Updated on 2023/09/21

写真a

 
Shigeaki Harayama
 
Organization
Research and Development Initiative Institute Professor
Contact information
The inquiry by e-mail is 《here
External link

Degree

  • 理学博士 ( 東京大学 )

Education

  • 1975.8
     

    The University of Tokyo   doctor course   withdrawn before completion

Research History

  • Chuo University Faculty of Science and Engineering

Research Areas

  • Life Science / Molecular biology

Papers

  • A truncated antenna mutant of the unicellular green alga Coccomyxa sp. strain Obi shows better biomass productivity than the wild-type strain under higher irradiance, at higher cell density, and in greater depth of culture

    Jumpei Hayakawa, Eiko Sato, Satsuki Takagi, Yoko Ide, Sousuke Imamura, Hiroyuki Koike, Yuki Kasai, Shigeaki Harayama

    Algal Research   103216 - 103216   2023.8

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    Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.algal.2023.103216

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  • Limited fatty-acid supply from the plastid and active catabolism of triacylglycerol prevent the accumulation of triacylglycerol in Coccomyxa sp. strain Obi grown under nitrogen-replete conditions

    Kaori Oyama, Izumi Matsuwaki, Maika Ito, Rei Iwahori, Haruka Nagata, Isuzu Nakamura, Ayaka Kondo, Akane Kodaka, Yurika Fuseya, Haruka Yamamoto, Yui Ueyama, Yoko Ide, Yuki Kasai, Shigeaki Harayama, Misako Kato

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   62   102620 - 102620   2022.3

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    The Coccomyxa sp. strain Obi is a unicellular green alga that accumulates > 40% triacylglycerol (TAG) per cell dry weight under nitrogen (N)-limiting conditions. In this study, we analyzed the de novo synthesis of TAG under N-replete and N-limiting conditions using C-14 tracers and quantitative reverse transcription PCR (RT-qPCR) approaches. In cells grown under N-replete conditions, the incorporation of photosynthetically fixed C-14 into TAG was < 5% of that incorporated into polar lipids, whereas the incorporation of exogenous C-14-fatty acids into TAG was approximately 50% of that incorporated into polar lipids. These results demonstrated that enzyme activities that convert fatty acids to TAG are appreciably expressed under N-replete conditions; however low fatty acid supply limits the TAG synthesis in N-replete cells. In agreement with this inference, the expression of FAT1 that encodes acyl-ACP thioesterase, which releases free fatty acids from acyl-ACP, was poorly expressed under N-replete conditions but induced under N-limiting conditions. Meanwhile, pulse-chase labeling experiments demonstrated a significant turnover of TAG under N-replete conditions. Taken together, we propose that both the limited carbon flux into TAG and active catabolism of TAG prevented the accumulation of TAG under N replete conditions, whereas the increased carbon flux into TAG and possibly decreased catabolism of TAG contributed to the enhanced accumulation of TAG under N-limiting conditions. Furthermore, pulse-chase experiments demonstrated that the rate of TAG synthesis was much slower than that of polar lipid synthesis under N-limiting conditions, partly because of the slow conversion of diacylglycerol to TAG.

    DOI: 10.1016/j.algal.2021.102620

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  • Application of 1.0-μm macroporous hollow fiber membrane for prevention of membrane fouling and enhancement of permeate flux in algae harvesting

    Hiroshi Yamamura, Takaki Matsumoto, Keisuke Goto, Qing Ding, Miho Kuroiwa, Shigeaki Harayama

    Bioresource Technology Reports   17   100895 - 100895   2022.2

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    DOI: 10.1016/j.biteb.2021.100895

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  • Nitrite as a causal factor for nitrate-dependent anaerobic corrosion of metallic iron induced by Prolixibacter strains. International journal

    Takao Iino, Nobuaki Shono, Kimio Ito, Ryuhei Nakamura, Kazuo Sueoka, Shigeaki Harayama, Moriya Ohkuma

    MicrobiologyOpen   10 ( 4 )   e1225   2021.8

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    Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate respiration- and Fe0 -corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate-reducing strains was enhanced by nitrate, which was not observed in the two strains unable to reduce nitrate. When the nitrate-reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20-fold compared to that in aseptic controls. This enhancement was not observed in cultures of the strains unable to reduce nitrate. The oxidation of Fe0 in the anaerobic cultures of nitrate-reducing strains occurred concomitantly with the formation of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0 - and carbon steel by the nitrate-reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.

    DOI: 10.1002/mbo3.1225

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  • A method for the preparation of electrocompetent cells to transform unicellular green algae, Coccomyxa (Trebouxiophyceae, Chlorophyta) strains Obi and KJ

    Hiroshi Tatara, Yusuke Sato, Yuki Kasai, Daisuke Hagiwara, Arisa Makino, Sousuke Imamura, Jun Abe, Yuya Yoshimitsu, Shigeaki Harayama

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   48   2020.6

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    Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm(-1) to the autospores yielded transformants at a frequency of > 10(-6) per input cell for strain KJ and > 10(-7) per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.

    DOI: 10.1016/j.algal.2020.101904

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  • Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ. International journal

    Yuya Yoshimitsu, Jun Abe, Shigeaki Harayama

    Biotechnology for biofuels   11 ( 1 )   326 - 326   2018.12

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    BackgroundOxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels>60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired.ResultsIn this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5ms) electric pulse at a high field strength (7500Vcm(-1)) followed by multiple 50-ms electric pulses at low field strength (250Vcm(-1)). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%.ConclusionsOur study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.

    DOI: 10.1186/s13068-018-1327-1

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  • An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis. International journal

    Hirohito Tsurumaru, Naofumi Ito, Koji Mori, Satoshi Wakai, Taku Uchiyama, Takao Iino, Akira Hosoyama, Hanako Ataku, Keiko Nishijima, Miyako Mise, Ai Shimizu, Takeshi Harada, Hiroshi Horikawa, Natsuko Ichikawa, Tomohiro Sekigawa, Koji Jinno, Satoshi Tanikawa, Jun Yamazaki, Kazumi Sasaki, Syuji Yamazaki, Nobuyuki Fujita, Shigeaki Harayama

    Scientific reports   8 ( 1 )   15149 - 15149   2018.10

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    Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe-0), are largely unknown. In this study, Fe-0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe-0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe-0, and oxidize it, generating hydrogen gas: Fe-0 + 2 H+ -> Fe2+ + H-2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

    DOI: 10.1038/s41598-018-33541-5

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  • Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa. International journal

    Yuki Kasai, Takuya Tsukahara, Fukiko Ikeda, Yoko Ide, Shigeaki Harayama

    Scientific reports   8 ( 1 )   11742 - 11742   2018.8

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    We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura(-)) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura(-)strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker-gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein)thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.

    DOI: 10.1038/s41598-018-30254-7

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  • Lipid productivity in TALEN-induced starchless mutants of the unicellular green alga Coccomyxa sp strain Obi

    Keita Takahashi, Yoko Ide, Jumpei Hayakawa, Yuya Yoshimitsu, Izumi Fukuhara, Jun Abe, Yuki Kasai, Shigeaki Harayama

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   32   300 - 307   2018.6

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    Starchless mutants of several green algae including Chlamydomonas reinhardtii and Scenedesmus obliquus showed improved lipid productivity. In this study, we isolated mutants of the unicellular green alga, Coccomyxa sp. strain Obi, defective in the gene for the large subunit of ATP: alpha-D-glucose-1-phosphate adenylyltransferase (AGPL) by means of a transcription activator-like effector nuclease (TALEN) as follows. First, two expression cassettes encoding left and right arms of TALEN for AGPL mutagenesis were introduced into strain Obi, and from two independent transformants of strain Obi carrying both expression cassettes, seven derivatives bearing different mutations in AGPL were isolated. When these mutants were grown in 1/2-diluted and 1/3-diluted minimal media, their lipid contents were significantly higher than those of their parental strains. The growth in 1/3-diluted medium was similar between the mutants and their parental strains, while the growth in 1/2-diluted medium was significantly lower for the mutants than for their parental strains. The lipid productivity, which is a crucial factor for commercial production of biofuels, was not statistically different between the parental and mutant strains. We concluded that the suppression of starch biosynthesis to increase lipid productivity in microalgae is a risky strategy as it could result in low biomass productivity.

    DOI: 10.1016/j.algal.2018.04.020

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  • Precise excision of a selectable marker gene in transgenic Coccomyxa strains by the piggyBac transposase

    Yuki Kasai, Kenta Matsuzaki, Fukiko Ikeda, Yuya Yoshimitsu, Shigeaki Harayama

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   27   152 - 161   2017.11

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    The piggyBac transposon isolated from the cabbage looper moth Trichoplusia ni, is integrated into the host genome, and then excised from it without leaving a footprint. The piggyBac transposon system has been used as a genomic engineering tool in a variety of organisms. In this study, we used two improved versions of the piggyBac transposase (PBase) to create marker-free transgenic strains of the unicellular green alga Coccomyxa sp. strain KJ as follows: Uracil-auxotrophic (Ura(-)) mutants of strain KJ defective in the gene for uridine monophosphate synthase (KJUMPS) were isolated on agar plates containing 5-fluoroorotic acid. Subsequently, cDNA of KJUMPS (cKJUMPS) was cloned between the promoter and terminator of the elongation factor 1 alpha gene to construct a cKJUMPS expression cassette. A DNA fragment carrying the cKJUMPS expression cassette flanked with piggyBac transposon terminal repeats was then constructed (TR_cKJUMPS) and introduced into an Ura-mutant, and Ura(+) transformants were isolated. One of the Ura(+) transformants was named strain TR2-7. Hyperactive PBase (hyPBase) is a mutant PBase with increased excision and integration frequencies. Herein, we synthesized a coding sequence for hyPBase (KJhyPBase), which has optimized codons for expression in strain KJ, and its expression cassette was introduced into strain TR2-7. Fourteen transformants stably carried the KJhyPBase expression cassette, and TR_cKJUMPS was excised from seven of these. We also introduced an expression cassette of KJhyPBase_Ex, which encodes the excision-competent/integration-defective R372A/K375A/D450N mutant of KJhyPBase (KJhyPBase_Ex), into strain TR2-7, and found that the excision frequency of TR_cKJUMPS in KJhyPBase_Ex transformants was significantly higher than that in KJhyPBase transformants. In further experiments, we purified His-tagged KJhyPBase_Ex, and transfected it into strain TR2-7 using electroporation. Under these conditions, TR_cKJUMPS was precisely excised at a frequency of 8.8x10(-8) cell(-1) .The present data extend applications of the present piggyBac transposase-catalyzed excision system in green algae.

    DOI: 10.1016/j.algal.2017.09.007

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  • Biofilm-based photobioreactor absorbing water and nutrients by capillary action. International journal

    Hayato Hamano, Shun Nakamura, Jumpei Hayakawa, Hideaki Miyashita, Shigeaki Harayama

    Bioresource technology   223   307 - 311   2017.1

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    Cells of the unicellular green alga, "Pseudochoricystis ellipsoidea", were uniformly spread on a cellulosic sheet or on a polytetrafluoroethylene (PTFE) membrane sheet superimposed on a cellulosic sheet at a density of 3.5-5.0 g dry weight per m(2), and the sheet was adhered to an inverted V-shaped acrylic plate of 10 cm in height. Several acrylic plates were placed side by side on a tray containing liquid medium at a depth of 0.6 cm, and illuminated from above with a light intensity of 300-340 mu mol m(-2) s(-1). Water and nutrients were supplied to cells by capillary action through the cellulosic sheet. Footprint biomass productivities of cells grown in atmospheric CO2 on this photobioreactor were 8-10 g m(-2) day(-1). This cultivation system is strongly energy-and labor-saving as it does not require mixing of culture fluid, irrigation of medium, and delivery of CO2-enriched air. (C) 2016 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.biortech.2016.10.088

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  • Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System. International journal

    Yuki Kasai, Shigeaki Harayama

    PloS one   11 ( 8 )   e0161733   2016.8

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    The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct markerfree transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble( linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wildtype strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

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  • Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea. International journal

    Yuki Kasai, Kohei Oshima, Fukiko Ikeda, Jun Abe, Yuya Yoshimitsu, Shigeaki Harayama

    Biotechnology for biofuels   8 ( 1 )   94 - 94   2015.6

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    Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea.Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator.Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

    DOI: 10.1186/s13068-015-0277-0

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  • Assessment of the biological invasion risks associated with a massive outdoor cultivation of the green alga, Pseudochoricystis ellipsoidea

    Izumi Matsuwaki, Shigeaki Harayama, Misako Kato

    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS   9   1 - 7   2015.5

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    Pseudochoricystis ellipsoidea MBIC 11204 is a unicellular green alga that accumulates, under certain stress conditions, lipids consisting primarily of triacylglycerols. The spread of P. ellipsoidea cells from an outdoor raceway pond into the surrounding area was investigated over 35 days by detecting the psbA gene of this alga in water-containing vessels installed at variable distances (between 0 and 150 m) from the raceway pond. psbA was detected even in vessels placed 150 m apart, demonstrating the ability of P. ellipsoidea to spread via wind. Based on this finding, we evaluated the survival and growth of P. ellipsoidea in various environmental water samples. P. ellipsoidea grew well in water containing >2 mg/L nitrate, but growth was not observed inwater containing 3 mg/L of ammonia if the nitrate concentration was <1 mg/L. The addition of nitrate to produce the final concentration of 3 mg/L improved the growth, demonstrating that nitrate is the growth-limiting nutrient in these water samples. (C) 2015 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.algal.2015.02.021

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  • Iron corrosion induced by nonhydrogenotrophic nitrate-reducing Prolixibacter sp. strain MIC1-1. International journal

    Takao Iino, Kimio Ito, Satoshi Wakai, Hirohito Tsurumaru, Moriya Ohkuma, Shigeaki Harayama

    Applied and environmental microbiology   81 ( 5 )   1839 - 46   2015.3

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    Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe-0) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe-0 oxidation. In this study, we describe Fe-0 corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe-0 as the sole electron donor. In addition, ferrous ion and L-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe-0-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe-0 concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO4 developed on the surface of the Fe-0 foils, and a layer of FeCO3 covered the FePO4 crystals. Wepropose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe-0 to reduce nitrate.

    DOI: 10.1128/AEM.03741-14

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  • Isolation of bacteria rapidly adhering to metal iron surface

    Wakai, S, Harayama, S

    Materials Technology   30 ( sup5 )   B38 - B43   2015

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  • Alcanivoraceae fam. nov.

    Golyshin, Peter N, Harayama, Shigeaki, Timmis, Kenneth N, Yakimov, Michail M

    Bergey's Manual of Systematics of Archaea and Bacteria   1 - 1   2015

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  • Alcanivorax

    Golyshin, Peter N, Harayama, Shigeaki, Timmis, Kenneth N, Yakimov, Michail M

    Bergey's Manual of Systematics of Archaea and Bacteria   1 - 7   2015

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  • Corrosion of iron by iodide-oxidizing bacteria isolated from brine in an iodine production facility. International journal

    Satoshi Wakai, Kimio Ito, Takao Iino, Yasuyoshi Tomoe, Koji Mori, Shigeaki Harayama

    Microbial ecology   68 ( 3 )   519 - 27   2014.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER  

    Elemental iodine is produced in Japan from underground brine (fossil salt water). Carbon steel pipes in an iodine production facility at Chiba, Japan, for brine conveyance were found to corrode more rapidly than those in other facilities. The corroding activity of iodide-containing brine from the facility was examined by immersing carbon steel coupons in "native" and "filter-sterilized" brine samples. The dissolution of iron from the coupons immersed in native brine was threefold to fourfold higher than that in the filter-sterilized brine. Denaturing gradient gel electrophoresis analyses revealed that iodide-oxidizing bacteria (IOBs) were predominant in the coupon-containing native brine samples. IOBs were also detected in a corrosion deposit on the inner surface of a corroded pipe. These results strongly suggested the involvement of IOBs in the corrosion of the carbon steel pipes. Of the six bacterial strains isolated from a brine sample, four were capable of oxidizing iodide ion (I-) into molecular iodine (I-2), and these strains were further phylogenetically classified into two groups. The iron-corroding activity of each of the isolates from the two groups was examined. Both strains corroded iron in the presence of potassium iodide in a concentration-dependent manner. This is the first report providing direct evidence that IOBs are involved in iron corrosion. Further, possible mechanisms by which IOBs corrode iron are discussed.

    DOI: 10.1007/s00248-014-0438-x

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  • Identification of novel non-metal haloperoxidases from the marine metagenome. International journal

    Hui-Jeong Gwon, Ide Teruhiko, Harayama Shigeaki, Sang-Ho Baik

    Journal of microbiology and biotechnology   24 ( 6 )   835 - 42   2014.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY  

    Haloperoxidase (HPO, E.C.1.11.1.7) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics, etc., in the presence of halides and H2O2. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from Pseudomonas putida ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with P. putida IF-3 sequences. Approximately 600 transformants screened for chimeric genes between P. putida ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with P. putida or Sinorhizobium or Streptomyces strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy. E. coli cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.

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  • Influence of extracellular polysaccharides (EPS) produced by two different green unicellular algae on membrane filtration in an algae-based biofuel production process International journal

    Takaki Matsumoto, Hiroshi Yamamura, Jyunpei Hayakawa, Yoshimasa Watanabe, Shigeaki Harayama

    WATER SCIENCE AND TECHNOLOGY   69 ( 9 )   1919 - 1925   2014.1

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    In the present study, two strains of green algae named S1 and S2, categorized as the same species of Pseudo-coccomyxa ellipsoidea but showing 99% homology, were cultivated under the same conditions and filtrated with a microfiltration membrane. On the basis of the results of the extracellular polysaccharides (EPS) characteristics of these two green algae and the degree of fouling, the influence of these characteristics on the performance of membrane filtration was investigated. There was no difference in the specific growth rate between the S1 and S2 strains; however, large differences were seen in the amount and quality of EPS between S1 and S2. When the S1 and S2 strains were filtered with a membrane, the trend in the increase in transmembrane pressure (TMP) was quite different. The filtration of the S1 strain showed a rapid increase in TMP, whereas the TMP of the filtration of the S2 strain did not increase at all during the operation. This clearly demonstrated that the characteristics of each strain affect the development of membrane fouling. On the basis of the detailed characterization of solved-EPS (s-EPS) and bound-EPS (b-EPS), it was clarified that s-EPS mainly contributed to irreversible fouling for both operations and the biopolymer-like organic matter contained in b-EPS mainly contributed to reversible fouling.

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  • Isolation and Screening of Surfactant-producing Bacteria from Indonesian Marine Environments and Its Application on Bioremediation

    Dwi Susilaningsih, Fumiyoshi Okazaki, Yopi, Yantyati Widyastuti, Shigeaki Harayama

    ANNALES BOGORIENSES-JOURNAL OF TROPICAL GENERAL BOTANY   17 ( 2 )   41 - 51   2013

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    Isolation and screening have been undertaken on oil-degrading microbes from Indonesian marine environments. During screening process it has been found many bacterial isolates capable of degrading crude oil. Hence, study has been focused on the biodiversity of biosurfactant-producing bacterial species in Indonesian marine environment and its function for remedial the pollutant in marine and soil areas. A total of 103 out of 463 isolates showed positive surfactant-degrading properties. By means of partial 16S rRNA gene analyses, it has been found that the majority of taxa are related to Alcanivorax, Pseudomonas, Bacillus, Bortetela, Brucella, Acenitobacter, Staphia, Lysobacter, and Talasosophira. Biosurfactant properties assay showed that they were capable of lowering the surface-and interfacial water tension from 74 mN/m to 40-65 mN/m and from 24 mN/m to 6-10 mN/m, respectively. In addition, most of the surfactants were capable of emulsifying hydrocarbon (crude oil) of 0.01 to 0.15 units, comparable to 0.08 units of synthetic surfactant (20% Tween). Further observation showed that the majority of the surfactants were able to degrade a long chain of alkane, but not branched alkane, with a recovering rate of 20-80%. The application of the surfactant towards oil polluted model beach was done in laboratory scale and showing the surfactant obtained from microbial broth cultures capable for recovering the oil pollutant significantly, compared to the control (without addition microbial broth).

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  • Enhanced production of astaxanthin in Paracoccus sp strain N-81106 by using random mutagenesis and genetic engineering

    Teruhiko Ide, Megumi Hoya, Toru Tanaka, Shigeaki Harayama

    BIOCHEMICAL ENGINEERING JOURNAL   65   37 - 43   2012.6

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    Astaxanthin is the most powerful antioxidant of all carotenoids and has great commercial value for use in the aquacultural, pharmaceutical, and food industries. The marine bacterium Paracoccus sp. strain N-81106 is one of the natural producers of astaxanthin, but its astaxanthin productivity is too low for economically feasible industrial production. We tried to improve the productivity of astaxanthin by this strain by a combination of (i) random mutagenesis and (ii) gene cloning/overexpression of astaxanthin-biosynthetic genes. In the first step, we isolated astaxanthin-overproducing mutants after random mutagenesis of strain N-81106. The mutants isolated, NG5 and NG9, showed greater production of astaxanthin than did the wild-type strain N-81106. The yield of astaxanthin in the mutants was about 17 times that in strain N-81106. In the second step, we cloned the astaxanthin-biosynthesis genes of strain N-81106 in Escherichia coli by using a broad-host-range vector, and we then mobilized the cloned genes from E. coli to strains N-81106, NG5, and NG9. The recombinant NG5 produced astaxanthin at 58 mg/l-56 times the production of the original strain. The yield was further increased by fed-batch fermentation to reach 480 mg/l culture fluid. (c) 2012 Elsevier B.V. All rights reserved.

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  • Genetic transformation of Pseudochoricystis ellipsoidea, an aliphatic hydrocarbon-producing green alga.

    Sousuke Imamura, Daisuke Hagiwara, Fumi Suzuki, Norihide Kurano, Shigeaki Harayama

    The Journal of general and applied microbiology   58 ( 1 )   1 - 10   2012.2

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    Pseudochoricystis ellipsoidea is a recently isolated unicellular green alga, which is classified within the family Trebouxiophyceae. This alga has a unique ability to synthesize and accumulate intracellularly a significant amount of aliphatic hydrocarbons. To elucidate molecular mechanisms of the hydrocarbon production in this organism, the development of genetic methods including DNA transformation methods are important. Towards the goal, we constructed several plasmids in which neomycin phosphotransferase II-encoding G418-resistant gene (nptII) is flanked by a P ellipsoidea-derived promoter and terminator. These plasmids were introduced into P ellipsoidea cells through particle-gun bombardment, and transformants were screened among G418-resistant cells by PCR amplification of plasmid-borne genes. Southern blot analysis demonstrated that the exogenous DNA was integrated into the genome of the transformants. Furthermore, the expression of nptII was confirmed at the transcript and protein levels by RTPCR and immunoblot analyses, respectively. These results clearly indicated that a genetic transformation system was successfully established for P ellipsoidea.

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  • Catabolism of Aromatic Hydrocarbons by Pseudomonas

    Harayama, SHIGEAKI, Timmis, KENNETH N

    Genetics of bacterial diversity   151 - 151   2012

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  • Alkane-degrading properties of Dietzia sp. H0B, a key player in the Prestige oil spill biodegradation (NW Spain).

    Shigeaki Harayama

    Journal of applied microbiology   2011.10

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  • Oleibacter marinus gen. nov., sp. nov., a bacterium that degrades petroleum aliphatic hydrocarbons in a tropical marine environment. International journal

    Maki Teramoto, Motoyuki Ohuchi, Ariani Hatmanti, Yeti Darmayati, Yantyati Widyastuti, Shigeaki Harayama, Yukiyo Fukunaga

    International journal of systematic and evolutionary microbiology   61 ( Pt 2 )   375 - 380   2011.2

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    Three Gram-negative, motile, mesophilic, aerobic, rod-shaped bacterial strains, designated 2O1(T), 1O14 and 1O18, were isolated from Indonesian seawater after enrichment with crude oil and a continuous supply of supplemented seawater. The strains exhibited high n-alkane-degrading activity, which indicated that the strains were important degraders of petroleum aliphatic, hydrocarbons in tropical marine environments. Phylogenetic analyses based on 16S rRNA gene sequences of members of the Gammaproteobacteria showed that the isolates formed a coherent and distinct cluster in a stable lineage containing Oceanobacter kriegii IFO 15467(T) (96.4-96.5 % 16S rRNA gene sequence similarity) and Thalassolituus oleivorans MIL-1(T). DNA G + C content was 53.0-53.1 mol%. The major fatty acids were C-16:0, C-16:1 omega 7 and C-18:1 omega 9 and the hydroxy fatty acids were C-12:0 3-OH and C-10:0 3-OH. The polar lipids were phosphatidylglycerol, a ninhydrin-positive phospholipid(s) and glycolipids. The major quinone was Q-9 (97-99%), which distinguished the isolates from Oceanobacter kriegii NBRC 15467(T) (Q-8; 91 %). On the basis of phenotypic, genotypic and chemotaxonomic data, including DNA-DNA hybridization, the isolates represent a novel genus and species, for which the name Oleibacter marinus gen. nov., sp. nov. is proposed. The type strain of Oleibacter marinus is 2O1(T) (=NBRC 105760(T) =BTCC B-675(T)).

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  • Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., mesophilic methanogens isolated from salty environments. International journal

    Koji Mori, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   61 ( Pt 1 )   138 - 143   2011.1

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    Two methane-producing archaea, designated Mic5c12(T) and Mic6c05(T), were isolated from sludge deposited in a crude oil storage tank and a tubercle on the interior of a pipe transporting natural gas-containing brine, respectively. The isolates were Gram-staining-variable, non-motile rods and grew only on H-2/CO2. Strain Mic6c05(T) produced methane from some alcohols without showing any growth; strain Mic5c12(T) did not utilize alcohols. The optimum growth conditions for strain Mic5c12(T) were 35 C, pH 6.5 and 0-0.68 M NaCl and for strain Mic6c05(T) were 40 degrees C, pH 6.0-7.5 and 0.34 M NaCl. Strain Mic5c12(T) was halotolerant and strain Mic6c05(T) was halophilic. Comparative 16S rRNA gene sequence analysis revealed that strains Mic5c12(T) and Mic6c05(T) belonged to the genus Methanobacterium and their closest relative was Methanobacterium subterraneum A8p(T) (97.3 and 97.9% 16S rRNA gene sequence similarity, respectively). The findings from the 16S rRNA gene sequence analyses were supported by analysis of McrA, the alpha subunit of methyl-coenzyme M reductase. On the basis of phylogenetic analyses and phenotypic characteristics, two novel species are proposed, Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., with type strains Mic5c12(T) (=NBRC 105198(T) =DSM 22353(T)) and Mic6c05(T) (=NBRC 105197(T) =DSM 21974(T)), respectively.

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  • A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites. International journal

    Kazuo Isobe, Keisuke Koba, Shingo Ueda, Keishi Senoo, Shigeaki Harayama, Yuichi Suwa

    Journal of microbiological methods   84 ( 1 )   46 - 51   2011.1

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    We modified and tuned a commercial model of a gas chromatography/mass spectrometry (GC/MS) instrument to develop a simple and rapid method for the simultaneous quantification of a variety of gas species. Using the developed method with the newly modified instrument, gas species such as H-2, N-2, O-2, CO, NO, CH4, CO2, and N2O, which are common components of microbial metabolism, were accurately identified based on their retention times and/or mass-to-charge ratios (m/z) in less than 2.5 min. By examining the sensitivities and dynamic ranges for the detection of H-2, N-2, O-2, CH4, CO2, and N2O, it was demonstrated that the method developed in this study was sufficient for accurately monitoring the production and the consumption of these gaseous species during microbial metabolism. The utility of the new method was demonstrated by a denitrification study with Pseudomonas aureofaciens ATCC 13985(T). This method will be suitable for a variety of applications requiring the identification of gaseous metabolites in microorganisms, microbial communities, and natural ecosystems. (C) 2010 Elsevier B.V. All rights reserved.

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  • Iron Corrosion by Methane-Producing Archaea (MPA) and Sulfate-Reducing Bacteria (SRB) Utilizing Metallic Iron as an Electron Donor

    Ito, Kimio, Wakai, Satoshi, Tsurumaru, Hirohito, Iino, Takao, Mori, Koji, Uchiyama, Taku, Miki, Osamu, Harayama, Shigeaki

    Corrosion Engineering   60 ( 9 )   336 - 336   2011

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  • Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., two new members of the family Dermatophilaceae, and reclassification of Dermatophilus chelonae (Masters et al. 1995) as Austwickia chelonae gen. nov., comb. nov.

    Moriyuki Hamada, Takao Iino, Takahiro Iwami, Shigeaki Harayama, Tomohiko Tamura, Ken-ichiro Suzuki

    The Journal of general and applied microbiology   56 ( 6 )   427 - 36   2010.12

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    Two Gram-positive bacteria, designated strains Aji5-31(T) and Ngc37-23(T), were isolated from the intestinal tracts of fishes. 16S rRNA gene sequence analysis indicated that both strains were related to the members of the family Dermatophilaceae, with 95.6-96.9% 16S rRNA gene sequence similarities. The family Dermatophilaceae contains 2 genera and 3 species: Dermatophilus congolensis, Dermatophilus chelonae and Kineosphaera limosa. However, it has been suggested that the taxonomic position of D. chelonae should be reinvestigated using a polyphasic approach, because the chemotaxonomic characteristics are not known (Stackebrandt, 2006; Stackebrandt and Schumann, 2000). Our present study revealed that strains Aji5-31(T), Ngc37-23(T) and D. chelonae NBRC 105200(T) should be separated from the other members of the family Dermatophilaceae on the basis of the following characteristics: the predominant menaquinone of strain Aji5-31(T) is MK-8(H-2), strain Ngc37-23(T) possesses iso- branched fatty acids as major components, and the menaquinone composition of D. chelonae is MK-8(H-4), MK-8 and MK-8(H-2) (5 : 3 : 2, respectively). On the basis of these distinctive phenotypic characteristics and phylogenetic analysis results, it is proposed that strains Aji5-31(T) and Ngc37-23(T) be classified as two novel genera and species of the family Dermatophilaceae. The names are Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., and the type strains are Aji5-31(T) (=NBRC 104925(T) = DSM 22762(T)) and Ngc37-23(T) (=NBRC 104926(T) = DSM 22761(T)), respectively. In addition, D. chelonae should be reassigned to a new genus of the family Dermatophilaceae with the name Austwickia chelonae gen. nov., comb. nov.

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  • Iron corrosion activity of anaerobic hydrogen-consuming microorganisms isolated from oil facilities.

    Koji Mori, Hirohito Tsurumaru, Shigeaki Harayama

    Journal of bioscience and bioengineering   110 ( 4 )   426 - 30   2010.10

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    The purpose of the present study was to test the hypothesis that anaerobic hydrogen-consuming microorganisms generally promote iron corrosion. We isolated 26 hydrogen-consuming microorganisms (acetogens, sulfate-reducing bacteria, and methanogens) from oil facilities in Japan using hydrogen as an electron donor. The iron corrosion activities of these microorganisms were examined using iron (Fe(0)) granules as the sole electron donor. Almost all the isolates consumed hydrogen that was chemically generated from iron granules but did not induce significant iron corrosion. The amount of corroded iron in the cultures of these organisms was less than 2-fold that in an abiotic chemical corrosion reaction. These results indicated that hydrogen consumption did not strongly stimulate iron corrosion. On the other hand, one isolate, namely, Methanococcus maripaludis Mic1c10, considerably corroded iron: this phenomenon was not accompanied by hydrogen consumption, methane formation, or cell growth. This finding also provided strong evidence that M. maripaludis Mic1c10 produced some material that caused iron to corrode. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

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  • Euzebya tangerina gen. nov., sp. nov., a deeply branching marine actinobacterium isolated from the sea cucumber Holothuria edulis, and proposal of Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov. International journal

    Midori Kurahashi, Yukiyo Fukunaga, Yayoi Sakiyama, Shigeaki Harayama, Akira Yokota

    International journal of systematic and evolutionary microbiology   60 ( Pt 10 )   2314 - 2319   2010.10

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    A tangerine-coloured, Gram-positive actinobacterial strain, designated F10(T), was isolated from the abdominal epidermis of a sea cucumber, Holothuria edulis, collected in seawater off the coast of Japan. A 16S rRNA gene sequence analysis indicated that strain F10(T) was a member of the class Actinobacteria and was most closely related to Nitriliruptor alkaliphilus ANL-iso2(T) (87.4% sequence similarity). Phylogenetic analyses showed that strain F10(T) represented a novel, deep-rooted, and distinct phylogenetic lineage within the class Actinobacteria and clustered with N. alkatiphilus and uncultured bacteria. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and rhamnose and galactose as the diagnostic cell-wall sugars. Strain F10(T) contained C-16:1 omega 7C, C-16:0 and C-17:1 omega 8c as the major cellular fatty acids. The predominant isoprenoid quinone was MK-9 (H-4). The G+C content of the DNA was 68.3 mol%. Based on data from the current polyphasic study, it is proposed that the new marine isolate be placed in a novel genus and be considered a novel species designated Euzebya tangerina gen. nov., sp. nov. within the new family, order and subclass Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov. in the class Actinobacteria. The type strain of Euzebya tangerina is F10(T) (=NBRC 105439(T) =KCTC 19736(T)).

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  • The potential of Cycloclasticus and Altererythrobacter strains for use in bioremediation of petroleum-aromatic-contaminated tropical marine environments.

    Maki Teramoto, Masahito Suzuki, Ariani Hatmanti, Shigeaki Harayama

    Journal of bioscience and bioengineering   110 ( 1 )   48 - 52   2010.7

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    Cycloclasticus sp. A5, which has been suggested to be a major degrader of petroleum aromatics spilled in temperate seas, showed higher degrading activities for petroleum aromatics, at both 25 degrees C and tropical sea temperature 30 degrees C, than the novel aromatic-degrading isolates, related to Altererythrobacter epoxidivorans (97.5% similarity in the almost full-length 16S rRNA gene sequence) and Rhodovulum iodosum (96.3% similarity), obtained after enrichment on crude oil in a continuous supply of Indonesian seawater. Cycloclasticus AS degraded petroleum aromatics at a similar rate or faster at 30 degrees C as compared to 25 degrees C, but its growth on acetate was severely inhibited at 30 degrees C. These results suggest that, although their abundance would be low in tropical seas not contaminated with aromatics, the Cycloclasticus strains could be major degraders of petroleum aromatics spilled in tropical seas. The 16S rRNA gene of the Cycloclasticus strains has been identified from Indonesian seawater, and the gene fragments showed 96.7-96.8% similarities to that of Cycloclasticus A5. Introducing Cycloclasticus AS may be an ecologically advantageous bioremediation strategy for petroleum-aromatic-contaminated tropical seas because strain AS would disappear at 30 degrees C after complete consumption of the aromatics. Altererythrobacter and Rhodovulum-related isolates grew well on pyruvate in 10% strength marine broth at 30 degrees C whereas Cycloclasticus AS did not grow well on acetate in the broth at 30 degrees C. These growth results, along with its petroleum-aromatic-degrading activity, suggest that the Altererythrobacter isolate could be an important petroleum-aromatic degrader in and around nutrient-rich tropical marine environments. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

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  • Direct cloning and expression of putative esterase genes from environmental DNA

    Takeshi Terahara, Kazutaka Yamada, Shinya Kurata, Toyokazu Yokomaku, Satoshi Tsuneda, Shigeaki Harayama

    ENZYME AND MICROBIAL TECHNOLOGY   47 ( 1-2 )   17 - 23   2010.7

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    Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32-80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli. Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p-nitrophenyl acetate, but almost undetectable for p-nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25-70 degrees C and at pH 8.0-9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA. (C) 2010 Elsevier Inc. All rights reserved.

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  • Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring and proposal of Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur bacteria. International journal

    Takao Iino, Koji Mori, Yoshihito Uchino, Tatsunori Nakagawa, Shigeaki Harayama, Ken-Ichiro Suzuki

    International journal of systematic and evolutionary microbiology   60 ( Pt 6 )   1376 - 1382   2010.6

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    A moderately thermophilic chemoheterotrophic bacterium, strain Mat9-16(T), was isolated from microbial mats developed in hot spring water streams from Yumata, Nagano, Japan. Cells of strain Mat9-16(T) were strictly anaerobic, Gram-stain-negative, non-sporulating, non-motile and short to long rods (2.0-15.5 mu m in length). Strain Mat9-16(T) grew fermentatively with optimum growth at 45 degrees C, pH 7.0-7.5 and 1% NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene revealed that strain Mat9-16(T) was affiliated with an uncultivated lineage, and the nearest cultivated neighbours were green sulfur bacteria belonging to the class Chlorobea with 77-83% sequence similarity. However, strain Mat9-16(T) could not grow phototrophically and did not possess light-harvesting structures, morphologically and genetically, such as the chlorosomes of green sulfur bacteria. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed for strain Mat9-16(T), to be named Ignavibacterium album gen. nov., sp. nov. (=NBRC 101810(T) =DSM 19864(T)). We also propose to place the cultivated bacterial lineage accommodating the sole representative Mat9-16(T) in a novel class, Ignavibacteria classis nov. In addition, we present a formal description of the phylum-level taxon 'Chlorobi' as Chlorobi phyl. nov.

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  • Iron-corroding methanogen isolated from a crude-oil storage tank. International journal

    Taku Uchiyama, Kimio Ito, Koji Mori, Hirohito Tsurumaru, Shigeaki Harayama

    Applied and environmental microbiology   76 ( 6 )   1783 - 8   2010.3

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    Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe-0) granules, which was then incubated anaerobically at 37 degrees C under an N-2-CO2 atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H-2 production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO3.

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  • Advances in the field of high-molecular-weight polycyclic aromatic hydrocarbon biodegradation by bacteria. International journal

    Robert A Kanaly, Shigeaki Harayama

    Microbial biotechnology   3 ( 2 )   136 - 64   2010.3

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    Interest in understanding prokaryotic biotransformation of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) has continued to grow and the scientific literature shows that studies in this field are originating from research groups from many different locations throughout the world. In the last 10 years, research in regard to HMW PAH biodegradation by bacteria has been further advanced through the documentation of new isolates that represent diverse bacterial types that have been isolated from different environments and that possess different metabolic capabilities. This has occurred in addition to the continuation of in-depth comprehensive characterizations of previously isolated organisms, such as Mycobacterium vanbaalenii PYR-1. New metabolites derived from prokaryotic biodegradation of four-and five-ring PAHs have been characterized, our knowledge of the enzymes involved in these transformations has been advanced and HMW PAH biodegradation pathways have been further developed, expanded upon and refined. At the same time, investigation of prokaryotic consortia has furthered our understanding of the capabilities of microorganisms functioning as communities during HMW PAH biodegradation.

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  • Iron corrosion under the enrichment culture of anaerobic microorganisms utilizing metallic iron as an electron donor Reviewed

    Kimio Ito, Taku Uchiyama, Takao Iino, Koji Mori, Satoshi Wakai, Hirohito Tsurumaru, Osamu Miki, Shigeaki Harayama

    Zairyo to Kankyo/ Corrosion Engineering   59 ( 8 )   298 - 304   2010

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    Iron corrosion under enrichment culture of anaerobic microorganisms utilizing metallic iron as an electron donor was investigated. Enrichment culture from residual water on bottom of a crude oil storage tank in Kyushu region caused severe iron corrosion. But the other two enrichment cultures from drain water of an oil field in Tohoku region and sediment at mouth of the Edogawa river did not corrode iron severely. Black ferrous sulfide film was detected in the corrosion products under the enrichment cultures from the drain water of the oil field in Tohoku region and the sediment at mouth of the Edogawa river. Therefore, sulfate reducing bacteria (SRB) were considered to be causative microorganisms to iron corrosion. But the ferrous sulfide film was not detected in the corrosion products under the enrichment culture from the crude oil storage tank in Kyushu region. Ferrous carbonate (FeCO3) was a main component of the corrosion products by the enrichment culture from the crude oil storage tank in Kyushu region. Therefore, iron corrosion under the enrichment culture from the crude oil storage tank in Kyushu region was considered to be not simply caused by the SRB. Open circuit potential of a carbon steel (SS400) coupon with corrosion products under the enrichment culture from the crude oil storage tank in Kyushu region was about 120 to 140 mV higher than that by the other two enrichment cultures or abiotic control in anaerobic artificial seawater deaerated by Ar gas. Therefore, Galvanic coupling between corrosion products layer and base metal was considered to be possible mechanism of iron corrosion under the enrichment culture of the crude oil storage tank in Kyushu region.

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  • Serinibacter salmoneus gen. nov., sp. nov., an actinobacterium isolated from the intestinal tract of a fish, and emended descriptions of the families Beutenbergiaceae and Bogoriellaceae. International journal

    Moriyuki Hamada, Takao Iino, Tomohiko Tamura, Takahiro Iwami, Shigeaki Harayama, Ken-Ichiro Suzuki

    International journal of systematic and evolutionary microbiology   59 ( Pt 11 )   2809 - 14   2009.11

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    A novel Gram-positive bacterium, designated Kis4-28(T), was isolated from the intestinal tract of a fish, and its taxonomic position was investigated by a polyphasic approach. The sample was collected from the coast of Tokyo Bay, Japan. Cells of strain Kis4-28(T) were rod-shaped, non-motile and non-sporulating. The peptidoglycan type of the isolate was A4 alpha; lysine was the diagnostic diamino acid. The only menaquinone detected was MK-8(H(4)), and the major fatty acids were anteiso-C(15:0) and C(16:0). Galactose was detected as a major cell-wall sugar. The polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G + C content was 70.7 mol%. 16S rRNA gene sequence analysis revealed that strain Kis4-28(T) and the type strain of Salana multivorans formed a monophyletic cluster with a 16S rRNA gene sequence similarity of 96.2 %. Strain Kis4-28(T) was clearly distinguishable from the genus Salana in terms of its chemotaxonomic characteristics. On the basis of the genotypic and phenotypic characteristics, a new genus and species is proposed for strain Kis4-28(T), with the name Serinibacter salmoneus gen. nov., sp. nov. The type strain of Serinibacter salmoneus is Kis4-28(T) (=NBRC 104924(T) = DSM 21801(T)). In addition, on the basis of 16S rRNA gene sequence analysis of the genus Serinibacter and related genera, emended descriptions of the families Beutenbergiaceae and Bogoriellaceae are proposed to accommodate the genera Beutenbergia, Salana and Serinibacter, and the genera Bogoriella and Georgenia, respectively.

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  • Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment. International journal

    Maki Teramoto, Masahito Suzuki, Fumiyoshi Okazaki, Ariani Hatmanti, Shigeaki Harayama

    Microbiology (Reading, England)   155 ( Pt 10 )   3362 - 3370   2009.10

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    Petroleum-hydrocarbon-degrading bacteria were obtained after enrichment on crude oil (as a 'chocolate mousse') in a continuous supply of Indonesian seawater amended with nitrogen, phosphorus and iron nutrients. They were related to Alcanivorax and Marinobacter strains, which are ubiquitous petroleum-hydrocarbon-degrading bacteria in marine environments, and to Oceanobacter kriegii (96.4-96.5% similarities in almost full-length 16S rRNA gene sequences). The Oceanobacter-related bacteria showed high n-alkane-degrading activity, comparable to that of Alcanivorax borkumensis strain SK2. On the other hand, Alcanivorax strains exhibited high activity for branched-alkane degradation and thus could be key bacteria for branched-alkane biodegradation in tropical seas. Oceanobacter-related bacteria became most dominant in microcosms that simulated a crude oil spill event with Indonesian seawater. The dominance was observed in microcosms that were unamended or amended with fertilizer, suggesting that the Oceanobacter-related strains could become dominant in the natural tropical marine environment after an accidental oil spill, and would continue to dominate in the environment after biostimulation. These results suggest that Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in the tropical sea.

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  • Lecanicillium and Verticillium species from Indonesia and Japan including three new species

    Nampiah Sukarno, Yuko Kurihara, Muhammad Ilyas, Wibowo Mangunwardoyo, Erny Yuniarti, Wellyzar Sjamsuridzal, Ju-Young Park, Rasti Saraswati, Shigeki Inaba, Yantyati Widyastuti, Katsuhiko Ando, Shigeaki Harayama

    MYCOSCIENCE   50 ( 5 )   369 - 379   2009.9

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    Forty-six Lecanicillium strains and one Verticillium strain were isolated from subterranean and epiphytic arthropods, soil, and other sources collected in Indonesia and Japan. These strains were identified as nine Lecanicillium and one Verticillium species including six undescribed species based on light microscopy and the sequences of the ITS-1 and ITS-2 regions including 5.8S ribosomal DNA. Four of the ten species (L. araneicola, L. kalimantanense, Lecanicillium sp. 4, and V. indonesiacum) were recovered from Indonesia, five of the ten (L. attenuatum, L. fusisporum, L. psalliotae, Lecanicillium sp. 1, and Lecanicillium sp. 3) were from Japan, and L. saksenae was from both countries. In this article, new species (L. araneicola, L. kalimantanense, and V. indonesiacum) and a new combination (L. saksenae) are proposed from the fungi isolated from epiphytic and subterranean arthropods collected in East Kalimantan.

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  • Phycisphaera mikurensis gen. nov., sp. nov., isolated from a marine alga, and proposal of Phycisphaeraceae fam. nov., Phycisphaerales ord. nov. and Phycisphaerae classis nov. in the phylum Planctomycetes.

    Yukiyo Fukunaga, Midori Kurahashi, Yayoi Sakiyama, Motoyuki Ohuchi, Akira Yokota, Shigeaki Harayama

    The Journal of general and applied microbiology   55 ( 4 )   267 - 75   2009.8

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    Three strains, FYK2301M01(T), FYK2301M18 and FYK2301M52, all being Gram-negative, spherical, motile and facultatively anaerobic, were isolated from a marine alga (Porphyra sp.) collected on Mikura Island, Japan. Colonies of the strains were circular and pink-pigmented on Marine Agar 2216 (Difco) at 25 degrees C. Cells of the strains reproduced by binary fission. The G+C content of the DNA was 73 mol%. The major isoprenoid quinone was MK-6. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains are the members of the WPS-1 group (Nogales et al., 2001) comprising no validly described taxa within the phylum Planctomycetes. The highest similarity value of the 16S rRNA gene sequences of the strains to those in the established bacterial taxa was only 78.7% to Planctomyces brasiliensis DSM 5305(T). From the taxonomic data obtained in this study, it is proposed that the new marine isolates be placed in a novel genus and species named Phycisphaera mikurensis gen. nov., sp. nov. within a new family, order and class Phycisphaeraceae fam. nov., Phycisphaerales ord. nov. and Phycisphaerae classis nov. in the phylum Planctomycetes. The type strain of Phycisphaera mikurensis is FYK2301M01(T) (= NBRC 102666(T) = KCTC 22515(T)).

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  • Paraoerskovia marina gen. nov., sp. nov., an actinobacterium isolated from marine sediment. International journal

    Shams Tabrez Khan, Shigeaki Harayama, Tomohiko Tamura, Katsuhiko Ando, Motoki Takagi, Shin-ya Kazuo

    International journal of systematic and evolutionary microbiology   59 ( Pt 8 )   2094 - 8   2009.8

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    A Gram-positive-staining, facultatively anaerobic bacterial strain, CTT-37(T), was isolated from a marine sediment sample collected from Tottori city, located on the shore of the Sea of Japan. A 16S rRNA gene sequence comparison indicated that the isolate represents a novel clade that clusters with members of the families Cellulomonadaceae and Sanguibacteraceae. Strain CTT-37(T) shared maximum 16S rRNA gene sequence similarity of 96.4% with Oerskovia paurometabola DSM 14281(T) and 96.2% with Oerskovia enterophila DSM 43852(T). The DNA-DNA hybridization value between strain CTT-37(T) and O. enterophila JCM 7350(T) was 10-12%. The following chemotaxonomic characteristics of strain CTT-37(T) were markedly different from those of strains in the genus Oerskovia. The cell wall contained L-serine in the peptidoglycan interpeptide bridge. The predominant menaquinone was MK-9 (H-4); other quinones detected were MK-9 and MK-9(H-2). The only polar lipid was phosphatidylglycerol and the G + C content of the DNA was 70 mol%. Differences in phenotypic characteristics and large phylogenetic distances between strain CTT-37(T) and all members of the genus Oerskovia supported the classification of CTT-37(T) within a new genus and species, for which the name Paraoerskovia marina gen. nov., sp. nov. is proposed. The type strain of Paraoerskovia marina is CTT-37(T) (=NBRC 104352(T)=DSM 21750(T)).

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  • Physicochemical characters of a tyrosinase inhibitor produced by Streptomyces roseolilacinus NBRC 12815.

    Takuji Nakashima, Kozue Anzai, Natsumi Kuwahara, Hisayuki Komaki, Shinji Miyadoh, Shigeaki Harayama, Ma Diarey Bordon Tianero, Junichi Tanaka, Akihiko Kanamoto, Katsuhiko Ando

    Biological & pharmaceutical bulletin   32 ( 5 )   832 - 6   2009.5

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    We examined the biological activities present in Streptomyces strains preserved at the National Institute of Technology and Evaluation Biological Resource Center, and found a metabolite of Streptomyces roseolilacinus NBRC 12815 that showed a potent anti-tyrosinase activity. The compounds with anti-tyrosinase activity were purified by several chromatographic procedures. Final HPLC analysis revealed at least two anti-tyrosinase compounds with different retention times (12815A and B). The identification of two anti-tyrosinase compounds was performed with instrumental analysis and database search. The results obtained suggest that the active compounds are SF 2583A and B. Compound 12815A (IC50 values; about 9 mu M) showed more potent tyrosinase inhibition than compound 12815B (IC50 values; about 1086 mu M). The only structural difference between 12815A and B is the presence of an additional chloric atom. In addition, the activity of 12815A was markedly decreased under acidic conditions, resulting in irreversible inactivation. However, the inactivated 12815A still exhibited residual activity when exposed to detergent, Tween 80. These results suggest that the chlorine and the hydration water are very important in the exertion of anti-tyrosinase activity by 12815A.

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  • Iamia majanohamensis gen. nov., sp. nov., an actinobacterium isolated from sea cucumber Holothuria edulis, and proposal of Iamiaceae fam. nov. International journal

    Midori Kurahashi, Yukiyo Fukunaga, Yayoi Sakiyama, Shigeaki Harayama, Akira Yokota

    International journal of systematic and evolutionary microbiology   59 ( Pt 4 )   869 - 73   2009.4

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    A novel, Gram-positive bacterial strain, F12(T), was isolated from the abdominal epidermis of a sea cucumber, Holothuria edulis, collected from seawater off the coast of Japan. According to 16S rRNA gene sequence analysis, this strain represents a novel, deep-rooting lineage within the class Actinobacteria and clusters with uncultured bacteria and Acidimicrobium ferrooxidans. Compared to species with validly published names, the highest 16S rRNA gene sequence similarity (89.8%) was to Acidimicrobium ferrooxidans DSM 10331(T). Phylogenetic analyses showed that strain F12(T) represents a distinct phylogenetic lineage related closely to the genus Acidimicrobium. Strain F12(T) contained MK-9(H-6) as the major menaquinone, whilst 17 : 0, 17 : 1 omega 8c, 15 : 0 and 16 : 0 were the major cellular fatty acids. The cell-wall peptidoglycan of strain F12(T) was composed of meso-diaminopimelic acid as the diagnostic diamino acid, alanine and glutamic acid (1 : 2 : 1). The cell-wall sugars detected were rhamnose, mannose, arabinose, galactose and xylose. The G + C content of the DNA was 74.4 mol%. From the taxonomic data obtained in this study, the name lamia majanohamensis gen. nov., sp. nov. is proposed for the isolate, with type strain F12T (=NBRC 102561(T)=DSM 19957(T)). The name lamiaceae fam. nov. is also proposed for the distinct phyletic line represented by the genus lamia.

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  • Lacticigenium naphtae gen. nov., sp. nov., a halotolerant and motile lactic acid bacterium isolated from crude oil. International journal

    Takao Iino, Ken-Ichiro Suzuki, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   59 ( Pt 4 )   775 - 80   2009.4

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    A novel lactic acid bacterium, strain MIC1-18(T), was isolated from crude oil collected at an oil-water well in Akita, Japan. Cells of strain MIC1-18(T) were found to be facultatively anaerobic, mesophilic, neutrophilic, Gram-negative, non-sporulating, motile by means of peritrichous flagella and oval rods, 1.8-2.5 mu m long. Optimum growth was observed at 30 degrees C, pH 7.0 and 3% (w/v) NaCl. Strain MIC1-18(T) produced acid from L-arabinose, ribose, glucose, fructose, mannose, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, trehalose, gentiobiose and 5-ketogluconate. L-Lactic acid was the major end product from glucose. The major cellular fatty acid was C-16:1 omega 7c. The cell-wall murein type was A4 alpha containing Lys-Glu. The G + C content of the genomic DNA was 37.8 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that strain MIC1-18(T) was accommodated as a member of the lactic acid bacteria of the low-G + C content Gram-positive bacteria; the closest neighbour of this organism was Atopococcus tabaci CCUG 48253(T), with only 90.0% sequence similarity. On the basis of the phenotypic features and phylogenetic position, a novel genus and species, Lacticigenium, naphtae gen. nov., sp. nov., are proposed for strain MIC1-18(T) (=NBRC 101988(T)=DSM 19658(T)).

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  • Molecular diversity of bacterial chitinases in arable soils and the effects of environmental factors on the chitinolytic bacterial community

    Takeshi Terahara, Seishi Ikeda, Chiaki Noritake, Kiwamu Minamisawa, Katsuhiko Ando, Satoshi Tsuneda, Shigeaki Harayama

    SOIL BIOLOGY & BIOCHEMISTRY   41 ( 3 )   473 - 480   2009.3

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    The molecular diversity of bacterial chitinases in the bulk soils of arable land was investigated using culture-independent methods. The results demonstrate that bacterial chitinases in arable soils are highly diverse and comprise unique groups when their sequences were compared to those in public databases. The diversity of bacterial chitinases in arable soil was further evaluated using conventional phylogenetic analysis, the UniFrac analysis of the phylogenetic data, and the multidimensional scaling (MDS) analysis of T-RFLP profiles to elucidate the relationship between the diversity of bacterial chitinases and soil characteristics. These analyses indicate that environmental factors such as soil type and pH are responsible for shaping the composition of bacterial chitinases. (C) 2008 Elsevier Ltd. All rights reserved.

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  • Hirsutella proturicola sp nov isolated from a proturan, Baculentulus densus (Protura, Hexapoda)

    Yuko Kurihara, Takashi Shirouzu, Seiji Tokumasu, Shigeaki Harayama

    MYCOSCIENCE   50 ( 1 )   56 - 62   2009.1

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    A new species of Hirsutella, H. proturicola, isolated from a subterranean proturan (Baculentulus densus; Protura, Hexapoda), is described and illustrated. Hirsutella proturicola is characterized by producing monoblastic phialides of 24-51.5 x 2.5-5 mu m with a slightly roughened neck, fusiform and curved conidia of 9-18 x 2.5-4 mu m that have a truncate base and a papillate projection often capped with sheath-like mucilage, and pluricellular, globose to subglobose chlamydospores of 21-48 x 21-41.5 mu m. This species is morphologically and phylogenetically close to H. rostrata, an acaropathogenic species, but can be distinguished from the size of the phialides and the size and shape of the conidia.

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  • Arsenicicoccus piscis sp. nov., a mesophilic actinobacterium isolated from the intestinal tract of a fish

    Hamada, Moriyuki, Iino, Takao, Iwami, Takahiro, Tamura, Tomohiko, Harayama, Shigeaki, Suzuki, Ken-ichiro

    Actinomycetologica   23 ( 2 )   40 - 45   2009

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  • Acanthopleuribacter pedis gen. nov., sp. nov., a marine bacterium isolated from a chiton, and description of Acanthopleuribacteraceae fam. nov., Acanthopleuribacterales ord. nov., Holophagaceae fam. nov., Holophagales ord. nov. and Holophagae classis nov. in the phylum 'Acidobacteria'. International journal

    Yukiyo Fukunaga, Midori Kurahashi, Kensuke Yanagi, Akira Yokota, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   58 ( Pt 11 )   2597 - 601   2008.11

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    Strain FYK2218(T) was isolated from a specimen of the chiton Acanthopleura japonica, which had been collected from a beach on the Boso peninsula in Japan. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strain belonged to the phylum 'Acidobacteria'. The most closely related type strains to strain FYK2218(T) were Holophaga foetida TMBS4(T) (83.6% 16S rRNA gene sequence similarity) and Geothrix fermentans H-5(T) (83.6%) in subdivision 8 of the 'Acidobacteria'. Cells of FYK2218(T) were motile, rod-shaped, Gram-negative, mesophilic and strictly aerobic. The G+C content of the strain was 56.7 mol%. The strain had isoprenoid quinones MK-6 and MK-7 as major components. Major fatty acids of the strain were iso-C(15:0), iso-C(17:0), C(16:0) and C(20:5)omega 93c (cis-5,8,11,14,17-eicosapentaenoic acid). From the taxonomic data obtained in this study, it is proposed that the new marine isolate be placed into a novel genus and species named Acanthopleuribacter pedis gen. nov., sp. nov. within the new family, order and class Acanthopleuribacteraceae fam. nov., Acanthopleuribacterales ord. nov. and Holophagae classis nov. The family Holophagaceae fam. nov. is also described. The type strain of Acanthopleuribacter pedis is FYK2218(T) (=NBRC 101209(T) =KCTC 12899(T)).

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  • PCR detection of type I polyketide synthase genes in myxobacteria. International journal

    Hisayuki Komaki, Ryosuke Fudou, Takashi Iizuka, Daisuke Nakajima, Koei Okazaki, Daisuke Shibata, Makoto Ojika, Shigeaki Harayama

    Applied and environmental microbiology   74 ( 17 )   5571 - 4   2008.9

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    The diversity of type I modular polyketide synthase (PKS) was explored by PCR amplification of DNA encoding ketosynthase and acyltransferase domains in myxobacteria. The sequencing of the amplicons revealed that many PKS genes were distantly related to the published sequences. Thus, myxobacteria may be excellent resources for novel and diverse polyketides.

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  • Taxonomic distribution of Streptomyces species capable of producing bioactive compounds among strains preserved at NITE/NBRC. International journal

    Kozue Anzai, Michiyo Ohno, Takuji Nakashima, Natsumi Kuwahara, Rieko Suzuki, Tomohiko Tamura, Hisayuki Komaki, Shinji Miyadoh, Shigeaki Harayama, Katsuhiko Ando

    Applied microbiology and biotechnology   80 ( 2 )   287 - 95   2008.8

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    The taxonomic distribution of Streptomyces species capable of producing bioactive compounds was investigated. Nine hundred and six strains were tested for the following four biological activities: antimicrobial, anti-tyrosinase, antioxidant, and hemolytic. Approximately 30% of strains tested showed antimicrobial activities, except for anti-Escherichia coli activity, which was present in only a few strains, while the rates of positivity for the anti-tyrosinase, antioxidant, and hemolytic activities were much lower. The distribution of Streptomyces strains capable of producing bioactive compounds was analyzed by the taxonomy based on 16S rRNA gene sequences. Moreover, the strains of Streptomyces hygroscopicus tested were divided into two clades in the phylogenetic tree, and all of the strains belonging to one clade showed antibacterial and antifungal activities. For detection of polyenes, the UV-visible spectra of metabolic extracts in the strains showing antifungal activities were measured. It was suggested that Streptomyces strains produce universal active compounds under different growth conditions. Further information on the relationship between the microbial taxonomy and the bioactive compounds produced would be useful for the utilization of industrial microorganisms.

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  • Fulvibacter tottoriensis gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from marine sediment. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   58 ( Pt 7 )   1670 - 4   2008.7

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    A novel bacterium, MTT-39(T), was isolated from a sample of marine sediment collected at Tottori on the coast of the Sea of Japan. Cells were Gram-negative, rod-shaped and non-motile. The bacterium formed yellowish brown colonies on marine agar 2216. Although the 16S rRNA gene sequence of strain MTT-39(T) classified this strain as a member of the family Flavobacteriaceae, the maximum sequence similarity obtained was only 91.5% (with Kordia algicida OT-1(T)). In the maximum-likelihood tree based on 1 6S rRNA gene sequences, the novel bacterium clustered with the type strains of Kordia algicida, Lutibacter litoralis, Tenacibaculum maritimum and Polaribacter filamentus. The novel strain exhibited the following characteristics: the predominant fatty acids in cells grown on artificial seawater-based tryptic; soya agar were iso-C(15:1), iso-C(15:0) and iso-C(15:0) 3-OH, the major respiratory quinone was MK-6 and the DNA G + C content was 35 mol%. On the basis of its distinct phenotypic traits and the phylogenetic distance between this marine isolate and other recognized taxa, strain MTT-39(T) represents a novel genus and species of the family Flavobacteriaceae, for which the name fulvibacter tottoriensis gen. nov., sp. nov. is proposed. The type strain of the type species is MTT-39T (=NBRC 102624(T) =KCTC 22214(T) = CGMCC 1.7058(T)).

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  • Calditerrivibrio nitroreducens gen. nov., sp. nov., a thermophilic, nitrate-reducing bacterium isolated from a terrestrial hot spring in Japan. International journal

    Takao Iino, Tatsunori Nakagawa, Koji Mori, Shigeaki Harayama, Ken-Ichiro Suzuki

    International journal of systematic and evolutionary microbiology   58 ( Pt 7 )   1675 - 9   2008.7

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    A moderately thermophilic, nitrate-reducing bacterium, strain Yu37-1(T), was isolated from hot spring water from Yumata, Nagano, Japan. Cells of strain Yu37-1(T) were strictly anaerobic, Gram-negative, non-sporulating, motile by means of a single polar flagellum, vibrio-shaped and 1.4-2.0 microm long. The temperature and pH for optimum growth were 55 degrees C and pH 7.0-7.5, respectively. Strain Yu37-1(T) grew best in basal medium without the addition of NaCl. Acetate, pyruvate, lactate, fumarate, succinate, malate, yeast extract, peptone and Casamino acids were utilized as electron donors, with nitrate as the only electron acceptor. Ammonium was the end product from nitrate. The G+C content of the genomic DNA was 35.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that strain Yu37-1(T) could be accommodated in the family Deferribacteraceae and that its closest neighbours were members of the five genera of the family Deferribacteraceae, namely Deferribacter, Denitrovibrio, Flexistipes, Geovibrio and Mucispirillum, with similarities of only 83.2-86.2 %. The growth temperature and salinity range for growth of strain Yu37-1(T) differed from those of the phylogenetically related organisms. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed, Calditerrivibrio nitroreducens gen. nov., sp. nov. Strain Yu37-1(T) (=NBRC 101217(T) =DSM 19672(T)) is the type strain of Calditerrivibrio nitroreducens.

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  • Two groups of thermophilic amino acid aminotransferases exhibiting broad substrate specificities for the synthesis of phenylglycine derivatives. International journal

    Daisuke Koma, Toshiya Sawai, Ryotaro Hara, Shigeaki Harayama, Kuniki Kino

    Applied microbiology and biotechnology   79 ( 5 )   775 - 84   2008.7

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    Thirty two thermophilic amino acid aminotransferases (AATs) were expressed in Escherichia coli as soluble and active proteins. Based on their primary structures, the 32 AATs were divided into four phylogenetic groups (classes I, II, IV, and V). The substrate specificities of these AATs were examined, and 12 AATs were found capable of synthesizing ring-substituted phenylglycine derivatives such as hydroxyl-, methoxy-, and fluorophenylglycines. Eleven out of the 12 AATs were enzymes belonging to two phylogenetic groups namely, one subgroup of the class I family and the class IV family. AATs in these two groups may thus be useful for the synthesis of a variety of ring-substituted phenylglycine derivatives.

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  • Complete genome sequence of the soil actinomycete Kocuria rhizophila. International journal

    Hiromi Takarada, Mitsuo Sekine, Hiroki Kosugi, Yasunori Matsuo, Takatomo Fujisawa, Seiha Omata, Emi Kishi, Ai Shimizu, Naofumi Tsukatani, Satoshi Tanikawa, Nobuyuki Fujita, Shigeaki Harayama

    Journal of bacteriology   190 ( 12 )   4139 - 46   2008.6

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    The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae, a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transfer, reflecting the small genome size. The presence of probable metabolic pathways for the transformation of phenolic compounds generated from the decomposition of plant materials, and the presence of a large number of genes associated with membrane transport, particularly amino acid transporters and drug efflux pumps, may contribute to the organism's utilization of root exudates, as well as the tolerance to various organic compounds.

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  • Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids. International journal

    Kazutaka Yamada, Takeshi Terahara, Shinya Kurata, Toyokazu Yokomaku, Satoshi Tsuneda, Shigeaki Harayama

    Environmental microbiology   10 ( 4 )   978 - 87   2008.4

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    We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

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  • Enhanced Diastereoselective Synthesis of L-threo-3,4-dihydroxyphenylserine by Low-specific L-threonine aldolase mutants

    Sang-Ho Baik, Hui-Jeong Gwon, Sang-Yong Lim, Dong-Ho Kim, Shigeaki Harayama

    FASEB JOURNAL   22   2008.4

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  • Sneathiella glossodoripedis sp. nov., a marine alphaproteobacterium isolated from the nudibranch Glossodoris cincta, and proposal of Sneathiellales ord. nov. and Sneathiellaceae fam. nov. International journal

    Midori Kurahashi, Yukiyo Fukunaga, Shigeaki Harayama, Akira Yokota

    International journal of systematic and evolutionary microbiology   58 ( Pt 3 )   548 - 52   2008.3

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    A novel marine bacterium, designated strain MKT133(T), was isolated from the foot epidermis of a nudibranch, Glossodoris cincta (Mollusca), collected in seawater off the coast of Japan at a depth of 4 m. This bacterium was Gram-negative, motile, mesophilic and strictly aerobic, with small rod-shaped cells. Colonies of the strain after 4-5 days incubation on marine agar 2216 at 30 degrees C were less than 1 mm in diameter. The strain required salt for growth and contained Q-10 as the predominant respiratory quinone, C(18 : 1) omega 7c, C(16 : 0) and C(17 : 1) as major cellular fatty acids and C(14 : 0) 3-OH as a hydroxy fatty acid. 16S rRNA gene sequence analysis showed that the isolate had highest similarity to Sneathiella chinensis, with 97.2 % sequence similarity to the type strain. Our phylogenetic analysis also revealed that this clade represents a distinct lineage and forms a deep branch with less than 90 % 16S rRNA gene sequence similarity to the members of the eight known orders within the Alphaproteobacteria. Sufficient differences exist to distinguish this strain from Sneathiella chinensis. The name Sneathiella glossodoripedis sp. nov. is proposed, with the type strain MKT133(T) (=IAM 15419(T) =KCTC 12842(T)). The novel order Sneathiellales ord. nov. and family Sneathiellaceae fam. nov. are proposed for the distinct phyletic line represented by the genus Sneathiella.

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  • Paracoccus marinus sp. nov., an adonixanthin diglucoside-producing bacterium isolated from coastal seawater in Tokyo Bay. International journal

    Shams Tabrez Khan, Shinichi Takaichi, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   58 ( Pt 2 )   383 - 6   2008.2

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    Two novel marine, Gram-negative, non-motile, catalase- and oxidase-positive, aerobic bacteria were isolated from coastal seawater in Tokyo Bay. Analysis of almost-complete 16S rRNA gene sequences showed that the two isolates are members of the genus Paracoccus, sharing highest 16S rRNA gene sequence similarity (96.5 %) with Paracoccus aminophilus NBRC 16710(T). The DNA-DNA reassociation values between P. aminophilus NBRC 16710(T) and these isolates were only 10-20 %, in contrast to the high DNA relatedness between the two isolates (89 %). At least 1 % (w/v) NaCl was required for growth. Cellular fatty acid profiles revealed C(18 : 1)omega7c as the major component and C(10 : 0) 3-OH as the major hydroxy fatty acid. Ubiquinone-10 was detected as the major respiratory quinone. The G+C content of the genomic DNA of both strains was 69 mol%. On the basis of DNA-DNA hybridization data and physiological and chemotaxonomic characteristics, it is proposed that these strains should be placed in a novel species, Paracoccus marinus sp. nov. The type strain is KKL-A5(T) (=NBRC 100637(T) =CIP 108500(T)); KKL-B9 (=NBRC 100640) is a reference strain.

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  • Hydrocarbonoclastic bacteria from Jakarta bay and Seribu islands

    Darmayati, Yeti, Harayama, Shigeaki, Yamazoe, Atsushi, Hatmanti, Ariani, Nuchsin, Ruyitno, Kunarso, Djoko Hadi, others

    Marine Research in Indonesia   33 ( 1 )   55 - 64   2008

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  • Emended descriptions of the genus Lewinella and of Lewinella cohaerens, Lewinella nigricans and Lewinella persica, and description of Lewinella lutea sp. nov. and Lewinella marina sp. nov. International journal

    Shams Tabrez Khan, Yukiyo Fukunaga, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 12 )   2946 - 2951   2007.12

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    Two strains, MKG-38(T) and FYK2402M69(T), were isolated from a marine sediment sample and a sea snail, respectively, both collected on the Pacific coast of Japan. Phylogeny of these new isolates based on 16S rRNA gene sequences indicated that they are members of the genus Lewinella. Morphological, physiological and biochemical properties of these two isolates, together with the type strains of the three previously described species of the genus Lewinella, were characterized. The new isolates were Gram-negative, aerobic, rod-shaped, chemo-organotrophic and able to degrade starch and CM-cellulose. A comparative polyphasic study showed that these two isolates represent two novel species of the genus Lewinella, for which the names Lewinella marina sp. nov. (type strain, MKG-38(T) =NBRC 102633(T) =NCIMB 14312(T)) and Lewinella lutea sp. nov. (type strain, FYK2402M69(T) =NBIRC 102634(T) =NCIMB 14313(T)) are proposed. Emended descriptions of the genus Lewinella (Sly et al. 1998) and of Lewinella cohaerens, Lewinella nigricans and Lewinella persica are also proposed.

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  • A high-throughput and generic assay method for the determination of substrate specificities of thermophilic alpha-aminotransferases. International journal

    Toshiya Sawai, Daisuke Koma, Ryotaro Hara, Kuniki Kino, Shigeaki Harayama

    Journal of microbiological methods   71 ( 1 )   32 - 8   2007.10

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    For the determination of substrate specificities of thermophilic alpha-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic omega-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP+ to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading. (C) 2007 Published by Elsevier B.V.

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  • Oscillibacter valericigenes gen. nov., sp. nov., a valerate-producing anaerobic bacterium isolated from the alimentary canal of a Japanese corbicula clam. International journal

    Takao Iino, Koji Mori, Kenji Tanaka, Ken-Ichiro Suzuki, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 8 )   1840 - 1845   2007.8

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    A mesophilic, strictly anaerobic bacterium, strain Sjm18-20(T), was isolated from the alimentary canal of a Japanese corbicula clam. Cells of strain Sjm18-20(T) were Gram-negative, nonsporulating, straight to slightly curved rods, 2.5-6.0 mu m long, and were motile with oscillatory movements by means of peritrichous flagella. Cells elongated to 30 pm after prolonged cultivation. Optimum growth was observed at 30 degrees C and pH 6.0-6.5. Growth occurred below 4.0 % (w/v) NaCl. Strain Sjm18-20(T) produced acid from D-glucose and a few pentoses such as L-arabinose, D-ribose and D-xylose. n-Valeric acid was the major end product from glucose. The genomic DNA G+C content of strain Sjm18-20(T) was 52.9 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that strain Sjm18-20(T) could be accommodated in clostriclial cluster IV of the low-G+C-content Gram-positive bacteria and that the closest neighbour of this organism (92.6-92.9 % similarity) was the cloned 16S rRNA gene sequence of a not-yet cultured bacterium, thought to represent Oscillospira guilliermondii. The nearest cultivated neighbours of strain Sjm18-20(T) were Clostridium orbiscindens DSM6740(T) and Clostridium viride T2-7(T), with sequence similarities of 91.3 and 89.1 %, respectively. On the basis of phenotypic features and phylogenetic position, it is proposed that this isolate represents a novel species in a new genus, Oscillibacter valericigenes gen. nov., sp. nov. The type strain of Oscillibacter valericigenes is Sjm18-20(T) (=NBRC 101213(T) =DSM18026(T)).

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  • Sediminitomix flava gen. nov., sp. nov., of the phylum Bacteroidetes, isolated from marine sediment. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 8 )   1689 - 1693   2007.8

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    A novel bacterium designated Mok-1-85(T) was isolated from a marine sediment sample collected from Okinawa Island, Japan. Cells of strain Mok-11-85(T) stained Gram-negative, were catalase- and oxidase-positive and were non-motile. In a neighbour-joining tree based on 16S rRNA gene sequences, the novel strain clustered with the genus Flammeovirga, a member of the family 'Flammeovirgaceae'. The novel isolate shared low 16S rRNA gene sequence similarities (<= 86%) with the members of the genus Flammeovirga and other related taxa. The major isoprenoid quinone was MK-7 and the predominant fatty acids of this organism were iso-C-15:0, C-16:1 omega 5c and C-16:0 3-OH. The G + C content of the DNA was 38 mol%. Combined phylogenetic and physiological data showed that strain Mok-1-85(T) represents a novel genus and species for which the name Sediminitomix flava gen. nov., sp. nov. is proposed. The type strain is Mok-1-85(T) (=NBRC 101625(T)=KCTC 12970(T)=CIP 109411(T)).

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  • A novel nuclear export inhibitor JBIR-02, a new piericidin discovered from Streptomyces sp. ML55. International journal

    Jun-Ya Ueda, Takushi Togashi, Susumu Matukura, Aya Nagai, Takuji Nakashima, Hisayuki Komaki, Kozue Anzai, Shigeaki Harayama, Takayuki Doi, Takashi Takahashi, Tohru Natsume, Yasutomo Kisu, Naoki Goshima, Nobuo Nomura, Motoki Takagi, Kazuo Shin-Ya

    The Journal of antibiotics   60 ( 7 )   459 - 62   2007.7

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    A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 mu M.

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  • Paraferrimonas sedimenticola gen. nov., sp. nov., a marine bacterium of the family Ferrimonadaceae. International journal

    Shams Tabrez Khan, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 7 )   1493 - 1498   2007.7

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    Three strains (Mok-106(T), Mok-142 and Mok-143) were isolated from marine sediment samples collected from the coast of Okinawa Island, Japan. On the basis of 16S rRNA gene sequence comparisons, the isolates were affiliated with the family Ferrimonadaceae; Shewanella denitrificans and Ferrimonas balearica were the closest relatives, having sequence similarities of 93.7 and 93.0 %, respectively. The novel isolates shared high levels of 16S rRNA gene sequence similarity with each other (98.7-99.3 %) and the results of DNA-DNA hybridization indicated that the three strains belong to the same species. The cells were rod-shaped, motile by means of single polar flagellum and formed colonies that produced a rose-coloured pigment within 6 days incubation at 25 degrees C. The isolates grew in the presence of 0.5-4.0 % (w/v) NaCl and at 15-40 degrees C. The major fatty acids were iso-13: 0, iso-15:0, 16:0, 18: 1 omega 7c and summed feature 3 (116: 1 omega 7c and/or iso-15: 0 2-OH). Menaquinone-6, menaquinone-7 and ubiquinone-8 were the major quinones and the major polar lipids were phosphaticlylethanolamine and phosphatidylglycerol. The DNA G + C content was 50-51 mol%. Phylogenetic and phenotypic analyses of these isolates suggested that they belong to a novel genus and species of the family Ferrimonadaceae, for which the name Paraferrimonas sedimenticola gen. nov., sp. nov. is proposed. The type strain is Mok-106(T) (= NBRC 101628(T) =CIP 109284(T)).

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  • Nocardia amamiensis sp. nov., isolated from a sugar-cane field in Japan. International journal

    Hideki Yamamura, Tomohiko Tamura, Yayoi Sakiyama, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 7 )   1599 - 1602   2007.7

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    An actinomycete, strain TT 00-78(T), was isolated from soil from a sugar-cane field on Amami Island in Japan, using an SDS/yeast extract pre-treatment method, and the taxonomy was studied using a polyphasic approach. The chemotaxonomic and morphological characterizations clearly demonstrated that the strain belongs to the genus Nocardia. 16S rRNA gene sequencing studies showed that the strain was closely related to the type strains of Nocardia pneumoniae (98.6%), Nocardia araoensis (98.1%), Nocardia arthritidis (97.9%) and Nocardia beijingensis (97.7%). However, the results of DNA-DNA hybridization and physiological and biochemical tests showed that strain TT 00-78(T) could be differentiated from its closest phylogenetic relatives both genotypically and phenotypically. Therefore this strain represents a novel species of the genus Nocardia, for which the name Nocardia arnarniensis sp. nov. is proposed. The type strain is TT 00-78(T) (=NBRC 102102(T) = DSM 45066(T) =KCTC 19208(T)).

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  • Method for selective isolation of Mycobacteria using olive oil emulsified with SDS. International journal

    Hideki Yamamura, Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   71 ( 6 )   1553 - 6   2007.6

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    This paper describes the demulsification of olive-oil/ SDS emulsion (DOSE) method for selective isolation of environmental mycobacteria. A soil sample was suspended in olive oil and centrifuged. The supernatant was emulsified on plates together with SDS solution. After incubation, the colonies that had developed on the plates were surrounded by clear zones. The isolates were identified as genus Mycobacterium, and as belonging to a fast-growing group, by 16S rRNA gene sequence analysis.

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  • Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4. International journal

    Makoto Urai, Hiroshi Yoshizaki, Hirosi Anzai, Jun Ogihara, Noriyuki Iwabuchi, Shigeaki Harayama, Michio Sunairi, Mutsuyasu Nakajima

    Carbohydrate research   342 ( 7 )   927 - 32   2007.5

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    Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C-19 branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using H-1 and C-13 NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure:Mucoidan beta-D-Glcp 1 down arrow 3 [4)-beta-D-Glcp-(1 -> 3)-beta-D-GlcpNAc-(1 -> 4)-alpha-D-GlcpA-(1 -> 3)-alpha-L-Fucp-(1 ->](n)(c) 2007 Elsevier Ltd. All rights reserved.

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  • Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4. International journal

    Makoto Urai, Hiroshi Yoshizaki, Hirosi Anzai, Jun Ogihara, Noriyuki Iwabuchi, Shigeaki Harayama, Michio Sunairi, Mutsuyasu Nakajima

    Carbohydrate research   342 ( 7 )   933 - 42   2007.5

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    Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C-19 branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1: 1: 1: 1: 1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D H-1 and C-13 NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure:PR4 FACEPS[GRAPHICS][4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp-(1 -> 3)-beta-D-GlcpA-(1 ->](n)(c) 2007 Elsevier Ltd. All rights reserved.

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  • Synthesis of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) with thermostabilized low-specific L-threonine aldolase from Streptomyces coelicolor A3(2). International journal

    Sang-Ho Balk, Hideki Yoshioka, Hideaki Yukawa, Shigeaki Harayama

    Journal of microbiology and biotechnology   17 ( 5 )   721 - 7   2007.5

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    Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, Of L-threonine aldolase (L-TA) from Streptomyces coelicolor A3(2) (SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A284 mutant and F181 in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at 60 degrees T was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at 6 degrees C was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml Of L-threo-DOPS. This result showed that H44 L- TA is more effective than the wild-type L-TA for the mass production Of L-threo-DOPS.

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  • Galbibacter mesophilus gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 5 )   969 - 973   2007.5

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    A Gram-negative, yellow-pigmented, rod-shaped bacterial strain (Mok-17(T)) was isolated from marine sediment sampled in Okinawa Island, Japan. Based on analysis of the almost complete sequence of its 16S rRNA gene, strain Mok-17(T) was found to belong to the family Flavobacteriaceae. Strain Mok-17(T) showed highest 16S rRNA gene sequence similarity (91%) to Leeuwenhoekiella marinoflava and Robiginitalea biformata. In a phylogenetic tree based on the 16S rRNA gene, strain Mok-17(T) formed a deep branch distinct from all other organisms in the family Flavobacteriaceae. The major quinone was MK-6 and the major fatty acids were iso-15:0, iso-15:1, iso-17:0 3-OH and summed feature 3 (16:1 omega 7c and/or iso-15:0 2-OH). The DNA G + C content was 37 mol%. The phylogenetic distance to the type strains of all recognized species in the family Flavobacteriaceae and the phenotypic properties of strain Mok-17(T) supported its classification as representing a novel species in a new genus, for which the name Galbibacter mesophilus gen. nov., sp. nov. is proposed. The type strain is Mok-17(T) (=NBRC 101624(T)=CIP 109219(T)).

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  • New aureothin derivative, alloaureothin, from Streptomyces sp. MM23. International journal

    Jun-ya Ueda, Junko Hashimoto, Aya Nagai, Takuji Nakashima, Hisayuki Komaki, Kozue Anzai, Shigeaki Harayama, Takayuki Doi, Takashi Takahashi, Kazuo Nagasawa, Tohru Natsume, Motoki Takagi, Kazuo Shin-ya

    The Journal of antibiotics   60 ( 5 )   321 - 4   2007.5

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    A new polypropionate alloaureothin (1) possessing a nitro group, together with a known polypropionate aureothin (2), was isolated from mycelium of Streptomyces sp. MM23. The structure was determined on the basis of spectroscopic data. I exhibited growth inhibitory effect against human fibrosarcoma HT1080 cells with an IC50 value of 30 mu M.

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  • Sediminibacter furfurosus gen. nov., sp. nov. and Gilvibacter sediminis gen. nov., sp. nov., novel members of the family Flavobacteriaceae. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   57 ( Pt 2 )   265 - 269   2007.2

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    Two Gram-negative, chemoheterotrophic, non-motile strains, Mok-1-36(T) and MAOS-86(T), were isolated from marine-sediment samples collected from the coasts of Okinawa island and the city of Odawara in Japan, respectively, Phylogenetic studies based on 16S rRNA gene sequences indicated that Mok-1-36(T) and MAOS-86(T) were members of the family Flavobacteriaceae, clustering with members of the genera Ulvibacterand Vitellibacter, respectively. Strains Mok-1-36(T) and MAOS-86(T) shared pairwise 16S rRNA gene sequence similarities of 93.5 and 89.1% with the type strains of Ulvibacter litoralis and Vitellibacter vladivostokensis, respectively. Phylogenetic distinctiveness and phenotypic differences from their phylogenetic neighbours indicated that these strains represent two novel species and genera within the family Flavobacteriaceae, for which the names Sediminibacter furfurosus gen. nov., sp. nov. (MAOS-86(T)) and Gilvibacter sediminis gen. nov., sp. nov. (Mok-1-36(T)) are proposed. The type strain of Sediminibacter furfurosus is MAOS-86(T) (=NBRC 101622(T) = CIP 109285(T)) and the type strain of Gilvibacter sediminis is Mok-1-36(T) (= NBRC 101626(T) = CIP 109286(T)).

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  • Identification and characterization of o-xylene-degrading Rhodococcus spp. which were dominant species in the remediation of o-xylene-contaminated soils. International journal

    Hironori Taki, Kazuaki Syutsubo, Richard G Mattison, Shigeaki Harayama

    Biodegradation   18 ( 1 )   17 - 26   2007.2

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    Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.

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  • Family II. Alcanivoraceae fam. nov

    Golyshin, PETER N, Harayama, SHIGEAKI, Timmis, KENNETH N, Yakimov, MICHAIL M

    Bergey's Manual® of Systematic Bacteriologyvol   2   295 - 295   2007

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  • Major carotenoid isolated from Paracoccus schoinia NBRC 100637T is adonixanthin diglucoside. International journal

    Shinichi Takaichi, Takashi Maoka, Naoshige Akimoto, Shams Tabrez Khan, Shigeaki Harayama

    Journal of natural products   69 ( 12 )   1823 - 5   2006.12

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    The structure of a novel major carotenoid glycoside (nearly 80% of total carotenoids) from a newly isolated bacterium, Paracoccus schoinia NBRC 100637(T), was determined to be adonixanthin diglucoside using spectral data. By contrast, carotenoid diglycosides are rare and are usually minor carotenoids in nature. The minor carotenoids in this bacterium included astaxanthin diglucoside, zeaxanthin diglucoside, canthaxanthin, echinenone, zeaxanthin, beta-cryptoxanthin, and beta-carotene.

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  • Ferrimonas futtsuensis sp. nov. and Ferrimonas kyonanensis sp. nov., selenate-reducing bacteria belonging to the Gammaproteobacteria isolated from Tokyo Bay. International journal

    Tatsunori Nakagawa, Takao Iino, Ken-Ichiro Suzuki, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   56 ( Pt 11 )   2639 - 2645   2006.11

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    Two novel mesophilic, facultatively anaerobic, selenate-reducing bacteria, designated strains FUT3661(T) and Asr22-7(T), were isolated from a sediment sample and the alimentary tract of littleneck clams, respectively. Both sources of the samples were collected from the coast of Tokyo Bay, Japan. Cells were Gram-negative rods and motile by means of a polar flagellum. The strains reduced selenate to elemental selenium (Se-0) and also reduced iron(III) oxyhydroxicle, iron(III) citrate, arsenate, manganese(IV) oxide, elemental sulfur and oxygen and used lactate, pyruvate, yeast extract, tryptone and Casamino acids as electron donors and carbon sources. The strains contained both menaquinone (MK-7) and ubiquinones (Q-7 and Q-8) as isoprenoid quinones. The major fatty acids were C-16:0 and C(16:1)w9c. The G + C content of the genomic DNA was 58.1 mol% for strain FUT3661(T) and 57.2 mol% for strain Asr22.7(T). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were related to members of the genus Ferrimonas (< 94-0 % similarities), although the two novel strains formed a separate lineage. 16S rRNA gene sequence similarity between strains FUT3661(T) and Asr22.7(T) was 96 %. On the basis of this polyphasic analysis, it was concluded that strains FUT3661(T) and Asr22.7(T) represent two novel species within the genus Ferrimonas, for which the names Ferrimonas futtsuensis sp. nov. (type strain FUT3661(T) = NBRC 101558(T) = DSM 18154(T)) and Ferrimonas kyonanensis sp. nov. (type strain Asr22.7(T) = NBRC 101286(T) = DSM 18153(T)) are proposed.

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  • Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation. International journal

    Daisuke Koma, Toshiya Sawai, Shigeaki Harayama, Kuniki Kino

    Applied microbiology and biotechnology   73 ( 1 )   172 - 80   2006.11

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    Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 degrees C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 degrees C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.

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  • Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols. International journal

    Xue Peng, Hironori Taki, Syoko Komukai, Mitsuo Sekine, Kaneo Kanoh, Hiroaki Kasai, Seon-Kang Choi, Seiha Omata, Satoshi Tanikawa, Shigeaki Harayama, Norihiko Misawa

    Applied microbiology and biotechnology   71 ( 6 )   824 - 32   2006.8

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    Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN 14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichiacoli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN 14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methytnaphthalene, 2-hydroxymethylnaphthalene, xylene-alpha,alpha'-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.

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  • Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2. International journal

    Makoto Urai, Hirosi Anzai, Jun Ogihara, Noriyuki Iwabuchi, Shigeaki Harayama, Michio Sunairi, Mutsuyasu Nakajima

    Carbohydrate research   341 ( 6 )   766 - 775   2006.5

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    A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).

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  • Sandarakinotalea sediminis gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   56 ( Pt 5 )   959 - 963   2006.5

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    Four Gram-negative, orange-coloured, aerobic, heterotrophic bacteria were isolated from sediment samples collected on the Pacific coast of Japan near the cities of Toyohashi and Katsuura. 16S rRNA gene sequence analysis indicated that these strains form a distinct lineage within the family Flavobacteriaceae. The four isolates shared 99.9-100 % 16S rRNA gene sequence similarity with each other and showed 88-90.9 % similarity with their neighbours in the family Flavobacteriaceae. The four strains also shared high DNA-DNA reassociation values of 67-99 % with each other. All the strains grew at 37 degrees C but not at 4 degrees C, and degraded gelatin, starch and DNA. The major fatty acids were i-C15:0, a-C15:0, i-C16:0 and i-C17:0 3-OH. However, two common fatty acids of members of the Flavobacteriaceae, i-C15:1 and a-C15:1, were absent in these strains. The DNA G+C contents of the four strains were in the range 35-37 mol%. On the basis of the polyphasic evidence, it was concluded that these strains should be classified as a novel genus and a novel species in the family Flavobacteriaceae, for which the name Sandarakinotalea sediminis gen. nov., sp. nov. is proposed. The type strain of Sandarakinotalea sediminis is CKA-5T (=NBRC 100970T=LMG 23247T).

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  • Sediminicola luteus gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   56 ( Pt 4 )   841 - 845   2006.4

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    The taxonomic position of four Gram-negative, rod-shaped, golden-yellow-coloured bacteria isolated from marine sediments was determined. Analysis of the almost complete 16S rRNA gene sequences indicated that these isolates belong to the family Flavobacteriaceae. An unclassified bacterium, NBRC 15975, was found to be the closest relative, showing 16S rRNA gene sequence similarity of 93 %; other related genera shared only 87.9-90.5 % similarity. In contrast, the four isolates shared high levels of 16S rRNA gene sequence similarity (99.3-99.7 %) and high DNA-DNA reassociation values (93-104 %). The isolates could be differentiated phenotypically from other genera by the abilities to reduce nitrate and to degrade gelatin, casein and starch. The only respiratory quinone was MK-6, and the major fatty acids were iso-C(15 : 0), iso-C(15 : 1), anteiso-C(15 : 0), iso-C(17 : 1)omega9c and iso-C(17 : 0) 3-OH. The DNA G+C content was 38-40 mol%. Differentiating phenotypic characteristics and large phylogenetic distances between the isolates and previously published genera indicated that the isolates constitute a novel genus, for which the name Sediminicola gen. nov. is proposed. The type species is Sediminicola luteus sp. nov. (type strain CNI-3T = NBRC 100966T = LMG 23246T).

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  • Erratum: Pseudovibrio ascidiaceicola sp. nov., isolated from ascidians (sea squirts) (International Journal of Systematic and Evolutionary Microbioloy (2006) 56 (343-347)) Reviewed

    Yukiyo Fukunaga, Midori Kurahashi, Kenji Tanaka, Kensuke Yanagi, Akira Yokota, Shigeaki Harayama

    International Journal of Systematic and Evolutionary Microbiology   56 ( 4 )   923   2006.4

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    DOI: 10.1099/00207713-56-4-923

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  • Pseudovibrio ascidiaceicola sp. nov., isolated from ascidians (sea squirts). International journal

    Yukiyo Fukunaga, Midori Kurahashi, Kenji Tanaka, Kensuke Yanagi, Akira Yokota, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   56 ( Pt 2 )   343 - 347   2006.2

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    Two bacterial strains, F423T and F10102, were isolated from two ascidians, Polycitor proliferus and Botryllidae sp., respectively, which were collected from a beach on the Boso peninsula in Japan. Cells of both isolates were motile, rod-shaped and formed star-shaped aggregates in the early stage of exponential growth, but were coccoid in stationary growth phase. The results of 16S rRNA gene sequence analysis, fatty acid analysis, DNA-DNA hybridization experiments and physiological and biochemical tests indicated that the two strains were members of a novel species of the genus Pseudovibrio for which the name Pseudovibrio ascidiaceicola sp. nov. is proposed. The type strain is F423T (=NBRC 100514T=IAM 15084T=DSM 16392T=KCTC 12308T).

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  • Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4. International journal

    Mitsuo Sekine, Satoshi Tanikawa, Seiha Omata, Mika Saito, Takatomo Fujisawa, Naofumi Tsukatani, Takahisa Tajima, Tomohiro Sekigawa, Hiroki Kosugi, Yasunori Matsuo, Rika Nishiko, Kohsuke Imamura, Mio Ito, Hitomi Narita, Shinichi Tago, Nobuyuki Fujita, Shigeaki Harayama

    Environmental microbiology   8 ( 2 )   334 - 46   2006.2

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    Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.

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  • Krokinobacter gen. nov., with three novel species, in the family Flavobacteriaceae. International journal

    Shams Tabrez Khan, Yasuyoshi Nakagawa, Shigeaki Harayama

    International journal of systematic and evolutionary microbiology   56 ( Pt 2 )   323 - 328   2006.2

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    Five strains belonging to the family Flavobacteriaceae were isolated from marine-sediment samples collected in Sagami and Tokyo bays on the Pacific coastline of Japan. The five isolates formed a coherent and novel genus-level lineage within the family Flavobacteriaceae. The most closely related species with a validly published name was Cellulophaga lytica. The five isolates were rod-shaped, Gram-negative, aerobic, catalase- and oxidase-positive, flexirubin-negative and yellow-pigmented. The dominant fatty acids were branched or hydroxy acids, i.e. i-C(15:0), i-C(15:1) and i-C(17:0) 3-OH. These strains degraded gelatin, casein, DNA and Tween 80. The G+C content of their DNAs ranged between 33 and 39 mol%. Although analysis of the 16S rRNA gene sequence similarity divided these strains into two subgroups with a 2.3% sequence difference, the results of DNA-DNA hybridization indicated the grouping of these strains into three distinct species. On the basis of phenotypic and genotypic analyses, the novel genus Krokinobacter is proposed, with Krokinobacter genikus sp. nov., containing three of the strains, as the type species. The type strain is Cos-13T (=NBRC 100811T=CIP 108744T). The names Krokinobacter eikastus sp. nov. (type strain PMA-26T=NBRC 100814T=CIP 108743T) and Krokinobacter diaphorus sp. nov. (type strain MSKK-32T=NBRC 100817T=CIP 108745T) are proposed for the other two isolates.

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  • Sequence diversity of type-II polyketide synthase genes in Streptomyces

    Komaki, Hisayuki, Harayama, Shigeaki

    Actinomycetologica   20 ( 2 )   42 - 48   2006

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  • Entomopathogenic fungi isolated from laboratory-reared Baculentulus densus (Acerentomidae, Protura)

    Kurihara, Yuko, Machida, Ryuichiro, Fukui, Makiko, Okuda, Toru, Harayama, Shigeaki

    Edaphologia   80   25 - 28   2006

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  • Bacterial phylogeny reconstruction from molecular sequences

    Harayama, Shigeaki, Kasai, Hiroaki

    Molecular identification, systematics, and population structure of Prokaryotes   105 - 139   2006

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  • Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state. International journal

    Sang-Ho Baik, Fabrice Michel, Nushin Aghajari, Richard Haser, Shigeaki Harayama

    Applied and environmental microbiology   71 ( 6 )   3285 - 93   2005.6

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    A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.

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  • The soil flagellate Heteromita globosa accelerates bacterial degradation of alkylbenzenes through grazing and acetate excretion in batch culture.

    Shigeaki Harayama

    Microbial ecology   2005.1

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    DOI: 10.1007/s00248-003-0226-5

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  • Effects of chloramphenicol on photosynthesis, protein profiles and transketolase activity under extremely high CO2 concentration in an extremely-high-CO2-tolerant green microalga, Chlorococcum littorale.

    Akira Satoh, Norihide Kurano, Shigeaki Harayama, Shigetoh Miyachi

    Plant & cell physiology   45 ( 12 )   1857 - 62   2004.12

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    An extremely-high-CO2-tolerant alga, Chlorococcum littorale, showed high quantum efficiency of PSII (PhiII) in the light at 40% CO2, as well as at 5% CO2. However, PhiII decreased greatly when chloramphenicol (CAP) was added at 40% CO2, while no such decrease was observed at 5% CO2. Cycloheximide showed no effect on PhiII at either 5% or 40% CO2. The amount of a 76 kDa polypeptide (p76) on SDS-PAGE decreased markedly in the presence of CAP at 40% CO2 but not at 5% CO2. A partial amino acid sequence of p76 was 71-100% identical (10-14 identical residues out of 14 amino acids determined) to those of transketolases (TKLs) reported in higher plants and a cyanobacterium. In agreement with these observations, the TKL activity in C. littorale was decreased by CAP at 40% CO2, but not at 5% CO2. The transient decrease in TKL activity caused by CAP under 40% CO2 was well correlated with that in PhiII. These results indicate that the addition of CAP directly or indirectly influences the stability of TKL in C. littorale at 40% CO2, but not at 5% CO2, and that photosynthetic activity was reduced by a decrease in TKL activity.

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  • Microbial communities in oil-contaminated seawater. International journal

    Shigeaki Harayama, Yuki Kasai, Akihiro Hara

    Current opinion in biotechnology   15 ( 3 )   205 - 14   2004.6

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    Although diverse bacteria capable of degrading petroleum hydrocarbons have been isolated and characterized, the vast majority of hydrocarbon-degrading bacteria, including anaerobes, could remain undiscovered, as a large fraction of bacteria inhabiting marine environments are uncultivable. Using culture-independent rRNA approaches, changes in the structure of microbial communities have been analyzed in marine environments contaminated by a real oil spill and in micro- or mesocosms that mimic such environments. Alcanivorax and Cycloclasticus of the gamma-Proteobacteria were identified as two key organisms with major roles in the degradation of petroleum hydrocarbons. Alcanivorax is responsible for alkane biodegradation, whereas Cycloclasticus degrades various aromatic hydrocarbons. This information will be useful to develop in situ bioremediation strategies for the clean-up of marine oil spills.

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  • Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol. International journal

    Kouhei Ohnishi, Akiko Okuta, Jiansong Ju, Tohru Hamada, Haruo Misono, Shigeaki Harayama

    Journal of biochemistry   135 ( 3 )   305 - 17   2004.3

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    3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.

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  • Cloning and functional analysis of alkB genes in Alcanivorax borkumensis SK2. International journal

    Akihiro Hara, Sang-ho Baik, Kazuaki Syutsubo, Norihiko Misawa, Theo H M Smits, Jan B van Beilen, Shigeaki Harayama

    Environmental microbiology   6 ( 3 )   191 - 7   2004.3

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    Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. To identify the genes responsible for alkane degradation in this organism, two putative genes for alkane hydroxylases were cloned from Alcanivorax borkumensis SK2. They were named alkB1 and alkB2. These genes were subsequently disrupted in A. borkumensis SK2, and the growth phenotypes of the disruptants were examined. The results indicate that the alkB1 gene is responsible for the degradation of short-chain n-alkanes. A double mutant defective in both alkB1 and alkB2 was still able to grow on medium-chain n-alkanes, indicating that genes other than alkB1 and alkB2 are also involved in n-alkane hydroxylation by A. borkumensis SK2.

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  • Construction of chimeric catechol 2,3-dioxygenase exhibiting improved activity against the suicide inhibitor 4-methylcatechol. International journal

    Akiko Okuta, Kouhei Ohnishi, Shigeaki Harayama

    Applied and environmental microbiology   70 ( 3 )   1804 - 10   2004.3

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    Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.

    DOI: 10.1128/aem.70.3.1804-1810.2004

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  • Single-stranded DNA family shuffling. International journal

    Osamu Kagami, Miho Kikuchi, Shigeaki Harayama

    Methods in enzymology   388   11 - 21   2004

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    DOI: 10.1016/s0076-6879(04)88002-9

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  • Effective DNA shuffling methods for enzyme evolution

    Kagami, Osamu, Baik, Sang-Ho, Harayama, Shigeaki

    Enzyme functionality. Design, engineering, and screening   425 - 441   2004

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  • Catabolism of PAHS

    Kasai, Yuki, Harayama, Shigeaki

    Pseudomonas   463 - 490   2004

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  • Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp. strain A5. International journal

    Yuki Kasai, Kazutoshi Shindo, Shigeaki Harayama, Norihiko Misawa

    Applied and environmental microbiology   69 ( 11 )   6688 - 97   2003.11

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    Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.

    DOI: 10.1128/aem.69.11.6688-6697.2003

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  • Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation. International journal

    Akihiro Hara, Kazuaki Syutsubo, Shigeaki Harayama

    Environmental microbiology   5 ( 9 )   746 - 53   2003.9

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    Alcanivorax is an alkane-degrading marine bacterium which propagates and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. In order to understand why Alcanivorax overcomes other bacteria under such cultural conditions, competition experiments between Alcanivorax indigenous to seawater and the exogenous alkane-degrading marine bacterium, Acinetobacter venetianus strain T4, were conducted. When oil-containing seawater supplemented with nitrogen and phosphorus nutrients was inoculated with A. venetianus strain T4, this bacterium was the dominant population at the early stage of culture. However, its density began to decrease after day 6, and Alcanivorax predominated in the culture after day 20. The crude-oil-degrading profiles of both bacteria were therefore investigated. Alcanivorax borkumensis strain ST-T1 isolated from the Sea of Japan exhibited higher ability to degrade branched alkanes (pristane and phytane) than A. venetianus strain T4. It seems that this higher ability of Alcanivorax to degrade branched alkanes allowed this bacterium to predominate in oil-containing seawater. It is known that some marine zooplanktons produce pristane and Alcanivorax may play a major role in the biodegradation of pristane in seawater.

    DOI: 10.1046/j.1468-2920.2003.00468.x

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  • Identification by the phage-display technique of peptides that bind to H7 flagellin of Escherichia coli. International journal

    Teruhiko Ide, Sang-Ho Baik, Takao Matsuba, Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   67 ( 6 )   1335 - 41   2003.6

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    The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library. The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin. An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner. Among them, a D1 phage clone showed the highest binding affinity to the H7 flagellin. We synthesized the D1 peptide (LHIHRPTLSIQG) corresponding to the peptide-encoding region of the D1 phage clone. The synthetic peptide showed micro-molar affinity (EC(50) value=1.9 microM) for the H7 flagellin. Furthermore, this D1 peptide interacted more specifically with the H7 flagellin than with the other flagellins (H1, H5, H12, or H23) of E. coli. In situ hybridization clearly showed that the peptide only detected those cells harboring the H7 flagellin gene (fliC). The peptide may specifically bind to the H7 flagellin on the cell surface. These results suggest that the phage-display technique could be used as a tool for identifying peptides as an alternative to using a ligand as a diagnostic reagent in food products or in clinical testing.

    DOI: 10.1271/bbb.67.1335

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  • Significantly enhanced stability of glucose dehydrogenase by directed evolution.

    Shigeaki Harayama

    Applied microbiology and biotechnology   2003.5

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    DOI: 10.1007/s00253-002-1215-1

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  • Intersubunit interaction and catalytic activity of catechol 2,3-dioxygenases. International journal

    Akiko Okuta, Kouhei Ohnishi, Sakiko Yagame, Shigeaki Harayama

    The Biochemical journal   371 ( Pt 2 )   557 - 64   2003.4

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    Catechol 2,3-dioxygenases (C23Os; EC 1.3.11.2) form a large protein family that is divided into several subgroups. Amino acid sequences of C23Os belonging to subgroup I.2.A and those belonging to I.2.B are found to be approx. 50% identical. When the central parts of the C23O sequences belonging to I.2.B were fused with the N-terminal and C-terminal sequences of I.2.A C23O, the hybrid enzymes were not active. To understand why these hybrid C23Os were inactive, hybrids between XylE(P) (C23O found in a Pseudomonas strain; subgroup I.2.A) and XylE(S) (C23O found in a Sphingomonas strain; subgroup I.2.B) were constructed. HB3-C23O consisted mostly of the XylE(S) sequence, except that its C-terminal end was derived from XylE(P). While HB3-C23O was not active, HB4-C23O, carrying shorter C-terminal XylE(P) sequences than HB3-C23O, was active. This observation indicated that certain amino acid residues at the C-terminus were crucial for C23O activity in the hybrid forms of enzymes between XylE(P) and XylE(S). According to the crystal structure of XylE(P), the C-terminal region is involved in the formation of a quaternary structure. Amino acid differences between HB3-C23O and HB4-C23O included the specific beta-strand for oligomerization. Thus the quaternary structures of active C23Os, XylE(S), XylE(P) and HB4-C23O, as well as that of inactive HB3-C23O, were examined. Active enzymes XylE(S), XylE(P) and HB4-C23O were homotetrameric, while HB3-C23O existed only as a monomer. We concluded that hybrids of subgroups I.2.A and I.2.B were often inactive because of a defect in their oligomerization.

    DOI: 10.1042/bj20021657

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  • Activity and population structure of nitrifying bacteria in an activated-sludge reactor containing polymer beads. International journal

    Jun-ichi Tanaka, Kazuaki Syutsubo, Kazuya Watanabe, Hitoshi Izumida, Shigeaki Harayama

    Environmental microbiology   5 ( 4 )   278 - 86   2003.4

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    Photo-crosslinked polymer beads were introduced into a laboratory activated-sludge unit containing municipal sewage sludge and the effect on nitrifying capacity was examined. The ammonia load started at a nitrogen-loading rate of 0.02 kg m(-3) day(-1) and was increased stepwise. It was found that the bead-containing unit could almost completely oxidize ammonia (over 95%) up to a nitrogen-loading rate of 0.216 kg m(-3) day(-1), whereas the maximum loading rate of the control unit (without polymer beads) was 0.096 kg m(-3) day(-1). The nitrifying potential of suspended and bead-associated organisms in the bead-containing unit was measured under different loading conditions. It was found that the bead-associated organisms exhibited high specific activities under high loading conditions and that the contribution of the bead-associated organisms to nitrification was greater than that of the suspended solids under these conditions. The bacterial population dynamics in the suspended solids and bead-associated organisms were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments and by fluorescence in situ hybridization with group-specific probes. Among the known nitrifying organisms, ammonia-oxidizing beta-proteobacteria and Nitrospira-related organisms were detected by these approaches. A comparison of the activity dynamics and population dynamics, however, suggested a possibility that other organisms may also have been involved in the nitrification process under high loading conditions.

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  • Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric, and ferulic acids. International journal

    Xue Peng, Norihiko Misawa, Shigeaki Harayama

    Applied and environmental microbiology   69 ( 3 )   1417 - 27   2003.3

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    Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The beta-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.

    DOI: 10.1128/aem.69.3.1417-1427.2003

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  • Isolation and phylogenetic characterization of bacteria capable of inducing differentiation in the green alga Monostroma oxyspermum. International journal

    Yoshihide Matsuo, Makoto Suzuki, Hiroaki Kasai, Yoshikazu Shizuri, Shigeaki Harayama

    Environmental microbiology   5 ( 1 )   25 - 35   2003.1

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    Many green algae cannot develop normally when they are grown under axenic conditions. Monostroma oxyspermum, for example, proliferates unicellularly in an aseptic culture, but develops into a normal foliaceous gametophyte in the presence of some marine bacteria. More than 1000 bacterial strains were isolated from marine algae and sponges and assayed for their ability to induce the morphogenesis of unicellular M. oxyspermum. Fifty bacterial strains exhibiting morphogenesis-inducing activity against unicellular M. oxyspermum were isolated. The partial gyrB (approximately 1.2 kbp) and 16S rDNA (approximately 1.4 kbp) sequences of about 40 active strains were determined, and their phylogenetic relationships were analysed. All these strains were located within the Cytophaga-Flavobacterium-Bacteroides (CFB) complex, and most of these strains were clustered in a clade comprising Zobellia uliginosa. On the other hand, these bacteria also exhibited morphogenetic activity against germ-free spores of Ulva pertusa, Ulva conglobata and Enteromorpha intestinalis. Moreover, these bacteria induced the release of spores from the leafy young gametophyte of M. oxyspermum. These results indicate that strains belonging to several groups in the CFB complex play an important role in the normal development of green algae in the marine coastal environment.

    DOI: 10.1046/j.1462-2920.2003.00382.x

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  • Functional and structural analyses of trichloroethylene-degrading bacterial communities under different phenol-feeding conditions: laboratory experiments.

    Shigeaki Harayama

    Applied microbiology and biotechnology   2003.1

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  • Hydroxylation of ionized aromatics including carboxylic acid or amine using recombinant Streptomyces lividans cells expressing modified biphenyl dioxygenase genes

    Shindo, Kazutoshi, Nakamura, Ryoko, Chinda, Ikuko, Ohnishi, Yasuo, Horinouchi, Sueharu, Takahashi, Haruko, Iguchi, Kazuo, Harayama, Shigeaki, Furukawa, Kensuke, Misawa, Norihiko

    Tetrahedron   59 ( 11 )   1895 - 1900   2003

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  • Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium. International journal

    Robert A Kanaly, Shigeaki Harayama, Kazuya Watanabe

    Applied and environmental microbiology   68 ( 12 )   5826 - 33   2002.12

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    A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.

    DOI: 10.1128/aem.68.12.5826-5833.2002

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  • Bacteria belonging to the genus cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. International journal

    Yuki Kasai, Hideo Kishira, Shigeaki Harayama

    Applied and environmental microbiology   68 ( 11 )   5625 - 33   2002.11

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    To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 10(3) cells/g of grains before crude oil was added to the tanks and increased to 3 x 10(6) cells/g of grains after crude oil was added. The number increased further after 14 days to 10(8) cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 x 10(6) cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.

    DOI: 10.1128/aem.68.11.5625-5633.2002

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  • Stable augmentation of activated sludge with foreign catabolic genes harboured by an indigenous dominant bacterium. International journal

    Kazuya Watanabe, Maki Teramoto, Shigeaki Harayama

    Environmental microbiology   4 ( 10 )   577 - 83   2002.10

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    Comamonas sp. rN7 is a phenol-degrading bacterium that represents the dominant catabolic population in activated sludge. The present study examined the utility of this bacterium for establishing foreign catabolic genes in phenol-digesting activated sludge. The phc genes coding for phenol hydroxylase and its transcriptional regulators of C. testosteroni R5 were integrated into the chromosome of strain rN7. The specific phenol-oxygenating activity of a resultant transformant designated rN7(R503) was three times higher than the activity of strain rN7, and the phc genes were stably inherited by rN7(R503) grown in a non-selective laboratory medium. Inoculation of phenol-acclimatized activated sludge with rN7(R503) resulted in a high phenol-oxygenating activity and improved resistance to phenol-shock loading compared to sludge inoculated with either no cells, rN7 or R5. Quantitative competitive polymerase chain reaction (PCR) showed that the phc genes were retained in the rN7(R503)-inoculated sludge at a density of more than 108 copies per ml of mixed liquor for more than 35 days, whereas those in the R5-inoculated sludge disappeared rapidly. No transfer of the phc genes to other indigenous populations was apparent in the rN7(R503)-harbouring sludge. From these results, we concluded that the phenol treatment of the activated sludge was enhanced by the phc genes harboured by the rN7(R503) population. This study suggests a possible bioaugmentation strategy for stably utilizing foreign catabolic genes in natural ecosystems.

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  • The bacterivorous soil flagellate Heteromita globosa reduces bacterial clogging under denitrifying conditions in sand-filled aquifer columns. International journal

    Richard G Mattison, Hironori Taki, Shigeaki Harayama

    Applied and environmental microbiology   68 ( 9 )   4539 - 45   2002.9

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    An exopolymer (slime)-producing soil bacterium Pseudomonas sp. (strain PS+) rapidly clogged sand-filled columns supplied with air-saturated artificial groundwater containing glucose (500 mg liter(-1)) as a sole carbon source and nitrate (300 mg liter(-1)) as an alternative electron acceptor. After 80 days of operation under denitrifying conditions, the effective porosity and saturated hydraulic conductivity (permeability) of sand in these columns had fallen by 2.5- and 26-fold, respectively. Bacterial biofilms appeared to induce clogging by occluding pore spaces with secreted exopolymer, although there may also have been a contribution from biogas generated during denitrification. The bacterivorous soil flagellate Heteromita globosa minimized reductions in effective porosity (1.6-fold) and permeability (13-fold), presumably due to grazing control of biofilms. Grazing may have limited growth of bacterial biomass and hence the rate of exopolymer and biogas secretion into pore spaces. Evidence for reduction in biogas production is suggested by increased nitrite efflux from columns containing flagellates, without a concomitant increase in nitrate consumption. There was no evidence that flagellates could improve flow conditions if added once clogging had occurred (60 days). Presumably, bacterial biofilms and their secretions were well established at that time. Nevertheless, this study provides evidence that bacterivorous flagellates may play a positive role in maintaining permeability in aquifers undergoing remediation treatments.

    DOI: 10.1128/aem.68.9.4539-4545.2002

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  • Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies. International journal

    Kazuya Watanabe, Hiroyuki Futamata, Shigeaki Harayama

    Antonie van Leeuwenhoek   81 ( 1-4 )   655 - 63   2002.8

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    Molecular ecological approaches have detected diverse microorganisms that occur in response to pollution and bioremediation; however, most of these organisms have not been isolated, and their physiological traits are poorly understood. One important objective in current bioremediation studies would therefore be an assessment of the physiology and functions of the diverse microbial population at a polluted site. Among the parameters relating to the diversity of the microbial catabolic potential, e.g., substrate specificity, inducer specificity, number of catabolic routes and kinetics of catabolic enzymes, our studies have focused on the kinetic diversity of phenol-degrading bacteria. In one example, a kinetic analysis allowed functionally important phenol-degrading bacteria to be identified in activated sludge; this information could be used to improve the performance of phenol-degrading activated sludge. In an analysis of phenol-degrading bacteria present in trichloroethylene (TCE)-contaminated aquifer soil, the kinetic data could be linked to group-specific monitoring of their phenol-hydroxylase genes. The results have suggested that one group of phenol-degrading bacteria can effectively contribute to TCE bioremediation, while other groups work poorly. Based on this information, we have succeeded in developing a high-performance TCE-degrading bioreactor. We suggest that a careful analysis of the diversity of microbial catabolic potential, particularly of the kinetic traits, may facilitate the development of new bioremediation strategies.

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  • An AraC/XylS family member at a high level in a hierarchy of regulators for phenol-metabolizing enzymes in Comamonas testosteroni R5. International journal

    Maki Teramoto, Kouhei Ohnishi, Shigeaki Harayama, Kazuya Watanabe

    Journal of bacteriology   184 ( 14 )   3941 - 6   2002.7

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    Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.

    DOI: 10.1128/jb.184.14.3941-3946.2002

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  • Phylogenetic study of the genus Oceanospirillum based on 16S rRNA and gyrB genes: emended description of the genus Oceanospirillum, description of Pseudospirillum gen. nov., Oceanobacter gen. nov. and Terasakiella gen. nov. and transfer of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb. nov. and Marinobacterium stanieri comb. no. International journal

    Masataka Satomi, Bon Kimura, Tohru Hamada, Shigeaki Harayama, Tateo Fujii

    International journal of systematic and evolutionary microbiology   52 ( Pt 3 )   739 - 747   2002.5

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    The phylogenetic relationships of Oceanospirillum strains were analysed by using the nucleotide sequences of 16S rRNA and gyrB genes. Results from sequence analysis demonstrated that the Oceanospirillum core group consisted of four species, Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum, with enough distance to separate them as different species. However, four other Oceanospirillum species occupied taxonomic positions separate from the Oceanospirillum core group: Oceanospirillum jannaschii, Oceanospirillum japonicum and Oceanospirillum kriegii in the gamma-Proteobacteria and Oceanospirillum pusillum in the alpha-Proteobacteria. Oceanospirillum jannaschii clustered with Marinobacterium georgiense, Pseudomonas iners and Pseudomonas stanieri on the basis of phylogenetic analysis of 16S rRNA and gyrB genes. The other three species did not cluster with known genera. Also, the sequence similarity values of the gyrB genes between the three subspecies of Oceanospirillum maris and those between the two subspecies of Oceanospirillum beijerinckii were above 99%. The close relationships between the subspecies of Oceanospirillum maris and of Oceanospirillum beijerinckii were further supported by similar physiological properties and high DNA-DNA hybridization values, suggesting that these subspecies should not be regarded as valid. From these results, Oceanospirillum sensu stricto should be defined to consist of Oceanospirillum linum, Oceanospirillum maris, Oceanospirillum beijerinckii and Oceanospirillum multiglobuliferum. We propose to create the following new genera: Pseudospirillum gen. nov. for Oceanospirillum japonicum as Pseudospirillum japonicum comb. nov.; Oceanobacter gen. nov. for Oceanospirillum kriegii as Oceanobacter kriegii comb. nov.; and Terasakiella gen. nov. for Oceanospirillum pusillum as Terasakiella pusilla comb. nov. The transfer is proposed of Oceanospirillum jannaschii and Pseudomonas stanieri to Marinobacterium as Marinobacterium jannaschii comb. nov. and Marinobacterium stanieri comb. nov. Furthermore, Pseudomonas iners should be reclassified as a strain of Marinobacterium georgiense. Finally, the subspecies of Oceanospirillum maris (O. maris subsp. maris, O. maris subsp. hiroshimense and O. maris subsp. williamsae) and Oceanospirillum beijerinckii (O. beijerinckii subsp. beijerinckii and O. beijerinckii subsp. pelagicum) should be combined as Oceanospirillum maris and Oceanospirillum beijerinckii, respectively.

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  • Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria. International journal

    Noriyuki Iwabuchi, Michio Sunairi, Makoto Urai, Chiaki Itoh, Hiroshi Anzai, Mutsuyasu Nakajima, Shigeaki Harayama

    Applied and environmental microbiology   68 ( 5 )   2337 - 43   2002.5

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    Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.

    DOI: 10.1128/aem.68.5.2337-2343.2002

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  • Predominant growth of Alcanivorax strains in oil-contaminated and nutrient-supplemented sea water. International journal

    Yuki Kasai, Hideo Kishira, Tetsuya Sasaki, Kazuaki Syutsubo, Kazuya Watanabe, Shigeaki Harayama

    Environmental microbiology   4 ( 3 )   141 - 7   2002.3

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    We found that bacteria closely related to Alcanivorax became a dominant bacterial population in petroleum-contaminated sea water when nitrogen and phosphorus nutrients were supplied in adequate quantity. The predominance of Alcanivorax bacteria was demonstrated under three experimental conditions: (i) in batch cultures of sea water containing heavy oil; (ii) in columns packed with oil-coated gravel undergoing a continuous sea water flow; and (iii) in a large-scale tidal flux reactor that mimics a beach undergoing tidal cycles with fresh sea water. These results suggest that bacteria related to Alcanivorax are major players in the bioremediation of oil-contaminated marine environments.

    DOI: 10.1046/j.1462-2920.2002.00275.x

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  • On line database of the culture collection of marine microorganisms at Marine Biotechnology Institute Culture collection (MBIC)

    Shigeaki Harayama

    2002

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  • Hydroxylation of various molecules including heterocyclic aromatics using recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes

    Misawa, Norihiko, Shindo, Kazutoshi, Takahashi, Haruko, Suenaga, Hikaru, Iguchi, Kazuo, Okazaki, Hiroshi, Harayama, Shigeaki, Furukawa, Kensuke

    Tetrahedron   58 ( 47 )   9605 - 9612   2002

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  • DNA shuffling and family shuffling for in vitro gene evolution. International journal

    Miho Kikuchi, Shigeaki Harayama

    Methods in molecular biology (Clifton, N.J.)   182   243 - 57   2002

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  • An effective peptide screening system using recombinant fluorescent bacterial surface display

    Yoshida, Hiromi, Baik, Sang-Ho, Harayama, Shigeaki

    Biotechnology letters   24 ( 20 )   1715 - 1722   2002

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  • Oxygenation reactions of various tricyclic fused aromatic compounds using Escherichia coli and Streptomyces lividans transformants carrying several arene dioxygenase genes.

    Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   2001.11

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    DOI: 10.1271/bbb.65.2472

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  • The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-xylene uptake.

    Shigeaki Harayama

    Journal of bacteriology   2001.11

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    DOI: 10.1128/jb.183.22.6662-6666.2001

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  • Group-specific monitoring of phenol hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation.

    Shigeaki Harayama

    Applied and environmental microbiology   2001.10

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    DOI: 10.1128/aem.67.10.4671-4677.2001

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  • Biotransformation of phenanthrene and 1-methoxynaphthalene with Streptomyces lividans cells expressing a marine bacterial phenanthrene dioxygenase gene cluster.

    Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   2001.8

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    DOI: 10.1271/bbb.65.1774

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  • Photo-oxidation of biodegraded crude oil and toxicity of the photo-oxidized products.

    Shigeaki Harayama

    Chemosphere   2001.8

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    DOI: 10.1016/s0045-6535(00)00292-7

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  • PhcS represses gratuitous expression of phenol-metabolizing enzymes in Comamonas testosteroni R5.

    Shigeaki Harayama

    Journal of bacteriology   2001.7

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    DOI: 10.1128/jb.183.14.4227-4234.2001

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  • Environmental biotechnology

    Shigeaki Harayama

    Current Opinion in Biotechnology   2001.6

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    DOI: 10.1016/s0958-1669(00)00213-5

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  • Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

    Shigeaki Harayama

    Environmental microbiology   2001.6

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    DOI: 10.1046/j.1462-2920.2001.00204.x

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  • Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil-spill accident.

    Shigeaki Harayama

    Environmental microbiology   2001.4

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    DOI: 10.1046/j.1462-2920.2001.00185.x

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  • Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

    Shigeaki Harayama

    Journal of microbiological methods   2001.4

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    DOI: 10.1016/s0167-7012(01)00220-2

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  • Biodegradation of n-alkylcycloalkanes and n-alkylbenzenes via new pathways in Alcanivorax sp. strain MBIC 4326.

    Shigeaki Harayama

    Applied and environmental microbiology   2001.4

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    DOI: 10.1128/aem.67.4.1970-1974.2001

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  • Bacterial populations occuring in a trichloroethylene-contaminated aquifer during methane injection.

    Shigeaki Harayama

    Environmental microbiology   2001.3

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    DOI: 10.1046/j.1462-2920.2001.00178.x

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  • Time-of-flight mass spectrometric analysis of high-molecular-weight alkanes in crude oil by silver nitrate chemical ionization after laser desorption.

    Shigeaki Harayama

    Analytical chemistry   2001.3

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    DOI: 10.1021/ac0009797

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  • Enhanced mineralization of benzo[a]pyrene in the presence of nonaqueous phase liquids.

    Shigeaki Harayama

    Environmental toxicology and chemistry   2001.3

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    DOI: 10.1897/1551-5028(2001)020<0498:emobap>2.0.co;2

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  • Diversity in kinetics of trichloroethylene-degrading activities exhibited by phenol-degrading bacteria.

    Shigeaki Harayama

    Applied microbiology and biotechnology   2001.3

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    DOI: 10.1007/s002530000500

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  • Analysis of long-side-chain alkylaromatics in crude oil for evaluation of their fate in the environment.

    Shigeaki Harayama

    Environmental science & technology   2001.1

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    DOI: 10.1021/es001165a

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  • ICB database: the gyrB database for identification and classification of bacteria.

    Shigeaki Harayama

    Nucleic acids research   2001.1

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    DOI: 10.1093/nar/29.1.344

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  • The predatory soil flagellate Heteromita globosa stimulates toluene biodegradation by a Pseudomonas sp.

    Shigeaki Harayama

    FEMS microbiology letters   2001.1

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    DOI: 10.1111/j.1574-6968.2001.tb09443.x

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  • [SWISS-PROT: the curated protein sequence database on Internet]

    Shigeaki Harayama

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   2001.1

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  • Molecular diversity of pMMO and sMMO in a TCE-contaminated aquifer during bioremediation

    Baker, Paul W, Futamata, Hiroyuki, Harayama, Shigeaki, Watanabe, Kazuya

    FEMS microbiology ecology   38 ( 2-3 )   161 - 167   2001

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  • Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov.

    Suzuki, Makoto, Nakagawa, Yasuyoshi, Harayama, Shigeaki, Yamamoto, Satoshi

    International journal of systematic and evolutionary microbiology   51 ( 5 )   1639 - 1652   2001

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    DOI: 10.1099/00207713-51-5-1639

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  • Reclamation of an activated-sludge microbial consortium by selective biostimulation.

    Shigeaki Harayama

    Applied microbiology and biotechnology   2000.11

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    DOI: 10.1007/s002530000424

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  • Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude oil storage cavities.

    Shigeaki Harayama

    Applied and environmental microbiology   2000.11

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    DOI: 10.1128/aem.66.11.4803-4809.2000

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  • Relationships between colony morphotypes and oil tolerance in Rhodococcus rhodochrous.

    Shigeaki Harayama

    Applied and environmental microbiology   2000.11

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    DOI: 10.1128/aem.66.11.5073-5077.2000

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  • Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. International journal

    Satoshi Yamamoto, Hiroaki Kasai, Dawn L Arnold, Robert W Jackson, Alan Vivian, Shigeaki Harayama

    Microbiology (Reading, England)   146 ( Pt 10)   2385 - 2394   2000.10

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    Phylogenetic analysis of the genus Pseudomonas: was conducted by using the combined gyrB and rpoD nucleotide sequences of 31 validly described species of Pseudomonas: (a total of 125 strains). Pseudomonas: strains diverged into two major clusters designated intrageneric cluster I (IGC I) and intrageneric cluster II (IGC II). IGC I was further split into two subclusters, the 'P: aeruginosa complex', which included P: aeruginosa, P: alcaligenes, P: citronellolis, P: mendocina, P: oleovorans and P: pseudoalcaligenes, and the 'P: stutzeri complex', which included P: balearica and P: stutzeri. IGC II was further split into three subclusters that were designated the 'P: putida complex', the 'P: syringae complex' and the 'P: fluorescens complex'. The 'P: putida complex' included P: putida and P: fulva. The 'P: syringae complex' was the cluster of phytopathogens including P: amygdali, P: caricapapayae, P: cichorii, P: ficuserectae, P: viridiflava and the pathovars of P. savastanoi and P. syringae. The 'P. fluorescens complex' was further divided into two subpopulations, the 'P. fluorescens lineage' and the 'P. chlororaphis lineage'. The 'P. fluorescens lineage' contained P. fluorescens biotypes A, B and C, P. azotoformans, P. marginalis pathovars, P. mucidolens, P. synxantha and P. tolaasii, while the 'P. chlororaphis lineage' included P. chlororaphis, P. agarici, P. asplenii, P. corrugata, P. fluorescens biotypes B and G and P. putida biovar B. The strains of P. fluorescens biotypes formed a polyphyletic group within the 'P. fluorescens complex'.

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  • Rapid mineralization of benzo[a]pyrene by a microbial consortium growing on diesel fuel.

    Shigeaki Harayama

    Applied and environmental microbiology   2000.10

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    DOI: 10.1128/aem.66.10.4205-4211.2000

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  • Starvation improves survival of bacteria introduced into activated sludge.

    Shigeaki Harayama

    Applied and environmental microbiology   2000.9

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    DOI: 10.1128/aem.66.9.3905-3910.2000

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  • Barnacle cement proteins. Importance of disulfide bonds in their insolubility.

    Shigeaki Harayama

    The Journal of biological chemistry   2000.9

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  • Conservative Structure of the Plaque Matrix Protein of Mussels in the Genus Mytilus.

    Shigeaki Harayama

    Marine biotechnology (New York, N.Y.)   2000.7

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  • Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria.

    Shigeaki Harayama

    Journal of bacteriology   2000.4

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    DOI: 10.1128/jb.182.8.2059-2067.2000

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  • A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli.

    Shigeaki Harayama

    Journal of bacteriology   2000.4

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    DOI: 10.1128/jb.182.8.2134-2141.2000

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  • The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp. strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA. International journal

    Masayuki Shimao, Tsuyoshi Tamogami, Shinsuke Kishida, Shigeaki Harayama

    Microbiology (Reading, England)   146 ( Pt 3)   649 - 657   2000.3

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    A 5.7 kbp SphI fragment containing the polyvinyl alcohol (PVA) dehydrogenase gene pvaA and its 1.9 kbp 5'-flanking region was cloned from the PVA-degrading bacterium Pseudomonas sp. VM15C. The pvaB gene, encoding oxidized PVA hydrolase, was found in the region upstream of pvaA. Sequence data and expression studies indicated that pvaA and B constitute an operon in the order pvaBA. The pvaB gene encoded a protein of 379 amino acid residues (40610 Da), and a lipoprotein signal sequence and the lipase consensus sequence, Gly-X-Ser-X-Gly, characteristic of the active-site serine region in serine hydrolases, were detected in the deduced amino acid sequence. The pvaB product with the pvaA product constituted an enzyme system for the cleavage of PVA molecules. The pvaA product introduced beta-diketone groups into the PVA molecule, and the pvaB product hydrolysed these beta-diketone groups in oxidized PVA. The pvaB product also hydrolysed 4,6-nonanedione at a low rate, but not acetylacetone or 5-nonanone. It was completely inhibited by PMSF and was concluded to be a serine hydrolase. There were no proteins showing high similarity to the pvaB product in the databases, but minor similarity to a number of serine hydrolases including polyhydroxyalkanoate depolymerases was apparent.

    DOI: 10.1099/00221287-146-3-649

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  • An effective family shuffling method using single-stranded DNA.

    Shigeaki Harayama

    Gene   2000.2

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    DOI: 10.1016/s0378-1119(99)00547-8

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  • Crystal ball: leading scientists in the field of environmental microbiology consider the technical and conceptual developments that they believe will drive innovative research during the first years of the new millennium.

    Shigeaki Harayama

    Environmental microbiology   2000.2

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    DOI: 10.1046/j.1462-2920.2000.00099.x

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  • Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences.

    Shigeaki Harayama

    Journal of clinical microbiology   2000.1

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  • Fate of crude oil by the combination of photooxidation and biodegradation

    Dutta, Tapan K, Harayama, Shigeaki

    Environmental science & technology   34 ( 8 )   1500 - 1505   2000

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  • Intrageneric relationships among Micromonospora species deduced from gyrB-based phylogeny and DNA relatedness.

    Kasai, Hiroaki, Tamura, Tomohiko, Harayama, Shigeaki

    International journal of systematic and evolutionary microbiology   50 ( 1 )   127 - 134   2000

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    DOI: 10.1099/00207713-50-1-127

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  • Phylogenetic analysis of genus Marinilabilia and related bacteria based on the amino acid sequences of gyrB and emended description of Marinilabilia salmonicolor with Marinilabilia agarovorans as its subjective synonym.

    Shigeaki Harayama

    International journal of systematic bacteriology   1999.10

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    DOI: 10.1099/00207713-49-4-1551

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  • Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1999.10

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    DOI: 10.1007/s004380051117

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  • Petroleum biodegradation in marine environments.

    Shigeaki Harayama

    Journal of molecular microbiology and biotechnology   1999.8

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  • Novel family shuffling methods for the in vitro evolution of enzymes.

    Shigeaki Harayama

    Gene   1999.8

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    DOI: 10.1016/s0378-1119(99)00240-1

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  • An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

    Shigeaki Harayama

    Applied and environmental microbiology   1999.7

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  • Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, an intermediate of the phenanthrene-degradative pathway of Nocardioides sp. strain KP7.

    Shigeaki Harayama

    Journal of bacteriology   1999.2

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  • Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization.

    Shigeaki Harayama

    International journal of systematic bacteriology   1999.1

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    DOI: 10.1099/00207713-49-1-87

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  • Cultured mussel foot cells expressing byssal protein genes

    Takeuchi, Yasuhiro, Inoue, Koji, Miki, Daisuke, Odo, Satoshi, Harayama, Shigeaki

    Journal of Experimental Zoology   283 ( 2 )   131 - 136   1999

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  • Characterization of genes for enzymes involved in the phenanthrene degradation in Nocardioides sp. KP7

    Saito, Atsushi, Iwabuchi, Tokuro, Harayama, Shigeaki

    Chemosphere   38 ( 6 )   1331 - 1337   1999

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    DOI: 10.1016/s0045-6535(98)00534-7

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  • Use of wastewater sludge for the amendment of crude oil bioremediation in meso-scale beach simulating tanks

    Maki, H, Sasaki, T, Sasaki, E, Ishihara, M, Goto, M, Harayama, S

    Environmental technology   20 ( 6 )   625 - 632   1999

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  • Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge.

    Shigeaki Harayama

    Applied and environmental microbiology   1998.11

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  • Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes.

    Shigeaki Harayama

    International journal of systematic bacteriology   1998.7

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    DOI: 10.1099/00207713-48-3-813

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  • PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes.

    Shigeaki Harayama

    Gene   1998.6

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    DOI: 10.1016/s0378-1119(98)00153-x

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  • Population dynamics of phenol-degrading bacteria in activated sludge determined by gyrB-targeted quantitative PCR.

    Shigeaki Harayama

    Applied and environmental microbiology   1998.4

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  • Biochemical and molecular characterization of 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7.

    Shigeaki Harayama

    The Journal of biological chemistry   1998.4

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    DOI: 10.1074/jbc.273.14.8332

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  • Proton transfer in benzyl alcohol dehydrogenase during catalysis: alternate proton-relay routes.

    Shigeaki Harayama

    Biochemistry   1998.3

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    DOI: 10.1021/bi970726g

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  • Artificial evolution by DNA shuffling.

    Shigeaki Harayama

    Trends in biotechnology   1998.2

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    DOI: 10.1016/s0167-7799(97)01158-x

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  • Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp.

    Shigeaki Harayama

    Applied and environmental microbiology   1998.2

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  • Biochemical and genetic characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase from a phenanthrene-degrading Nocardioides strain.

    Shigeaki Harayama

    Journal of Bacteriology   1998.2

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  • Vanadium as an internal marker to evaluate microbial degradation of crude oil

    Sasaki, Tetsuya, Maki, Hideaki, Ishihara, Masami, Harayama, Shigeaki

    Environmental science & technology   32 ( 22 )   3618 - 3621   1998

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  • Construction of the gyrB Database for the Identification and Classification of Bacteria.

    Shigeaki Harayama

    Genome informatics. Workshop on Genome Informatics   1998

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  • Isolation and characterization of marine Nocardiodes capable of growing and degrading phenanthrene at 42 C

    Iwabuchi, Tokuro, Inomata-Yamauchi, Yukie, Katsuta, Atsuko, Harayama, Shigeaki

    Journal of Marine Biotechnology   6   86 - 90   1998

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  • Rapid estimation of population densities of uncultured bacteria in the environment

    Watanabe, Kazuya, Harayama, Shigeaki

    Microbes and environments   13 ( 3 )   123 - 127   1998

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    Publishing type:Research paper (scientific journal)   Publisher:Japanese Society of Microbial Ecology{\textperiodcentered} The Japanese Society of Soil Microbiology  

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  • Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7.

    Shigeaki Harayama

    Journal of bacteriology   1997.10

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    DOI: 10.1128/jb.179.20.6488-6494.1997

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  • Polycyclic aromatic hydrocarbon bioremediation design.

    Shigeaki Harayama

    Current opinion in biotechnology   1997.6

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    DOI: 10.1016/s0958-1669(97)80002-x

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  • A possible hybrid zone in the Mytilus edulis complex in Japan revealed by PCR markers

    Shigeaki Harayama

    Marine Biology   1997.4

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    DOI: 10.1007/s002270050072

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  • Physicochemical Properties and Biodegradability of Crude Oil

    Shigeaki Harayama

    Environmental Science & Technology   1997.1

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    DOI: 10.1021/es950961r

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  • Expression of two major byssal protein genes during larval development of the mussel Mytilus galloprovincialis

    Takeuchi, Yasuhiro, Inoue, Koji, Miki, Daisuke, Odo, Satoshi, Harayama, Shigeaki

    Fisheries science   63 ( 4 )   648 - 649   1997

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  • Comparison of the biodegradability of four crude oil samples by a single bacterial species and by a microbial population

    Sugiura, K, Ishihara, M, Harayama, S

    Environ. Sci. Technol   31   45 - 51   1997

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  • Application of wastewater sludge to microbial degradation of crude oil

    Maki, Hideaki, Ishihara, M, Harayama, S

    In Situ and On-site Bioremediation   4   371 - 375   1997

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  • Adhesive protein cDNA sequence of the mussel Mytilus coruscus and its evolutionary implications.

    Shigeaki Harayama

    Journal of molecular evolution   1996.10

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    DOI: 10.1007/bf02339009

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  • Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp. strain VM15C.

    Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   1996.7

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    DOI: 10.1271/bbb.60.1056

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  • Cloning, sequencing and sites of expression of genes for the hydroxyarginine-containing adhesive-plaque protein of the mussel Mytilus galloprovincialis.

    Shigeaki Harayama

    European journal of biochemistry   1996.7

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    DOI: 10.1111/j.1432-1033.1996.0172u.x

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  • Characteristics of transposon insertion mutants of mandelic acid-utilizing Pseudomonas putida strain A10L.

    Shigeaki Harayama

    Bioscience, biotechnology, and biochemistry   1996.7

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    DOI: 10.1271/bbb.60.1051

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  • Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products.

    Shigeaki Harayama

    International journal of systematic bacteriology   1996.4

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    DOI: 10.1099/00207713-46-2-506

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  • Construction of bacterial consortia that degrade Arabian light crude oil

    Komukai-Nakamura, Syoko, Sugiura, Keiji, Yamauchi-Inomata, Yukie, Toki, Haruhisa, Venkateswaran, Kasthuri, Yamamoto, Satoshi, Tanaka, Hirki, Harayama, Shigeaki

    Journal of fermentation and bioengineering   82 ( 6 )   570 - 574   1996.1

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    DOI: 10.1016/s0922-338x(97)81254-8

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  • Degradation of crude oil by marine bacteria International journal

    Harayama, Sh

    Oceanographic Literature Review   11 ( 43 )   1162 - 1162   1996

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  • Bioremediation of Oil-Contaminated Coastal Zone

    Shigeaki Harayama

    Restoration and Rehabilitation of the Desert Environment   1996

  • Long-chain n-alkanol dehydrogenase from Pseudomonas putida

    Nagashima, Hiroaki, Inoue, Jun, Sasaki, Etsuro, Yamamoto, Satoshi, Sasaki, Yoshio, Yamauchi-Inomata, Yukie, Harayama, Shigeaki

    Journal of fermentation and bioengineering   82 ( 4 )   328 - 333   1996

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  • Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus.

    Shigeaki Harayama

    The Biological bulletin   1995.12

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    DOI: 10.2307/1542155

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  • PCR Amplification and Direct Sequencing of gyrB Genes with Universal Primers and Their Application to the Detection and Taxonomic Analysis of Pseudomonas putida Strains.

    Shigeaki Harayama

    Applied and environmental microbiology   1995.10

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  • Sequential enrichment of microbial populations exhibiting enhanced biodegradation of crude oil.

    Shigeaki Harayama

    Canadian journal of microbiology   1995.9

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    DOI: 10.1139/m95-106

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  • Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.

    Shigeaki Harayama

    Journal of biochemistry   1995.6

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    DOI: 10.1093/oxfordjournals.jbchem.a124853

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  • Biochemical characterization of a Ca(2+)-dependent lectin from the hemolymph of a photosymbiotic marine bivalve, Tridacna derasa (Röding).

    Shigeaki Harayama

    Journal of biochemistry   1995.5

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  • Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida.

    Shigeaki Harayama

    European journal of biochemistry   1995.4

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    DOI: 10.1111/j.1432-1033.1995.tb20445.x

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  • Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

    Shigeaki Harayama

    Journal of bacteriology   1995.3

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    DOI: 10.1128/jb.177.5.1196-1201.1995

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  • Phylogenetic identification and in situ detection of Pseudomonas bacteria

    Shigeaki Harayama

    Pseudomonas 1995   1995

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  • Experimental simulation of bioremediation of spilled oil

    Shigeaki Harayama

    Bioremediation: the Tokyo ‘94 workshop   1995

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  • Evaluation of crude oil degradation in beach simulator system

    Shigeaki Harayama

    Green Age   1995

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  • Characterization in vitro of the hydroxylase component of xylene monooxygenase, the first enzyme of the TOL-plasmid-encoded pathway for the mineralization of toluene and xylenes

    Shaw, Jeffrey P, Harayama, Shigeaki

    Journal of Fermentation and bioengineering   79 ( 3 )   195 - 199   1995

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  • Biodegradation potentials of Oil-Degrading Microorganisms

    Venkateswaran, Kasthuri, Harayama, Shigeaki

    Geotechnical Applications. Chapter   9   309 - 334   1995

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  • Oil degradation in microcosms and mesocosms

    Ishihara, Masami, Sugiura, Keiji, Asaumi, Masayoshi, Harayama, Shigeaki, Goto, Masafumi, Sasaki, Etsuro

    Microbial processes for bioremediation   1995

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  • Conversion of pBR322-based plasmids into broad-host-range vectors by using the Tn3 transposition mechanism.

    Shigeaki Harayama

    Journal of bacteriology   1994.11

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    DOI: 10.1128/jb.176.21.6566-6571.1994

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  • Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

    Shigeaki Harayama

    Journal of bacteriology   1994.10

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    DOI: 10.1128/jb.176.19.6074-6081.1994

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  • Codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid TOL pWW0 of Pseudomonas putida.

    Shigeaki Harayama

    Journal of molecular evolution   1994.4

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    DOI: 10.1007/bf00163150

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  • Regulation of the rpoN, ORF102 and ORF154 genes in Pseudomonas putida.

    Shigeaki Harayama

    FEMS microbiology letters   1994.1

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    DOI: 10.1016/0378-1097(94)90010-8

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  • Low growth yield of a marine Pseudomonas grown on phenanthrene: A general phenomenon in bacteria grown on polycyclic aromatic hydrocarbons.

    Iwabuchi, T, Harayama, Shigeaki

    J Mar Biotechnol   2   11 - 14   1994

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  • In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.

    Shigeaki Harayama

    The EMBO journal   1993.8

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  • Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO. A member of the zinc-containing long chain alcohol dehydrogenase family.

    Shigeaki Harayama

    The Journal of biological chemistry   1993.5

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  • Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1993.5

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  • Molecular mechanisms of genetic adaptation to xenobiotic compounds.

    Shigeaki Harayama

    Microbiological reviews   1992.12

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  • Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

    Shigeaki Harayama

    European journal of biochemistry   1992.10

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    DOI: 10.1111/j.1432-1033.1992.tb17260.x

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  • 4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer.

    Shigeaki Harayama

    The Journal of biological chemistry   1992.9

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  • Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWW0. Metabolic and mechanistic implications.

    Shigeaki Harayama

    Biochemical Journal   1992.5

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    DOI: 10.1042/bj2830789

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  • cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily.

    Shigeaki Harayama

    European journal of biochemistry   1992.2

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    DOI: 10.1111/j.1432-1033.1992.tb16612.x

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  • Comparison of two hydrocarbon-monooxygenases of Pseudomonas putida. International journal

    Shigeaki Harayama

    Pseudomonas. Molecular Biology, and Biotechnology   1992

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  • Functional and evolutionary relationships among diverse oxygenases.

    Shigeaki Harayama

    Annual review of microbiology   1992

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    DOI: 10.1146/annurev.mi.46.100192.003025

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  • Mutational modification of the substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida

    Shigeaki Harayama

    Pseudomonas: Molecular Biology and Biotechnology   1992

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  • Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases.

    Shigeaki Harayama

    Journal of bacteriology   1991.12

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    DOI: 10.1128/jb.173.23.7540-7548.1991

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  • Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

    Shigeaki Harayama

    Journal of Bacteriology   1991.11

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    DOI: 10.1128/jb.173.22.7219-7227.1991

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  • Induction kinetics of RNA and proteins in exponentially growing organisms.

    Shigeaki Harayama

    Biochemical and biophysical research communications   1991.10

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    DOI: 10.1016/s0006-291x(05)81152-0

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  • DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism.

    Shigeaki Harayama

    Molecular microbiology   1991.10

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    DOI: 10.1111/j.1365-2958.1991.tb02091.x

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  • Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1, 2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

    Neidle, Ellen L, Hartnett, CHRISTOPHER, Ornston, L Nicholas, Bairoch, A, Rekik, Monique, Harayama, Shigeaki

    Journal of Bacteriology   173 ( 17 )   5385 - 5395   1991.9

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    DOI: 10.1128/jb.173.17.5385-5395.1991

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  • Divergent evolution of chloroplast-type ferredoxins.

    Shigeaki Harayama

    FEBS letters   1991.7

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    DOI: 10.1016/0014-5793(91)80730-q

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  • Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system.

    Shigeaki Harayama

    Journal of bacteriology   1991.3

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    DOI: 10.1128/jb.173.5.1690-1695.1991

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  • Identification and characterization of porins in Pseudomonas aeruginosa.

    Shigeaki Harayama

    The Journal of biological chemistry   1991.1

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  • A Pseudomonas putida strain able to degrade m-toluate in the presence of 3-chlorocatechol

    Wasserfallen, Alain, Rekik, Monique, Harayama, Shigeaki

    Bio/Technology   9 ( 3 )   296 - 296   1991

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  • Growth-phase-dependent expression of the Pseudomonas putida TOL plasmid pWW0 catabolic genes.

    Shigeaki Harayama

    Journal of bacteriology   1990.12

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1128/jb.172.12.6651-6660.1990

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  • Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida.

    Shigeaki Harayama

    European journal of biochemistry   1990.8

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    DOI: 10.1111/j.1432-1033.1990.tb19179.x

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  • Effect of motility and chemotaxis on the invasion of Salmonella typhimurium into HeLa cells.

    Shigeaki Harayama

    Microbial pathogenesis   1990.7

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    DOI: 10.1016/0882-4010(90)90039-s

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  • The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1990.3

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    DOI: 10.1007/bf00280375

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  • Nucleotide and deduced amino acid sequence of the RpoN sigma-factor of Pseudomonas putida.

    Shigeaki Harayama

    Nucleic acids research   1989.12

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    DOI: 10.1093/nar/17.23.10125

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  • Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida.

    Shigeaki Harayama

    Journal of bacteriology   1989.11

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    DOI: 10.1128/jb.171.11.6251-6258.1989

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  • Bacterial aromatic ring-cleavage enzymes are classified into two different gene families.

    Harayama, S, Rekik, Monique

    Journal of Biological Chemistry   264 ( 26 )   15328 - 15333   1989.9

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  • Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

    Shigeaki Harayama

    Journal of bacteriology   1989.9

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    DOI: 10.1128/jb.171.9.5048-5055.1989

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  • Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions.

    Shigeaki Harayama

    Journal of bacteriology   1989.8

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    DOI: 10.1128/jb.171.8.4326-4333.1989

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  • A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK.

    Shigeaki Harayama

    Gene   1989.5

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    DOI: 10.1016/0378-1119(89)90310-7

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  • Evolutionary relationships between catabolic pathways for aromatics: conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1987.12

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    DOI: 10.1007/bf00325689

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  • Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants.

    Shigeaki Harayama

    Infection and immunity   1987.9

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  • Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.

    Shigeaki Harayama

    Journal of bacteriology   1987.2

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    DOI: 10.1128/jb.169.2.558-564.1987

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  • Genetic manipulation of catabolic pathways of Pseudomonas.

    Mermod, N, Ramos, RL, Harayama, S, Engesser, KH, Knackmuss, H-J, Timmis, KN

    Dechema-Monographien   105   163 - 178   1987

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  • Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

    Shigeaki Harayama

    Journal of bacteriology   1986.8

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1128/jb.167.2.455-461.1986

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  • Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1986.2

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    DOI: 10.1007/bf00331641

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  • Experimental evolution of plasmid specified functions

    Harayama, S, Ramos, JL, Timmis, KN

    Antibiotic resistance genes: ecology, transfer and expression. Banbury Report   24   389 - 402   1986

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  • Structure, function and evolution of catabolic genes of TOL and NAH plasmids

    Shigeaki Harayama

    Genetics of Industrial Microorganisms   1986

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  • New route to bacterial production of indigo

    Mermod, N, Harayama, S, Timmis, KN

    Bio/Technology   4 ( 4 )   321 - 321   1986

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  • Characterization of light-harvesting mutants of Rhodopseudomonas sphaeroides. I. Measurement of the efficiency of energy transfer from light-harvesting complexes to the reaction center.

    Shigeaki Harayama

    Archives of biochemistry and biophysics   1985.1

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    DOI: 10.1016/0003-9861(85)90612-5

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  • Analysis and manipulation of plasmid-encoded pathways for the catabolism of aromatic compounds by soil bacteria

    Timmis, KN, Lehrbach, PR, Harayama, S, Don, RH, Mermod, N, Bas, S, Leppik, R, Weightman, AJ, Reineke, W, Knackmuss, HJ

    Plasmids in bacteria   719 - 739   1985

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  • Catabolic plasmids: their analysis and utilization in the manipulation of bacterial metabolic activities

    Harayama, S, Don, RH

    Genetic engineering: principles and methods   283 - 307   1985

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  • Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

    Shigeaki Harayama

    Journal of bacteriology   1984.10

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  • Structure of the Trg protein: Homologies with and differences from other sensory transducers of Escherichia coli.

    Shigeaki Harayama

    Proceedings of the National Academy of Sciences of the United States of America   1984.6

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    DOI: 10.1073/pnas.81.11.3287

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  • Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

    Shigeaki Harayama

    Journal of bacteriology   1984.5

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  • Does Tn10 transpose via the cointegrate molecule?

    Shigeaki Harayama

    Molecular & general genetics : MGG   1984

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    DOI: 10.1007/bf00425556

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  • Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1984

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    DOI: 10.1007/bf00436198

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  • The E. coli K-12 chromosome flanked by two IS10 sequences transposes.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1984

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    DOI: 10.1007/bf00327923

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  • Genetic engineering systems for Pseudomonas and their use in the analysis and manipulation of metabolic pathways.

    Harayama, S

    Transferable Antibiotic Resistance: Plasmids and Gene Manipulation   361 - 372   1984

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  • Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning.

    Shigeaki Harayama

    Journal of Bacteriology   1983.1

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  • Search for bacterial strains in laboratory stocks using a microcomputer

    UYEDA, Taro QP, HARAYAMA, Shigeaki

    The Japanese Journal of Genetics   58 ( 3 )   251 - 255   1983

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  • Sensory transduction in bacterial chemotaxis.

    Shigeaki Harayama

    International review of cytology   1983

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  • Cloning of trg, a gene for a sensory transducer in Escherichia coli.

    Shigeaki Harayama

    Journal of bacteriology   1982.10

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  • A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli.

    Shigeaki Harayama

    Journal of bacteriology   1982.9

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  • Studies on the export of the maltose-binding protein and the LamB protein.

    Shigeaki Harayama

    Annales de microbiologie   1982.1

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  • Special features of chemotaxis towards maltose.

    Shigeaki Harayama

    Annales de microbiologie   1982.1

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  • A simple method for constructing recA strains in Escherichia coli

    HARAYAMA, Shigeaki, IINo, Tetsuo

    The Japanese Journal of Genetics   57 ( 2 )   189 - 191   1982

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    Publishing type:Research paper (scientific journal)   Publisher:The Genetics Society of Japan  

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  • The complex nature of methyl-accepting chemotaxis proteins of enteric bacteria

    Hazelbauer, Gerald L, Engstr{\"o}m, Peter, Harayama, Shigeaki

    Biochemistry of S-adenosylmethionine and Related Compounds   83 - 90   1982

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    Publisher:Springer  

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  • Energy is required for maturation of exported proteins in Escherichia coli.

    Shigeaki Harayama

    European journal of biochemistry   1981.5

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1111/j.1432-1033.1981.tb05323.x

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  • Methyl-accepting chemotaxis protein III and transducer gene trg.

    Shigeaki Harayama

    Journal of Bacteriology   1981.1

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  • Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1981

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/bf00271194

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  • Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1981

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/bf00268420

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  • High frequency mobilization of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperature-sensitive for maintenance.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1980

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/bf00267351

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  • Relation of chemotactic response to the amount of receptor: evidence for different efficiencies of signal transduction.

    Shigeaki Harayama

    Journal of bacteriology   1979.6

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  • Mutants in transmission of chemotactic signals from two independent receptors of E. coli.

    Shigeaki Harayama

    Cell   1979.3

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/0092-8674(79)90035-7

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  • Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

    Shigeaki Harayama

    Molecular & general genetics : MGG   1979.3

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/bf00270005

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  • Preferential behavior in bacterial chemotaxis

    Shigeaki Harayama

    Chemoreception and preference behavior   1979

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  • Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

    Shigeaki Harayama

    Journal of bacteriology   1977.7

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  • FERRIC ION AS PHOTORECEPTOR OF PHOTO- PHOBOTAXIS IN NON-PIGMENTED RHODOSPIRILLUM RUBRUM

    Shigeaki Harayama

    Photochemistry and Photobiology   1977.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1111/j.1751-1097.1977.tb09130.x

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  • Phototactic response of aerobically cultivated Rhodospirillum rubrum.

    Shigeaki Harayama

    Journal of general microbiology   1976.5

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1099/00221287-94-1-173

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  • 油脂生産性が増加した真核微細藻類変異体及びその利用

    笠井 由紀,阿部 淳,井出 曜子

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    Application no:特願2021-049173  Date applied:2021.3.23

    Announcement no:特開2022-147782  Date announced:2022.10.6

    Applicant (Organization):学校法人中央大学,株式会社デンソー

  • 油脂生産性が増加した緑藻変異体及びその利用

    早川 准平,井出 曜子

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    Application no:特願2019-202496  Date applied:2019.11.7

    Announcement no:特開2020-074772  Date announced:2020.5.21

    Applicant (Organization):学校法人中央大学,株式会社デンソー

  • クロロフィル含有量が減少し、且つ油脂生産性が増加した緑藻変異体及びその利用

    早川 准平,井出 曜子

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    Application no:特願2019-202388  Date applied:2019.11.7

    Announcement no:特開2020-096583  Date announced:2020.6.25

    Applicant (Organization):学校法人中央大学,株式会社デンソー

  • 油脂分解能が減少し、且つ油脂生産性が増加した緑藻変異体及びその利用

    早川 准平、井出 曜子

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    Application no:特願2018-209336  Date applied:2018.11.7

    Announcement no:特開2020-074703  Date announced:2020.5.21

    Registration no:特許第7269580号  Date registered:2023.4.26 

    Applicant (Organization):学校法人中央大学,株式会社デンソー

  • 微細藻類のエネルギー低消費型培養方法

    中村駿,濱野 隼史

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    Application no:特願2015-233187  Date applied:2015.11.30

    Announcement no:特開2017-099301  Date announced:2017.7.27

    Registration no:特許第6670015号  Date registered:2020.3.3 

    Applicant (Organization):学校法人中央大学