, 800000, 800000

Ⅲ.社会連携活動実績

1.公的団体の委員等(審議会、国家試験委員、他大学評価委員,科研費審査委員等)

  • Japanese Society of Plant Physiologists, Treasurer, Jan. 2004, Apr. 2005, Society
  • Japanese Society of Plant Physiologists, Secretary, Jan. 1992, Dec. 1993, Society


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