研究者情報

マスター

アカウント（マスター）

• 氏名

  鷲尾　健司(ワシオ　ケンジ), ワシオ　ケンジ

所属（マスター）

• 地球環境科学研究院　環境生物科学部門　環境分子生物学分野

所属（マスター）

• 地球環境科学研究院　環境生物科学部門　環境分子生物学分野

独自項目

syllabus

• 2021, 環境分子生物学特論Ⅱ, Advanced Course in Environmental Molecular Biology II, 修士課程, 環境科学院, 植物、微生物、環境応答、分子機構、進化、光合成、ストレス耐性 Plants, microorganisms, response to environments, molecular mechanisms, evolution, photosynthesis, resistance to environmental stress
• 2021, 分子生物学基礎論, Fundamental Course in Molecular Biology, 修士課程, 環境科学院, 分子生物学，転写，翻訳，微生物の群集構造解析，微生物の呼吸活性，電子顕微鏡観察，遺伝子操作，バイオフィルム形成，植物ストレス耐性，バイオセンサー，昆虫免疫系，昆虫体表脂質, 動物細胞 molecular biology, transcription, translation, bacterial culture, bacterial community structure analysis, bacterial respiration activity, electron microscopic observation, genetic manipulation, biofilm formation, stress tolerance of plants, biosensor, insect immune system, insect body surface lipids, animal cells
• 2021, 生物多様性概論, Introduction to Biological Diversity, 学士課程, 理学部, 現代生物科学，21世紀に生物科学が解決しなければならない課題，生物の多様性，系統，進化，生物の形態，生命活動の多様性
• 2021, 英語演習, English Seminar, 学士課程, 全学教育, 細胞、遺伝学、分子生物学、ゲノム、進化、生物の多様性、生態学、行動、感覚
• 2021, 環境生物学Ⅰ, Environmental Biology I, 学士課程, 理学部
• 2021, ISP生物科学実習Ⅱ・a, ISP Biological Laboratory Course II･a, 学士課程, 理学部, 生物の環境応答と適応
• 2021, ISP生物科学実習Ⅱ・b, ISP Biological Laboratory Course II･b, 学士課程, 理学部, 生物の環境応答と適応
• 2021, 環境生物学実習, Laboratory Course in Environmental Biology, 学士課程, 理学部, 生物の環境応答と適応

researchmap

プロフィール情報

所属

• 環境科学院 生物圏科学専攻 分子生物学コース, 助教

学位

• 博士(理学)（北海道大学）

プロフィール情報

• 姓

  鷲尾, ワシオ

• 名

  健司, ケンジ

• ID各種

  200901053419008197

所属

• 環境科学院 生物圏科学専攻 分子生物学コース, 助教

業績リスト

研究キーワード

• 植物科学   環境科学   分子生物学   植物生理学   

研究分野

• ライフサイエンス / 植物分子、生理科学
• ライフサイエンス / 応用分子細胞生物学
• 環境・農学 / 環境影響評価

経歴

• 2007年 - 現在 北海道大学 地球環境科学研究院 環境生物科学部門 助教
• 2005年 - 2007年 北海道大学 地球環境科学研究院 環境生物科学部門 助手
• 1993年 - 2005年 北海道大学 地球環境科学研究科 生物科学専攻 助手
• 1992年 - 1993年 北海道大学 理学部 生物学科 助手

学歴

• 1988年 - 1992年   北海道大学   理学研究科   植物学専攻 博士後期課程
• 1986年 - 1988年   北海道大学   理学研究科   植物学専攻 修士課程
• 1982年 - 1986年   北海道大学   理学部   生物学科

委員歴

• 2011年 - 2012年   北海道植物学会   幹事
• 2007年 - 2011年   社団法人日本植物学会 北海道支部   幹事

受賞

• 2002年 北海道大学クラーク記念財団 海外派遣助成
   

  受賞者: 鷲尾 健司

論文

• Gene cloning and characterization of a vanadium-dependent bromoperoxidase from the red alga Laurencia saitoi, a producer of brominated diterpenoids and triterpenoids

  Kensuke Kaneko, Daiki Kobayashi, Shiro Masaki, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  JOURNAL OF APPLIED PHYCOLOGY 2023年04月 

  The gene encoding vanadium-dependent bromoperoxidase (V-BPO) was cloned for the first time from the red alga Laurencia saitoi, which produces pharmaceutically promising brominated diterpenoids and triterpenoids. The molecular weight of V-BPO from L. saitoi (LsVBPO1) was the highest (77.0 kDa) among previously reported V-BPOs from Laurencia with a peptide insertion Asn194-Ser221 containing short Gln repeats. It shares approximately 60% amino acid sequence identity with V-BPOs from L. nipponica (LnVBPO1 and LnVBPO2) and L. okamurae (LoVBPO1a and LoVBPO2a). Heterologously expressed LsVBPO1 in Escherichia coli was partially purified and exhibited low but significant bromination activity of 38 U mg(-1) protein using monochlorodimedone. The pH optimum was 8.0, which was more alkaline than that for LnVBPOs and LoVBPO2a (pH 7.0). The K-m for H2O2 was 0.04 mM, comparable to LnVBPO1 (0.026 mM), LnVBPO2 (0.025 mM), and LoVBPO2a (0.014 mM). LsVPBO1 retained its bromination activity until 45 degrees C for 20 min. When incubated at 55 degrees C for 20 min, catalytic activity decreased rapidly, as shown for LnVBPO1 and LoVBPO2a (retained at 45 degrees C, decreased at 55 degrees C) and LnVBPO2 (retained at 55 degrees C, decreased at 65 degrees C). Unlike other V-BPOs from Laurencia (LnVBPO1, LnVBPO2, and LoVBPO2a), dialysis and concentration during purification process were rapidly inactivated LsVBPO1, suggesting its structural instability.
• Nostosin G and Spiroidesin B from the Cyanobacterium Dolichospermum sp. NIES-1697

  Chin-Soon Phan, Jakia Jerin Mehjabin, Andrea Roxanne J. Anas, Masahiro Hayasaka, Reiko Onoki, Juting Wang, Taiki Umezawa, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  Journal of Natural Products 2022年08月10日
• Characterization of vanadium-dependent bromoperoxidases involved in the production of brominated sesquiterpenes by the red alga Laurencia okamurae

  Takafumi Ishikawa, Kenji Washio, Kensuke Kaneko, Xiao Rong Tang, Masaaki Morikawa, Tatsufumi Okino

  Applied Phycology 3 1 120 - 131 2022年07月20日 [査読有り][通常論文]
  
• Wewakazole B, a Cytotoxic Cyanobactin from the Cyanobacterium Moorea producens Collected in the Red Sea

  Julius Adam V. Lopez, Sultan S. Al-Lihaibi, Walied M. Alarif, Ahmed Abdel-Lateff, Yasuyuki Nogata, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino

  JOURNAL OF NATURAL PRODUCTS 79 4 1213 - 1218 2016年04月 [査読有り][通常論文]
   

  A mass spectrometry (MS)-guided isolation has led to the purification of a new cyanobactin, wewakazole B (1), along with the known compound curacin D from a Red Sea Moorea producens. The planar structure of 1 was elucidated using a combination of NMR and MS techniques. After ozonolysis and acid hydrolysis, the absolute configurations of the amino acid components of 1 were determined by chiral-phase LC-MS and HPLC analyses. Notably, compound 1 exhibited cytotoxic activity toward human MCF7 breast cancer cells (IC50 = 0.58 mu M) and human H460 lung cancer cells (IC50 = 1.0 mu M) and was also found to be inactive in a siderophore assay.
• An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds

  Keita Sutoh, Kenji Washio, Ryozo Imai, Masamitsu Wada, Tomonori Nakai, Daisuke Yamauchi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 5 747 - 759 2015年05月 [査読有り][通常論文]
   

  The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.
• 紅海のシアノバクテリアMoorea producensから得られた新規環状ドデカペプチド Wewakazole B

  Julius Adam Velasco Lopez, Sultan S. Al-Lihaibi, Walied M. Alarif, Ahmed Abdel-Lateff, 鷲尾 健司, 森川 正章, 沖野 龍文

  天然有機化合物討論会講演要旨集 57 PosterP23  天然有機化合物討論会実行委員会 2015年 

  シアノバクテリアは、新規生物活性物質の有望な探索源である。¹ 特に、Moorea producens（旧名Lyngbya majuscula）からは新規化合物が報告され続けている。例えば、チューブ重合阻害物質であるcuracin A、アクチン重合促進物質であるhectochlorin、神経活性物質であるjamaicamide B、antillatoxin B、kalkitoxin^2-6などが挙げられる。本研究では、紅海で採集したシアノバクテリアの化合物のプロファイルをLC/MSにより分析し、データベース（MarinLit）で解析した。その結果、新規化合物の存在が示唆されたM. producens から環状ドデカペプチドwewakazole B (1)を単離し、構造を決定した。 単離・精製 赤褐色で糸状のシアノバクテリアをサウジアラビアのジェッダでスキューバにより採集した。サンプルの一部をRNAlater 溶液中で保存し、標準的な16S rRNA配列決定に使用した。シアノバクテリア特異的プライマーの106Fと1509Rおよび内部プライマーの359Fと781R^7,8 を用いたところ、M. producensと同定された。また、メタノール抽出物を酢酸エチルと水、ついでブタノールと水で溶媒分画した。各画分をMCF-7乳ガン細胞に対する細胞毒性、トリプシン阻害活性、タテジマフジツボAmphibalanus amphitriteキプリス幼生に対する付着阻害活性などの生物活性試験に供すると共に、LC/MS分析を実施し、データベース (MarinLit⁹)で検索した。その結果、どの画分も活性を示さなかったが、酢酸エチル画分にみられたピーク([M+H]⁺ m/z 1127) が新規物質と予想され、単離を目指すこととした。シリカゲルカラムクロマトグラフィー (hexane-EtOAc、EtOAc-MeOH) により８画分に分離され、LC/MSにより目的のピークはEtOAc-MeOH (3:1)で溶出された画分７に含まれることが示された。さらに、逆相のHPLC (0-20 min 30 – 80% MeCN, 20-40 min 80% MeCN, Cosmosil 5C₁₈-AR, 10 × 250 mm, 3 mL/min, UV 210 nm) で得られた画分をさらに精製したところ(0-20 min 50 – 70% MeCN, Cosmosil 5C₁₈-MS, 4.6 × 250 mm, 1 mL/min, UV 210 nm) 、0.4 mg の 1が得られた。 構造決定 分子式C₅₈H₇₀N₁₂O₁₂がHRESIMS ([M+H]⁺ m/z 1127.5310, ∆ = 0.39 ppm)によって得られ、不飽和度３０であった。¹H NMRスペクトルには複数のアミドプロトンおよび芳香族プロトン(δ_H ≥ 7.0)とアミノ酸残基の α-プロトン(δ_H 4-6)、さらに３つの鋭いシングレット(δ_H 2.41, 2.53, 8.02)が観測された。これらMSとNMRのデータを用いてあらためてMarinLitによって検索したところ、wewakazole¹⁰ に類似していると考えられ、上記の鋭いシングレットピークは２個のメチルオキサゾール(MeOxz)のメチルプロトンとオキサゾール(Oxz)のメチンプロトン１個と帰属された。HSQC データにより８個のα-メチン、２組の一置換フェニル基、６個のメチル、１３個のメチレンとさらにもう１個のメチンプロトンが認められた。Wewakazole のHSQC と比較すると1ではδ_C 140.6と δ_H 8.02の間にHSQC相関がなかった。しかし、HMBCでは¹J_CH 相関 (210 Hz) が観測され、オキサゾールであることが示された。COSY、 TOCSYおよびHMBCスペクトルにより９個のアミノ酸残基と３個 (View PDFfor the rest of the abstract.)
• cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica

  Kensuke Kaneko, Kenji Washio, Taiki Umezawa, Fuyuhiko Matsuda, Masaaki Morikawa, Tatsufumi Okino

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 8 1310 - 1319 2014年08月 [査読有り][通常論文]
   

  The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their K-m values for Br- were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.
• 紅藻ソゾ属Laurenciaのブロモペルオキシダーゼ

  金子 賢介, 鷲尾 健司, 梅澤 大樹, 小林 大毅, 湯 暁蓉, 松田 冬彦, 森川 正章, 沖野 龍文

  天然有機化合物討論会講演要旨集 56 Poster53  天然有機化合物討論会実行委員会 2014年 

  紅藻ウラソゾ (Laurencia nipponica) から含臭素環状エーテル laurencin (1) が単離されて以来 ^{1, 2)}、ソゾ属からは、現在までに 600 を超える多様なハロゲン化合物 (主に臭素付加化合物) が単離・報告されている ³⁾。これらソゾ由来含臭素化合物の生合成における鍵反応は、環形成を伴った臭素付加反応と考えられている。 村井らは、同過程の触媒酵素の候補として、ブロモペルオキシダーゼ (BPO) をウラソゾから部分精製し ⁴⁾、1 の推定生合成前駆体 laurediol (2) ⁵⁾ を用いた臭素付加・環形成実験に供した。その結果、部分精製 BPO は、2 から deacetyllaurencin (3) への臭素付加・環形成を触媒することが示された (Scheme 1) ⁴⁾。酵素生成物である 3 は、1 の生合成最終前駆体であることから、1 の臭素付加・環形成過程は、BPO により触媒されることが示唆された。一方で、ウラソゾ BPO の構造および生化学的知見は、現在に至るまで明らかにされていない。 他の紅藻 Corallina 属などでは、BPOの結晶構造が解明されており、活性中心にバナジン酸 (VO₄^3-) を要求するバナジウム依存型 BPO (VBPO) であると報告されている ⁶⁾。そこで、本研究では、ソゾ由来の臭素付加酵素の同定のため、ウラソゾより VBPO 遺伝子クローニングを行った。また、ウラソゾ VBPO組み換えタンパク質を調製し、生化学的特性の評価とソゾ由来臭素化合物の推定前駆体を用いた in vitro 臭素付加反応を行い、生合成における役割を評価した。 Scheme 1. In vitro enzymatic bromination of laurediol using partially purified BPO from L. nipponica ⁴⁾. ウラソゾ由来 VBPO の cDNA クローニングと組み換えタンパク質の調製 RACE PCR 法によって、ウラソゾ VBPO cDNA クローニングを行った結果、二種類のウラソゾVBPO 全長配列 (LnVBPO1およびLnVBPO2, 95% identity) を取得した。両遺伝子を、それぞれ大腸菌発現系 BL21 (DE3) pLysS に導入後、可溶画分を抽出し、硫安沈殿と DE52 陰イオン交換クロマトグラフィによって、LnVBPO1 および LnVBPO2組み換えタンパク質を精製した。得られたタンパク質をSDS-PAGE に供したところ、いずれも 77 kDa の位置に単一のバンドとして泳動された。また、両組み換えタンパク質は、ゲルろ過において最大で750 kDa 程度であり、10 量体を形成することが推定された。VBPO においては、紅藻 Corallina 由来 VBPO も 12 量体を形成することが知られている ^7,8) 。 次に、藻体由来の BPO と LnVBPOs 組み換えタンパク質との同一性を検証するため、同様な精製方法を用いて、ウラソゾ藻体から BPO の精製を試みた。藻体由来 BPO は、色素成分との分離が困難であり、部分精製に留まった。組み換え LnVBPOs と部分精製 BPO をSDS-PAGE に供したところ、ともに 75 kD (View PDFfor the rest of the abstract.)
• Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology and its molecular mechanisms

  Kohei Shimada, Yoshikane Itoh, Kenji Washio, Masaaki Morikawa

  CHEMOSPHERE 87 3 226 - 233 2012年04月 [査読有り][通常論文]
   

  In natural environments, bacteria often exist in close association with surfaces and interfaces. There they form "biofilms", multicellular aggregates held together by an extracellular matrix. The biofilms confer on the constituent cells high resistance to environmental stresses and diverse microenvironments that help generate cellular heterogeneity. Here we report on the ability of Pseudomonas stutzeri T102 biofilm-associated cells, as compared with that of planktonic cells, to degrade naphthalene and survive in petroleum-contaminated soils. In liquid culture system. T102 biofilm-associated cells did not degrade naphthalene during initial hours of incubation but then degraded it faster than planktonic cells, which degraded naphthalene at a nearly constant rate. This delayed but high degradation activity of the biofilms could be attributed to super-activated cells that were detached from the biofilms. When the fitness of T102 biofilm-associated cells was tested in natural petroleum-contaminated soils, they were capable of surviving for 10 wk; by then T102 planktonic cells were mostly extinct. Naphthalene degradation activity in the soils that had been inoculated with T102 biofilms was indeed higher than that observed in soils inoculated with T102 planktonic cells. These results suggest that inoculation of contaminated soils with P. stutzeri T102 biofilms should enable bioaugmentation to be a more durable and effective bioremediation technology than inoculation with planktonic cells. (C) 2012 Elsevier Ltd. All rights reserved.
• A Truncated Form of SpoT, Including the ACT Domain, Inhibits the Production of Cyclic Lipopeptide Arthrofactin, and Is Associated with Moderate Elevation of Guanosine 3 ',5 '-Bispyrophosphate Level in Pseudomonas sp MIS38

  Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 10 1880 - 1888 2011年10月 [査読有り][通常論文]
   

  Arthrofactin is a biosurfactant produced by Pseudomonas sp. MIS38. We have reported that transposon insertion into spoT (spoT::Tn5) causes moderate accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) and abrogates arthrofactin production. To analyze the linkage of SpoT function and ablation of arthrofactin production, we examined the spoT::Tn5 mutation. The results showed that spoT::Tn5 is not a null mutation, but encodes separate segments of SpoT. Deletion of the 3' region of spoT increased the level of arthrofactin production, suggesting that the C-terminal region of SpoT plays a suppressive role. We evaluated the expression of a distinct segment of SpoT. Forced expression of the C-terminal region that contains the ACT domain resulted in the accumulation of ppGpp and abrogated arthrofactin production. Expression of the C-terminal segment also reduced MIS38 swarming and resulted in extensive biofilm formation, which constitutes the phenocopy of the spoT::Tn5 mutant.
• Sustainable Biodegradation of Phenol by Acinetobacter calcoaceticus P23 Isolated from the Rhizosphere of Duckweed Lemna aoukikusa

  Fumiko Yamaga, Kenji Washio, Masaaki Morikawa

  ENVIRONMENTAL SCIENCE & TECHNOLOGY 44 16 6470 - 6474 2010年08月 [査読有り][通常論文]
   

  Phenol-degrading bacteria were isolated from the rhizosphere of duckweed (Lemna aoukikusa) using an enrichment culture method. One of the isolates, P23, exhibited an excellent ability to degrade phenol and attach to a solid surface under laboratory conditions. Phylogenetic analysis revealed that P23 belongs to the genera Acinetobacter and has the highest similarity to Acinetobacter calcoaceticus. P23 rapidly colonized on the surface of sterilized duckweed roots and formed biofilms, indicating that the conditions provided by the root system of duckweed are favorable to P23. A long-term performance test (160 h) showed that continuous removal of phenol can be attributed to the beneficial symbiotic interaction between duckweed and P23 P23 is the first growth-promoting bacterium identified from Lemna aoukikusa. The results in this study suggest the potential usefulness of dominating a particular bacterium in the rhizosphere of duckweeds to achieve efficient and sustainable bioremediation of polluted water
• Identification and Characterization of the Genes Responsible for the Production of the Cyclic Lipopeptide Arthrofactin by Pseudomonas sp MIS38

  Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 5 992 - 999 2010年05月 [査読有り][通常論文]
   

  Pseudomonas sp. MIS38 produces an effective biosurfactant named arthrofactin, which is a cyclic lipopeptide synthesized by a mega complex composed of three nonribosomal peptide synthetases. In order to gain insight into the control mechanism of arthrofactin production, a Tn5 mutant library was constructed and screened for arthrofactin-deficient mutants. Along with a number of mutations that occurred in the arthrofactin synthetase operon, three other mutants harbored distinct Tn5 insertions in the genes encoding SyrF-like protein (arfF), heat shock protein (htpG), and (p)ppGpp synthetase/hydrolase (spoT). Epistasis analyses revealed that spoT functions early in the arthrofactin production pathway. We also found that spoT affects MIS38 swarming, biofilm formation, and the cell morphology.
• The Role of Urease Activity on Biofilm Formation by Staphylococcus sp T-02 Isolated from the Toilet Bowl

  Kaihei Oki, Kenji Washio, Daigo Matsui, Shinichi Kato, Yoshihiko Hirata, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 3 583 - 589 2010年03月 [査読有り][通常論文]
   

  Urolith, which consists of dirty yellow-colored attachments on the toilet bowl, is associated with a variety of odorous chemicals, including ammonia, and causes disadvantages in daily life. Although largely it is derived from microorganisms, little is known about the microbial processes underlying the formation of urolith. In order to gain insight into the types and the activities of microorganisms present in urolith, culturable bacteria were isolated, identified, and physiologically characterized. One of the isolates exhibited higher ability to produce ammonia when it was grown in artificial urine medium. Phylogenetic and physiological analyses indicated that this strain (T-02) belonged to a new group of Staphylococcus species, showing combined phenotypes as between S. lentus and S. xylosus. T-02 exhibited high urease activity and was capable of growing in the urinary condition by forming robust biofilms. The results of this study indicate that T-02 has successfully adapted itself to the environment of urolith.
• Flexible exportation mechanisms of arthrofactin in Pseudomonas sp MIS38

  S. P. Lim, N. Roongsawang, K. Washio, M. Morikawa

  JOURNAL OF APPLIED MICROBIOLOGY 107 1 157 - 166 2009年07月 [査読有り][通常論文]
   

  To obtain further insights into transportation mechanisms of a most effective biosurfactant, arthrofactin in Pseudomonas sp. MIS38. A cluster genes arfA/B/C encodes an arthrofactin synthetase complex (ArfA/B/C). Downstream of the arfA/B/C lie genes encoding a putative periplasmic protein (ArfD, 362 aa) and a putative ATP-binding cassette transporter (ArfE, 651 aa), namely arfD and arfE, respectively. The arfA/B/C, arfD, and arfE form an operon suggesting their functional connection. Gene knockout mutants ArfD:Km, ArfE:Km, ArfD:Tc/ArfE:Km, and gene overexpression strains MIS38(pME6032_arfD/E) and ArfE:Km(pME6032_arfD/E) were prepared and analysed for arthrofactin production profiles. It was found that the production levels of arthrofactin were temporally reduced in the mutants or increased in the gene overexpression strains, but they eventually became similar level to that of MIS38. Addition of ABC transporter inhibitors, glibenclamide and sodium ortho-vanadate dramatically reduced the production levels of arthrofactin. This excludes a possibility that arthrofactin is exported by diffusion with the aid of its own high surfactant activity. ArfD/E is not an exclusive but a primary exporter of arthrofactin during early growth stage. Reduction in the arthrofactin productivity of arfD and arfE knockout mutants was eventually rescued by another ABC transporter system. Effects of arfD and arfE overexpression were evident only for 1-day cultivation. Multiple ATP dependent active transporter systems are responsible for the production of arthrofactin. Pseudomonas bacteria are characterized to be endued with multiple exporter and efflux systems for secondary metabolites including antibiotics, plant toxins, and biosurfactants. The present work demonstrates exceptionally flexible and highly controlled transportation mechanisms of a most effective lipopeptide biosurfactant, arthrofactin in Pseudomonas sp. MIS38. Because lipopeptide biosurfactants are known to enhance efficacy of bioactive compounds and arfA/B/C/D/E orthologous genes are also found in plant pathogenic P. fluorescens and P. syringae strains, the knowledge would also contribute to develop a technology controlling plant diseases.
• Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments

  Saori Iijima, Kenji Washio, Ryota Okahara, Masaaki Morikawa

  MICROBIAL BIOTECHNOLOGY 2 3 361 - 369 2009年05月 [査読有り][通常論文]
   

  In order to save natural resources and supply good fishes, it is important to improve fish-farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish-farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture-based methods for the identification and characterization of biofilm-forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm-dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.
• Production of sophorolipid biosurfactant by Pichia anomala

  Jiraporn Thaniyavarn, Tanusta Chianguthai, Polakit Sangvanich, Niran Roongsawang, Keji Washio, Masaaki Morikawa, Suthep Thaniyavarn

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 8 2061 - 2068 2008年08月 [査読有り][通常論文]
   

  Biosurfactant production by Pichia anomala PY1, a thermotorelant strain isolated from fermented food, was examined as grown in media containing various carbon and nitrogen sources. The optimal conditions for biosurfactant production included 4% soybean oil as carbon source at pH 5.5 at 30 degrees C for 7d. Under these conditions, the surface tension of the medium decreased to 28mN/m with oil displacement measured at 69.43 cm(2). Comparative studies of biosurfactant production in media containing glucose or soybean oil were performed. The biosurfactants obtained were isolated and purified by chromatographic methods. The molecular weights of samples were further investigated by mass spectrometry. In medium containing glucose, biosurfactants of molecular weights of 675, 691, and 707 were obtained, while those isolated from medium containing soybean oil were of molecular weights of 658, 675, and 691. These results reveal that sophorolipid compounds containing fatty acids of C20 and C18:1 were produced from both media.
• Identification of alkane hydroxylase genes in Rhodococcus sp strain TMP2 that degrades a branched alkane

  Daisuke Takei, Kenji Washio, Masaaki Morikawa

  BIOTECHNOLOGY LETTERS 30 8 1447 - 1452 2008年08月 [査読有り][通常論文]
   

  Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.
• Functional analysis of a pyoverdine synthetase from Pseudomonas sp MIS38

  Slew Ping Lim, Niran Roongsawang, Kenji Washio, Masaaki Morikawa

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 8 2002 - 2009 2007年08月 [査読有り][通常論文]
   

  Fluorescent Pseudomonas sp. MIS38 produces a cyclic lipopeptide, arthr4actin. Arthrofactin is synthesized by a unique nonribosomal peptide synthetase (NRPS) with dual C/E-domains. In this study, another class of cyclic peptide, pyoverdine, was isolated from MIS38, viz., Pvd38. The main fraction of Pvd38 had an m/z value of 1,064.57 and contained Ala, Glu, Gly, (OHOrn), Ser, and Thr at a ratio of 2:1:1:(1):1:1 in the peptide part, suggesting a new structure compound. A gene encoding NRPS for the chromophore part of Pvd38 was identified, and we found that it contained a conventional E-domain. Gene disruption completely impaired the production of Pvd38, demonstrating that the synthetase is functional. This observation allows us to conclude that different NRPS systems with dual C/Edomains (in arthrofactin synthetase) and a conventional E-domain (in pyoverdine synthetase) are both functional in MIS38.
• In vivo characterization of tandem C-terminal thioesterase domains in arthrofactin synthetase

  Niran Roongsawang, Kenji Washio, Masaaki Morikawaa

  CHEMBIOCHEM 8 5 501 - 512 2007年03月 [査読有り][通常論文]
   

  Mocrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase WO from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C-terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site-directed mutagenesis was introduced at the putotive active-site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1.S89A, ArfC_Te1.-S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_7e2:S92A, ArfC_7e2:S92A/D118A, and AnFC Delta 7e2 was reduced by 95% without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_7e2 might hove been added during the evolution of Arf in order to improve macrocyclization efficiency.
• Common mechanisms regulating expression of rice aleurone genes that contribute to the primary response for gibberellin

  Kenji Washio, Masaaki Morikawa

  BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1759 10 478 - 490 2006年10月 [査読有り][通常論文]
   

  During germination of cereal seeds, aleurone cells respond to gibberellins (GA) by synthesizing and secreting hydrolytic enzymes that mobilize the reserved nutrients. It has been shown that products of early GA response genes, like a transcription factor GAMyb, act as key molecules leading to this regulation. Pivotal roles of GAMyb on expression of hydrolase genes have been well documented, whereas regulation of GAMyb expression itself remains obscure. In order to understand virtual mechanisms of the GA-mediated expression of genes, it is important to know how GA control expression of early GA response genes. Using an aleurone transient expression system of rice (Oryza sativa L.), we examined GA responsive domains of early GA response genes in the aleurone, such as GAMyb and OsDof3. The upstream promoter could not confer GA response. Extensive analyses revealed the presence of enhancer-like activities in a large first intron. In Arabidopsis, intron enhancers have been identified in MADS-box homeotic genes, AGAMOUS (AG) and FLOWERING LOCUS C (FLC), in which large introns should not only confer proper gene expressions, but also associate with gene silencing by covalent modifications of both DNA and historic. These evidences prompt us to assign that chromatin-based control might be important for initial GA action. Based on this assumption, we have identified DNA methylation of the GAMyb locus in germinated rice seeds. (c) 2006 Elsevier B.V All rights reserved.
• Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases

  N Roongsawang, SP Lim, K Washio, K Takano, S Kanaya, M Morikawa

  FEMS MICROBIOLOGY LETTERS 252 1 143 - 151 2005年11月 [査読有り][通常論文]
   

  Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are L-peptidyl donors, D-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from L to D-form of incorporating amino acid acceptor occurs during or after peptide bond formation. L-peptidyl donors and D-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
• Functional dissections between GAMYB and dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone

  K Washio

  PLANT PHYSIOLOGY 133 2 850 - 863 2003年10月 [査読有り][通常論文]
   

  In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules. Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB. In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes. Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure. In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa). Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA. The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region. The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation. The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes.
• Cloning and characterization of cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza

  M Nishikoori, K Washio, A Hase, N Morita, H Okuyama

  PHYSIOLOGIA PLANTARUM 113 2 241 - 248 2001年10月 [査読有り][通常論文]
   

  A cDNA clone of the glycosylphosphatidylinositol (GPI)-anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5' rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate-deficient ( - P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI-anchoring (omega) site. The absence of a dibasic motif upstream of the putative omega site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using - P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate-deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of - P plants and this time-dependent occurrence in mRNA levels of the PAP in - P plants was also observed in their protein and activity levels.
• Identification of Dof proteins with implication in the gibberellin-regulated expression of a peptidase gene following the germination of rice grains

  K Washio

  BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1520 1 54 - 62 2001年07月 [査読有り][通常論文]
   

  Type III carboxypeptidase (CPD3) is one of the hydrolytic enzymes whose expression is up-regulated by gibberellins (GA) in the aleurones. of germinated cereal grains. A number of pyrimidine boxes and a sequence resembling the gibberellic acid response element (GARE) are observed in the region upstream of the transcription initiation site of the CPD3 gene, showing a characteristic of cereal GA-responsive genes. Transient gene expression assays in germinated rice aleurone demonstrated that the CPD3 promoter was able to confer hormonally responses on the expression of the reporter gene. By southwestern screening, several cDNAs encoding the Dof class proteins were isolated from a rice aleurone library. Each mRNA accumulation for five novel members of Dof proteins (OsDof1-5) occurs with a different time course and in a tissue-specific manner following the germination of grains. Of these, the expression of the OsDof3 gene is abundant in aleurones where it precedes that of the CPD3 gene, implying that this is an early response gene of GA. The OsDof3 protein, expressed in Escherichia coli, selectively bound AAAG motifs of the pyrimidine boxes through the DNA-binding activity of its Dof domain. Co-expression experiments in aleurones suggested that the OsDof3 protein should play a regulatory role in the expression of the CPD3 gene under the control of CIA. (C) 2001 Elsevier Science B.V. All rights reserved.
• cDNA encoding Dof-proteins that are present in germinated aleurone cells (PGR 99.107).

  Washio K

  Plant Physiology 120 1205  1999年 [査読有り][通常論文]
  
• The glycosylphosphatidylinositol-anchored phosphatase from Spirodela oligorrhiza is a purple acid phosphatase

  H Nakazato, T Okamoto, M Nishikoori, K Washio, N Morita, K Haraguchi, GA Thompson, H Okuyama

  PLANT PHYSIOLOGY 118 3 1015 - 1020 1998年11月 [査読有り][通常論文]
   

  We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, C.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53-62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of 5. oligorrhiza plants incubated with the GPI-specific precursor [(3)H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.
• ORGAN-SPECIFIC AND HORMONE-DEPENDENT EXPRESSION OF GENES FOR SERINE CARBOXYPEPTIDASES DURING DEVELOPMENT AND FOLLOWING GERMINATION OF RICE GRAINS

  K WASHIO, K ISHIKAWA

  PLANT PHYSIOLOGY 105 4 1275 - 1280 1994年08月 [査読有り][通常論文]
   

  Several cDNA clones encoding either serine carboxypeptidases or related proteins of Oryza sativa L. were identified, and the abundance of the corresponding mRNA in immature and germinated grains was examined. The deduced amino acid sequence of each cDNA included key sequences, such as a pentapeptide (G-X-S-X-G/A) that is conserved among many serine carboxypeptidases, and the putative protein products were classified as two general and one novel type of cereal serine carboxypeptidases. Two general types exhibited considerable homology to type I and type III carboxypeptidases of cereal plants. The novel type encoded a serine carboxypeptidase-like protein that was very similar to type III carboxypeptidases of barley and wheat but had slight differences in both the N- and the C-terminal sequences. The mRNAs of each of these carboxypeptidases were observed in immature grains, and they decreased during maturation. The abundance of mRNA for each class of carboxypeptidase increased again following germination with the same time course and in a tissue-specific manner. The mRNAs for type I and type III-like carboxypeptidases were abundant in germinated embryos composed of leaf, root, and scutellum, whereas the mRNA for type III carboxypeptidase was conspicuous in endosperm that contained the aleurone layer. Altered amounts of mRNA in deembryonated half-grains in response to phytohormones, such as gibberellic acid and abscisic acid, were only detectable in the case of type III carboxypeptidase. Southern blot analysis using rice genomic DNA revealed the simple organization of each gene for these three classes of carboxypeptidases.
• CLONING AND SEQUENCING OF THE GENE FOR TYPE-I CARBOXYPEPTIDASE IN RICE

  K WASHIO, K ISHIKAWA

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1199 3 311 - 314 1994年04月 [査読有り][通常論文]
  
• STRUCTURE AND EXPRESSION DURING THE GERMINATION OF RICE SEEDS OF THE GENE FOR A CARBOXYPEPTIDASE

  K WASHIO, K ISHIKAWA

  PLANT MOLECULAR BIOLOGY 19 4 631 - 640 1992年07月 [査読有り][通常論文]
   

  The carboxypeptidase gene from rice and corresponding cDNA clones were isolated. The Sal I 11.2 kb fragment of DNA cloned from a size-fractionated genome library contained eight introns and an open reading frame that encoded 500 amino acids (M(r) 55445). The structure deduced for the carboxypeptidase from rice was very similar to those of type III serine carboxypeptidases from barley and wheat. The extent of homology of the amino acid sequence to that of these carboxypeptidases from barley and wheat was 92.3% and 87.2%, respectively. The accumulation of mRNA for the rice carboxypeptidase was conspicuous in germinating endosperms that contained aleurone layers, but levels were lower in leaves and roots. The abundance of the mRNA in endosperms was enhanced by gibberellic acid (GA) and accumulation of the mRNA was inhibited by abscisic acid (ABA). The rice gene for carboxypeptidase contained some pyrimidine boxes (TCCTTTTC) in the 5' flanking region, which are a characteristic of a GA-responsive gene.
• QUANTITATIVE COMPARISON OF FERN-LEAF DEVELOPMENT AND FERTILITY WITH RESPECT TO ALTITUDE IN THE TIROL, CENTRAL EUROPEAN ALPS, AUSTRIA

  T SATO, G GRABHERR, K WASHIO

  JOURNAL OF BIOGEOGRAPHY 16 5 449 - 455 1989年09月 [査読有り][通常論文]
  
• THERMOTROPIC PROPERTIES OF LIPIDS EXTRACTED FROM PLASMA-MEMBRANE AND TONOPLAST ISOLATED FROM CHILLING-SENSITIVE MUNG BEAN (VIGNA-RADIATA [L] WILCZEK)

  S YOSHIDA, K WASHIO, J KENRICK, G ORR

  PLANT AND CELL PHYSIOLOGY 29 8 1411 - 1416 1988年12月 [査読有り][通常論文]
  

MISC

• 紅藻マギレソゾLaurencia saitoi由来ブロモペルオキシダーゼの臭素化反応

  正木志良, 金子賢介, 小林大毅, 石川高史, 西川慶祐, 森本善樹, 鷲尾健司, 森川正章, 沖野龍文 日本農芸化学会大会講演要旨集(Web) 2016 4B032 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  
• 紅藻ミツデソゾLaurencia okamurae由来ブロモペルオキシダーゼの性状解析

  石川高史, 金子賢介, 湯暁蓉, 鷲尾健司, 森川正章, 沖野龍文 化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM) 2016 ROMBUNNO.1B17 2016年 [査読無し][通常論文]
  
• 紅藻ソゾ属3種のブロモペルオキシダーゼの活性評価

  金子賢介, 小林大毅, 鷲尾健司, 森川正章, 沖野龍文 日本化学会講演予稿集 95th (4) 1409 2015年03月11日 [査読無し][通常論文]
  
• 紅藻ウラソゾLaurencia nipponica由来バナジウム依存型ブロモペルオキシダーゼの性状解析

  金子賢介, 鷲尾健司, 梅澤大樹, 松田冬彦, 森川正章, 沖野龍文 日本水産学会大会講演要旨集 2014 119 2014年03月27日 [査読無し][通常論文]
  
• The chromatin structure and seed function of land plants.

  Washio K International Journal of Plant Biology & Research 2 1015 2014年 [査読有り][招待有り]
  
• 紅藻ウラソゾLaurencia nipponica由来バナジウム依存型ブロモペルオキシダーゼの性状解析

  金子賢介, 鷲尾健司, 梅澤大樹, 松田冬彦, 森川正章, 沖野龍文 マリンバイオテクノロジー学会大会講演要旨集 15th 68 2013年06月01日 [査読無し][通常論文]
  
• The prediction of climate change and rice production in Japan.

  Washio K Journal of Rice Research 2 e103 2013年 [査読有り][招待有り]
  
• Unique microbiological characteristics of Pseudoalteromonas spp. that form biofilms in the sea water environment : Their possible contribution to water purification and fish farming.

  Iijima S, Okahara R, Washio K, Morikawa M Bulletin of the Society of Sea Water Science 66 186 -190 2012年 [査読有り][通常論文]
  
• 1Hp04 Marinobacter属細菌と共存する新属海洋細菌の混合バイオフィルム形成(発酵生理学・発酵工学,一般講演)

  森川 正章, 栄木 悠, 柞木田 なつみ, 鷲尾 健司 日本生物工学会大会講演要旨集 63 54 -54 2011年
• Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

  Niran Roongsawang, Kenji Washio, Masaaki Morikawa INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 12 (1) 141 -172 2011年01月 [査読有り][通常論文]
   

  Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.
• The role of urease activity on biofilm and urolith formation by Staphylococcus sp T-02 that was isolated from a toilet bowl

  K. Oki, K. Washio, D. Matsui, Y. Hirata, M. Morikawa JOURNAL OF BIOTECHNOLOGY 150 S247 -S247 2010年11月 [査読無し][通常論文]
  
• Pseudomonas stutzeriバイオフィルムのナフタレン分解活性と土壌安定性

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章 日本農芸化学会大会講演要旨集 2008 39 2008年03月05日 [査読無し][通常論文]
  
• S-C5 バイオフィルムを利用した持続的環境浄化技術の基盤開発(シンポジウムC「バイオフィルム研究の様々な切り口-分子、細胞、生態-」,第24回大会シンポジウム)

  森川 正章, 鷲尾 健司 日本微生物生態学会講演要旨集 (24) 193 -193 2008年
• 持続的環境浄化技術を拓くウキクサと根圏微生物の共生系

  山賀文子, 鷲尾健司, 森川正章 化学と生物 46 (10) 682 -688 2008年 [査読無し][招待有り]
   

  微生物活性を利用した環境浄化法は，地球への負荷が少ない技術として広く認知されている．しかし，実用的技術としては，有用微生物の現場での定着率の低さによるコスト高や浄化後に危惧される微生物の異常増殖や生態系の攪乱などの問題が常に指摘されてきた．近年，この問題を解決する一つの方向性として根圏微生物の活用が注目されている．植物の光合成作用と微生物の汚染物質分解活性をリンクさせるこの手法の一例を紹介するとともに，今後の可能性を展望する．
• Analysis of transcription factors regulating gibberellin-inducible expression of a gene for a proteinase, Rep1 in rice seeds

  Keita Sutoh, Ryozo Imai, Kenji Washio, Tomonori Nakai, Daisuke Yamauchi PLANT AND CELL PHYSIOLOGY 48 S191 -S191 2007年 [査読無し][通常論文]
  
• Pseudomonas sutzeriバイオフィルムのナフタレン分解活性と土壌安定性

  嶋田恒平, 片岡剛文, 鷲尾健司, 森川正章 環境バイオテクノロジー学会大会プログラム講演要旨集 31st 14 2007年 [査読無し][通常論文]
  
• Expression of rice germination-control genes that accompany with the change of DNA modification.

  K Washio, M Morikawa PLANT AND CELL PHYSIOLOGY 47 S147 -S147 2006年 [査読無し][通常論文]
  
• 枯草菌バイオフィルム形成遺伝子の探索

  森川正章, 河合良尚, 相原 悠, 鷲尾健司, 高野和文, 金谷茂則 生物工学会誌 84 488 -490 2006年 [査読無し][招待有り]
  
• mRNAを鋳型としないペプチド合成反応：セントラルドグマを凌ぐ微生物の営みに学ぶこと

  鷲尾健司, 森川正章 化学と生物 44 (2) 85 -92 2006年 [査読無し][招待有り]
  
• Dissection of DNA methylation status in gibberellin-primary response genes that involve the germination of rice seeds.

  K Washio, M Morikawa PLANT AND CELL PHYSIOLOGY 46 S130 -S130 2005年 [査読無し][通常論文]
  
• 種子が目覚めるとき：発芽を促すジベレリンの作用

  鷲尾健司 先端シンポジウム 植物科学の新展開 -分子から群集まで広視野研究をめざす- プログラム要旨集 84 -92 2004年 [査読無し][招待有り]
  
• RICE GRAIN DOF PROTEINS POSITIVELY REGULATE THE EXPRESSION OF GIBBERELLIN-RESPONSIVE GENES, COORDINATED WITH THE FUNCTION OF GAMYB :

  WASHIO Kenji Plant and cell physiology 42 s34 2001年
• CHARACTERIZATION OF DOF PROTEINS RELATED TO THE GIBBERELLIN REGULATED EXPRESSION OF GENES FOLLOWING THE GERMINATION OF RICE (Oryza sativa L.) GRAINS :

  WASHIO Kenji Plant and cell physiology 41 s131 2000年
• IDENTIFICATION OF LOW-MOLECULAR-WEIGHT DNA-BINDING PROTEINS THAT INTERACT WITH AN A/T-RICH PROMOTER SEQUENCE OF A GIBBERELLIN-INDUCIBLE GENE OF RICE (Oryza sativa)

  WASHIO Kenji Plant and cell physiology 39 S50 -S50 1998年05月
• PROMOTER ANALYSIS OF A GIBBERELLIN-INDUCIBLE GENE OF RICE (Oryza sativa) AND IDENTIFICATION OF ALEURONE PROTEINS THAT INTERACT WITH ITS 5'-POSITIVE REGURATORY REGION

  WASHIO Kenji Plant and cell physiology 38 s137 1997年03月
• EXPRESSION ANALYSIS OF A GENE FOR RICETYPE III CARBOXYPEPTIDASE USING OAT ALEURONE PROTOPLASTS

  WASHIO Kenji, ISHIKAWA Koh Plant and cell physiology 36 S84 1995年03月

書籍等出版物

• Biofilms in bioremediation, current research and emerging technologies: Using microbial biofilms to enhance the phytoremediation of contaminants in soil and water. Part A: A trial for sustainable phenol degradation by duckweed-colonizing biofilms.

  Morikawa M, Yamaga F, Suzuki K, Kurashina K, Miwa K, Washio K (担当:共著範囲:Chapter 13, pp233–240)
  Caister Academic Press 2015年
• Biofilms in bioremediation, current research and emerging technologies: Comparison of the degradation activity of biofilm-associated versus planktonic cells.

  Morikawa M, Washio K (担当:共著範囲:Chapter 12, pp221–232)
  Caister Academic Press 2015年
• バイオフィルムの基礎と制御 バイオサーファクタントを利用したバイオフィルムの形成と阻害

  鷲尾健司, 森川正章 (担当:共著範囲:pp278-287)
  (株)エヌ・ティー・エス 2008年
• レーヴン/ジョンソン 生物学 原著第7版

  鷲尾健司 (担当:共訳範囲:下巻、第36章、pp755-766)
  培風館 2007年
• Plant nutrition-for sustainable food production and environment: Characterization and cDNA cloning of the GPI-anchored phosphatase from Spirodela oligorrhiza.

  Nakazato H, Okamoto T, Nishikoori M, Washio K, Morita N, Haraguchi K, Thompson GA Jr, Okuyama H (担当:共著範囲:vol13, pp229-230)
  Kluwer Academic Publishers, Dordrecht, The Netherlands 1997年

講演・口頭発表等

• Study on bromination enzyme from the red alga Laurencia nipponica.  [招待講演]

  Kaneko K, Washio K, Umezawa T, Morikawa M, Matsuda F, Okino T

  Gordon Research Conferences, Marine Natural Products 2012年02月 シンポジウム・ワークショップパネル（指名）
• Staphylococcus属細菌のバイオフィルム形成における尿素分解酵素の役割  [招待講演]

  大木海平, 鷲尾健司, 松井大悟, 平田善彦, 森川正章

  平成22年度北海道腸内細菌叢研究会総会 2010年09月 シンポジウム・ワークショップパネル（指名）
• Generation of D-amino acid during biosynthesis of the cyclic lipopeptide arthrofactin by nonribosomal peptide synthetases.  [招待講演]

  Washio K, Roongsawang N, Morikawa M

  BIT 1st Annual World Congress of Catalytic Asymmetric Synthesis 2010 2010年05月 シンポジウム・ワークショップパネル（指名）
• ウキクサと根圏細菌の相利共生作用による汚染浄化法  [招待講演]

  森川正章, 鷲尾健司

  水処理生物学会46回大会 2009年11月 シンポジウム・ワークショップパネル（指名）
• Biofilm formation and effective production of menaquinone-7 (Vitamin K2) by Bacillus subtilis.  [招待講演]

  Washio K, Morikawa M

  Japan-Argentina Workshop (JST/MYNCyT) 2009年08月 シンポジウム・ワークショップパネル（公募）
• Genetic analysis of synthetic regulation and transportation of lipopeptide type biosurfactant.  [招待講演]

  Morikawa M, Roongsawang N, Lim SP, Washio K

  Biosurfactant Workshop in Vietnam 2009年04月 シンポジウム・ワークショップパネル（指名）
• Sustainable bioremediation technology by utilizing biofilms.  [招待講演]

  Morikawa M, Yamaga F, Shimada K, Washio K

  Int. Symposium on Biotechnological Approaches to Environmental Science for Energy Production and Sustainability 2009年02月 シンポジウム・ワークショップパネル（指名）
• Efficacy of biofilm formation by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology.  [招待講演]

  Morikawa M, Shimada K, Washio K

  BIT 1st World Congress of ibio-2008 2008年05月 シンポジウム・ワークショップパネル（指名）
• 環境微生物の生存戦略−バイオフィルム−  [招待講演]

  森川正章, 相原 悠, 山崎和彦, 鷲尾健司

  日本生化学会北海道支部 第44回支部例会 2007年07月 口頭発表（基調）
• 種子が目覚めるとき 発芽を促すジベレリンの作用  [招待講演]

  鷲尾健司

  先端シンポジウム 植物科学の新展開 -分子から群集まで広視野研究をめざす- 2003年10月 シンポジウム・ワークショップパネル（指名）

所属学協会

• 北海道植物学会   日本分子生物学会   日本農芸化学会   米国植物生理学会   日本植物生理学会   Japanese Society of Plant Physiology   

Works（作品等）

• 海産藻類が生産する含臭素化合物の生合成解析

  鷲尾健司  2010年 - 現在
• 高等植物の成長システムの理解と機能応用

  鷲尾健司  1994年 - 現在

共同研究・競争的資金等の研究課題

• 船底防汚塗料候補物質含ハロゲン化合物の酵素反応による合成

  日本学術振興会：科学研究費助成事業

  研究期間 : 2012年04月 -2016年03月 

  代表者 : 沖野 龍文, 松田 冬彦, 梅澤 大樹, 野方 靖行, 鷲尾 健司

   

  含ハロゲン化合物であるローレンシンを生産するウラソゾから、その生合成酵素の一つとして環化と臭素化を触媒するバナジウム依存型ブロモペルオキシダーゼのクローニングに成功し、組換え酵素を用いてその臭素化活性を明らかにした。含ハロゲンセスキテルペノイドであるローリンテロールを生産するミツデソゾから、バナジウム依存型ブロモペルオキシダーゼのクローニングに成功し、その性状を明らかにした。さらに、含ハロゲントリテルペノイドを生産するマギレソゾから、同酵素のクローニングに成功し、その性状を明らかにした。ブロモアレンを有するC15化合物であるオマエザレンの構造を報告すると共に、その全合成に成功した。
• 植物種子に脱水耐性をもたらすゲノム機能の解明

  日本学術振興会：科学研究費補助金 基盤研究(C)

  研究期間 : 2011年04月 -2014年03月 

  代表者 : 鷲尾健司
• 高度好熱菌性油田細菌の原始的なアルカン代謝および生態に関する研究

  日本学術振興会：科学研究費補助金 基盤研究(B)

  研究期間 : 2010年04月 -2014年03月 

  代表者 : 森川正章
• バイオフィルム形成分子機構を切り口とした微生物未知機能の解明

  日本学術振興会：科学研究費補助金 基盤研究(B)

  研究期間 : 2007年04月 -2010年03月 

  代表者 : 森川正章
• ジベレリン酸シグナリングによる遺伝子稼働化を促す分子機構の解析

  日本学術振興会：科学研究費補助金 基盤研究(C)

  研究期間 : 2004年04月 -2006年03月 

  代表者 : 鷲尾健司
• オオムギ糊粉層細胞表層におけるジベレリン酸受容体の検索

  日本学術振興会：科学研究費補助金 奨励研究(A)

  研究期間 : 1996年04月 -1997年03月 

  代表者 : 鷲尾健司
• ジベレリン酸によるイネ・III型カルボキシペプチダーゼ遺伝子の発現制御の解析

  日本学術振興会：科学研究費補助金 奨励研究(A)

  研究期間 : 1994年04月 -1995年03月 

  代表者 : 鷲尾健司

産業財産権

• 特願2008-099213:新規水草根圏微生物  2008年04月07日

  森川正章, 鷲尾健司, 山賀文子

その他

• 2011年 - 2011年  新エネルギー・産業技術総合開発機構 受託研究 

  微生物機能を活用した環境調和型製造基盤技術開発 バイオフィルム工学による微生物のデザイン化
• 2009年 - 2009年  科学技術振興機構 戦略的国際科学技術協力推進事業 

  日本－アルゼンチン共催WS 農業・食糧生産に関するバイオサイエンス・バイオテクノロジー
• 2006年 - 2006年  日本学術振興会 拠点大学交流事業 

  微生物の生物化学的研究分野 耐熱性微生物資源の開発と利用
• 2006年 - 2006年  科学技術振興機構 サイエンスパートナーシッププログラム事業 

  講座型学習活動 生命と環境を科学する
• 2003年 - 2003年  科学技術振興機構 サイエンスパートナーシッププログラム事業 

  教育連携講座 北海道の農作物とバイオテクノロジー