研究者データベース

曾根 輝雄(ソネ テルオ)
農学研究院 連携研究部門 連携推進分野
教授

基本情報

所属

  • 農学研究院 連携研究部門 連携推進分野

職名

  • 教授

ホームページURL

J-Global ID

研究キーワード

  • 応用微生物学   植物—微生物相互作用   Molecular Plant Microbe Interactions   

研究分野

  • ライフサイエンス / 応用微生物学
  • 環境・農学 / 植物保護科学
  • 環境・農学 / 植物保護科学

職歴

  • 2017年08月 - 現在 北海道大学 大学院農学研究院 教授
  • 2008年 - 2012年 放送大学 非常勤講師
  • 2005年 - 2008年 酪農学園大学 非常勤講師
  • 2003年 - 2007年 北海道大学大学院農学研究科 講師
  • 2003年 - 2007年 Lecturer
  • 2007年 - 北海道大学大学院農学研究院 准教授
  • 2004年 - 2006年 大阪大学生物工学国際交流センター 招へい助教授
  • 2002年 - 2005年 札幌科学技術専門学校生物工学科 非常勤講師
  • 2001年 - 2003年 北海道大学大学院農学研究科 助手
  • 2001年 - 2003年 Research Associate
  • 1997年 - 1998年 Department of Botany, University of Britich Columbia Postdoctoral Fellow

学歴

  •         - 1997年   北海道大学   農学研究科   博士課程(農芸化学専攻)
  •         - 1997年   北海道大学
  •         - 1994年   北海道大学   農学研究科   修士課程(農芸化学専攻)
  •         - 1994年   北海道大学
  •         - 1992年   北海道大学   農学部   農芸化学科
  •         - 1992年   北海道大学

所属学協会

  • 日本農芸化学会   日本生物工学会   日本生物工学会北日本支部   糸状菌分子生物学研究会   日本植物病理学会   

研究活動情報

論文

  • Shiori Yamamoto, Wataru Kitagawa, Motoki Nakano, Hiroshi Asakura, Tatsuya Nakayama, Eriko Iwabuchi, Teruo Sone, Kozo Asano
    Current microbiology 79 8 217 - 217 2022年06月15日 
    Gentamicin is an important antibiotic for the treatment of opportunistic infections in the clinical field. Gentamicin-resistant bacteria have been detected in livestock animals and can be transmitted to humans through the food supply or direct contact. We have previously revealed that gentamicin-resistant Escherichia coli are distributed at a comparatively high rate from beef cattle in Japan, but few studies have focused on the molecular epidemiology of gentamicin-resistant bacteria. To understand these bacteria, this study examined the prevalence of various gentamicin resistance genes in gentamicin-resistant E. coli isolates from beef cattle feces. Of the 239 gentamicin-resistant E. coli isolates, the presence of the aacC2, aadB, or aac(3)-VIa genes was confirmed in 147, 84, and 8 isolates, respectively. All aac(3)-VIa-harboring isolates had an MIC value of 64 μg/mL for gentamicin and exhibited resistance to 11 antibiotic agents. An analysis of the representative aac(3)-VIa-harboring E. coli strain GC1-3-GR-4 revealed that the aac(3)-VIa gene was present on the IncA/C plasmid together with the aadA and blaCMY genes. Furthermore, the upstream region of the aac(3)-VIa gene contained the aadA gene and the class 1 integron-integrase gene (intI1). The aac(3)-VIa gene was detected for the first time in Japan and is expected to be able to transfer between bacteria via the IncA/C plasmid and integron. These results reveal the expansion of the distribution or diversity of gentamicin resistance genes in Japan.
  • Pradabrat Prajanket, Kim-Chi Thi Vu, Jun Arai, Worawan Sornkom, Ayumi Abe, Teruo Sone
    Bioscience, Biotechnology, and Biochemistry 1 - 4 2020年07月30日 [査読有り][通常論文]
  • Muhammad Akhid Syib’li, Aoi Kodama, Ayumi Abe, Teruo Sone
    Journal of General Plant Pathology 86 4 250 - 256 2020年07月 [査読有り][通常論文]
  • Tetsuo Nakano, Yuki Ohara, Hiroshi Fujita, Akira Ainai, Ei-Tora Yamamura, Tadaki Suzuki, Hideki Hasegawa, Teruo Sone, Kozo Asano
    DNA and Cell Biology 39 9 1730 - 1740 2020年06月22日 [査読有り][通常論文]
     
    Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.
  • Shiori Yamamoto, Wataru Kitagawa, Motoki Nakano, Hiroshi Asakura, Eriko Iwabuchi, Teruo Sone, Kozo Asano
    Microbiology resource announcements 9 20 2020年05月14日 [査読有り][通常論文]
     
    Escherichia coli is a common reservoir for antimicrobial resistance genes that can be easily transformed to possess multidrug resistance through plasmid transfer. To understand multidrug resistance plasmids, we report the plasmid sequences of four large plasmids carrying a number of genes related to antimicrobial resistance that were found in E. coli strains isolated from beef cattle.
  • Shiori Goto, Tsubasa Ohbayashi, Kazutaka Takeshita, Teruo Sone, Yu Matsuura, Peter Mergaert, Yoshitomo Kikuchi
    Microbes and Environments 35 4 n/a - n/a 2020年
  • Goto Shiori, Ohbayashi Tsubasa, Takeshita Kazutaka, Sone Teruo, Matsuura Yu, Mergaert Peter, Kikuchi Yoshitomo
    Microbes and environments 35 4 n/a  日本微生物生態学会 / 日本土壌微生物学会 / Taiwan Society of Microbial Ecology / 植物微生物研究会 / 極限環境微生物学会 2020年 

    Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.

  • Tsubasa Ohbayashi, Ryo Futahashi, Mia Terashima, Quentin Barrière, Florian Lamouche, Kazutaka Takeshita, Xian-Ying Meng, Yasuo Mitani, Teruo Sone, Shuji Shigenobu, Takema Fukatsu, Peter Mergaert, Yoshitomo Kikuchi
    The ISME Journal 13 6 1469 - 1483 2019年06月 [査読有り][通常論文]
     
    In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.
  • Souichiro Kato, Kaoru Wada, Wataru Kitagawa, Daisuke Mayumi, Masayuki Ikarashi, Teruo Sone, Kozo Asano, Yoichi Kamagata
    Microbes and environments 34 1 95 - 98 2019年03月30日 [査読有り][通常論文]
     
    Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.
  • Tetsuo Nakano, Ei-Tora Yamamura, Hiroshi Fujita, Teruo Sone, Kozo Asano
    Bioscience, Biotechnology, and Biochemistry 82 11 1889 - 1901 2018年11月02日 [査読有り][通常論文]
  • Kato S, Yamagishi A, Daimon S, Kawasaki K, Tamaki H, Kitagawa W, Abe A, Tanaka M, Sone T, Asano K, Kamagata Y
    Applied and environmental microbiology 84 19 2018年10月01日 [査読有り][通常論文]
     
    Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.
  • Takeshita Kazutaka, Tamaki Hideyuki, Ohbayashi Tsubasa, Meng Xian-Ying, Sone Teruo, Mitani Yasuo, Peeters Charlotte, Kikuchi Yoshitomo, Vandamme Peter
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 68 7 2370 - 2374 2018年07月 [査読有り][通常論文]
     
    A Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacterium, designated strain RPE64T, was isolated from the gut symbiotic organ of the bean bug Riptortus pedestris, collected in Tsukuba, Japan, in 2007. 16S rRNA gene sequencing showed that this strain belongs to the Burkholderia glathei clade, exhibiting the highest sequence similarity to Burkholderia peredens LMG 29314T (100 %), Burkholderia turbans LMG 29316T (99.52 %) and Burkholderia ptereochthonis LMG 29326T (99.04 %). Phylogenomic analyses based on 107 single-copy core genes and Genome blast Distance Phylogeny confirmed B. peredens LMG 29314T, B. ptereochthonis LMG 29326T and several uncultivated, endophytic Burkholderia species as its nearest phylogenetic neighbours. Digital DNA-DNA hybridization experiments unambiguously demonstrated that strain RPE64T represents a novel species in this lineage. The G+C content of its genome was 63.2 mol%. The isoprenoid quinone was ubiquinone 8 and the predominant fatty acid components were C16 : 0, C18 : 1ω7c and C17 : 0 cyclo. The absence of nitrate reduction and the capacity to grow at pH 8 clearly differentiated strain RPE64T from related Burkholderia species. Based on these genotypic and phenotypic characteristics, strain RPE64T is classified as representing a novel species of the genus Burkholderia, for which the name Burkholderia insecticola sp. nov. is proposed. The type strain is RPE64T (=NCIMB 15023T=JCM 31142T).
  • Andre Ohara, Yui Tashika, Ayumi Abe, Teruo Sone
    Journal of General Plant Pathology 84 3 176 - 188 2018年05月01日 [査読有り][通常論文]
     
    Appressorium differentiation, one of the most important steps in pathogenesis by the rice blast fungus, Pyricularia oryzae, is strongly coordinated with the cell cycle. In this study, we identified an ortholog gene of 53BP1, which encodes a signal transducer protein that participates in G2-M cell cycle checkpoint in higher eukaryotes, in the genome of P. oryzae and characterized the phenotype of deletion and overexpression mutants. Deletion mutants showed no significant deficiency in vegetative growth compared to wild-type and complemented strains, even on the media containing DNA-damaging agents. However, these null mutants had abnormal appressoria and formed more appressoria per conidium than in the wild type and were unable to penetrate the epidermis of rice leaves. eGFP-fused Mop53BP1 and qRT-PCR analyses revealed that Mop53BP1 is expressed during the first hours of appressorium formation. In addition, in overexpression mutants, Mop53BP1 localized to nuclei during all stages of appressorium maturation and penetration, and the mutants were resistant to the microtubule inhibitor benomyl, suggesting that Mop53BP1 is nuclear protein and may have some role related to microtubules.
  • Worawan Sornkom, Shinsuke Miki, Saori Takeuchi, Ayumi Abe, Kozo Asano, Teruo Sone
    MOLECULAR PLANT PATHOLOGY 18 8 1138 - 1149 2017年10月 [査読有り][通常論文]
     
    In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (pls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.
  • Siriporn Chaikaew, Sasitorn Baipong, Teruo Sone, Apinun Kanpiengjai, Naradorn Chui-chai, Kozo Asano, Chartchai Khanongnuch
    JOURNAL OF MICROBIOLOGY 55 9 720 - 729 2017年09月 [査読有り][通常論文]
     
    The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041(T). Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.
  • Souichiro Kato, Yoshiki Kanata, Wataru Kitagawa, Teruo Sone, Kozo Asano, Yoichi Kamagata
    Scientific reports 7 1 1965 - 1965 2017年05月16日 [査読有り][通常論文]
     
    Biological incorporation of cesium ions (Cs+) has recently attracted significant attention in terms of the possible applications for bioremediation of radiocesium and their significant roles in biogeochemical cycling. Although high concentrations of Cs+ exhibit cytotoxicity on microorganisms, there are a few reports on the promotive effects of Cs+ on microbial growth under K+-deficient conditions. However, whether this growth-promoting effect is a common phenomenon remains uncertain, and direct correlation between growth promotion and Cs+ uptake abilities has not been confirmed yet. Here, we validated the growth promotive effects of Cs+ uptake under K+-deficient conditions using an Escherichia coli strain with an inducible expression of the Kup K+ transporter that has nonspecific Cs+ transport activities (strain kup-IE). The strain kup-IE exhibited superior growth under the Cs+-supplemented and K+-deficient conditions compared to the wild type and the kup null strains. The intracellular Cs+ levels were significantly higher in strain kup-IE than in the other strains, and were well correlated with their growth yields. Furthermore, induction levels of the kup gene, intracellular Cs+ concentrations, and the growth stimulation by Cs+ also correlated positively. These results clearly demonstrated that Cs+ incorporation via Kup transporter restores growth defects of E. coli under K+-deficient conditions.
  • Kazutaka Takeshita, Yu Matsuura, Hideomi Itoh, Ronald Navarro, Tomoyuki Hori, Teruo Sone, Yoichi Kamagata, Peter Mergaert, Yoshitomo Kikuchi
    Microbes and Environments 30 4 321 - 329 2015年12月22日 [査読有り][通常論文]
     
    A number of phytophagous stinkbugs (order Heteroptera: infraorder Pentatomomorpha) harbor symbiotic bacteria in a specific midgut region composed of numerous crypts. Among the five superfamilies of the infraorder Pentatomomorpha, most members of the Coreoidea and Lygaeoidea are associated with a specific group of the genus Burkholderia, called the “stinkbugassociated beneficial and environmental (SBE)” group, which is not vertically transmitted, but acquired from the environment every host generation. A recent study reported that, in addition to these two stinkbug groups, the family Largidae of the superfamily Pyrrhocoroidea also possesses a Burkholderia symbiont. Despite this recent finding, the phylogenetic position and biological nature of Burkholderia associated with Largidae remains unclear. Based on the combined results of fluorescence in situ hybridization, cloning analysis, Illumina deep sequencing, and egg inspections by diagnostic PCR, we herein demonstrate that the largid species are consistently associated with the “plant-associated beneficial and environmental (PBE)” group of Burkholderia, which are phylogenetically distinct from the SBE group, and that they maintain symbiosis through the environmental acquisition of the bacteria. Since the superfamilies Coreoidea, Lygaeoidea, and Pyrrhocoroidea are monophyletic in the infraorder Pentatomomorpha, it is plausible that the symbiotic association with Burkholderia evolved at the common ancestor of the three superfamilies. However, the results of this study strongly suggest that a dynamic transition from the PBE to SBE group, or vice versa, occurred in the course of stinkbug evolution.
  • Toyoyuki Ose, Azusa Oikawa, Yukiko Nakamura, Katsumi Maenaka, Yuya Higuchi, Yuki Satoh, Shiho Fujiwara, Makoto Demura, Teruo Sone, Masakatsu Kamiya
    JOURNAL OF BIOMOLECULAR NMR 63 2 229 - 235 2015年10月 [査読有り][通常論文]
  • Worawan Sornkom, Kozo Asano, Teruo Sone
    Engineering Journal 19 3 85 - 93 2015年06月05日 [査読有り][通常論文]
     
    Rice blast disease, caused by Magnaporthe oryzae, is a major rice disease over the world. Recent studies have identified avirulence effectors from the blast fungus that trigger rice immune against the pathogen invasion after specific interaction with resistance (R) proteins in rice. AVR-Pia is one of avirulence effectors that correspond to Pia-resistant protein, inducing hypersensitive response (HR). Enhanced Green fluorescent protein (eGFP) was used as a reporter of AVR-Pia expression in this study. We synthesized expression vector containing native promoter of AVR-Pia (PRR) fused to eGFP gene. Rice sheath assay was done to observe the fluorescence and the signal was found in appressoria and invasive hypha of M. oryzae, suggesting that AVR-Pia is expressed in appressorium and invasive hyphae after penetration. Although, rice sheath assay is a reliable way to study rice-pathogen interaction, it is a consuming-time method. Onion epidermis was tested to check the availability to use as model system instead of rice sheath. After inoculation M. oryzae containing PPR::eGFP on onion epidermis, fluorescence was observed in appressoria and invasive hypha of transformants similar to observation on rice sheath. Therefore, onion epidermis can be used as plant cell model to study M. oryzae effectors expression by fluorescence observation.
  • Teruo Sone, Yumiko Haraguchi, Aki Kuwahara, Toyoyuki Ose, Megumi Takano, Ayumi Abe, Michiko Tanaka, Isao Tanaka, Kozo Asano
    Protein and Peptide Letters 22 1 63 - 72 2015年 [査読有り][通常論文]
     
    Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.
  • Shiori Yamamoto, Motoki Nakano, Wataru Kitagawa, Michiko Tanaka, Teruo Sone, Katsuya Hirai, Kozo Asano
    MICROBES AND ENVIRONMENTS 29 2 136 - 144 2014年07月 [査読有り][通常論文]
     
    The emergence of multiple-antibiotic-resistance bacteria is increasing, which is a particular concern on livestock farms. We previously isolated 1,347 antimicrobial-resistant (AMR) Escherichia coli strains from the feces of beef cattle on 14 Japanese farms. In the present study, the genetic backgrounds and phylogenetic relationships of 45 AMR isolates were characterized by the chromosome phylotype, AMR phenotype, AMR genotype, and plasmid type. These isolates were classified into five chromosome phylotypes, which were closely linked to the farms from which they were isolated, suggesting that each farm had its own E. coli phylotype. AMR phenotype and plasmid type analyses yielded 8 and 14 types, all of which were associated with the chromosomal phylotype and, thus, to the original farms. AMR genotype analysis revealed more variety, with 16 types, indicating both inter- and intra-farm diversity. Different phylotype isolates from the same farm shared highly similar plasmid types, which indicated that plasmids with AMR genes could be transferred between phylotypes, thereby generating multi-antibiotic-resistant microorganisms. This ecological study demonstrated that the chromosome phylotype was strongly correlated with the farm from which they were isolated, while the AMR phenotype, genotype, and plasmid type were generally correlated with the chromosome phylotype and farm source.
  • Yuki Satoh, Shinsuke Miki, Toyoyuki Ose, Azusa Oikawa, Katsumi Maenaka, Ryouhei Terauchi, Kozo Asano, Teruo Sone
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 4 680 - 686 2014年04月 [査読有り][通常論文]
     
    The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.
  • Sri Pudjiraharti, Midori Ohtani, Nanami Takano, Ayumi Abe, Puspita Lisdiyanti, Michiko Tanaka, Teruo Sone, Kozo Asano
    JOURNAL OF ANTIBIOTICS 67 2 137 - 141 2014年02月 [査読有り][通常論文]
     
    The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 degrees C, optimum pH between 5.5 and 6.0 and remained stable up to 70 degrees C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.
  • Kentaro Funo, Wataru Kitagawa, Michiko Tanaka, Teruo Sone, Kozo Asano, Yoichi Kamagata
    Genome Announcements 2 1 2014年 [査読有り][通常論文]
     
    Tomitella biformata AHU 1821T was isolated and cultured from a permafrost ice wedge, aged presumably about 25,000 years, in the Fox permafrost tunnel (64.952°N 147.617°W), Alaska. These genome data provide the basis for investigating T. biformata AHU 1821T, identified as a long-term survivor of the extremely cold and closed environment.
  • Takahiro Kugo, Wataru Kitagawa, Yoshinori Shimomura, Takuya Yamagishi, Michiko Tanaka, Teruo Sone, Kozo Asano, Yoichi Kamagata
    Genome Announcements 2 2 2014年 [査読有り][通常論文]
     
    Methylophilus sp. strain OH31 was isolated from the sediment of the Ohno pond at Hokkaido University. Strain OH31 utilizes methanol as its energy source. Here, we present the draft genome sequence of Methylophilus sp. strain OH31.
  • Hiroaki Nakata, Hiroaki Sakurai, Taizo Nagura, Michiko Tanaka, Teruo Sone, Kozo Asano
    MYCOSCIENCE 54 4 247 - 251 2013年07月 [査読有り][通常論文]
     
    An alpha-galactosidase for raffinose synthesis by a reverse hydrolysis reaction was searched from intracellular of endophric fungi. About 500 strains were screened, one strain was selected and identified as Paraphaeosphaeria sp. Partially purified alpha-galactosidase (1 U/ml in the reaction mixture), 100 g/L galactose, and 500 g/L sucrose at 60 degrees C for 48 h resulted in synthesis of 13.3 g/L raffinose and 4.6 g/L planteose at a ratio of about 3:1. The data suggest that alpha-galactosidase was able to synthesize raffinose at a ratio higher than that of alpha-galactosidases derived from other fungi. (C) 2012 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
  • Ida Bagus Wayan Gunam, Kenta Yamamura, I Nengah Sujaya, Nyoman Semadi Antara, Wayan Redi Aryanta, Michiko Tanaka, Fusao Tomita, Teruo Sone, Kozo Asano
    Journal of microbiology and biotechnology 23 4 473 - 82 2013年04月 [査読有り][通常論文]
     
    The desulfurization ability of Sphingomonas subarctica T7b was evaluated using resting and immobilized cells with dibenzothiophene (DBT), alkyl DBTs, and commercial light gas oil (LGO) as the substrates. The resting cells of S. subarctica T7b degraded 239.2 mg of the initial 250 mg of DBT/l (1.36 mM) within 24 h at 27 degrees C, while 127.5 mg of 2-hydroxybiphenyl (2-HBP)/l (0.75 mM) was formed, representing a 55% conversion of the DBT. The DBT desulfurization activity was significantly affected by the aqueous-to-oil phase ratio. In addition, the resting cells of S. subarctica T7b were able to desulfurize alkyl DBTs with long alkyl chains, although the desulfurization rate decreased with an increase in the total carbon number of the alkylated DBTs. LGO with a total sulfur content of 280 mg/l was desulfurized to 152 mg/l after 24 h of reaction. Cells immobilized by entrapment with polyvinyl alcohol (PVA) exhibited a high DBT desulfurization activity, including repeated use for more than 8 batch cycles without loss of biodesulfurization activity. The stability of the immobilized cells was better than that of the resting cells at different initial pHs, higher temperatures, and for DBT biodesulfurization in successive degradation cycles. The immobilized cells were also easily separated from the oil and water phases, giving this method great potential for oil biodesulfurization.
  • Teruo Sone, Saori Takeuchi, Shinsuke Miki, Yuki Satoh, Keisuke Ohtsuka, Ayumi Abe, Kozo Asano
    FEMS microbiology letters 339 2 102 - 9 2013年02月 [査読有り][通常論文]
     
    AVR-Pia, an avirulence gene in the genome of the rice blast fungus Magnaporthe oryzae, triggers a hypersensitive reaction in rice cultivars harbouring the resistance gene Pia. The copy number of AVR-Pia was revealed to vary from one to three among M. oryzae isolates avirulent to Pia rice, and three copies of the gene were located on a single chromosome in strain Ina168, from which the gene was originally cloned. The spontaneous avr-Pia mutant originated from Ina168, named Ina168m95-1, which lacks the AVR-Pia gene, and was therefore used to elucidate the molecular mechanism of the deletion of all three copies of AVR-Pia. Screening and analysis of cosmid clones indicated that two copies of the DNA-type transposon Occan (Occan(9E12) and Occan(3A3) ) were located on the same chromosome, and three copies of AVR-Pia were located in between the two Occan elements. Ina168m95-1 contains a conserved Occan element, named Occan(m95-1) , between sequences homologous to the 5'-flanking region of Occan(3A3) and the 3'-flanking region of Occan(9E12) . In addition, sequence polymorphisms indicated a homologous recombination between Occan(3A3) and Occan(9E12) , which resulted in Occan(m95-1) . Based on these observations, we propose the hypothesis that homologous recombination in the two Occan elements leads to the deletion of AVR-Pia in Ina168m95-1.
  • Ni Wayan Arya Utami, Teruo Sone, Michiko Tanaka, Cindy H Nakatsu, Akihiko Saito, Kozo Asano
    Bioscience of microbiota, food and health 32 4 167 - 78 2013年 [査読有り][通常論文]
     
    The yacon (Smallanthus sonchifolius) tuber was examined with regard to its prebiotic effects compared with commercialized fructo-oligosaccharides (FOS). A feed containing 10% yacon tuber, which is equivalent to 5% commercialized FOS in terms of the amount of fructo-oligosaccharides (GF2, GF3 and GF4), was administrated to rats for 28 days. The yacon diet changed the intestinal microbial communities beginning in the first week, resulting in a twofold greater concentration of cecal short-chain fatty acids (SCFAs). The SCFA composition differed, but the cecal pH in rats fed yacon tuber was equal to that in rats fed FOS. Serum triglycerides were lower in rats fed yacon compared with rats fed FOS and the control diet. Cecal size was greater with the yacon tuber diet compared with the control diet. The abundant fermentation in the intestines created a selective environment for the intestinal microbiota, which included Lactobacillus acidophilus, Bifidobacterium pseudolongum, Bifidobacterium animalis and Barnesiella spp. according to identification with culture-independent analysis, 16S rRNA gene PCR-DGGE combined with cloning and sequencing. Barnesiella spp. and B. pseudolongum were only found in the rats fed the yacon diet, while L. acidophilus and B. animalis were found in abundance in rats fed both the yacon and FOS diets. The genus Barnesiella has not previously been reported to be associated with yacon or FOS fermentation. We concluded that the physiological and microbiological effects of the yacon tuber were different from those of FOS. Differences in cecal size, blood triglycerides and microbial community profiles including their metabolites (SCFAs) between the yacon tuber and FOS were shown to be more greatly affected by the yacon tuber rather than FOS.
  • Yamamoto S, Nakano M, Tanaka M, Kitagawa W, Sone T, Asano K
    International Journal of Antimicrobial Agents 42 S84  2013年 [査読有り][通常論文]
  • Lotis Escobin-Mopera, Midori Ohtani, Sachie Sekiguchi, Teruo Sone, Ayumi Abe, Michiko Tanaka, Vithaya Meevootisom, Kozo Asano
    Journal of bioscience and bioengineering 113 5 562 - 7 2012年05月 [査読有り][通常論文]
     
    Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.
  • 岩井 崇郎, 伊藤 由美, 工藤 綾子, 田中 みち子, 阿部 歩, 曾根 輝雄, 城間 力, 徳安 健, 矢部 希見子, 長島 實, 浅野 行蔵
    バイオマス科学会議発表論文集 7 46 - 47 一般社団法人日本エネルギー学会 2012年 
    Rice straw is a new resource of second-generation bioethanol. We estimated that endophyte (microorganisms living in plant without sympton) secrete cell wall degrading enzyme when invade into host plant and they may be useful enzyme resources. Result of screening of endophytes, extracellular enzyme from 4 strains increased xylan saccharification. Especially, enzymes cocktail of commercial enzymes and enzymes from Fusarium sp. E12-4CA can degrade xylan more than 90%. Crude enzymes from 4 endophytes indicated high hemicellulase activities, so the possibility of synergistic saccharification of commercial enzymes and the crude enzymes.
  • Edmundo Sanchez, Kozo Asano, Teruo Sone
    JOURNAL OF GENERAL PLANT PATHOLOGY 77 4 239 - 242 2011年07月 [査読無し][通常論文]
     
    From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.
  • Sri Pudjiraharti, Nobuchika Takesue, Taiki Katayama, Puspita Lisdiyanti, Muhamad Hanafi, Michiko Tanaka, Teruo Sone, Kozo Asano
    Journal of bioscience and bioengineering 111 6 671 - 4 2011年06月 [査読有り][通常論文]
     
    An actinomycete that excretes inulin fructotransferase to the culture supernatant was able to produce di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) from inulin, with the greatest rate of enzyme activity at 65°C and at a pH of 5.5. Through chemotaxonomic and 16S rRNA gene analysis, this strain was identified as genus Nonomuraea in the Streptosporangiaceae family. This is the first report of an inulin fructotransferase producer in this family.
  • Yudai Okuyama, Hiroyuki Kanzaki, Akira Abe, Kentaro Yoshida, Muluneh Tamiru, Hiromasa Saitoh, Takahiro Fujibe, Hideo Matsumura, Matt Shenton, Dominique Clark Galam, Jerwin Undan, Akiko Ito, Teruo Sone, Ryohei Terauchi
    The Plant journal : for cell and molecular biology 66 3 467 - 79 2011年05月 [査読有り][通常論文]
     
    The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.
  • Sali Atanga Ndindeng, Shinsuke Miki, Ayumi Abe, Kozo Asano, Teruo Sone
    JOURNAL OF GENERAL PLANT PATHOLOGY 76 6 377 - 381 2010年12月 [査読無し][通常論文]
     
    To detect and count DNA double-strand breaks (DSBs) during the life cycle of Magnaporthe oryzae, we constructed an expression vector for EGFP-Rhm51 fusion protein and introduced it into strain Ina86-137. EGFP-Rhm51 foci were detected, and the number of foci in mycelia treated with mitomycin C, which induces DSBs, was higher than in untreated samples, indicating that the foci enabled the visualization of DSBs during the life cycle. EGFP-Rhm51 foci were observed at all stages of the asexual cycle including when invasive hyphae formed in an intact rice leaf sheath, demonstrating that M. oryzae undergoes DSBs during vegetative and parasitic growth.
  • Taiki Katayama, Tomoko Kato, Michiko Tanaka, Thomas A Douglas, Anatoli Brouchkov, Ayumi Abe, Teruo Sone, Masami Fukuda, Kozo Asano
    International journal of systematic and evolutionary microbiology 60 Pt 12 2803 - 7 2010年12月 [査読有り][通常論文]
     
    Gram-reaction-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated AHU1821(T) and AHU1820, were isolated from an ice wedge in the Fox permafrost tunnel, Alaska. The strains were psychrophilic, growing at -5 to 27°C. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences indicated that the ice-wedge isolates formed a clade distinct from other mycolic-acid-containing bacteria within the suborder Corynebacterineae. The cell wall of strains AHU1821(T) and AHU1820 contained meso-diaminopimelic acid, arabinose and galactose, indicating chemotype IV. The muramic acids in the peptidoglycan were glycolated. The predominant menaquinone was MK-9(H(2)). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified glycolipid. The major fatty acids were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)), octadecenoic acid (C(18 : 1)) and tetradecanoic acid (C(14 : 0)). Tuberculostearic acid was present in relatively small amounts (1 %). Strains AHU1821(T) and AHU1820 contained mycolic acids with 42-52 carbons. The DNA G+C content of the two strains was 69.3-71.6 mol% (T(m)). 16S rRNA, rpoB and recA gene sequences were identical between strains AHU1821(T) and AHU1820 and those of the gyrB gene showed 99.9 % similarity. Based on phylogenetic and phenotypic evidence, strains AHU1821(T) and AHU1820 represent a single novel species of a novel genus, for which the name Tomitella biformata gen. nov., sp. nov. is proposed. The type strain of Tomitella biformata is AHU1821(T) (=DSM 45403(T) =NBRC 106253(T)).
  • Mami Takahashi, Taketo Ashizawa, Kazuyuki Hirayae, Jouji Moriwaki, Teruo Sone, Ryoichi Sonoda, Masako Tsujimoto Noguchi, Susumu Nagashima, Kouji Ishikawa, Michiyoshi Arai
    Phytopathology 100 6 612 - 8 2010年06月 [査読有り][通常論文]
     
    We analyzed the avirulence gene AVR-Pita1 in Japanese rice blast isolates to determine how they gain virulence toward rice cultivars containing the Pita resistance gene. An avirulent isolate, OS99-G-7a (G7a), from a Japanese commercial field contained two paralogs of AVR-Pita1, designated as AVR-Pita1(JA) and AVR-Pita1(JB). Analysis of virulent, independent mutants derived from G7a, a single avirulent progenitor strain, indicated that AVR-Pita1(JA) was functional but AVR-Pita1(JB) was nonfunctional. The most frequent mutation was loss of AVR-Pita1(JA). Analyses of field isolates collected from diverse areas in Japan revealed that most of the AVR-Pita1 genes carried by Japanese isolates were identical to AVR-Pita1(JA) or AVR-Pita1(JB). The relationship between these major paralogs in Japanese isolates and the virulence of the strains carrying them indicate that AVR-Pita1(JA) is functional but AVR-Pita1(JB) is not, as is the case in G7a. Isolates that show virulence toward rice cultivars containing the Pita gene are presumed to have evolved virulence from avirulent origins via loss of AVR-Pita1(JA), except for one case in which virulence resulted from a base substitution. In this study, we discuss the properties and specificities of Japanese rice blasts that relate to virulence against Pita-containing rice. Furthermore, we present a method to amplify AVR-Pita1(JA) and AVR-Pita1(JB) separately and, specifically, to monitor functional AVR-Pita1 in Japan.
  • Noriko Ishida, Daisuke Irikura, Kazuhiro Matsuda, Seiji Sato, Teruo Sone, Michiko Tanaka, Kozo Asano
    Journal of bioscience and bioengineering 109 4 341 - 5 2010年04月 [査読有り][通常論文]
     
    A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.
  • Ayumi Abe, Kozo Asano, Teruo Sone
    Bioscience, biotechnology, and biochemistry 74 7 1325 - 31 2010年 [査読有り][通常論文]
     
    In order to establish the molecular taxonomy of the genus Rhizopus, all described species of the genus were collected and the nucleotide sequences of the internal transcribed spacer of the rRNA gene (rDNA ITS), actin, and translation elongation factor 1alpha (EF-1alpha) were determined. Quantitative real-time PCR revealed that R. americanus had a R. stolonifer-type ITS sequence as the dominant sequence type, although it had three different types of ITS sequences in a single genome. Phylogenetic analysis and gene genealogy concordance phylogenetic species recognition (GCPSR) identified eight species in the genus, whereas recent morphological taxonomy includes 10 species. R. niveus is proposed to be re-classified as R. delemar, and R. sexualis and R. americanus are re-classified as R. stolonifer.
  • Martin Patrick Ongol, Michiko Tanaka, Teruo Sone, Kozo Asano
    FOOD RESEARCH INTERNATIONAL 42 8 893 - 898 2009年10月 [査読無し][通常論文]
     
    A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein, rimM, for specific detection of Streptococcus thermophilus was developed. The designed real-time PCR primers and probe were specific for S. thermophilus JCM20026, LMG6896, LMG18311, OJT101, OJT102 but not Enteroccocus spp., Lactococcus lactis subsp. lactis, and Streptococcus salivarius which are phylogenetically closely related to S. thennophilus and are difficult to identify using culture-based methods. The linear range of the developed real-time PCR method was from 2.7 to 8.6 log CFU ml(-1) with an amplification efficiency of 96%. Minor differences (about 0.4 log CFU ml(-1)) were observed between counts of S. thermophilus obtained by culture and real-time PCR method in plain yoghurt and yoghurt containing fruits. Therefore, the developed real-time PCR method could be of potential application in specific detection and accurate enumeration of S. thermophilus in a wide range of dairy products. (C) 2009 Elsevier Ltd. All rights reserved.
  • Ayumi Abe, Kozo Asano, Teruo Sone
    Bioscience, biotechnology, and biochemistry 73 8 1860 - 2 2009年08月 [査読有り][通常論文]
     
    A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.
  • Li-Jun Ma, Ashraf S Ibrahim, Christopher Skory, Manfred G Grabherr, Gertraud Burger, Margi Butler, Marek Elias, Alexander Idnurm, B Franz Lang, Teruo Sone, Ayumi Abe, Sarah E Calvo, Luis M Corrochano, Reinhard Engels, Jianmin Fu, Wilhelm Hansberg, Jung-Mi Kim, Chinnappa D Kodira, Michael J Koehrsen, Bo Liu, Diego Miranda-Saavedra, Sinead O'Leary, Lucila Ortiz-Castellanos, Russell Poulter, Julio Rodriguez-Romero, José Ruiz-Herrera, Yao-Qing Shen, Qiandong Zeng, James Galagan, Bruce W Birren, Christina A Cuomo, Brian L Wickes
    PLoS genetics 5 7 e1000549  2009年07月 [査読有り][通常論文]
     
    Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.
  • Charin Thawornkuno, Michiko Tanaka, Teruo Sone, Kozo Asano
    Bioscience, biotechnology, and biochemistry 73 6 1435 - 8 2009年06月 [査読有り][通常論文]
     
    Asaccharobacter celatus AHU1763 is a Gram-positive, obligate anaerobic, non-spore forming, rod-shaped bacteria that was successfully isolated from rat cecal content. Daizein was converted to equol via dihydrodaidzein by this bacterium. A crude enzyme that converted daidzein to dihydrodaidzein was detected mainly in the culture supernatant. The ability of this enzyme dropped after the culture supernatant was exposed to a normal atmospheric environment for even 5 min. Furthermore, the enzyme responsible for changing dihydrodaidzein to equol was detected mainly in the cell debris, which required anaerobic conditions for its activity.
  • Noriko Ishida, Daisuke Irikura, Kazuhiro Matsuda, Seiji Sato, Teruo Sone, Michiko Tanaka, Kozo Asano
    Current microbiology 58 6 535 - 40 2009年06月 [査読有り][通常論文]
     
    A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.
  • TAKESUE Nobuchika, SONE Teruo, TANAKA Michiko, TOMITA Fusao, ASANO Kozo
    Journal of Bioscience and Bioengineering 107 6 623 - 9 2009年06月 [査読有り][通常論文]
     
    Di-D-fructofuranosyl 2,6':2',6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.
  • Shinsuke Miki, Kotaro Matsui, Hideki Kito, Keisuke Otsuka, Taketo Ashizawa, Nobuko Yasuda, Satoru Fukiya, Junko Sato, Kazuyuki Hirayae, Yoshikatsu Fujita, Toshihiko Nakajima, Fusao Tomita, Teruo Sone
    Molecular plant pathology 10 3 361 - 74 2009年05月 [査読有り][通常論文]
     
    In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.
  • Hideki Kito, Ayumi Abe, I-Nengah Sujaya, Yuji Oda, Kozo Asano, Teruo Sone
    Bioscience, biotechnology, and biochemistry 73 4 861 - 4 2009年04月23日 [査読有り][通常論文]
     
    Twenty-one strains of Amylomyces rouxii isolated from starters of Asian fermented foods were divided into two groups, lactic acid (LA) and fumaric and malic acid (FMA) producers, by organic acid productivity in liquid culture. Phylogenetic analysis based on the ldhB gene, ribosomal RNA encoding DNA (rDNA) internal transcribed spacer (ITS) sequence, and genome-wide amplified fragment length polymorphism (AFLP) revealed that A. rouxii was grouped into two major clusters as to organic acid accumulation, corresponding to Rhizopus oryzae and Rhizopus delemar. These observations suggest that the species A. rouxii is composed of two distinct types, derived from R. oryzae or R. delemar via domestication in the starters.
  • Martin Patrick Ongol, Takeshi Iguchi, Michiko Tanaka, Teruo Sone, Hiroaki Ikeda, Kozo Asano, Takashi Nishimura
    Bioscience, biotechnology, and biochemistry 72 11 2847 - 57 2008年11月 [査読有り][通常論文]
     
    Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.
  • 加藤 里佳, 白鳥 優子, 曾根 輝雄, 比良 徹, 原 博, 浅野 行蔵
    日本生物工学会大会講演要旨集 20 0 117 - 117 公益社団法人日本生物工学会 2008年07月11日 [査読無し][通常論文]
  • Kimiko Minamida, Kyohei Ota, Megumi Nishimukai, Michiko Tanaka, Ayumi Abe, Teruo Sone, Fusao Tomita, Hiroshi Hara, Kozo Asano
    International journal of systematic and evolutionary microbiology 58 Pt 5 1238 - 40 2008年05月 [査読有り][通常論文]
     
    An obligately anaerobic and equol-producing bacterium, designated strain do03T, was isolated from the caecal content of a rat. Cells were Gram-positive, non-spore-forming rods. The results from a phylogenetic analysis based on 16S rRNA gene sequences showed that strain do03T formed a separate line of descent in the phylogenetic cluster of the family Coriobacteriaceae. The strain was unable to metabolize glucose or other carbohydrates as sole carbon sources; growth was enhanced in the presence of arginine. The cell wall contained meso-diaminopimelic acid. The major fatty acid was C18 : 1cis9 (54.0 %). The strain had one unidentified predominant (91.9 %) quinone that was not menaquinone, methylmenaquinone, demethylmenaquinone, ubiquinone or rhodoquinone. The DNA G+C content was 63 mol%. The data presented in this work show that strain do03T differs from members of the related recognized genera Eggerthella and Denitrobacterium at both the phylogenetic and phenotypic level. Therefore, the strain constitutes a novel genus and species, for which the name Asaccharobacter celatus gen. nov., sp. nov. is proposed. The type strain of the type species is do03T (=JCM 14811T=DSM 18785T=AHU 1763T).
  • TAKESUE Nobuchika, SONE Teruo, TANAKA Michiko, TOMITA Fusao, ASANO Kozo
    Enzyme and Microbial Technology 41 6-7 673 - 676 2007年11月 [査読無し][通常論文]
     
    We developed di-D-fructofranosyl-2,6':2',6-anhydride (DFA IV) production system with single culture of Bacillus subtilis directly from sucrose. This system can avoid the purification procedure of levan which organic solvent was used for precipitation. The levan fructotransferase (LFTase) gene was cloned from Arthrobacter nicotinovorans GS-9 (AHU1840, FERM P-15285) and expressed in levan producing B. subtilis 168. LFTase activity was detected in the culture supernatant of the transformant with maximal activity of 0.062 U/ml after 15.5 h post induction. Then sucrose was added as substrate and incubated. About 78 h after addition of sucrose, 20.5 g/l of DFA TV was produced from 139.3 g/l of sucrose consumed. The yield of DFA IV from sucrose was 14.7 wt.%. (C) 2007 Elsevier Inc. All rights reserved.
  • Ayumi Abe, Yuji Oda, Kozo Asano, Teruo Sone
    MYCOLOGIA 99 5 714 - 722 2007年09月 [査読無し][通常論文]
     
    The zygomycete Rhizapus oryzae currently is identified by sporangiophore morphology and growth temperature, but heterogeneity of the species has been reported. We examined the suitability of organic acid production as an effective taxonomic character for reclassification of the species. Strains were divided into two groups, LA (lactic acid producer) and FMA (fumaric-malic acid producers) according to organic acid production. These grouping were confirmed as phylogenetically distinct because analyses of rDNA ITS, lactate dehydrogenase B, actin, translation elongation factor-la and genome-wide AFLP resolved the same two exclusive clusters, corresponding with the organic acid grouping. Reclassification of strains in the FMA group as R delemar was proposed.
  • Martin Patrick Ongol, Yuki Sawatari, Yoshiko Ebina, Teruo Sone, Michiko Tanaka, Fusao Tomita, Atsushi Yokota, Kozo Asano
    International journal of food microbiology 116 3 358 - 66 2007年05月30日 [査読有り][通常論文]
     
    Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+ -ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+ -ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P < or = 0.05) reduced H+ -ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (> or = 10(8) CFU/ml) of both Bifidobacteruim breve JCM 1192(T) and Bifidobacteruim breve JCM 7017 during storage at 10 degrees C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+ -ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage.
  • Chayaporn Saranpuetti, Michiko Tanaka, Teruo Sone, Kozo Asano, Fusao Tomita
    Journal of bioscience and bioengineering 102 5 452 - 6 2006年11月 [査読有り][通常論文]
     
    The allelopathic substance lepidimoide (Lp), which exhibits multiple functions in the growth and development of plants, was produced by Colletotrichum sp. AHU9748 from okra polysaccharide. Okra polysaccharide has the repeating structure (1-->4)-O-alpha-(d-galactopyranosyluronic acid)-(1-->2)-O-alpha-l-rhamnopyranose in its hexasaccharide repeating unit of its main chain. To determine the enzymes essential for Lp production, the supernatant of a culture broth was fractionated by repeated column chromatographies to identify two serial fractions responsible for Lp production and non-Lp production by measuring Lp production together with beta-galactosidase (beta-gal), rhamnogalacturonan lyase (RG-lyase) and acetylesterase (AE) activities, which we hypothesized to be necessary for Lp production from the structure of Lp. We confirmed the presence of these three enzymatic activities in the highest-Lp-producing fraction. The addition of purified RG-lyase to fractions producing no or a small amount of Lp demonstrated that beta-gal and RG-lyase activities are necessary for Lp production. The N-terminal amino acid sequences of the three separated proteins on SDS-PAGE confirmed the presence of enzymes identical to beta-gal, RG-lyase and AE in the Lp-producing fractions.
  • Ayumi Abe, Yuji Oda, Kozo Asano, Teruo Sone
    Bioscience, biotechnology, and biochemistry 70 10 2387 - 93 2006年10月 [査読有り][通常論文]
     
    In order to establish the molecular phylogeny of the genus Rhizopus, three molecules of the ribosomal RNA-encoding DNA (rDNA), complete 18S, internal transcribed spacer (ITS)1-5.8S-ITS2, and 28S D1/D2 regions of all the species of the genus were sequenced. Phylogenetic trees showed three major clusters corresponding to the three groups in the current morphological taxonomy, microsporus-group, stolonifer-group, and R. oryzae. R. stolonifer var. lyococcos was clustered independently from the major clusters. R. schipperae clustered differently in all trees. Strains of R. sexualis had multiple ITS sequences. A. rouxii clustered with R. oryzae. These results indicate the possibility of molecular identification of species groups using rDNA sequencing. Reclassification of the genus might be appropriate.
  • Kimiko Minamida, Michiko Tanaka, Ayumi Abe, Teruo Sone, Fusao Tomita, Hiroshi Hara, Kozo Asano
    Journal of bioscience and bioengineering 102 3 247 - 50 2006年09月 [査読有り][通常論文]
     
    Isoflavones (mainly daidzein and genistin) belong to the flavonoid group of compounds and are classified as phytoestrogens. In the intestine, daidzin is converted to daidzein by beta-glucosidase, and then daidzein is converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by enzymes of intestinal bacteria. We isolated, for the first time, an anaerobic gram-positive rod-shaped strain capable of producing equol from daidzein. Its 16S rDNA gene sequence (1428 bp) showed 99% similarity with that of the human intestinal bacterium SNU-Julong 732 (AY310748) and 93% similarity with that of Eggerthella lenta ATCC 25559(T) (AF292375). This strain converted daidzein to equol via dihydrodaidzein in an equol-assay medium anaerobically. The addition of butyric acid and arginine increased the conversion ratio of daidzein to equol 4.7- and 4.5-fold, respectively.
  • Ida Bagus Wayan Gunam, Yosuke Yaku, Makoto Hirano, Kenta Yamamura, Fusao Tomita, Teruo Sone, Kozo Asano
    Journal of bioscience and bioengineering 101 4 322 - 7 2006年04月 [査読有り][通常論文]
     
    Sphingomonas subarctica T7b was isolated from soil in Toyotomi, Hokkaido, Japan as an organism capable of desulfurizing aromatic hydrocarbons in light gas oil (LGO) through enrichment culture. S. subarctica T7b could grow on mineral salt sulfur-free (MSSF) medium with the n-tetradecane oil phase containing dibenzothiophene (DBT), alkyl dibenzothiophenes (alkyl DBTs) or alkyl benzothiophenes (alkyl BTs) as the sole sulfur source and desulfurize these compounds, but could not utilize the tetradecane as a carbon source. This is the first report of a gram-negative bacterium which can desulfurize 4,6-dibutyl DBT and 4,6-dipentyl DBT. The desulfurized product of DBT produced by this strain was 2-hydroxybiphenyl, as in the case of other DBT-desulfurizing bacteria. S. subarctica T7b could desulfurize LGO and the sulfur content was decreased to 41% within 36 h.
  • Ayumi Abe, Evelyn B Elegado, Teruo Sone
    Current microbiology 52 3 210 - 5 2006年03月 [査読有り][通常論文]
     
    We have constructed pDESTR, a destination vector of gateway system especially for gene targeting and disruption in filamentous fungi. The vector was constructed by removing the multicloning site of pGEM-T easy vector, and inserting hygromycin phosphotransferase gene construct from pCB1004, and a gateway vector conversion cassette. In order to construct a DNA for gene disruption, only an inverse-polymerase chain reaction (PCR) amplification of the restricted, target sequence is needed. After the amplification with a 5'CACC-tagged primer and an ordinary primer, the DNA fragment will be inserted into pENTR/D-TOPO vector and then transferred into pDESTR through LR-recombination reaction. The resulting vector has the disruption construct, after being digested with the restriction enzyme used for the inverse-PCR. The effectiveness of this vector was assessed in Neurospora crassa. The use of pDESTR will therefore simplify the construction of a targeting vector, where multiple ligation steps are usually needed.
  • Kimiko Minamida, Chikako Asakawa, I Nengah Sujaya, Maki Kaneko, Ayumi Abe, Teruo Sone, Hiroshi Hara, Kozo Asano, Fusao Tomita
    Journal of bioscience and bioengineering 101 2 149 - 56 2006年02月 [査読有り][通常論文]
     
    Changes in the intestinal microbiota of 10 human subjects with long-term ingestion of 3 g/d difructose anhydride III (DFA III; 4 persons, 2 months; 3 persons, 6 months; and 3 persons, 12 months) were examined by denaturing gradient gel electrophoresis (DGGE). According to the answers to questionnaires, the subjects were divided into two groups (constipated and normal). The DGGE profile was different for every individual and each subject had unique profiles of intestinal microbiota. In the DGGE profiles of constipated subjects, the intensities of bands related to Bacteroides spp. increased. Moreover, the DFA III-assimilating bacteria, Ruminococcus sp. were isolated from subjects who ingested DFA III for 12 months. These strains showed 95% similarity of their 16S rDNA sequences with that of Ruminococcus obeum ATCC 29174(T) (X85101) and produced large amounts of acetic acid. DFA III ingestion for 2 months tended to increase total organic acids in feces, and tended to decrease fecal pH and the secondary bile acid (SBA) ratio in total bile acids. The SBA ratio in total bile acids corresponded to fecal pH. The production of SBA was decreased by low pH in vitro. These results indicated that DFA III ingestion in humans tended to lower intestinal pH, inhibited bile acid 7alpha-dehydroxylation activities and also tended to decrease the SBA ratios in total bile acids. Moreover, as another cause for the decrease in the SBA ratio in total bile acids, it was suggested that the number of bile acid 7alpha-dehydroxylating bacteria were decreased by DFA III ingestion.
  • Evelyn Elegado, Asumi Iwasaki, Marites Sales, Chizu Ishii, Fusao Tomita, Teruo Sone
    Journal of General Plant Pathology 72 1 16 - 19 2006年02月 [査読無し][通常論文]
     
    The first two recombinational repair genes of Magnaporthe grisea were cloned. Analysis of the deduced amino acid sequences revealed that Rhm52 and Rhm54 are Saccharomyces cerevisiae RAD52 and RAD54 homologs, respectively. Phenotypic complementation testing of these genes showed their function in recombinational repair. Both genes were in single copies in M. grisea genome. Expression of these genes was induced by methyl methanesulfonate and ultraviolet radiation as known for other homologs of the RAD52 epistasis group. Higher induction of both genes by oxidative stress and heat shock indicated the probability for recombinational repair during the infection cycle of M. grisea. © The Phytopathological Society of Japan and Springer-Verlag 2006.
  • Abu Sadeque Md Selim, Piyanuch Boonkumklao, Teruo Sone, Apinya Assavanig, Masaru Wada, Atsushi Yokota
    Applied and environmental microbiology 71 8 4214 - 9 2005年08月 [査読有り][通常論文]
     
    A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345(T) and Lactobacillus fermentum JCM 1173(T), and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 x 10(3) cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10(4) cells/g feces on day 4 and 10(5) cells/g feces on days 11 to 18. However, cell populations of 10(6) to 10(7) cells/g feces were detected in the second trial. The total bacterial count, measured by 4',6-diamidino-2-phenylindole (DAPI) staining, was approximately 10(11) cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.
  • Kimiko Minamida, Maki Kaneko, Midori Ohashi, I Nengah Sujaya, Teruo Sone, Masaru Wada, Atsushi Yokota, Hiroshi Hara, Kozo Asano, Fusao Tomita
    Journal of bioscience and bioengineering 99 6 548 - 54 2005年06月 [査読有り][通常論文]
     
    The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.
  • Kimiko Minamida, Kazuki Shiga, I Nengah Sujaya, Teruo Sone, Atsushi Yokota, Hiroshi Hara, Kozo Asano, Fusao Tomita
    Journal of bioscience and bioengineering 99 3 230 - 6 2005年03月 [査読有り][通常論文]
     
    The effects of difructose anhydride III (di-D-fructofuranose-1,2':2,3'-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.
  • Masashi Tsuda, Mai Sasaki, Takao Mugishima, Kazusei Komatsu, Teruo Sone, Michiko Tanaka, Yuzuru Mikami, Jun'ichi Kobayashi
    Journal of natural products 68 2 273 - 6 2005年02月 [査読有り][通常論文]
     
    Three new pyrrolidine alkaloids, scalusamides A-C (1-3), were isolated from the cultured broth of the fungus Penicillium citrinum, which was separated from the gastrointestine of a marine fish, and the structures were elucidated by spectroscopic data. The absolute stereochemistry of C-2 in the pyrrolidine unit was determined by HPLC analysis of a Marfey's derivative of the hydrolysate of 1, while that of 2 and 3 was assigned by comparison of spectroscopic data of 3 and reductive products of 1 and 2. On the other hand, each of 1-3 was found to be a mixture of epimers at C-7. Scalusamide A (1) exhibited antifungal and antibacterial activities.
  • Masashi Tsuda, Yuu Kasai, Kazusei Komatsu, Teruo Sone, Michiko Tanaka, Yuzuru Mikami, Jun'ichi Kobayashi
    Organic letters 6 18 3087 - 9 2004年09月02日 [査読有り][通常論文]
     
    [structure: see text] A novel pentacyclic alkaloid, citrinadin A (1), was isolated from the cultured broth of the fungus Penicillium citrinum, which was separated from a marine red alga, and the structure was elucidated by spectroscopic data. The relative stereochemistry of the pentacyclic core was assigned on the basis of NOESY data and (1)H-(1)H coupling constants, and the presence of an N,N-dimethyl-L-valine residue in 1 was determined by chiral HPLC analysis of the hydrolysate.
  • A Abe, IN Sujaya, T Sone, K Asano, Y Oda
    FOOD TECHNOLOGY AND BIOTECHNOLOGY 42 3 169 - 173 2004年07月 [査読無し][通常論文]
     
    When potato pulp was mixed with Indonesian starter ragi tape and incubated, both lactic acid and ethanol were gradually formed and attained certain concentrations during 2 days of fermentation. Viable counts of fungi in fresh weight matter, yeasts and lactic acid bacteria after fermentation were 10(5), 10(7) and 10(5) CFU/g, respectively. Denaturing gradient gel electrophoresis of the PCR-amplified internal transcribed spacer of 18S-28S rRNA genes detected Amylomyces rouxii-Rhizopus oryzae, Mucor indicus, Candida tropicalis and Saccharomycopsis fibuligera and revealed that Amylomyces rouxii-Rhizopus oryzae dominated throughout the fermentation period. Amylomyces rouxii cannot be discriminated from the lactic acid-accumulating group of Rhizopus oryzae because the amplified sequences of these fungi were shown to be identical. Morphological characteristics were then studied for Rhizopus-like fungi isolated from fermented potato pulp. Those strains that had produced an enormous number of chlamydospores in the aerial and substrate mycelium were identified as Amylomyces rouxii. The microflora of fermented potato pulp was similar to that made from glutinous rice, namely tape ketan.
  • K Saito, A Abe, IN Sujaya, T Sone, Y Oda
    FOOD SCIENCE AND TECHNOLOGY RESEARCH 10 2 224 - 226 2004年05月 [査読無し][通常論文]
     
    Amytomyces rouxii, the filamentous fungus widely used in the production of Asian fermented foods, is closely related to certain strains of Rhizopus oryzae secreting lactic acid. Among seven strains of A. rouxii, CBS 438.76(T) most vigorously produced both lactic acid and ethanol from glucose, starch, and pectin in liquid media. When this strain was grown on apple peels and successively mixed with potato pulp, the concentration of lactic acid produced was lower than that produced by Rhizopus oryzae NBRC 4707. However, the growth of A. rouxii CBS 438.76(T) acidified the pulp to less than pH 4, the level found in conventional silage fermented by lactic acid bacteria. A. rouxii may be preferable to R. oryzae for recycling potato pulp and other agricultural by-products into food materials because this fungus was being consumed long before written history, which attests to its safety for humans.
  • 南田公子, 金子真紀, 天野みどり, イヌガ スジャーヤ, 曽根輝雄, 和田大, 横田篤, 原博, 浅野行蔵
    日本農芸化学会大会講演要旨集 2004 36  2004年03月05日 [査読無し][通常論文]
  • IN Sujaya, NS Antara, T Sone, Y Tamura, WR Aryanta, A Yokota, K Asano, F Tomita
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 20 2 143 - 150 2004年03月 [査読無し][通常論文]
     
    Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer ( ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae ( 35 strains), Candida glabrata ( six strains), Pichia anomala ( three strains) and Issatchenkia orientalis ( seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.
  • MINAMIDA K, SUJAYA I N, TAMURA A, SHIGEMATSU N, SONE T, YOKOTA A, ASANO K, BENNO Y, TOMITA F
    Journal of Bioscience and Bioengineering 98 4 244 - 50 2004年 [査読有り][通常論文]
     
    Di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) was shown to enhance Ca absorption in rat and human intestine. The effects of DFA III administration (9 g per day for 4 weeks that corresponded to 3-fold the optimal dosage of DFA III) on human intestinal microbiota were studied using denaturing gradient gel electrophoresis (DGGE). The major groups of human intestinal microbiota reported previously: the Bacteroides, the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum group (Clostridium cluster IV), and the Bifidobacterium group were detected. The similarity of 30 DGGE profiles based on the V3 region (before and after administration to the 15 subjects) of the 16S rDNA were calculated using Pearson's correlation based on numbers, positions and intensity of bands, and then a dendrogram of DGGE profiles was constructed by the unweighted pair group method using arithmetic average (UPGMA) clustering method. By these analyses, no difference in DGGE profiles after DFA III administration was observed in healthy subjects, while two subjects with chronic constipation showed different profiles, namely on numbers, positions and the intensity of some bands. Their stools were softer and stool frequencies increased and they obtained relief from constipation.
  • Ayumi Abe, Teruo Sone, I Nengah Sujaya, Katsuichi Saito, Yuji Oda, Kozo Asano, Fusao Tomita
    Bioscience, biotechnology, and biochemistry 67 8 1725 - 31 2003年08月 [査読有り][通常論文]
     
    Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.
  • M Tsuda, T Mugishima, K Komatsu, T Sone, M Tanaka, Y Mikami, M Shiro, M Hirai, Y Ohizumi, J Kobayashi
    TETRAHEDRON 59 18 3227 - 3230 2003年04月 [査読無し][通常論文]
     
    A new pentacyclic oxindole alkaloid, speradine A (1), was isolated from the cultured broth of a fungus Aspergillus tamarii, which was separated from driftwood at a seashore in Okinawa. The structure and relative stereochemistry were determined by spectroscopic data and a single crystal X-ray diffraction analysis. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • Hideki Kito, Yosuke Takahashi, Junko Sato, Satoru Fukiya, Teruo Sone, Fusao Tomita
    Current genetics 42 6 322 - 31 2003年03月 [査読有り][通常論文]
     
    We investigated a DNA fragment and its flanking region deleted in the spontaneous Pi-a virulent mutant of Magnaporthe grisea Ina168. A new transposon-like sequence was identified from a region adjacent to the deleted fragment and was named Occan. Occan contained a 2,259-bp ORF interrupted by one 63-bp intron and had both a TA dinucleotide and 77 bp of perfect inverted repeats at both termini, without direct repeats. These features indicated that Occan is a member of the Fot1 family. RT-PCR analysis confirmed the expression of the putative transposase and the presence of an intron. Southern analysis of pulse-field gel electrophoresis-separated chromosomes indicated that Occan was dispersed in all chromosomes of the rice pathogen, Ina168. Copy numbers of Occan were also preserved in a host-specific manner amongst M. grisea isolates. In particular, rice pathogens contained a large number of the element inserted into their genome. Phylogenetic analysis with other known members of the Fot1 family revealed that Occan was dissimilar to any other known elements and it is thus proposed that Occan be separated to a new subfamily.
  • Masashi Tsuda, Takao Mugishima, Kazusei Komatsu, Teruo Sone, Michiko Tanaka, Yuzuru Mikami, Jun'ichi Kobayashi
    Journal of natural products 66 3 412 - 5 2003年03月 [査読有り][通常論文]
     
    Two new 10-membered macrolides, modiolides A (1) and B (2), and a new linear pentaketide, modiolin (3), were isolated from the cultured broth of a fungus Paraphaeosphaeria sp. (N-119), which was separated from a marine horse mussel, and the structures were elucidated by spectroscopic data.
  • Piyanuch Niamsup, I Nengah Sujaya, Michiko Tanaka, Teruo Sone, Satoshi Hanada, Yoichi Kamagata, Saisamorn Lumyong, Apinya Assavanig, Kozo Asano, Fusao Tomita, Atsushi Yokota
    International journal of systematic and evolutionary microbiology 53 Pt 1 263 - 268 2003年01月 [査読有り][通常論文]
     
    Five strains of thermotolerant lactic acid bacteria (G 12, G 22, G 35T, G 43 and G 44) isolated from chicken faeces were characterized taxonomically. The strains were facultatively anaerobic, Gram-positive, catalase-negative, non-motile, non-spore-forming rods. They were heterofermentative lactobacilli that produced DL-lactic acid. Growth of the strains occurred at 45 degrees C but not at 15 degrees C. The optimum temperature for growth was 42 degrees C, as determined from the specific growth rate. The highest permissive temperatures for growth were 50 degrees C for strain G35T and 48 degrees C for the other four strains. DNA G+C content of the strains was between 49 and 51 mol%. Complex fatty acid patterns of the strains showed the presence of C14:0, C16:0, sometimes C18:0, C18:1 and C19:0 cyclo in the cell walls. Phylogenetic analysis of the 16S rRNA gene sequences of the five strains placed them in the Lactobacillus caseil Pediococcus group, with Lactobacillus fermentum as their closest relative (about 95% sequence similarity). DNA-DNA hybridization data indicated that the thermotolerant strains were not L. fermentum. Taken together, the findings of this study show that the five strains isolated from chicken faeces represent a novel species within the genus Lactobacillus, for which the name Lactobacillus thermotolerans is proposed (G 35T = DSM 14792T =JCM 11425T).
  • Haruto Kumura, Kentaro Takagaki, Teruo Sone, Maki Tsukahara, Tetsuya Tanaka, Kei-ichi Shimazaki
    Bioscience, biotechnology, and biochemistry 66 6 1370 - 3 2002年06月 [査読有り][通常論文]
     
    To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on beta-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both alpha(s)- and beta-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.
  • Satoru Fukiya, Takao Kuge, Tomomi Tanishima, Teruo Sone, Takashi Kamakura, Isamu Yamaguchi, Fusao Tomita
    Bioscience, biotechnology, and biochemistry 66 3 663 - 6 2002年03月 [査読有り][通常論文]
     
    We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.
  • The production of lepidimoide by endophytic fungus from polysaccharide extracted from Abelmoshus sp. – Identification of the product and the producing organism
    Biotechnology for Sustainable Utilization of Biological Resources in the Tropics 15 82 - 87 2002年 [査読無し][通常論文]
  • Endophytic fungi isolated from Ulmus davidiana and their association with the host plant
    Biotechnology for Sustainable Utilization of Biological Resources in the Tropics 15 72 - 76 2002年 [査読無し][通常論文]
  • S Fukiya, M Kodama, H Kito, T Sone, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 7 1464 - 1473 2001年07月 [査読無し][通常論文]
     
    Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasuciamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.
  • JW Bok, T Sone, LB Silverman-Gavrila, RR Lew, FJ Bowring, DEA Catcheside, AJF Griffiths
    FUNGAL GENETICS AND BIOLOGY 32 3 145 - 158 2001年04月 [査読無し][通常論文]
     
    Bok, J.-W., Sone, T., Silverman-Gavrila, L. B., Lew, R. R., Bowring, F. J., Catcheside, D. E. A., and Griffiths, A. J. F. 2001. Structure and function analysis of the calcium-related gene spray in Neurospora crassa, Fungal Genetics and Biology 32, 145-158. The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca2+ via calcineurin. (C) 2001 Academic Press.
  • H Kumura, T Sone, K Shimazaki, E Kobayashi
    JOURNAL OF DAIRY RESEARCH 67 4 631 - 636 2000年11月 [査読無し][通常論文]
  • T Sone, S Fukiya, M Kodama, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 64 8 1733 - 1736 2000年08月 [査読無し][通常論文]
     
    The 8-kb repeat unit of M, grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two hinds of short tandemly repeated elements.
  • T Sone, F Tomita
    ADVANCES IN RICE BLAST RESEARCH 15 221 - 233 2000年 [査読無し][通常論文]
  • T Sone, AJF Griffiths
    FUNGAL GENETICS AND BIOLOGY 28 3 227 - 237 1999年12月 [査読無し][通常論文]
     
    The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca2+, suggesting that characterization of this gene might clarify the mechanism of Ca2+-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia, RFLP analysis revealed that the cloned DNA fragment mapped to the fu locus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDCl protein, implicated in manganese homeostasis, The fr mutant was sensitive to Mn2+, and a revertant allele whose product differs by one amino acid was tolerant to Mn2+. Mn2+ depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The fr mutant was also sensitive to calcineurin inhibitors. These results suggest that fr is involved in Mn2+ homeostasis and point to a role for Mn2+ in Neurospora branching. (C) 1999 Academic Press.
  • T Sone, T Abe, M Suto, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 1 81 - 86 1997年01月 [査読無し][通常論文]
     
    We have identified karyotypic mutants of Magnaporthe grisea isolate Ina168, The mutants have a karyotype with an extra band of chromosome-v in addition to six bands of the normal karyotype in contour-clamped homogeneous electric field (CHEF) electrophoresis, We named a karyotype of the original isolate as alpha, and that of mutants as alpha', The mutation has not affected the DNA fingerprint pattern, host specific pathogenicity (pathogenic race), or crossing abilities with the isolate Guy11, Karyotypic analyses of isolates from a repetitive single-spore isolation indicated that the frequency of mutation was 12.5% and that the mutation was limited in the manner of alpha to alpha', and never reverted, Hybridization analyses showed that chromosome-nu originated from the chromosome in band IIIb, of type a and might be produced by deletion.
  • SONE T, ABE T, YOSHIDA N, SUTO M, TOMITA F
    Annals of the Phytopathological Society of Japan 63 3 155 - 163 1997年 [査読無し][通常論文]
  • 曽根 輝雄, 冨田 房男
    化学と生物 34 10 676 - 682 Japan Society for Bioscience, Biotechnology, and Agrochemistry 1996年10月25日
  • T SONE, M SUTO, F TOMITA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 57 7 1228 - 1230 1993年07月 [査読無し][通常論文]
     
    We cloned a repetitive sequence to show RFLPs in the genome of Magnaporthe grisea, a fungal pathogen responsible for rice blast. As the sequence was 0.8 kb in length and dispersed in the genome, it was named MGSR1 (for Magnaporthe grisea short repeat 1). MGSR1 was conserved highly in the genome of rice pathogens, but poorly in the genome of pathogens of grasses other than rice. And the RFLPs, displayed with the sequence, could distinguish between clonal lineages in rice-pathogenic isolates. The nucleotide sequence showed the presence of an internal promoter of RNA polymerase III, a 3'-poly(T), and an 8-bp direct repeat in it.

その他活動・業績

特許

  • アルカリジェネス属の微生物の生菌を主成分とする植物発根促進剤
    特許出願2004−117303

受賞

  • 2013年11月 日本農芸化学会北海道支部 日本農芸化学会北海道支部奨励賞
     イネいもち病菌の病原性変異に関する分子遺伝学的研究 
    受賞者: 曾根 輝雄

共同研究・競争的資金等の研究課題

  • イネいもち病菌におけるQoI剤耐性変異メカニズムの解明~いたちごっこからの脱却~
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 曾根 輝雄, 阿部 歩
     
    本研究では,イネいもち病菌におけるストロビルリン系殺菌剤(QoI剤)耐性変異の発生,遺伝,蔓延のメカニズムを明らかにすることで,薬剤の導入→耐性菌の発生→新薬剤の導入の「いたちごっこ」からの脱却を目指すことを目的としている. まず,mtDNA cytbコドン143変異の発生とDNA損傷乗り越え合成の関係の解析のため,いもち病菌のオルタナティブオキシダーゼの欠損株の作成を行った.2回相同組換えにより遺伝子の破壊を試みているが,破壊候補株の生育が芳しくなく,取得に至っていない.CRISPR-Cas9など,ゲノム編集を用いることでより効率的に得ることを検討したい. 一方で,プロトプラスト融合により得られた野生型ミトコンドリアと変異型ミトコンドリアの混在株は,分生子形成によりホモプラスミー化し,一方のミトコンドリアに大きく偏る傾向があることが明らかとなった.こちらに関しては,融合株をそのまま使用して数の変化が起こるかを検討したい. また,ミトコンドリアの可視化のため,蛍光タンパク質による可視化を行った.ミトコンドリアに局在するタンパク質として,クエン酸シンターゼを用い,eGFP蛍光タンパク質をC末端に融合させたタンパク質をイネいもち病菌Ina86-137株にに発現させた.形質転換株のGFP蛍光とmitotrackerによるミトコンドリアの蛍光染色との比較により,GFPによりいもち病菌のミトコンドリアが可視化できたことがわかった.一方,分生子形成過程におけるミトコンドリアの定量のため,分生子形成初期のタイミングを検討し,培地上での分生子形成誘導処理後6時間後に初期分生子形成が見られることがわかった.今後は,分生子形成初期のミトコンドリアの定量を行い,どのようにミトコンドリアが伝播するかを明らかにしたい.
  • 植物耐酸性研究の新展開:菌根形成による耐酸性獲得の生理・分子機構
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 江澤 辰広, 曾根 輝雄, 増田 税
     
    II. 菌根菌耐酸性遺伝子のスクリーニング 昨年度と同様の実験系---酸性硫酸塩土壌のpHを3.5-4.9の範囲で4段階に調整した土壌を用いたナイロンメッシュ区画ポットを利用して栽培---において新規に入手したR. clarus RF1株の同種地理的隔離株であるCK001株を接種したミヤコグサを7週間栽培した。その後、それぞれのpH区から採取したAM菌の外生菌糸からRNAを抽出し、比較トランスクリプトーム解析に供試した。RF1株の際と同様の手法でpHの低下に伴って直線的に発現上昇する48個の遺伝子群を抽出したが、RF1株と共通の遺伝子はそのうちのわずか14個であった。RF1株において耐酸性に関わる重要遺伝子として抽出された原形質膜型マグネシウム輸送体遺伝子は土壌pHには反応せず、代わりにオルガネラ局在が予想されるマグネシウム輸送体遺伝子の発現がpHの低下に伴って発現上昇していた。RF1株は強酸性を示す酸性硫酸塩土壌から分離された株であるのに対し、CK001株は熱帯泥炭土壌から分離された株であり、土壌酸性への応答の相違は、両株の生育環境の相違と関係しているものと推定された。 III. 共生ウイルスの耐酸性における役割 前項と同様の実験系を用いてCK001株を接種したミヤコグサを栽培し、その外生菌糸からRNAを抽出した。一本鎖RNAウイルスを標的にRNA-Seqを行った。これまの予備的な試験においてCK001株で見つかっていたR. clarus Mitovirus 4およびR. clarus Mitovirus 5は今回も検出されたものの、前回同定され、他の地理的隔離株からも検出されているR. clarus Mitovirus 3は検出されなかった。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 中馬 いづみ, 荒添 貴之, 曾根 輝雄, レ ディン・ドン, グエン ティ・タィン・ガ, クマグン クリスチャン, スリーウォンチャイ タニー, スプリング オトマー
     
    イネいもち病菌非病原力遺伝子AVR-Piaの変異頻度比較実験を行った。イネ菌野生株(AVR-Pia非保有)に対し、AVR-Piaを転移因子の多い領域に導入したta#126と転移因子の少ない領域に導入したzt#1を作出し、Pia保有抵抗性イネ品種に対する接種試験を行った。抵抗性品種の葉上に形成された病原性獲得変異菌由来の病斑数より変異頻度を比較したところ、ta#126の方がzt#1より頻度が高かった。分離した変異菌における変異機構は、いずれも導入したAVR-Piaの欠失変異で、欠失領域の両端には転移因子が存在していた。欠失領域の大きさは転移因子間の距離が一因となっていることが示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2011年04月 -2014年03月 
    代表者 : 曾根 輝雄, 寺内 良平, 尾瀬 農之, 尾瀬 農之
     
    いもち病はイネの最重要病害である.いもち病の防除は主に農薬と抵抗性品種を使って行われている.しかし,抵抗性品種がどのようにしてその抵抗性を発揮するかについて,まだ完全に理解されていない.本研究はイネのいもち病抵抗性遺伝子Piaの抵抗性発現のメカニズムについて,いもち病菌のAVR-Pia遺伝子との相互作用を詳細に明らかにしようとしたものである.本研究の結果,いもち病菌はイネに侵入する前からAVR-Pia遺伝子を発現しはじめること,分泌後AVR-Piaタンパクは二量体を形成し,イネのPiaタンパク質と相互作用することがわかった.これらの知見は,いもち病抵抗性を理解するのに役立つものと期待される.
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2011年 -2013年 
    代表者 : 曾根 輝雄
     
    いもち病はイネの最重要病害である.いもち病防除の重要な問題はイネ品種の抵抗性を打破するいもち病菌の突然変異である.いもち病の突然変異は主にDNAの組換えを介する.本研究の目的はいもち病菌のDNA組換えを担う遺伝子の働きを解明し,突然変異を抑えながら防除する方法を開発することである.本研究の結果,イネいもち病菌が病原性を発揮するためにはDNA修復タンパク複合体が重要な役割を果たしていることが明らかとなった.その複合体形成を阻害する薬剤をスクリーニングする系を構築することが出来た.本研究は突然変異も抑えられるいもち病の新たな防除法につながるものとなるだろう.
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2008年 -2010年 
    代表者 : 曾根 輝雄
     
    イネいもち病菌の非病原性遺伝子AVR-Piaの単離と同定に成功した.AVR-Pia遺伝子は中国産菌株を使って解析されたものと同じ遺伝子であった.AVR-Pia遺伝子はイネいもち病菌のイネ感染時にのみ発現し,親和性相互作用ではその産物はイネ細胞内の侵入菌糸のBICから宿主細胞へと分泌されていることが示唆された.また,Ina168m95-1におけるAVR-Pia遺伝子の欠失は,2コピーのトランスポゾンOccan間の相同組換えであることが示唆された.日本菌株においては,AVR-Piaの有無をPCRで調べることにより病原性の判定が可能であることがわかった.
  • フィターゼとその遺伝子に関する研究
    その他の研究制度
    研究期間 : 2008年
  • Studies on phytases and their genes
    The Other Research Programs
    研究期間 : 2008年
  • メタロチオネイン分解酵素による水産廃棄物からの脱カドミウム技術の開発
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2004年 -2005年 
    代表者 : 浅野 行蔵, 曾根 輝雄
     
    生体内でカドミウムはメタロチオネイン(重金属結合低分子量蛋白質)と結合している。これを特異的に分解すれば水産廃棄物中のカドミウムをイオン化し分離できる。酵素分解を用いれば環境にやさしい水産廃棄物の脱カドミウム処理が可能になる。 システイン・リッチなタンパク質であるメタロチオネインを特異的・積極的に分解する酵素は新規性が高く、今までにない性質が期待できる。我々の探索によって細菌Arthrobacter nicotnovorans 23-0-11株がカドミウム遊離能を持つ菌体外プロテアーゼを生成することを発見した。 目的のプロテアーゼを単離するためA.nicotinovorans 23-0-11株の培養上清を2種類のイオン交換カラムクロマトグラフィーにかけて酵素を精製した。精製酵素の分子量は27kDaで、市販のプロテアーゼと比較して、ホタテ貝ウロからカドミウムを効率的に遊離させる特徴を示した。 精製酵素の至適pHは7.0で至適温度は50℃であり、pH3.0-9.0、60℃以下で安定であった。阻害剤の影響から本酵素は中性セリンプロテアーゼに分類される酵素であると推定した。 遺伝子のクローニングを行うために精製酵素をプロテアーゼで断片化して4ヶ所の内部アミノ酸配列を得た。N末端を含めた5ヶ所にプライマーを設計してPCRを行い、部分配列を増幅した。それをもとに全長の遺伝子とアミノ酸配列を決定した。アミノ酸配列にはセリンプロテアーゼで保存されているモチーフが含まれていた。アミノ酸配列の相同性を検索したところArthrobacter sp.FB24の未同定タンパク質と69%の相同性を示した。既知プロテアーゼとの相同性は30%未満であり、新規プロテアーゼと考えられる。 クローニングした酵素遺伝子を大腸菌に導入し、複合タンパク質として発現させた。複合タンパク質は封入体になって不溶化したため、現在ベクターと発現条件の検討をし直して活性のある形での大量発現を行っている。
  • イネいもち病菌はいつどこで変異するのか
    日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2004年 -2005年 
    代表者 : 曾根 輝雄
     
    1.新たな遺伝子のクローニング 新たに非相同末端再結合に関わる遺伝子であるKu70,Ku80のいもち病菌ホモログ,Khm70,Khm80をPCRにより,日本産イネいもち病菌Ina168株より取得した.これらはこれまでにクローニングしていた遺伝子と同様,アカパンカビ(Neurospora crassa)のホモログと最も高い相同性を示した. 2.N.crassa変異株の相補による機能の確認 これまでにクローニングしたRhm52,Rhm54,Mhm11,Khm70,Khm80をハイグロマイシン耐性プラスミドベクターpCSN43-DESTに連結し,それぞれN.crassaの変異株mus-11,mus-25,mus-23,mus-51,mus-52に導入し,変異の相補により遺伝子の機能を確認した.その結果,Rhm52,Rhm54,Khm80は相補が確認され,機能を持った遺伝子であることが示唆された.Mhm11,Rhm70は相補が見られなかったが,これらは複合体を形成し機能する遺伝子であり,その複合体の形成に問題があることが考えられた. 3.欠損変異株の作成 昨年度開発したベクターをpDESTRと命名し,さらに同様の,ビアラフォス耐性遺伝子を選択マーカーとして持ったベクター,pBARSTを作成した.これらのベクターを使用し,Inverse PCR法によりRhm54,Rhm52,Khm70,Khm80破壊コンストラクトを作成した.10株の形質転換体から,欠損変異株を得ることができ,ベクターの有効性が証明された.
  • 赤カビ毒(DON, NIV)のアセチル化酵素の特性の解明とその応用
    研究期間 : 2005年
  • Studies on trichothecene acetyl transferases
    研究期間 : 2005年
  • いもち病菌の病原性レース変異機構に関する研究〜非病原性遺伝子と遺伝的組換え〜
    日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 1999年 -2001年 
    代表者 : 冨田 房男, 安田 伸子, 中島 敏彦, 石井 千津, 曾根 輝雄, 辻本 雅子, 犬飼 剛
     
    1.非病原性遺伝子の解析 K59に対する病原性変異株が取得できなかったため,既存の愛知旭に対する病原性変異株Ina168m95-1を用い,親株Ina168r95-1とのゲノムDNA多型を検索した.その結果変異株Ina168m95-1に特異的に欠失したDNA断片PM01を得た.PM01と非病原性遺伝子Avr-piaは強く連鎖していることが示された.PM01の周辺領域を含む,3つのコスミドクローンを用いて変異株を形質転換したところ1つのクローンでその変異が相補され,Avr-piaが含まれていることが示唆された. 一方,交配可能な2つのイネ菌株を用い交配後代菌の病原性の分離を調べた.その結果,イネ品種「石狩白毛」に対して非病原性を示す菌株と病原性を示す菌株の比は1:1に適合し,「石狩白毛」に対する非病原性は1個の遺伝子により支配されていることが明らかとなった。同様にして,イネ品種「八反3号」に対する非病原性遺伝子も同定した.RFLP解析、RAPD分析をすることにより、イネ品種分子マーカーを選抜し,染色体地図上にマッピングした.イネ品種「八反3号」に対する非病原性遺伝子Avr-Hattan3近傍の物理地図を作製するため,Avr-Hattan3と密接に連鎖するマーカーを用いて,いもち病菌のBACライブラリーを選抜し,約600kbにわたるBACコンティグを作成した. 2.組換えに関する遺伝子の解析 Degenerate PCR法を用いて,いもち病菌のRad50,Rad52,Rad54,mre11のいもち病菌ホモログのクローニングを行い,さらに上記のゲノムライブラリーより,全長のRad52,Rad54,Mre11遺伝子を含むクローンを得た.これらの塩基配列を解析したところ,アカパンカビの相同遺伝子と高い相同性を示した.これらの遺伝子をそれぞれRhm52,Rhm54,Mhm11と命名した.これらのクローンを用いてアカパンカビの相同遺伝子の変異株を形質転換したところ,Rhm52,Rhm54遺伝子はその変異を相補し,ホモログであることが確認された.このうちRhm54遺伝子については,MMS処理によりその転写が誘導されることが確認された.
  • Rhizopus属菌の分子系統解析とその応用
    研究期間 : 2001年
  • Molcular taxonomy of the genus Rhizopus and its application
    研究期間 : 2001年
  • いもち病菌の染色体再編成による病原性レース変異機構の解析
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 1998年 -2000年 
    代表者 : 曽根 輝雄
  • いもち病菌の分子遺伝学的解析による病原性レース変異機構の解明
    研究期間 : 1991年
  • Molecular genetic analysis of Magnaporthe grisea host specificity and mutations
    研究期間 : 1991年

教育活動情報

主要な担当授業

  • 人口・食料・環境学総論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 農学院
    キーワード : 人口爆発、人口論、アグロエコシステム、動物生態学、経済成長理論、食料問題、人口成長率、農業技術、GMO、飢餓、飢饉、外部性、効率性、衡平性、食料の利用権、感染症、育種、土壌、アフリカ
  • ワンダーフォーゲル実習Ⅰ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : Please see the English version.
  • 事前・事後演習Ⅰ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : Please see the English version.
  • 微生物生態学特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 農学院
    キーワード : 微生物、共生、寄生、培養、難培養、植物、昆虫、エネルギー獲得、生物間相互作用、分子機構、進化、生態系
  • ワンダーフォーゲル実習Ⅳ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 世界各地の食資源問題,農村振興、富良野市、平取町、ワイン、トマト、観光
  • 事前・事後演習Ⅳ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 北海道の農業・農村問題、富良野市、平取町、ワイン、トマト、農村観光
  • 食資源と健康
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 健康,食,栄養学,環境,人類生態学,人間栄養学,脂質,酸化,糖鎖生物学,蛋白質,腸内環境,機能性食品、ポストハーベスト加工技術
  • ワンダーフォーゲル実習Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 発展途上国,農業生産,食品加工,環境問題
  • 事前・事後演習Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 東南アジアの食資源問題
  • 食資源特別演習
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : ワイン,醸造用ブドウ栽培,発酵,熟成,流通,官能検査
  • 大学院共通授業科目(一般科目):複合領域
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : ワイン,北海道,農業,環境,醸造,マーケティング,食
  • 食資源生産論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 作物,畜産物,水産物,GAP, バイオテクノロジー,発酵生産,食の安全,HACCP, 機能性食品,健康,エネルギー
  • 持続的生物生産技術特論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : 生物生産,バイオテクノロジー,植物,動物,魚類,水産,微生物,植物病理学,育種,繁殖,遺伝資源,生物多様性, 遺伝子組み換え生物(GMO),エネルギー,持続性,地球温暖化,環境ストレス,土壌,水,薬剤耐性,栄養分循環、バイオリファイナリー
  • 大学院共通授業科目(一般科目):自然科学・応用科学
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : Agroecosystem, Biological diversity, Domestication, Livestock and human, Genetic diversity, Landscape, Useful enzyme,genetic engineering, GMO
  • 食資源特別講義
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 国際食資源学院
    キーワード : Agroecosystem, Biological diversity, Domestication, Livestock and human, Genetic diversity, Landscape, Useful enzyme,genetic engineering, GMO
  • 環境と人間
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 食のバリューチェーン、Society 5.0、エネルギー、異分野融合、持続可能性、フィールド、ロバストネス(強靱性)
  • 環境と人間
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 共生、寄生、進化、生物生産、植物、哺乳動物、昆虫、微生物、ウィルス、分子生物学、生態学、環境保全
  • 生物学実験Ⅰ
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 微生物学、細菌、糸状菌、培養、観察、分類
  • 応用菌学
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 農学部
    キーワード : 代謝制御、アミノ酸、核酸、醸造、発酵食品、酵素、抗生物質、微生物利用
  • 一般教育演習(フレッシュマンセミナー)
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 食料、安全、作物生産、放射能汚染、食品微生物、酵素利用技術、毒物、ガン、アレルギー、メタボリックシンドローム、腸内細菌叢、保健機能食品

大学運営

学内役職歴

  • 2021年4月1日 - 2023年3月31日 大学院国際食資源学院副学院長

委員歴

  • 2017年01月 - 現在   日本農芸化学会   英文誌編集委員
  • 2016年01月 - 現在   日本植物病理学会   英文誌編集委員
  • 2010年 - 2012年   日本農芸化学会   北海道支部庶務幹事   日本農芸化学会
  • 2009年 - 2011年   日本農芸化学会   代議員   日本農芸化学会
  • 2008年 - 2010年   日本植物病理学会   北海道部会実行幹事   日本植物病理学会


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