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Master

Affiliation (Master)

  • Faculty of Environmental Earth Science　Environmental Biology　Environmental Molecular Biology

Affiliation (Master)

  • Faculty of Environmental Earth Science　Environmental Biology　Environmental Molecular Biology

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Profile and Settings

Affiliation

  • Graduate School of Environmental Science, Division of Biosphere Science, Course of Molecular Biology

Degree

  • Doctor(Science)（Hokkaido University）

Profile and Settings

  • Name (Japanese)

    Washio

  • Name (Kana)

    Kenji

  • Name

    200901053419008197

Affiliation

  • Graduate School of Environmental Science, Division of Biosphere Science, Course of Molecular Biology

Achievement

Research Interests

  • Plant Science   Environmental Science   Molecular Biology   Plant Physiology   

Research Areas

  • Life sciences / Plants: molecular biology and physiology
  • Life sciences / Applied molecular and cellular biology
  • Environmental science/Agricultural science / Environmental impact assessment

Research Experience

  • 2007 - Today Hokkaido University Section of Environmental Biology, Faculty of Environmental Earth Science Assistant Professor
  • 2005 - 2007 Hokkaido University Section of Environmental Biology, Faculty of Environmental Earth Science Lecturer
  • 1993 - 2005 Hokkaido University Division of Biosciences, Graduate School of Environmental Earth Science Lecturer
  • 1992 - 1993 Hokkaido University Department of Biology, Faculty of Science Lecturer

Education

  • 1988 - 1992  Hokkaido University  Graduate School of Science  Doctor Course of Botany
  • 1986 - 1988  Hokkaido University  Graduate School of Science  Master Course of Botany
  • 1982 - 1986  Hokkaido University  Faculty of Science  Department of Biology

Awards

  • 2002 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

Published Papers

  • Kensuke Kaneko, Daiki Kobayashi, Shiro Masaki, Kenji Washio, Masaaki Morikawa, Tatsufumi Okino
    JOURNAL OF APPLIED PHYCOLOGY 0921-8971 2023/04 
    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.
  • 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 0163-3864 2022/08/10
  • Takafumi Ishikawa, Kenji Washio, Kensuke Kaneko, Xiao Rong Tang, Masaaki Morikawa, Tatsufumi Okino
    Applied Phycology 3 (1) 120 - 131 2022/07/20 [Refereed][Not invited]
  • 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 0163-3864 2016/04 [Refereed][Not invited]
     
    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.
  • Keita Sutoh, Kenji Washio, Ryozo Imai, Masamitsu Wada, Tomonori Nakai, Daisuke Yamauchi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 (5) 747 - 759 0916-8451 2015/05 [Refereed][Not invited]
     
    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.
  • Lopez Julius Adam Velasco, Al-Lihaibi Sultan S., Alarif Walied M., Abdel-Lateff Ahmed, Washio Kenji, Morikawa Masaaki, Okino Tatsufumi
    Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 57 PosterP23  2015 
    Cyanobacteria are prolific sources of novel bioactive compounds. The chemical profiles we obtained using LC-MS and checked with the MarinLit database showed that there are potential new compounds from Moorea producens samples obtained from the Red Sea. In this presentation the isolation and structure elucidation of a new cyclic dodecapeptide, wewakazole B (1), is reported. The brownish-red filamentous sample was obtained by SCUBA near Jeddah, Saudi Arabia. A small portion of the sample was preserved in RNAlater and identified as M. producens by standard 16S rRNA gene sequencing using cyanobacteria-specific primers 106F and 1509R, and internal primers 359F and 781R (Martinez-Murcia et al., 1995; Nubel et al., 1997). The bulk of the sample was extracted with methanol followed by solvent partition using EtOAc and water, then, BuOH. These extracts were tested for antifouling activity against the barnacle larvae Amphibalanus amphitrite, cytotoxicity against MCF-7 breast cancer cells, and trypsin inhibition. LC-MS was carried out in parallel for dereplication in conjunction with the MarinLit database. The extracts were found to be inactive but a unique [M+H]+ m/z of 1127 was detected in the EtOAc extract and was pursued. A series of open column chromatography and HPLCs afforded 0.4 mg of 1. The molecular formula, C58H70N12O12, determined by HRESIMS ([M+H]+ m/z1127.5310, ∆ = 0.39 ppm), suggested thirty unsaturations. Dereplication using mass and 1H NMR data showed wewakazole (Nogle et al., 2003) as the nearest candidate. HSQC, COSY, and TOCSY revealed nine common and three modified amino acid residues which were connected by HMBC and ROESY correlations acquiring five fragments. Connections to the oxazole and methyloxazole fragments proved to be a challenge due to the lack of correlations towards neighboring amino acids. All possible combinations were considered that lead to the most logical planar structure and cyclic sequence. Chiral LC-MS and HPLC analysis showed that all amino acid residues possess the L- configuration. The wewakazoles are the only cyclic peptides from marine cyanobacteria having both Oxz and MeOxz. Their function and bioactivity require further investigation.
  • Kensuke Kaneko, Kenji Washio, Taiki Umezawa, Fuyuhiko Matsuda, Masaaki Morikawa, Tatsufumi Okino
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (8) 1310 - 1319 0916-8451 2014/08 [Refereed][Not invited]
     
    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.
  • Kaneko Kensuke, Washio Kenji, Umezawa Taiki, Kobayashi Daiki, Tang Xiaorong, Matsuda Fuyuhiko, Morikawa Masaaki, Okino Tatsufumi
    Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 56 Poster53  2014 
    The genus of red alga Laurencia is one of the richest producers of halogenated compounds (mostly brominated) 3). Murai et al 4) and Butler et al 9) showed enzymatic bromination of Laurencia C15acetogenins including laurencin (1) 1,2)and sesquiterpenoids snyderols using partially purified bromoperoxidase (BPO) from L. nipponica and vanadium-dependent BPO (VBPO) from a mixture of red algae including L. pacifica. However, since the lack of information of sequences, the structural information and biochemical aspects of BPO from L. nipponica and VBPO from L. pacifica remains unknown. Here, we report cDNA cloning of VBPOs from L. nipponicaand in vitro enzymatic bromination of putative biosynthetic precursors using recombinant enzymes. Two VBPOs (LnVBPO1 and LnVBPO2) were cloned from L. nipponica and recombinant LnVBPOs were prepared. Recombinant LnVBPOs were subjected to enzyme activity assay. A general substrate, monochlorodimedone (MCD), was utilized for Km determination for H2O2 and , essential for bromination of organic substrate. Km for H2O2was 0.03 mM in both LnVBPOs, Km for were 0.53 mM for LnVBPO1 and 0.35 mM for LnVBPO2, respectively. A derivative of putative precursor of laurencin (1), TMS-capped (3E, 6R, 7R)-laurediol (4) was subjected to LnVBPOs enzymatic bromination and gave similar MS/MS spectrum of deacetylaurencin (3). Since the compound 3 is considered to be the final precursor of 1, it is suggested that VBPO catalyzes bromination in Laurenciametabolites. We also cloned VBPOs from other Laurencia spp. which produce brominated sesquiterpenoids (L. okamurae) and triterpenoids (L. saitoi). We will discuss in vitro enzymatic bromination of these two LaurenciaVBPOs.
  • Kohei Shimada, Yoshikane Itoh, Kenji Washio, Masaaki Morikawa
    CHEMOSPHERE 87 (3) 226 - 233 0045-6535 2012/04 [Refereed][Not invited]
     
    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.
  • Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (10) 1880 - 1888 0916-8451 2011/10 [Refereed][Not invited]
     
    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.
  • Fumiko Yamaga, Kenji Washio, Masaaki Morikawa
    ENVIRONMENTAL SCIENCE & TECHNOLOGY 44 (16) 6470 - 6474 0013-936X 2010/08 [Refereed][Not invited]
     
    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
  • Kenji Washio, Siew Ping Lim, Niran Roongsawang, Masaaki Morikawa
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 (5) 992 - 999 0916-8451 2010/05 [Refereed][Not invited]
     
    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.
  • Kaihei Oki, Kenji Washio, Daigo Matsui, Shinichi Kato, Yoshihiko Hirata, Masaaki Morikawa
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 (3) 583 - 589 0916-8451 2010/03 [Refereed][Not invited]
     
    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.
  • S. P. Lim, N. Roongsawang, K. Washio, M. Morikawa
    JOURNAL OF APPLIED MICROBIOLOGY 107 (1) 157 - 166 1364-5072 2009/07 [Refereed][Not invited]
     
    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.
  • Saori Iijima, Kenji Washio, Ryota Okahara, Masaaki Morikawa
    MICROBIAL BIOTECHNOLOGY 2 (3) 361 - 369 1751-7907 2009/05 [Refereed][Not invited]
     
    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.
  • Jiraporn Thaniyavarn, Tanusta Chianguthai, Polakit Sangvanich, Niran Roongsawang, Keji Washio, Masaaki Morikawa, Suthep Thaniyavarn
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 (8) 2061 - 2068 0916-8451 2008/08 [Refereed][Not invited]
     
    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.
  • Daisuke Takei, Kenji Washio, Masaaki Morikawa
    BIOTECHNOLOGY LETTERS 30 (8) 1447 - 1452 0141-5492 2008/08 [Refereed][Not invited]
     
    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.
  • Slew Ping Lim, Niran Roongsawang, Kenji Washio, Masaaki Morikawa
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (8) 2002 - 2009 0916-8451 2007/08 [Refereed][Not invited]
     
    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.
  • Niran Roongsawang, Kenji Washio, Masaaki Morikawaa
    CHEMBIOCHEM 8 (5) 501 - 512 1439-4227 2007/03 [Refereed][Not invited]
     
    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.
  • Kenji Washio, Masaaki Morikawa
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1759 (10) 478 - 490 0167-4781 2006/10 [Refereed][Not invited]
     
    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.
  • N Roongsawang, SP Lim, K Washio, K Takano, S Kanaya, M Morikawa
    FEMS MICROBIOLOGY LETTERS 252 (1) 143 - 151 0378-1097 2005/11 [Refereed][Not invited]
     
    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.
  • K Washio
    PLANT PHYSIOLOGY 133 (2) 850 - 863 0032-0889 2003/10 [Refereed][Not invited]
     
    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.
  • M Nishikoori, K Washio, A Hase, N Morita, H Okuyama
    PHYSIOLOGIA PLANTARUM 113 (2) 241 - 248 0031-9317 2001/10 [Refereed][Not invited]
     
    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.
  • K Washio
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1520 (1) 54 - 62 0167-4781 2001/07 [Refereed][Not invited]
     
    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 [Refereed][Not invited]
  • H Nakazato, T Okamoto, M Nishikoori, K Washio, N Morita, K Haraguchi, GA Thompson, H Okuyama
    PLANT PHYSIOLOGY 118 (3) 1015 - 1020 0032-0889 1998/11 [Refereed][Not invited]
     
    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.
  • K WASHIO, K ISHIKAWA
    PLANT PHYSIOLOGY 105 (4) 1275 - 1280 0032-0889 1994/08 [Refereed][Not invited]
     
    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.
  • K WASHIO, K ISHIKAWA
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1199 (3) 311 - 314 0304-4165 1994/04 [Refereed][Not invited]
  • K WASHIO, K ISHIKAWA
    PLANT MOLECULAR BIOLOGY 19 (4) 631 - 640 0167-4412 1992/07 [Refereed][Not invited]
     
    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.
  • T SATO, G GRABHERR, K WASHIO
    JOURNAL OF BIOGEOGRAPHY 16 (5) 449 - 455 0305-0270 1989/09 [Refereed][Not invited]
  • S YOSHIDA, K WASHIO, J KENRICK, G ORR
    PLANT AND CELL PHYSIOLOGY 29 (8) 1411 - 1416 0032-0781 1988/12 [Refereed][Not invited]

MISC

Books etc

  • 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 (Joint workChapter 13, pp233–240)
    Caister Academic Press 2015
  • Biofilms in bioremediation, current research and emerging technologies: Comparison of the degradation activity of biofilm-associated versus planktonic cells.
    Morikawa M, Washio K (Joint workChapter 12, pp221–232)
    Caister Academic Press 2015
  • 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 (Joint workpp278-287)
    (株)エヌ・ティー・エス 2008
  • レーヴン/ジョンソン 生物学 原著第7版
    鷲尾健司 (Joint translation下巻、第36章、pp755-766)
    培風館 2007
  • 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 (Joint workvol13, pp229-230)
    Kluwer Academic Publishers, Dordrecht, The Netherlands 1997

Presentations

  • Study on bromination enzyme from the red alga Laurencia nipponica.  [Invited]
    Kaneko K, Washio K, Umezawa T, Morikawa M, Matsuda F, Okino T
    Gordon Research Conferences, Marine Natural Products  2012/02
  • 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  [Invited]
    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  2010/09
  • Generation of D-amino acid during biosynthesis of the cyclic lipopeptide arthrofactin by nonribosomal peptide synthetases.  [Invited]
    Washio K, Roongsawang N, Morikawa M
    BIT 1st Annual World Congress of Catalytic Asymmetric Synthesis 2010  2010/05
  • ウキクサと根圏細菌の相利共生作用による汚染浄化法  [Invited]
    森川正章, 鷲尾健司
    水処理生物学会46回大会  2009/11
  • Biofilm formation and effective production of menaquinone-7 (Vitamin K2) by Bacillus subtilis.  [Invited]
    Washio K, Morikawa M
    Japan-Argentina Workshop (JST/MYNCyT)  2009/08
  • Genetic analysis of synthetic regulation and transportation of lipopeptide type biosurfactant.  [Invited]
    Morikawa M, Roongsawang N, Lim SP, Washio K
    Biosurfactant Workshop in Vietnam  2009/04
  • Sustainable bioremediation technology by utilizing biofilms.  [Invited]
    Morikawa M, Yamaga F, Shimada K, Washio K
    Int. Symposium on Biotechnological Approaches to Environmental Science for Energy Production and Sustainability  2009/02
  • Efficacy of biofilm formation by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology.  [Invited]
    Morikawa M, Shimada K, Washio K
    BIT 1st World Congress of ibio-2008  2008/05
  • 環境微生物の生存戦略−バイオフィルム−  [Invited]
    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  2007/07
  • 種子が目覚めるとき 発芽を促すジベレリンの作用  [Invited]
    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-  2003/10

Association Memberships

  • Botanical Society of Hokkaido   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY   American Society of Plant Biologists   日本植物生理学会   Japanese Society of Plant Physiology   

Works

  • Studies on biosynthesis mechanisms responsible the production of marine halogenated compounds.
    WASHIO Kenji 2010 - Today
  • Functional dissections of the adoptive growth in higher plants.
    WASHIO Kenji 1994 - Today

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2016/03 
    Author : Okino Tatsufumi, MATSUDA Fuyuhiko, UMEZAWA Taiki, NOGATA Yasuyuki, WASHIO Kenji
     
    Vanadium dependent bromoperoxidase (VBPO) was cloned from the red alga Laurencia nipponica which produces a halogenated compound, laurencin. The enzyme catalyzed cyclization and bromination of a precursor of laurencin. Bromination activity of recombinant enzyme was investigated. A similar type of VBPO was cloned from L. okamurae which produces a halogenated sesquiterpenoid, laurinterol. The recombinant enzyme showed bromination activity. In addition, another type of VBPO was cloned from L. saitoi which produces a halogenated triterpenoid. Its properties were investigated by using its recombinant protein. The structure of bromoallene containing C15 compound, omaezallene was reported. Its totaly syntehsis was successfully achieved.
  • 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)
    Date (from‐to) : 2011/04 -2014/03 
    Author : 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)
    Date (from‐to) : 2010/04 -2014/03 
    Author : 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)
    Date (from‐to) : 2007/04 -2010/03 
    Author : 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)
    Date (from‐to) : 2004/04 -2006/03 
    Author : 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)
    Date (from‐to) : 1996/04 -1997/03 
    Author : 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)
    Date (from‐to) : 1994/04 -1995/03 
    Author : 鷲尾健司

Industrial Property Rights

  • 特願2008-099213:新規水草根圏微生物  2008年/04/07
    森川正章, 鷲尾健司, 山賀文子

Others

  • 2011 -2011 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 -2009 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 -2006 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 -2006 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 -2003 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

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