Researcher Database

Atsushi Yokota

Researcher Profile and Settings


  • President/Vice-Presidents

Job Title

  • Executive/Vice-President


  • Doctor of Agriculture (Hokkaido University) (1984)(Hokkaido University)


J-Global ID

Research Interests

  • 応用微生物学   Microbial Biotechnology   Microbial Physiology   Applied Microbiology   

Research Areas

  • Life sciences / Applied microbiology

Academic & Professional Experience

  • 2000 - 2006 北海道大学 大学院農学研究科 応用生命科学専攻 分子生命科学講座 微生物資源生態学分野 教授
  • 2000 - 2006 Professor,Laboratory of Microbial Resources and Ecology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • 2006 - 北海道大学 大学院農学研究院 応用生命科学部門 分子生命科学分野 微生物生理学研究室 教授
  • 2006 - Professor,Laboratory of Microbial Physiology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Reseach Faculty of Agriculture, Hokkaido University
  • 1999 - 2000 北海道大学 大学院農学研究科 応用生命科学専攻 分子生命科学講座 微生物資源生態学分野 助教授
  • 1999 - 2000 Associate Professor,Laboratory of Microbial Resources and Ecology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • 1992 - 1999 Hokkaido University Faculty of Agriculture
  • 1992 - 1999 Associate Professor,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1991 - 1992 Hokkaido University Faculty of Agriculture
  • 1991 - 1992 Lecturer,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1989 - 1991 Hokkaido University Faculty of Agriculture
  • 1989 - 1991 Research Associate,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1984 - 1989 味の素株式会社 中央研究所 微生物化学部 研究員
  • 1984 - 1989 Researcher,Central Research Laboratory of Ajinomoto Co., Inc., Kawasaki, Japan


  •        - 1984  Hokkaido University
  •        - 1984  Hokkaido University  Graduate School, Division of Agriculture
  •        - 1981  Hokkaido University
  •        - 1981  Hokkaido University  Graduate School, Division of Agriculture
  •        - 1979  Hokkaido University  Faculty of Agriculture
  •        - 1979  Hokkaido University  Faculty of Agriculture

Association Memberships

  • (財)日本ビフィズス菌センター   (財)バイオインダストリー協会   日本応用糖質科学会   American Society for Microbiology   日本乳酸菌学会   日本生物工学会   日本農芸化学会   Japan Bifidus Foundation   Japan Bioindustry Association (JBA)   The Japanese Society of Applied Glycoscience   Japan Society for Lactic Acid Bacteria (JSLAB)   The Society for Biotechnology, Japan   and Agrochemistry   Biotechnology   Japan Society for Bioscience   

Research Activities

Published Papers

  • Mikiyasu Sakanaka, Morten Ejby Hansen, Aina Gotoh, Toshihiko Katoh, Keisuke Yoshida, Toshitaka Odamaki, Hiroyuki Yachi, Yuta Sugiyama, Shin Kurihara, Junko Hirose, Tadasu Urashima, Jin-Zhong Xiao, Motomitsu Kitaoka, Satoru Fukiya, Atsushi Yokota, Leila Lo Leggio, Maher Abou Hachem, Takane Katayama
    Science advances 5 (8) eaaw7696  2019/08 [Refereed][Not invited]
    The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.
  • Shinji Kato, Haruhi Tobe, Hiroki Matsubara, Mariko Sawada, Yasuko Sasaki, Satoru Fukiya, Naoki Morita, Atsushi Yokota
    Biochimica et biophysica acta. Molecular and cell biology of lipids 1864 (3) 403 - 412 0006-3002 2019/03 [Refereed][Not invited]
    Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131T. Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.
  • Kataoka N, Vangnai AS, Pongtharangkul T, Yakushi T, Wada M, Yokota A, Matsushita K
    Bioscience, Biotechnology, and Biochemistry 83 (2) 372 - 380 0916-8451 2019/02 [Refereed][Not invited]
  • Mikiyasu Sakanaka, Shingo Nakakawaji, Shin Nakajima, Satoru Fukiya, Arisa Abe, Wataru Saburi, Haruhide Mori, Atsushi Yokota
    Applied and environmental microbiology 84 (17) 0099-2240 2018/09/01 [Refereed][Not invited]
    Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of B. longum subsp. longumIMPORTANCE Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.
  • T. Shiraishi, S. Yokota, Y. Sato, T. Ito, S. Fukiya, S. Yamamoto, T. Sato, A. Yokota
    Beneficial Microbes 9 (4) 653 - 662 1876-2891 2018 [Refereed][Not invited]
    Lipoteichoic acid (LTA) is a cell surface molecule specific to Gram-positive bacteria. How LTA localises on the cell surface is a fundamental issue in view of recognition and immunomodulation in hosts. In the present study, we examined LTA localisation using strain JCM 1131T of Lactobacillus gasseri, which is a human intestinal lactic acid bacterium, during various growth phases by immunoelectron microscopy. We first evaluated the specificity of anti-LTA monoclonal antibody clone 55 used as a probe. The glycerophosphate backbone comprising almost intact size (20 to 30 repeating units) of LTA was required for binding. The antibody did not bind to other cellular components, including wall-teichoic acid. Immunoelectron microscopy indicated that LTA was embedded in the cell wall during the logarithmic phase, and was therefore not exposed on the cell surface. Similar results were observed for Lactobacillus fermentum ATCC 9338 and Lactobacillus rhamnosus ATCC 7469T. By contrast, membrane vesicles were observed in the logarithmic phase of L. gasseri with LTA exposed on their surface. In the stationary and death phases, LTA was exposed on cell wall-free cell membrane generated by autolysis. The dramatic alternation of localisation in different growth phases and exposure on the surface of membrane vesicles should relate with complicated interaction between bacteria and host.
  • Sarinya Tawthep, Satoru Fukiya, Ja-Young Lee, Masahito Hagio, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 124 (5) 514 - 522 1389-1723 2017/11 [Refereed][Not invited]
    Understanding the dynamics of secondary bile acid (SBA) formation in the gut by SBA-producing bacteria is important for host health, as SBAs have been shown to affect host pathophysiology and gut microbiota composition. However, our knowledge of SBA producers is limited in light of the diversity of gut microbes. Here, we isolated six novel SBA-producing bacteria from rat cecal contents, all of which were members of known species of gut microbes. Anaeros-tipes caccae DIO, Bacteroides nordii C5, Clostridioides difficile D7, and Clostridium cadaveris Gil were capable of oxidizing cholic acid and chenodeoxycholic acid into 7-oxo-derivatives with varying yields. B. nordii C5 and its type strain JCM 12987(T) had the highest molar yield, similar to 90%. Clostridium disporicum F4 and Clostridium subterminale C4 epimerized cholic acid into ursocholic acid with yields of similar to 85%; the corresponding type strains lacked epimerization activity. Further more, although not novel as an SBA producer, Clostridium scindens G10 that produced deoxycholic acid from cholic acid was isolated for the first time from rodents. These findings will contribute to elucidation of SBA formation in the gut. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Keita Nishiyama, Yuji Yamamoto, Makoto Sugiyama, Takashi Takaki, Tadasu Urashima, Satoru Fukiya, Atsushi Yokota, Nobuhiko Okada, Takao Mukai
    MBIO 8 (5) 2150-7511 2017/09 [Refereed][Not invited]
    Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the alpha 2,6-linked but not to the alpha 2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAc alpha 1-3(Fuc alpha 1-2) Gal beta] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates. IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the alpha 2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.
  • Masamichi Watanabe, Satoru Fukiya, Atsushi Yokota
    JOURNAL OF LIPID RESEARCH 58 (6) 1143 - 1152 0022-2275 2017/06 [Refereed][Not invited]
    In addition to functioning as detergents that aid digestion of dietary lipids in the intestine, some bile acids have been shown to exhibit antimicrobial activity. However, detailed information on the bactericidal activities of the diverse molecular species of bile acid in humans and rodents is largely unknown. Here, we investigated the toxicity of 14 typical human and rodent free bile acids (FBAs) by monitoring intracellular pH, membrane integrity, and viability of a human intestinal bacterium, Bifidobacterium breve Japan Collection of Microorganisms (JCM) 1192(T), upon exposure to these FBAs. Of all FBAs evaluated, deoxycholic acid (DCA) and chenodeoxycholic acid displayed the highest toxicities. Nine FBAs common to humans and rodents demonstrated that beta-hydroxy-type bile acids are more toxic than their oxoderivatives and beta-hydroxy-type epimers. In five rodent-specific FBAs, beta-muricholic acid and hyodeoxycholic acid showed comparable toxicities at a level close to DCA. Similar trends were observed for the membrane-damaging effects and bactericidal activities to Blautia coccoides JCM 1395(T) and Bacteroides thetaiotaomicron DSM 2079(T), commonly represented in the human and rodent gut microbiota. These findings will help us to determine the fundamental properties of FBAs and better understand the role of FBAs in the regulation of gut microbiota composition.-
  • Soya Maeda, Kumiko Shimizu, Chie Kihira, Yuki Iwabu, Ryuichi Kato, Makoto Sugimoto, Satoru Fukiya, Masaru Wada, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 123 (4) 437 - 443 1389-1723 2017/04 [Refereed][Not invited]
    Pyruvate dehydrogenase complex regulator (PdhR) is a transcriptional regulator that negatively regulates formation of pyruvate dehydrogenase complex (PDHc), NADH dehydrogenase (NDH)-2, and cytochrome boa oxidase in Escherichia coli. To investigate the effects of a PdhR defect on glucose metabolism, a pdhR deletion mutant was derived from the wild-type E. coli W1485 strain by A Red-mediated recombination. While no difference in the fermentation profiles was observed between the two strains under oxygen-sufficient conditions, under oxygen-limited conditions, the growth level of the wild-type strain was significantly decreased with retarded glucose consumption accompanied by by production of substantial amounts of pyruvic acid and acetic acid. In contrast, the mutant grew and consumed glucose more efficiently than did the wild-type strain with enhanced respiration, little by-production of pyruvic acid, less production yield and rates of acetic acid, thus displaying robust metabolic activity. As expected, increased activities of PDHc and NDH-2 were observed in the mutant. The increased activity of PDHc may explain the loss of pyruvic acid by production, probably leading to decreased acetic acid formation, and the increased activity of NDH-2 may explain the enhanced respiration. Measurement of the intracellular NAD(+)/NADH ratio in the mutant revealed more oxidative or more reductive intracellular environments than those in the wild-type strain under oxygen-sufficient and- limited conditions, respectively, suggesting another role of PdhR: maintaining redox balance in E. coli. The overall results demonstrate the biotechnological advantages of pdhR deletion in boosting glucose metabolism and also improve our understanding of the role of PdhR in bacterial physiology. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Hiroyuki Nagano, Kana Shibano, Yu Matsumoto, Atsushi Yokota, Masaru Wada
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 (6) 1156 - 1164 0916-8451 2017 [Refereed][Not invited]
    An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.
  • Atsushi Yokota, Kazunori Sawada, Masaru Wada
    AMINO ACID FERMENTATION 159 181 - 198 0724-6145 2017 [Refereed][Not invited]
    In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CSL), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPCR), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques. The pyk deletion and PEPCR (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CSL (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CSL mutant showed instabilities in growth and lysine yield. Surprisingly, the pyk deletion was found to increase biomass production in wildtype C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD660) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic
  • Masaki Yanase, Tohru Aikoh, Kazunori Sawada, Kotaro Ogura, Takuya Hagiwara, Keita Imai, Masaru Wada, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 122 (2) 160 - 167 1389-1723 2016/08 [Refereed][Not invited]
    Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 lysine from 100 g/L, glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (similar to 1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (138-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 a). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
  • Masaru Wada, Kazunori Sawada, Kotaro Ogura, Yuta Shimono, Takuya Hagiwara, Masakazu Sugimoto, Akiko Onuki, Atsushi Yokotal
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 121 (2) 172 - 177 1389-1723 2016/02 [Refereed][Not invited]
    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.
  • Tsukasa Shiraishi, Shinichi Yokota, Satoru Fukiya, Atsushi Yokota
    Bioscience of microbiota, food and health 35 (4) 147 - 161 2186-6953 2016 [Refereed][Not invited]
    Bacterial cell surface molecules are at the forefront of host-bacterium interactions. Teichoic acids are observed only in Gram-positive bacteria, and they are one of the main cell surface components. Teichoic acids play important physiological roles and contribute to the bacterial interaction with their host. In particular, lipoteichoic acid (LTA) anchored to the cell membrane has attracted attention as a host immunomodulator. Chemical and biological characteristics of LTA from various bacteria have been described. However, most of the information concerns pathogenic bacteria, and information on beneficial bacteria, including probiotic lactic acid bacteria, is insufficient. LTA is structurally diverse. Strain-level structural diversity of LTA is suggested to underpin its immunomodulatory activities. Thus, the structural information on LTA in probiotics, in particular strain-associated diversity, is important for understanding its beneficial roles associated with the modulation of immune response. Continued accumulation of structural information is necessary to elucidate the detailed physiological roles and significance of LTA. In this review article, we summarize the current state of knowledge on LTA structure, in particular the structure of LTA from lactic acid bacteria. We also describe the significance of structural diversity and biological roles of LTA.
  • Yu Matsumoto, Yoshiaki Yasutake, Yuki Takeda, Tomohiro Tamura, Atsushi Yokota, Masaru Wada
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 99 (17) 7137 - 7150 0175-7598 2015/09 [Refereed][Not invited]
    d-threo-3-Hydroxyaspartate dehydratase (d-THA DH) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme, isolated from a soil bacterium of Delftia sp. HT23. It catalyzes the dehydration of d-threo-3-hydroxyaspartate (d-THA) and l-erythro-3-hydroxyaspartate (l-EHA). To elucidate the mechanism of substrate stereospecificity, crystal structures of d-THA DH were determined in complex with various ligands, such as an inhibitor (d-erythro-3-hydroxyaspartate (d-EHA)), a substrate (l-EHA), and the reaction intermediate (2-amino maleic acid). The C (beta) -OH of l-EHA occupied a position close to the active-site Mg2+, clearly indicating a possibility of metal-assisted C (beta) -OH elimination from the substrate. In contrast, the C (beta) -OH of an inhibitor was bound far from the active-site Mg2+. This suggests that the substrate specificity of d-THA DH is determined by the orientation of the C (beta) -OH at the active site, whose spatial arrangement is compatible with the 3R configuration of 3-hydroxyaspartate. We also report an optically pure synthesis of l-threo-3-hydroxyaspartate (l-THA) and d-EHA, promising intermediates for the synthesis of beta-benzyloxyaspartate, by using a purified d-THA DH as a biocatalyst for the resolution of racemic dl-threo-3-hydroxyaspartate (dl-THA) and dl-erythro-3-hydroxyaspartate (dl-EHA). Considering 50 % of the theoretical maximum, efficient yields of l-THA (38.9 %) and d-EHA (48.9 %) as isolated crystals were achieved with > 99 % enantiomeric excess (e.e.). The results of nuclear magnetic resonance signals verified the chemical purity of the products. We were directly able to isolate analytically pure compounds by the recrystallization of acidified reaction mixtures (pH 2.0) and thus avoiding the use of environmentally harmful organic solvents for the chromatographic purification.
  • Hidehisa Shimizu, Nanako Baba, Takuma Nose, Ryoko Taguchi, Shinya Tanaka, Ga-Hyun Joe, Hideaki Maseda, Nobuhiko Nomura, Masahito Hagio, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka, Hitoshi Miyazaki
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 (6) 937 - 942 0916-8451 2015/06 [Refereed][Not invited]
    The signal molecule, 3-oxo-C-12-homoserine lactone (3-oxo-C-12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C-12-HSL concentrations (>200 mu M) on mammalian cellular functions because ~600 mu M 3-oxo-C-12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C-12-HSL concentration (30 mu M) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C-12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C-12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C-12-HSL.
  • Mikiyasu Sakanaka, Satoru Fukiya, Ryoko Kobayashi, Arisa Abe, Yosuke Hirayama, Yasunobu Kano, Atsushi Yokota
    FEMS MICROBIOLOGY LETTERS 362 (7) 0378-1097 2015/04 [Refereed][Not invited]
    Transposon mutagenesis systems are still under development in bifidobacteria, partly because intrinsic active insertion sequences are not well characterized in bifidobacteria. Here, we isolated an active insertion sequence, ISBlo11, from Bifidobacterium longum 105-A using a sacB-based counterselection system, which is generally used to screen for active insertion sequences from bacterial genomes. ISBlo11 is 1432 bp long and belongs to the IS3 family. It has a single ORF encoding a transposase and 25-bp inverted repeats at its termini. Full-length copies of ISBlo11 are specifically conserved among certain B. longum genomes and exist in different sites. Transposition analysis of an artificial ISBlo11 transposon using an Escherichia coli conjugation system revealed that ISBlo11 has adequate transposition activity, comparable to the reported activity of IS629, another IS3 family element initially isolated from Shigella sonnei. ISBlo11 also showed low transposition selectivity for non-conserved 3-or 4-bp target sequences. These characteristics of ISBlo11 seem suitable for the development of a new transposon mutagenesis system in bifidobacteria.
  • Masahito Hagio, Hidehisa Shimizu, Ga-Hyun Joe, Manami Takatsuki, Maiko Shiwaku, Hong Xu, Ja-Young Lee, Nobuyuki Fujii, Satoru Fukiya, Hiroshi Hara, Atsushi Yokota, Satoshi Ishizuka
    TOXICOLOGY LETTERS 232 (1) 246 - 252 0378-4274 2015/01 [Refereed][Not invited]
    Consumption of a high-fat diet increases some secondary bile acids (BAs) such as deoxycholic acid (DCA) in feces. DCA is derived from cholic acid (CA), a primary BA. We evaluated intestinal epithelial proliferation and BA metabolism in response to oral administration of cholic acid (CA) in rats to determine the influence of a CA diet on the responses of gut epithelia to gamma-rays. WKAH/HkmSlc rats were divided into two dietary groups: control diet or CA-supplemented (2 g/kg diet) diet. Some of the rats from each group were irradiated with gamma-rays, and epithelial cell proliferation in the colon was analyzed histochemically. Unirradiated CA-fed rats had high levels of DCA and CA in the sera, as well as the presence of taurocholic acid in their feces. Significant increases were observed in both epithelial proliferation and the number of epithelial cells in the colon of the CA-fed rats, and this effect was observed at 8 weeks after gamma-ray exposure. Furthermore, extracts from both cecal contents and sera of the unirradiated CA-fed rats promoted proliferation of IEC-6 cells. These results indicate that BAs in enterohepatic circulation promote proliferation and survival of the intestinal epithelium after receiving DNA damage. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
  • Yu Kanesaki, Hisayoshi Masutani, Mikiyasu Sakanaka, Yuh Shiwa, Takatomo Fujisawa, Yasukazu Nakamura, Atsushi Yokota, Satoru Fukiya, Tohru Suzuki, Hirofumi Yoshikawa
    Genome announcements 2 (6) 2014/12/18 [Refereed][Not invited]
    Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency.
  • Hidehisa Shimizu, Masahito Hagio, Hitoshi Iwaya, Ikuya Tsuneki, Ja-Young Lee, Satoru Fukiya, Atsushi Yokota, Hitoshi Miyazaki, Hiroshi Hara, Satoshi Ishizuka
    JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY 60 (6) 450 - 454 0301-4800 2014/12 [Refereed][Not invited]
    Obesity is increasingly becoming associated with increased risk of atherosclerosis. Serum levels of the bile acid deoxycholic acid (DCA) are elevated in mice with obesity induced by a high-fat (HF) diet. Therefore, we investigated the influence of DCA on the functions of vascular smooth muscle cells (VSMCs) because the initiation and progression of atherosclerosis are associated with VSMC proliferation and migration. DCA induced c-jun N-terminal kinase (JNK) activation whereas a JNK inhibitor prevented DCA-induced VSMC proliferation and migration. Based on these findings, we examined whether DCA promotes the expression of platelet-derived growth factor beta-receptor (PDGFR beta) that has a c-Jun binding site in its promoter region. The mRNA and protein expression levels of PDGFR beta were upregulated in VSMCs after a 24- and 48-h incubation with DCA, respectively. The effects of PDGF such as proliferation and migration of VSMCs were promoted after a 48-h incubation with DCA despite the absence of DCA during PDGF stimulation. These findings suggest that elevated serum concentrations of DCA are involved in the pathogenesis of atherosclerosis in HF-induced obesity.
  • Mikiyasu Sakanaka, Saki Tamai, Yosuke Hirayama, Ai Onodera, Hiroka Koguchi, Yasunobu Kano, Atsushi Yokota, Satoru Fukiya
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 118 (5) 489 - 495 1389-1723 2014/11 [Refereed][Not invited]
    Heterologous gene expression in bifidobacteria requires weak, strong, and inducible promoters depending on the objectives of different expression studies. Weak promoters in Escherichia colt can also be desirable for stable heterologous gene cloning. Here, we developed a reporter system using the Bifidobacterium longum alpha-galactosidase gene and investigated the activity and inducibility of seven bifidobacterial promoters in B. longum and their activities in E. colt. These studies revealed diverse promoter activities. Three promoters were highly active in B. longum, but only slightly active in E. coli. Among these, two phosphoketolase gene (xfp) promoters exhibited strong activity in B. longum cells grown on glucose. In contrast, the promoter activity of the fructose transporter operon (fruERFG) was strongly induced by carbohydrates other than glucose, including fructose, xylose, and ribose. These promoters will allow strong or highly inducible expression in bifidobacteria and stable gene cloning in E. con. In contrast to the functions of these promoters, the promoter of sucrose-utilization operon cscBA showed very high activity in E. coli but low activity in B. longum. Other three promoters were functional in both B. longum and E. con. In particular, two sucrose phosphorylase gene (scrP) promoters showed inducible activity by sucrose and raffinose in B. longum, indicating their applicability for regulated expression studies. The diverse promoter functions revealed in this study will contribute to enabling the regulated expression of heterologous genes in bifidobacteria research. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.
  • Takashi Narihiro, Aya Suzuki, Kazuaki Yoshimune, Tomoyuki Hori, Tamotsu Hoshino, Isao Yumoto, Atsushi Yokota, Nobutada Kimura, Yoichi Kamagata
    MICROBES AND ENVIRONMENTS 29 (2) 154 - 161 1342-6311 2014/07 [Refereed][Not invited]
    Metagenomic screening and conventional cultivation have been used to exploit microbial lipolytic enzymes in nature. We used an indigenous forest soil (NS) and oil-fed enriched soil (OS) as microbial and genetic resources. Thirty-four strains (17 each) of lipolytic bacteria were isolated from the NS and OS microcosms. These isolates were classified into the (sub) phyla Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria, all of which are known to be the main microbial resources of commercially available lipolytic enzymes. Seven and 39 lipolytic enzymes were successfully retrieved from the metagenomic libraries of the NS and OS microcosms, respectively. The screening efficiency (a ratio of positive lipolytic clones to the total number of environmental clones) was markedly higher in the OS microcosm than in the NS microcosm. Moreover, metagenomic clones encoding the lipolytic enzymes associated with Alphaproteobacteria, Deltaproteobacteria, Acidobacteria, Armatimonadetes, and Planctomycetes and hitherto-uncultivated microbes were recovered from these libraries. The results of the present study indicate that functional metagenomics can be effectively used to capture as yet undiscovered lipolytic enzymes that have eluded the cultivation-based method, and these combined approaches may be able to provide an overview of lipolytic organisms potentially present in nature.
  • Yuki Soma, Keigo Tsuruno, Masaru Wada, Atsushi Yokota, Taizo Hanai
    METABOLIC ENGINEERING 23 175 - 184 1096-7176 2014/05 [Refereed][Not invited]
    Overexpressiun of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc.
  • Ja-Young Lee, Hisashi Arai, Yusuke Nakamura, Satoru Fukiya, Masaru Wada, Atsushi Yokota
    JOURNAL OF LIPID RESEARCH 54 (11) 3062 - 3069 0022-2275 2013/11 [Refereed][Not invited]
    Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7 beta-HSDH revealed that the k(cat)/K-m value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon.
  • Yu Matsumoto, Yoshiaki Yasutake, Yuki Takeda, Tomohiro Tamura, Atsushi Yokota, Masaru Wada
    D-threo-3-Hydroxyaspartate dehydratase (D-THA DH) isolated from the soil bacterium Delftia sp. HT23 is a novel enzyme consisting of 380 amino-acid residues which catalyzes the conversion of D-threo-3-hydroxyaspartate to oxaloacetate and ammonia. D-THA DH also catalyzes the dehydration of L-threo-3-hydroxyaspartate, L-erythro-3-hydroxyaspartate and D-serine. The amino-acid sequence of D-THA DH shows significant similarity to that of two eukaryotic D-serine dehydratases derived from Saccharomyces cerevisiae and chicken kidney. D-THA DH is classified into the fold-type III group of pyridoxal enzymes and is the first example of a fold-type III dehydratase derived from a prokaryote. Overexpression of recombinant D-THA DH was carried out using a Rhodococcus erythropolis expression system and the obtained protein was subsequently purified and crystallized. The crystals of D-THA DH belonged to space group I4(1)22, with unit-cell parameters a = b = 157.3, c = 157.9 angstrom. Single-wavelength anomalous diffraction data were collected to a resolution of 2.0 angstrom using synchrotron radiation at the wavelength of the Br K absorption edge.
  • Haruko Sakurama, Masashi Kiyohara, Jun Wada, Yuji Honda, Masanori Yamaguchi, Satoru Fukiya, Atsushi Yokota, Hisashi Ashida, Hidehiko Kumagai, Motomitsu Kitaoka, Kenji Yamamoto, Takane Katayama
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (35) 25194 - 25206 0021-9258 2013/08 [Refereed][Not invited]
    Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N- tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Gal beta 1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAc beta 1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), and sialyllacto-N-tetraose a (Neu5Ac alpha 2-3Gal beta 1-3GlcNAc beta 1-3-Gal beta 1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-beta-lacto-N-bioside I (Gal beta 1-3-GlcNAc beta-pNP) and GalNAc beta 1-3GlcNAc beta-pNP.GalNAc beta 1-3-GlcNAc beta linkage has been found in O-mannosyl glycans of alpha-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.
  • Tsukasa Shiraishi, Shin-ichi Yokota, Naoki Morita, Satoru Fukiya, Satoru Tomita, Naoto Tanaka, Sanae Okada, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 79 (10) 3315 - 3318 0099-2240 2013/05 [Refereed][Not invited]
    We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131(T). The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.
  • Yosuke Hirayama, Mikiyasu Sakanaka, Hidenori Fukuma, Hiroki Murayama, Yasunobu Kano, Satoru Fukiya, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 (14) 4984 - 4994 0099-2240 2012/07 [Refereed][Not invited]
    Functional analysis of Bifidobacterium genes is essential for understanding host-Bifidobacterium interactions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover in Bifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-Delta repA, which lacks the plasmid replication gene repA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harbors repA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected in B. longum 105-A using the chromosomal full-length beta-galactosidase gene as a target. Markerless gene deletion was tested using the aga gene, which encodes alpha-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion of aga. Carbohydrate assimilation analysis and alpha-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-based aga-complemented strain. These functional analyses revealed that aga is the only gene encoding a functional alpha-galactosidase enzyme in B. longum 105-A.
  • Kazunori Sawada, Yui Kato, Keita Imai, Liyuan Li, Masaru Wada, Kazunobu Matsushita, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 113 (4) 467 - 473 1389-1723 2012/04 [Refereed][Not invited]
    We previously reported that a spontaneous H+-ATPase-defective mutant of Corynebacterium glutamicum, F172-8, derived from C. glutamicum ATCC 14067, showed enhanced glucose consumption and respiration rates. To investigate the genome-based mechanism of enhanced respiration rate in such C glutamicum mutants, A-1, an H+-ATPase-defective mutant derived from C. glutamicum ATCC 13032, which harbors the same point mutation as F172-8, was used in this study. A-1 showed similar fermentation profiles to F172-8 when cultured in a jar fermentor. Enzyme activity measurements, quantitative real-time PCR, and DNA microarray analysis suggested that A-1 enhanced malate:quinone oxidoreductase/malate dehydrogenase and L-lactate dehydrogenase/NAD(+)-dependent-lactate dehydrogenase coupling reactions, but not NADH dehydrogenase-II, for reoxidation of the excess NADH arising from enhanced glucose consumption. A-1 also up-regulated succinate dehydrogenase, which may result in the relief of excess proton-motive force (pmf) in the H+-ATPase mutant. In addition, the transcriptional level of cytochrome bd oxidase, but not cytochrome bc(1)-aa(3), also increased, which may help prevent the excess pmf generation caused by enhanced respiration. These results indicate that C. glutamicum possesses intriguing strategies for coping with NADH over-accumulation. Furthermore, these mechanisms are different from those in Escherichia coli, even though the two species use similar strategies to prevent excess pmf generation. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.
  • Chie Kihira, Yukari Hayashi, Naoki Azuma, Sakiko Noda, Soya Maeda, Satoru Fukiya, Masaru Wada, Kazunobu Matsushita, Atsushi Yokota
    JOURNAL OF BIOTECHNOLOGY 158 (4) 215 - 223 0168-1656 2012/04 [Not refereed][Not invited]
    The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo(3) oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted l-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant. (C) 2011 Elsevier B.V. All rights reserved.
  • Atsushi Yokota, Satoru Fukiya, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Satoshi Ishizuka
    Gut Microbes 3 (5) 455 - 459 1949-0984 2012 [Refereed][Not invited]
    Recently, we discovered that bile acid, a main component of bile, is a host factor that regulates the composition of the cecal microbiota in rats. Because bile secretion increases on a high-fat diet and bile acids generally have strong antimicrobial activity, we speculated that bile acids would be a determinant of the gut microbiota in response to a high-fat diet. The observed changes in the rat cecal microbiota triggered by cholic acid (the most abundant bile acid in human biliary bile) administration resemble those found in animals fed high-fat diets. Here, we discuss the rationale for this hypothesis by evaluating reported diet-induced gut microbiota alterations based on the postulate that bile acids worked as an underlying determinant. The identification of host factors determining the gut microbiota greatly contributes to understanding the causal relationships between changes in the gut microbiota and disease development, which remain to be elucidated. © 2012 Landes Bioscience.
  • Satoru Fukiya, Yosuke Hirayama, Mikiyasu Sakanaka, Yasunobu Kano, Atsushi Yokota
    Bioscience of microbiota, food and health 31 (2) 15 - 25 2186-6953 2012 [Refereed][Not invited]
    Bifidobacteria are well known as beneficial intestinal bacteria that exert health-promoting effects in humans. In addition to physiological and immunological investigations, molecular genetic technologies have been developed and have recently started to be applied to clarify the molecular bases of host-Bifidobacterium interactions. These technologies include transformation technologies and Escherichia coli-Bifidobacterium shuttle vectors that enable heterologous gene expression. In this context, a plasmid artificial modification method that protects the introduced plasmid from the restriction system in host bifidobacteria has recently been developed to increase transformation efficiency. On the other hand, targeted gene inactivation systems, which are vital for functional genomics, seemed far from being practically applicable in bifidobacteria. However, remarkable progress in this technology has recently been achieved, enabling functional genomics in bifidobacteria. Integrated use of these molecular genetic technologies with omics-based analyses will surely boost characterization of the molecular basis underlying beneficial effects of bifidobacteria. Applications of recombinant bifidobacteria to medical treatments have also progressed.
  • K. B. M. Saiful Islam, Satoru Fukiya, Masahito Hagio, Nobuyuki Fujii, Satoshi Ishizuka, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota
    GASTROENTEROLOGY 141 (5) 1773 - 1781 0016-5085 2011/11 [Not refereed][Not invited]
    BACKGROUND & AIMS: Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. METHODS: Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. RESULTS: Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7 alpha-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. CONCLUSIONS: Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.
  • Takayuki Maeda, Yuki Takeda, Tomoko Murakami, Atsushi Yokota, Masaru Wada
    JOURNAL OF BIOCHEMISTRY 148 (6) 705 - 712 0021-924X 2010/12 [Not refereed][Not invited]
    D-threo-3-hydroxyaspartate dehydratase (D-THA DH) was purified from the cell-free extract of the soil-isolated bacterium Delftia sp. HT23. The enzyme exhibited dehydratase activity towards D-threo-3-hydroxyaspartate, l-threo-3-hydroxyaspartate, l-erythro-3-hydroxyaspartate and d-serine. Absorption of the purified enzyme at 412 nm suggests that it contains pyridoxal 5'-phosphate (PLP) as a cofactor. The NH(2)-terminal and internal amino acid sequences showed significant similarity to hypothetical alanine racemase of genome-sequenced Delftia acidovorans SPH-1; however, the purified enzyme showed no alanine racemase activity. Using the sequence information of D. acidovorans SPH-1, the gene encoding d-THA DH was cloned. The deduced amino acid sequence, which belongs to the alanine racemase family, shows significant (26-36%) similarity to d-serine dehydratase of both Saccharomyces cerevisiae and chicken. In order to obtain purified d-THA DH efficiently, the gene was expressed in Escherichia coli. The recombinant enzyme was highly activated by divalent cations, such as Mn(2+), Co(2+) and Ni(2+). Site-directed mutagenesis experiment revealed that lysine 43 is an important residue involved in PLP binding and catalysis. This is the first reported enzyme that acts on d-THA. In addition, this enzyme is the first example of a prokaryotic dehydratase belonging to the fold-type III PLP-dependent enzyme family.
  • Takuya Suzuki, Megumi Nishimukai, Aki Shinoki, Hidenori Taguchi, Satoru Fukiya, Atsushi Yokota, Wataru Saburi, Takeshi Yamamoto, Hiroshi Hara, Hirokazu Matsui
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 58 (19) 10787 - 10792 0021-8561 2010/10 [Not refereed][Not invited]
    Gastrectomy often results in osteopenia and anemia because of calcium (Ca) and iron (Fe) malabsorption. Here, we investigated the effects of feeding epilactose, a non-digestible disaccharide, on gastrectomy-induced osteopenia, anemia, and Ca and Fe malabsorption in male Sprague Dawley rats. Totally gastrectomized or sham-operated rats were fed the control or epilactose (50 g/kg) diets for 30 days. Gastrectomy severely decreased intestinal Ca and Fe absorption, femoral bone strength, Ca content, hemoglobin concentration, and hematocrit. These decreases were partly or totally restored by feeding epilactose. Feeding epilactose increased the cecal tissue weight and the soluble Ca concentration and short-chain fatty acid pools of the cecal contents. Collectively, the increases in cecal mucosal area and/or soluble Ca concentration of the cecal contents, resulting from short-chain fatty acid production by intestinal microbes, are thought to be responsible for the epilactose-mediated promotion of Ca and Fe absorption in the gastrectomized rats.
  • Satoru Fukiya, Tomohiko Sugiyama, Yasunobu Kano, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 110 (2) 141 - 146 1389-1723 2010/08 [Not refereed][Not invited]
    The characteristics of mobile genetic elements in bifidobacteria are not well understood. We characterized an insertion sequence-like element of the IS200/IS605 family found in a size-increased cryptic plasmid in Bifidobacterium longum. During a plasmid profile analysis of B. longum BK strains, we encountered a 6.5-kbp cryptic plasmid pBK283 in B. longum BK28, the size of which has not been identified in bifidobacteria. Nucleotide sequence analysis indicated that an insertion sequence-like element was inserted into the 5.0-kbp pKJ50-like plasmid and resulted in a size increase of pBK283. The element, named ISBlo15, was 1593 bp in length and contained a single ORF encoding a putative transposase, which is similar to the transposase OrfB encoded by IS200/IS605 family elements. Several sequence characteristics, including conserved transposase motifs in OrfB and terminal palindromic sequences that differ from the typical terminal inverted repeats, strongly suggested that ISBlo15 is a member of the IS200/IS605 family. Sequences similar to ISBlo15 were widely distributed among the nine Bifidobacterium species tested, and those of highly homologous sequences were detected only in Bifidobacterium gallicum JCM8224(T). (C) 2010, The Society for Biotechnology, Japan. All rights reserved.
  • Kazunori Sawada, Susumu Zen-in, Masaru Wada, Atsushi Yokota
    METABOLIC ENGINEERING 12 (4) 401 - 407 1096-7176 2010/07 [Not refereed][Not invited]
    To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wile-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolypyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum. (C) 2010 Elsevier Inc. All rights reserved.
  • Hiroto Kikuchi, Hiroaki Sakurai, Taizo Nagura, Tsutomu Aritsuka, Fusao Tomita, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 109 (3) 240 - 243 1389-1723 2010/03 [Not refereed][Not invited]
    The newly established difructose anhydride IV (DFA IV) production system is comprised of the effective production of levan from sucrose by Serratia levanicum NN, the conversion of the levan into DFA IV by levan fructotransferase from Arthrobacter nicotinovorans GS-9, which is highly expressed in an Escherichia coli transformant, and a practical purification step. The chemical properties of DFA IV were also investigated. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.
  • Satoshi Ishizuka, Ami Iwama, Achmad Dinoto, Akarat Suksomcheep, Kohshi Maeta, Takanori Kasai, Hiroshi Hara, Atsushi Yokota
    MOLECULAR NUTRITION & FOOD RESEARCH 53 S62 - S67 1613-4125 2009/05 [Not refereed][Not invited]
    We evaluated the effects of Bifidobacterium breve JCM1192(T) and/or raffinose on epithelial proliferation in the rat small and large intestines. WKAH/Hkm Slc rats (4 wk old) were fed a control diet, a diet supplemented with either encapsulated B. breve (30 g/kg diet, 1.5 x 10(7) colony-forming unit/g capsule) or raffinose (30 g/kg diet), or a diet supplemented with both encapsulated B. breve and raffinose, for 3 wk. Epithelial proliferation in the small intestine, as assessed by bromodeoxyuridine immunohistochemistry, was increased only in the B. breve plus raffinose-fed group. We determined the number of bifidobacteria in cecal contents using fluorescence in situ hybridization and confirmed the presence of ingested B. breve only in the B. breve plus raffinose-fed group. This suggests that the ingested B. breve cells used raffinose and were activated in the small intestine, where they subsequently influenced epithelial proliferation. In conclusion, we found a prominent synbiotic effect of encapsulated B. breve in combination with raffinose on epithelial proliferation in rat small intestine but not in large intestine. To our knowledge, this is the first report of a synbiotic that affects epithelial proliferation.
  • Ogami, Shinichi, Hijikata, Shoichi, Tsukahara, Tamotsu, Mie, Yasuhiro, Matsuno, Toshihide, Morita, Naoki, Hara, Isao, Yamazaki, Koji, Inoue, Norio, Yokota, Atsushi, Hoshino, Tamotsu, Yoshimune, Kazuaki, Yumoto, Isao
    EXTREMOPHILES 13 (3) 491 - 504 1431-0651 2009/05 [Not refereed][Not invited]
    A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C-15, C-16 and C-17 chain lengths and C-15 chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) -> Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.
  • Tomoko Murakami, Takayuki Maeda, Atsushi Yokota, Masaru Wada
    JOURNAL OF BIOCHEMISTRY 145 (5) 661 - 668 0021-924X 2009/05 [Not refereed][Not invited]
    L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC, which catalyses the cleavage of L-threo-3-hydroxyaspartate (L-THA) to oxalacetate and ammonia, has been purified from the soil bacterium Pseudomonas sp. T62. In this report, the gene encoding L-THA DH was cloned and expressed in Escherichia coli, and the gene product was purified and characterized in detail. A 957-bp nucleotide fragment was confirmed to be the gene encoding L-THA DH, based on the agreement of internal amino acid sequences. The deduced amino acid sequence, which belongs to the serine/threonine dehydratase family, shows similarity to YKL218c from Saccharomyces cerevisiae (64), serine racemase from Schizosaccharomyces pombe (64) and Mus musculus (36), and biodegradative threonine dehydratase from E. coli (38). Site-directed mutagenesis experiments revealed that lysine at position 53 is an important residue for enzymatic activity. This enzyme exhibited dehydratase activity specific only to L-THA [K-m 0.54 mM, V-max 39.0 mol min(1) (mg protein)(1)], but not to other 3-hydroxyaspartate isomers, and exhibited no detectable serine/aspartate racemase activity. This is the first report of an amino acid sequence of the bacterial enzyme that acts on L-THA.
  • Satoru Fukiya, Miki Arata, Hiroko Kawashima, Daisuke Yoshida, Maki Kaneko, Kimiko Minamida, Jun Watanabe, Yoshio Ogura, Kiyohisa Uchida, Kikuji Itoh, Masaru Wada, Susumu Ito, Atsushi Yokota
    FEMS MICROBIOLOGY LETTERS 293 (2) 263 - 270 0378-1097 2009/04 [Not refereed][Not invited]
    Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019(T), which are known to possess 7 alpha-HSDH activity. The level of 7 alpha-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7 alpha-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.
  • Hiroto Kikuchi, Masanao Inoue, Hidetoshi Saito, Hiroaki Sakurai, Tsutomu Aritsuka, Fusao Tomita, Atsushi Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 107 (3) 262 - 265 1389-1723 2009/03 [Not refereed][Not invited]
    A practical, economical, and industrial process for the enzymatic production of difructose anhydride III (DFA III) was investigated for crude inulin prepared from chicory roots using Arthrobacter sp. H65-7 fructosyltransferase. A comparable level of DFA III production to that from commercial inulin was obtained using crude inulin, suggesting the feasibility of this production process. (C) 2009. The Society for Biotechnology, Japan. All rights reserved.
  • Takeshi Senoura, Hidenori Taguchi, Shigeaki Ito, Shigeki Hamada, Hirokazu Matsui, Satoru Fukiya, Atsushi Yokota, Jun Watanabe, Jun Wasaki, Susumu Ito
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 (2) 400 - 406 0916-8451 2009/02 [Not refereed][Not invited]
    Cellobiose 2-epimerase (CE, EC catalyzes the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilis NCTC 9343. The recombinant enzyme, expressed in Escherichia coli cells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, beta-mannobiose (4-O-beta-D-mannopyranosyl-D-mannose), and globotriose [O-alpha-D-galactopyranosyl-(1 -> 4)-O-beta-D-galactopyranosyl-(1 -> 4)-D-glucose]. The CE from B. fragilis showed less than 40% identity to reported functional CEs. It exhibited 44-63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacteria] genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.
  • Masaru Wada, Nowaki Hijikata, Ryo Aoki, Nobuchika Takesue, Atsushi Yokota
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 (11) 2959 - 2965 0916-8451 2008/11 [Not refereed][Not invited]
    We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H+-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H+-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H+-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mm for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H+-ATPase-defective mutant of C glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H+-ATPase defect is also effective in the fermentative production of other practical compounds.
  • Eriko Yahata, Yuki Sawatari, Hisashi Sugiyama, Akihiro Hanaoka, Atsushi Yokota, Haruo Saruyama
    FOOD SCIENCE AND TECHNOLOGY RESEARCH 14 (3) 285 - 292 1421-9972 2008/05 [Not refereed][Not invited]
    The lactic acid fermentation of instant Chinese noodle sheet by Laetobacillus plantarum NRIC 0380 changed the noodle quality as evaluated by texture and sensory tests. This interesting change was induced by only a short 2-h fermentation time, however the noodle quality subsequently degraded with prolongation of the fermentation of up to 24 h. SDS-PAGE analysis of proteins in the noodle sheet indicated no change in proteins extracted from the 2-h fermented noodle sheet compared to non-fermented noodle sheet. In contrast, native-PAGE analysis showed a shift in molecular weight of gluten proteins, with those extracted from the 2-h fermented noodle sheet having a higher molecular weight than those from non-fermented noodle sheet. These results strongly suggested a conformational change of gluten proteins in the noodle sheet caused by the short 2-h fermentation. The 2-h lactic acid fermentation decreased the pH from 8.5 to 7.5, but gluten proteins extracted from noodle sheet made with the addition of lactic acid to adjust the pH to 7.3 did not show this increase in molecular weight. Thus, the change from native state of gluten proteins does not seem to be induced by the presence of the lactic acid itself but by other factor(s) associated with lactic acid fermentation. On the other hand, the reduction of noodle quality by prolonged fermentation for up to 24 h seems to induce degradation of albumin and globulin proteins as revealed by SDS-PAGE analysis and also the change in the native state of gluten proteins as detected by native-PAGE analysis.
  • Masaru Wada, Kae Okabe, Michihiko Kataoka, Sakayu Shimizu, Atsushi Yokota, Hiroshi Takagi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 (2) 582 - 586 0916-8451 2008/02 [Not refereed][Not invited]
    The budding yeast Saccharomyces cerevisiae Sigma 1278b contains the MPR1 gene encoding N-acetyltransferase, which detoxifies the L-proline analog L-azetidine-2-carboxylate (AZC). Of 131 yeasts tested, AZC acetyltransferase activity was detected in 17 strains of 41 strains that showed AZC resistance. Degenerate-PCR analysis revealed that two strains, i.e., Candida saitoana AKU4533 and Wickerhamia fluorescens AKU4722, contained a DNA fragment highly homologous to MPR1. This indicates that AZC acetyltransferases are widely distributed in yeasts.
  • Liyuan Li, Masaru Wada, Atsushi Yokota
    PROTEOMICS 7 (23) 4317 - 4322 1615-9853 2007/12 [Not refereed][Not invited]
    We constructed a cytoplasmic proteome reference map for a glutamic acid producing Corynebacterium glutamicum ATCC 14067 by 2-DE and protein identification by MALDI-TOF-MS and PMF using genome database of the type strain ATCC 13032. The map allowed us to identify 166 protein spots representing 139 different proteins. A considerable strain difference was observed in the proteomic images between strains ATCC 14067 and ATCC 13032 grown under the glutamic acid production conditions, suggesting the importance of strain-specific reference map for proteomic analysis.
  • Masaru Wada, Kotomi Narita, Atsushi Yokota
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 76 (4) 819 - 825 0175-7598 2007/09 [Not refereed][Not invited]
    Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H+-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164-2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H+-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H+-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.
  • Liyuan Li, Masaru Wada, Atsushi Yokota
    PROTEOMICS 7 (18) 3348 - 3357 1615-9853 2007/09 [Not refereed][Not invited]
    F172-8, an H+-ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H+-ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies.
  • Yuki Sawatari, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 73 (12) 3909 - 3915 0099-2240 2007/06 [Not refereed][Not invited]
    We determined the maximum pH that allows growth (pHmax) for 34 strains of lactobacilli. High alkali tolerance was exhibited by strains of Lactobacillus casei, L. paracasei subsp. tolerans, L. paracasei subsp. paracasei, L. curvatus, L. pentosus, and L. plantarunt that originated from plant material, with pHmax values between 8.5 and 8.9. Among these, L. casei NRIC 1917 and L. paracasei subsp. tolerans NRIC 1940 showed the highest pHmax, at 8.9. Digestive tract isolates of L. gasseri, Ljohnsonii, L. reuteri, L. salivarius subsp. salicinius, and L. salivarius subsp. salivarius exhibited moderate alkali tolerance, with pHmax values between 8.1 and 8.5. Dairy isolates of L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and L. helveticus exhibited no alkali tolerance, with pHmax values between 6.7 and 7.1. Measurement of the internal pH of representative strains revealed the formation of transmembrane proton gradients (ApH) in a reversed direction (i.e., acidic interior) at alkaline external-pH ranges, regardless of their degrees of alkali tolerance. Thus, the reversed ApH did not determine alkali tolerance diversity. However, the ApH contributed to alkali tolerance, as the pHmax values of several strains decreased with the addition of nigericin, which dissipates ApH. Although neutral external-pH values resulted in the highest glycolysis activity in the presence of nigericin regardless of alkali tolerance, substantial glucose utilization was still detected in the alkali-tolerant strains, even in a pH range of between 8.0 and 8.5, at which the remaining strains lost most activity. Therefore, the alkali tolerance of glycolysis reactions contributes greatly to the determination of alkali tolerance diversity.
  • Martin Patrick Ongol, Yaki Sawatani, Yoshiko Ebina, Teruo Sone, Michiko Tanaka, Fusao Tomita, Atsushi Yokota, Kozo Asano
    INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY 116 (3) 358 - 366 0168-1605 2007/05 [Not refereed][Not invited]
    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 <= 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 (>= 10(8) CFU/ml) of both Bifidobacteruini breve JCM 1192(T) and Biflidobacteruint 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 W-ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during reffigerated storage. (c) 2007 Elsevier B.V. All rights reserved.
  • Yuki Sawatari, Tomomi Hirano, Atsushi Yokota
    JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 52 (6) 349 - 356 0022-1260 2006/12 [Not refereed][Not invited]
    Based on MRS medium, two types of food grade (FG) culture media (FG medium I and FG medium II) for the preparation of a concentrated starter culture of Lactobacillus plantarum NRIC 0380 to manufacture a new type of instant Chinese noodle, the fermented instant Chinese noodle, were developed using FG materials. FG medium I, which is for normal static culture, contains table sugar (sucrose), Yeast peptone standard type F, Sunsoft Q-17S (emulsifier), sodium acetate, trisodium citrate and MnSO4 . 4-5H(2)O. FG medium II was designed to be used for the pH-controlled jar fermentor culture conditions. Therefore, sodium acetate and trisodium citrate as a buffer to prevent acidification of medium were omitted from FG medium I. When L. plantarum NRIC 0380 was cultured under the pH-controlled jar fermentor culture conditions, the kinetics of growth, sugar consumption and lactic acid production in FG medium II were quite similar to those observed in the Difco Lactobacilli MRS Broth. Furthermore, growths of many lactobacilli strains isolated from various fermented foods in FG medium I were also quite similar to those observed in MRS medium. Therefore, simple and practical FG media for the culture of lactobacilli were successfully established.
  • Achmad Dinoto, Tatiana M. Marques, Kanta Sakamoto, Satoru Fukiya, Jun Watanabe, Susumu Ito, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72 (12) 7739 - 7747 0099-2240 2006/12 [Not refereed][Not invited]
    The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.
  • Sakiko Noda, Yuji Takezawa, Tomohiko Mizutani, Tomoaki Asakura, Eiichiro Nishiumi, Kazunori Onoe, Masaru Wada, Fusao Tomita, Kazunobu Matsushita, Atsushi Yokota
    JOURNAL OF BACTERIOLOGY 188 (19) 6869 - 6876 0021-9193 2006/10 [Not refereed][Not invited]
    The physiological changes in an F-1-ATPase-defective mutant of Escherichia coli W1485 growing in a glucose-limited chemostat included a decreased growth yield (60%) and increased specific rates of both glucose consumption (168%) and respiration (171%). Flux analysis revealed that the mutant showed approximately twice as much How in glycolysis but only an 18% increase in the tricarboxylic acid (TCA) cycle, owing to the excretion of acetate, where most of the increased glycolytic flux was directed. Genetic and biochemical analyses of the mutant revealed the downregulation of many TCA cycle enzymes, including citrate synthase, and the upregulation of the pyruvate dehydrogenase complex in both transcription and enzyme activities. These changes seemed to contribute to acetate excretion in the mutant. No transcriptional changes were observed in the glycolytic enzymes, despite the enhanced glycolysis. The most significant alterations were found in the respiratory-chain components. The total activity of NADH dehydrogenases (NDHs) and terminal oxidases increased about twofold in the mutant, which accounted for its higher respiration rate. These changes arose primarily from the increased (3.7-fold) enzyme activity of NDH-2 and an increased amount of cytochrome bd in the mutant. Transcriptional upregulation appeared to be involved in these phenomena. As NDH-2 cannot generate an electrochemical gradient of protons and as cytochrome bd is inferior to cytochrome bo(3) in this ability, the mutant was able to recycle NADH at a higher rate than the parent and avoid generating an excess proton-motive force. We discuss the physiological benefits of the alterations in the mutant.
  • P Kurdi, K Kawanishi, K Mizutani, A Yokota
    JOURNAL OF BACTERIOLOGY 188 (5) 1979 - 1986 0021-9193 2006/03 [Not refereed][Not invited]
    The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated Delta pH. The bile acid concentrations that dissipated Delta pH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (Delta psi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated Delta pH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the ApH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane ApH and A-If dissipation), potassium ion, and other cellular components and eventually cell death.
  • A Dinoto, A Suksomcheep, S Ishizuka, H Kimura, S Hanada, Y Kamagata, K Asano, F Tomita, A Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72 (1) 784 - 792 0099-2240 2006/01 [Not refereed][Not invited]
    To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192(T) cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Sic rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BID). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.
  • Y Sawatari, H Sugiyama, Y Suzuki, A Hanaoka, K Saito, H Yamauchi, S Okada, A Yokota
    FOOD MICROBIOLOGY 22 (6) 539 - 546 0740-0020 2005/12 [Not refereed][Not invited]
    A new-type of instant Chinese noodle was developed with the application of lactic acid fermentation by lactobacilli. Since the pH value of the noodle sheets is alkaline with kansui (around 8.5), alkaline tolerance is required for the lactobacilli to ferment noodle sheets. The screening of the lactobacilli strains suitable for the fermentation was conducted using 46 strains from 12 species (including subspecies) of lactobacilli. Several strains of Lactobacillus pentosus and Lactobacillus plantarum were found to be fermenters. Among these, L. plantarum NRIC 0380, that showed the highest fermentation rate and favorable modification of noodle, was selected as the best strain, and was employed for the pilot scale manufacture of instant Chinese noodle. During fermentation, L. plantarum NRIC 0380 produced lactic acid to about 11 g/kg noodle sheet after 24 It with a concomitant pH decrease from an initial of about 7.9 down to 3.9. Sensory test after rehydration with boiled water revealed that the fermented instant Chinese noodle sheets at pH 7.5 had increased hardness, elasticity and light sour taste. (c) 2005 Elsevier Ltd. All rights reserved.
  • ASM Selim, P Boonkumklao, T Sone, A Assavanig, M Wada, A Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 71 (8) 4214 - 4219 0099-2240 2005/08 [Not refereed][Not invited]
    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.
  • R Aoki, M Wada, N Takesue, K Tanaka, A Yokota
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 (8) 1466 - 1472 0916-8451 2005/08 [Not refereed][Not invited]
    Previously we reported that a mutant of Corynebactetium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 mu g/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 mu g/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 mu g/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.
  • K Minamida, IN Sujaya, A Tamura, N Shigematsu, T Sone, A Yokota, K Asano, Y Benno, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 98 (4) 244 - 250 1389-1723 2004/10 [Not refereed][Not invited]
    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.
  • 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 0959-3993 2004/03 [Not refereed][Not invited]
    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.
  • Journal of Applied Glycoscience 51 (4) 291 - 296 2004 [Not refereed][Not invited]
  • IN Sujaya, Y Tamura, T Tanaka, T Yamaki, T Ikeda, N Kikushima, H Yata, A Yokota, K Asano, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 96 (5) 438 - 447 1389-1723 2003/11 [Not refereed][Not invited]
    Recently, the use of the dry yeast of Zygosaccharomyces roaxii M2 for miso (soybean paste) fermentation has been established. A molecular monitoring method was developed and validated in this study to analyze the population of Z rouxii M2 during the fermentation. The method was based on the restriction patterns of internal transcribed spacer (ITS) regions of the rDNA using HaeIII and Hhal. Among the homologous ITS regions of Z roaxii strains, Z rouxii M2 produced diagnostic bands by which it can be differentiated from the other strains used. The specific restriction bands were due to the difference in nucleotide sequence of two different copies of ITS of Z. rouxii M2. Both ITS copies showed 94% sequence similarity but a 13-bp nucleotide substitution and a 19-bp deletion were found in the ITS1 region. Phylogenetic trees were constructed based on ITS and 18S rDNA sequences and it was found that the ITS sequences provide better resolution for the classification of Z rouxii M2. Since Z rouxii M2 is a promising strain for use in miso fermentation as a dry starter, the method developed is significant in terms of industrial application in monitoring the growth of Z rouxii M2 in miso fermentation.
  • P Kurdi, H Tanaka, HW van Veen, K Asano, F Tomita, A Yokota
    MICROBIOLOGY-SGM 149 (8) 2031 - 2037 1350-0872 2003/08 [Not refereed][Not invited]
    Cholic acid (CA) transport was investigated in nine intestinal Bifidobacterium strains. Upon energization with glucose, all of the bificlobacteria accumulated CA. The driving force behind CA accumulation was found to be the transmembrane proton gradient (DeltapH, alkaline interior). The levels of accumulated CA generally coincided with the theoretical values, which were calculated by the Henderson-Hasselbalch equation using the measured internal pH values of the bificlobacteria, and a pK(a) value of 6.4 for CA. These results suggest that the mechanism of CA accumulation is based on the diffusion of a hydrophobic weak acid across the bacterial cell membrane, and its dissociation according to the DeltapH value. A mixture of short-chain fatty acids (acetate, propionate and butyrate) at the appropriate colonic concentration (117 mM in total) reduced CA accumulation in Bifidobacterium breve JCM 1192 T. These short-chain fatty acids, which are weak acids, reduced the DeltapH, thereby decreasing CA accumulation in a dose-dependent manner. The bificlobacteria did not alter or modify the CA molecule. The probiotic potential of CA accumulation in vivo is discussed in relation to human bile acid metabolism.
  • K Saito, Y Sumita, Y Nagasaka, F Tomita, A Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 95 (5) 538 - 540 1389-1723 2003/05 [Not refereed][Not invited]
    The gene encoding an intracellular enzyme hydrolyzing di-D-fructofuranose 1,2': 2,3' dianhydride (DFA III) (DFA IIIase) was cloned from the genomic DNA of Arthrobacter sp. H65-7 for the first time. The single open reading frame (ORF) of the DFA IIIase gene consisted of 1368-bp encoding 455 amino acids. DFA IIIase showed a phylogenetically distinct position from other inulin-degrading enzymes and showed similarity only with inulin fructotransferases (depolymerizing) (inulase II, EC from Arthrobacter globiformis C11-1, Arthrobacter sp. A-6, and Arthrobacter sp. H65-7 (48.7-50.3%), and inulin fructotransferase (DFA I-producing) (EC from A. globiformis S14-3 (44.4%). An Escherichia coli transformant harboring a recombinant plasmid, pINB2, in which the DFA IIIase gene was fused with the P-galactosidase of pUC19 and under the control of the lac promoter, expressed DFA IIIase and the cloned enzyme produced inulobiose from DFA III similarly to the DFA IIIase of the wild-type strain, Arthrobacter sp. H65-7.
  • K Saito, Z Nishio, K Takata, T Kuwabara, A Yokota, H Yamauchi, Y Oda
    FOOD SCIENCE AND TECHNOLOGY RESEARCH 9 (1) 40 - 44 1344-6606 2003/02 [Not refereed][Not invited]
    We screened the bacteria affecting the quality of alkaline noodles to improve the noodles by biological methods. Brevibacterium helvolum B8 and Arthrobacter sp. B25 were selected as strains providing favorable effects on alkaline noodles from 158 bacteria able to grow under a high alkaline condition. The addition of these strains to alkaline noodles increased the degree of yellowness of the noodles and repressed the formation of organic acids which caused reduction in the pH. The increase of other bacteria in the noodles and the appearance of mold on the noodle surface were also suppressed, so that it was assumed that these bacteria had also the ability to prevent microbial spoilage of the noodles. These results suggested that B. helvolum B8 and Arthrobacter sp. B25 were useful in improving the appearance and retaining the quality of alkaline noodles.
  • P Niamsup, IN Sujaya, M Tanaka, T Sone, S Hanada, Y Kamagata, S Lumyong, A Assavanig, K Asano, F Tomita, A Yokota
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 53 (1) 263 - 268 1466-5026 2003/01 [Not refereed][Not invited]
    Five strains of thermotolerant lactic acid bacteria (G 12, G 22, G 35 T, G 43 and G 44) isolated from chicken faeces were characterized taxonomically. The strains were facultatively anaerobic, Grampositive, catalase-negative, non-motile, non-spore-forming rods. They were heterofermentative lactobacilli that produced DL-lactic acid. Growth of the strains occurred at 45 degreesC but not at 15 degreesC. The optimum temperature for growth was 42 degreesC, as determined from the specific growth rate. The highest permissive temperatures for growth were 50 degreesC for strain G 35 T and 48 degreesC 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 C-14:0, C-16:0, sometimes C-18:0, C-18:1 and C, 9: 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 35(T) = DSM 14792(T) =JCM 11425(T)).
  • K Saito, Y Oda, F Tomita, A Yokota
    FEMS MICROBIOLOGY LETTERS 218 (2) 265 - 270 0378-1097 2003/01 [Not refereed][Not invited]
    The gene encoding a 2,6-beta-D-fructan 6-levanbiohydrolase (LF2ase) (EC that converts levan into levanbiose was cloned from the genomic DNA of Streptomyces exfoliatus F3-2. The gene encoded a signal peptide of 37. amino acids and a mature protein of 482 amino acids with a total length of 1560 bp and was successfully expressed in Escherichia coli. The similarities of primary structure were observed with levanases from Clostridium acetobutylicum, Bacillus subtilis, B. stearothermophilus (51.0-54.3%) and with LF2ase from Microbacterium levaniformans (53.9%). The enzyme from S. exfoliatus F3-2 shared the conserved six domains and the completely conserved five amino acid residues with family 32 glycosyl hydrolases, which include levanase, inulinase, and invertase. These observations led to the conclusion that the enzyme belongs to family 32 glycosyl hydrolases. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
  • M Tanaka, M Yoshimura, M Suto, A Yokota, K Asano, E Sukara, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 93 (6) 531 - 536 1389-1723 2002/06 [Not refereed][Not invited]
    A highly potent allelopathic factor, lepidimoide, was initially extracted from mucilage of germinated cress seeds. Polysaccharide extracted from okra (Abelmoschus esculentum Moench) is considered to have a similar structure to lepidimoide as its repeating unit. We therefore initiated the screening of enzymes capable of degrading okra polysaccharide into lepidimoide from endophytes. We discovered an endophytic fungal strain AHU9748 isolated from Cole"s galeatus, which produced an oligosaccharide having similar properties to lepidimoide on thin layer chromatography. The physico-chemical data from ESI-MS, NMR spectra and other analyses also showed the purified product to be identical to lepidimoide. The strain AHU9748 was identified as a fungus belonging to the coelomycetes, closely related to the genus Colletotrichum, based on morphological characteristics and sequence analysis of the 18S rDNA and ITS region.
  • IN Sujaya, S Amachi, K Saito, A Yokota, K Asano, F Tomita
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 18 (3) 263 - 270 0959-3993 2002/04 [Not refereed][Not invited]
    Lactic acid bacteria (LAB) are among the integral microflora of ragi tape, a dry starter of Balinese rice wine, brem. The species diversity and population level of LAB present in different types of ragi tape were studied by colony hybridization using 16S and 23S rDNA targeted oligonucleotide probes. These probes were DB6, Lbc, Wgp, and Rpt, which were specific for Enterococcus faecium, Lactobacillus curvatus, Weissella spp., and Pediococcus pentosaceus, respectively. Results revealed that P. pentosaceus and Weissella spp. were the predominant LAB in ragi tape, whereas L. curvatus and E. faecium were associated specific to types of ragi tape. A 21-mer species-specific oligonucleotide probe, Rpt, that targets the 16S rDNA of P. pentosaceus was developed in this study and found to be highly specific to be used as an effective tool to enumerate population of this species in ragi tape and its population changes during rice wine production. It was detected that LAB showed active growth during the early stage of brem fermentation. A succession of growth of LAB population during the fermentation was observed in which the heterofermentative LAB, Weissella spp., grew first, followed by the proliferation of P. pentosaceus.
  • NS Antara, IN Sujaya, A Yokota, K Asano, WR Aryanta, F Tomita
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 18 (3) 255 - 262 0959-3993 2002/04 [Not refereed][Not invited]
    'Urutan' is a Balinese traditional fermented sausage, which is made of lean pork and fat mixed with spices, sugar, and salt. The mixture is stuffed into cleaned pig intestine and fermented under uncontrolled condition during sun drying for 5 days. The investigation showed that lactic acid bacteria (LAB) were the dominating bacteria during 'urutan' fermentation. Among the 71 isolates obtained, lactobacilli dominated by 77.5% and the other 22.5% were pediococci. Based on physiological characteristics, the isolates were classified into 13 groups: nine belonged to the lactobacilli and the other four were pediococci. One isolate representing each group was chosen randomly, and then was identified by 16S rDNA sequence comparison. Phylogenetic relationship positioned three groups to Lactobacillus plantarum and four groups were closely related to L. farciminis. Two groups were identified as obligate heterofermentative lactobacilli: one was L. fermentum and the other was distantly related to L. hilgardii. Two groups belonging to the pediococci were strains of Pediococcus acidilactici and the other two were closely related to P. pentosaceus. A dramatic succession occurred during fermentation of 'urutan'. Three species mainly dominated the process wherein the initial growth was started by L. plantarum then followed by the growth of P. acidilactici, and finally, L. farciminis was found to be predominant at the last stage of fermentation.
  • H Sekine, T Shimada, C Hayashi, A Ishiguro, F Tomita, A Yokota
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 57 (4) 534 - 540 0175-7598 2001/11 [Not refereed][Not invited]
    A mutant of Corynebacterim glutamicum ('Brevibacterium flavum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant. The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain. Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor. It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent. The respiration rate per cell of the mutant also increased to twice as much as that of the parent. However, the growth rate of the mutant was lower than that of the parent. Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid. Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose. Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant. On the other hand, the parent accumulated a-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products. It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent. Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.
  • IN Sujaya, S Amachi, A Yokota, K Asano, F Tomita
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 17 (4) 349 - 357 0959-3993 2001/06 [Not refereed][Not invited]
    One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.
  • GR Dedeles, A Abe, K Saito, K Asano, K Saito, A Yokota, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 90 (5) 515 - 521 1389-1723 2000/11 [Not refereed][Not invited]
    A soil isolate designated as YA-1 strain was selected for its ability to degrade nickel protoporphyrin disodium (NiPPDS). The strain was capable of utilizing NiPPDS as the sole source of carbon. This strain, a gram-negative aerobic rod, was identified as Pseudomonas azelaica YA-1 based on the result of its 16S rRNA analysis. Product analyses by HPLC showed that this strain can decompose the porphyrin ring to which a metal ion is bound. However, the use of whole bacterial cells cannot result in extensive NiPPDS degradation; therefore, the YA-I enzyme was extracted and purified. This NiPPDS-degrading enzyme named as protoporphyrinase was purified from P. azelaica YA-1 by ammonium sulfate fractionation and sequential chromatographies using DEAE Toyopearl 650 M, CM Toyopearl 650 M and Biogel P-60 columns, with a yield of 11.3% based on the enzyme activity and an overall purification of 498-fold. The molecular weight of this enzyme is estimated to be 39,000 Da by SDS-PAGE and 34,000 Ha by gel filtration. The optimum pH and temperature for the enzyme were 7.0 and 30 degreesC, respectively. The activity was stable at pH 2.0-11.0 and at temperatures below 50 degreesC. The enzyme activity was inactivated by ferric chloride, potassium ferricyanide, ZnCl2 and CdCl2.
  • P Kurdi, HW van Veen, H Tanaka, Mierau, I, WN Konings, GW Tannock, F Tomita, A Yokota
    JOURNAL OF BACTERIOLOGY 182 (22) 6525 - 6528 0021-9193 2000/11 [Not refereed][Not invited]
    Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.
  • A Yokota, M Veenstra, P Kurdi, HW van Veen, WN Konings
    JOURNAL OF BACTERIOLOGY 182 (18) 5196 - 5201 0021-9193 2000/09 [Not refereed][Not invited]
    The cholate-resistant Lactococcus lactis strain C41-2, derived from mild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux, However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363, The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2 carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux: rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L, lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.
  • SH Baik, K Saito, A Yokota, K Asano, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 90 (2) 168 - 173 1389-1723 2000/08 [Not refereed][Not invited]
    A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/gal alpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/gal alpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera Ho 8084.
  • A Saito, K Kondo, Kojima, I, A Yokota, F Tomita
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 66 (1) 252 - 256 0099-2240 2000/01 [Not refereed][Not invited]
    Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7, The optimal pH and temperature of the enzyme for levan degradation mere pH 5.5 and 60 degrees C, respectively, The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose, Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a navel 2,6-beta-D-fructan 6-levanbiohydrolase (EC
  • N Phay, T Higashiyama, M Tsuji, H Matsuura, Y Fukushi, A Yokota, F Tomita
    PHYTOCHEMISTRY 52 (2) 271 - 274 0031-9422 1999/09 [Not refereed][Not invited]
    A novel antifungal compound, fistulosin (octadecyl 3-hydroxyindole), was isolated from roots of Welsh onion (Allium fistulosum L.), and its structure was elucidated by spectroscopic means. This compound showed high activity against Fusarium oxysporum primarily inhibiting protein synthesis. (C) 1999 Elsevier Science Ltd. All rights reserved.
  • T Sakurai, N Cheeptham, T Mikawa, A Yokota, F Tomita
    JOURNAL OF ANTIBIOTICS 52 (5) 508 - 511 0021-8820 1999/05 [Not refereed][Not invited]
  • K Saito, T Hira, T Suzuki, H Hara, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 (4) 655 - 661 0916-8451 1999/04 [Not refereed][Not invited]
    Di-n-fructose-2,6':6,2'-dianhydride (DFA IV) is a disaccharide consisting of two fructose residues that can be prepared from levan by levan fructotransferase from Arthrobacter nicotinovorans GS-9, and it can be expected to have novel physiological functions from its unique structure. In this study, the effects of DFA IV on calcium absorption and the metabolism of DFA IV by intestinal microorganisms were studied in rats to examine the physiological functions of DFA IV. The apparent calcium absorption in rats fed with DFA IV was significantly higher than that in the control rats, and it seems that calcium absorption had almost been completed at the end of the small intestine. DFA IV also increased the calcium absorption in in vitro experiments, using everted jejunal and ileal sacs, and this result supports the finding obtained in the in vivo experiments. These results indicate that DFA IV may have a function for increasing the calcium absorption in the small intestine of rats. However, the effect in the large intestine could not be clearly observed because of the lack of calcium that reached there. The results of analyses of organic acids in the cecal and colonic contents and of DFA IV in the fecal, cecal, and colonic contents showed that the metabolism of DFA IV by microorganisms in the large intestine progressed gradually, and that DFA IV was converted mainly to acetate, butyrate, and lactate.
  • Studies on an antifungi antibiotic from Ellisiodothis inguinans L1588-A8
    Thai Journal of Biotechnology 1 37 - 45 1999 [Not refereed][Not invited]
  • Y Nagasaka, K Kurosawa, A Yokota, F Tomita
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 50 (3) 323 - 330 0175-7598 1998/09 [Not refereed][Not invited]
    Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of,glucoamylase (G1-G5) and a small amount of a-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and GS 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze ram starch.
  • S Amachi, K Ishikawa, S Toyoda, Y Kagawa, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 62 (8) 1574 - 1580 0916-8451 1998/08 [Not refereed][Not invited]
    A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic on near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermenters were measured. The relative ATPase activity of the mutant at pH 7.0 was 42% of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0, while the level of increase in the mutant was only 1.6. Northern and western blot analyses found that at pH 7.0 the transcriptional level and the amount of F(1)beta subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F(1)beta subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.
  • K Saito, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 (12) 2076 - 2079 0916-8451 1997/12 [Not refereed][Not invited]
    The gene encoding an extracellular levan fructotransferase, designated the lft gene, was cloned from the genomic DNA of Arthrobacter nicotinovorans GS-9, and expressed in Escherichia coli. It was found that a single open reading frame consisted of 1554 base pairs that encoded a polypeptide composed of a signal peptide of 33 amino acids and a mature protein of 484 amino acids (M-r 53,152), and it was also found that a putative ribosome-binding site was present in the upstream from the ORF. The primary structure had no significant similarity with those of inulin fructotransferases, but had low similarity to the catalytic regions of other fructosylhydrolases. The expression of the lft gene was increased on a plasmid, pLFT-BB1, in which the lft gene was fused with alpha-peptide of the lacZ gene of pUC18. An E. coli transformant carrying pLFT-BB1 expressed six times as much activity of levan fructotransferase as that of the original strain, A. nicotinovorans GS-9.
  • K Saito, H Goto, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 (10) 1705 - 1709 0916-8451 1997/10 [Not refereed][Not invited]
    A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism produced an extracellular enzyme that converted levan into DFA IV, This strain was identified as Arthrobacter nicotinovorans. The DFA IV-producing enzyme was specifically induced by levan, The enzyme was purified 60-fold from culture supernatant to give a single band on SDS-PAGE. The molecular weight of this enzyme was 52,000 by SDS-PAGE and a monomer by gel filtration, The enzyme gave DFA IV as a main product (> 75%), and fructose, levanbiose, and two unidentified oligosaccharides as minor products, and was identified as a novel levan fructotransferase.
  • K Saito, H Goto, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 (10) 1705 - 1709 0916-8451 1997/10 [Not refereed][Not invited]
    A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism produced an extracellular enzyme that converted levan into DFA IV, This strain was identified as Arthrobacter nicotinovorans. The DFA IV-producing enzyme was specifically induced by levan, The enzyme was purified 60-fold from culture supernatant to give a single band on SDS-PAGE. The molecular weight of this enzyme was 52,000 by SDS-PAGE and a monomer by gel filtration, The enzyme gave DFA IV as a main product (> 75%), and fructose, levanbiose, and two unidentified oligosaccharides as minor products, and was identified as a novel levan fructotransferase.
  • H Sakurai, A Yokota, Y Sumita, Y Mori, H Matsui, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 (6) 989 - 993 0916-8451 1997/06 [Not refereed][Not invited]
    Assimilation of DFA III by Arthrobacter sp, H65-7 was found to consist of two sequential enzyme steps, hydrolysis of DFA III to inulobiose (1-O-beta-D-fructofuranosyl-D-fructopyranose) and inulobiose to fructose, that is, alpha-(2-->3') and beta-(2'-->1) fructosidic linkages were split separately, The enzyme catalyzing the first step, named DFA III hydrolysis enzyme (DFA IIIase), has been purified from the cell-free extracts of Arthrobacter sp, H65-7 to an electrophoretically pure state by heat-treatment, ammonium sulfate fractionation, and chromatographies on DEAE-Toyopearl 650M, Butyl-Sepharose 4B and TSKgel G3000SW(XL), and Biophoresis III, The molecular weight of the purified enzyme was estimated to be 125,000 by gel filtration, and 61,000 by SDS-polyacrylamide gel electrophoresis, The enzyme showed the highest activity at pH 6.0 and 45 degrees C, and was stable from pH 4.5 to 10.0 and up to 60 degrees C. The K-m of this enzyme for DFA III was 12.5 mM, The enzyme also catalyzed the reverse reaction, inulobiose to DFA III.
  • A Yokota, M Henmi, N Takaoka, C Hayashi, Y Takezawa, Y Fukumori, F Tomita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 83 (2) 132 - 138 0922-338X 1997 [Not refereed][Not invited]
    An F-1-ATPase-defective mutant of Escherichia coli K-12, strain TBLA-1, showing an enhanced pyruvic acid productivity compared with its parental strain W1485lip2, was characterized physiologically to elucidate the mechanisms of the enhancement in productivity. Cells of strain TBLA-1 producing pyruvic acid showed a higher rate of the oxygen consumption and a higher b-type cytochromes content than cells of strain W1485lip2. The activities of the phosphotransferase system for glucose uptake measured in terms of the level of phosphoenolpyruvate-dependent phosphorylation of methyl alpha-D-glucopyranoside and 2-deoxy-D-glucose using toluenized cells were higher in strain TBLA-1 than in strain W1485lip2. Among the activities of all the glycolytic enzymes, those of phosphoglycerate kinase and pyruvate kinase-I were found to be higher in strain TBLA-1 than in strain W1485lip2 during the logarithmic growth phase. These characteristics of strain TBLA-1 are discussed in relation to its enhanced pyruvic acid productivity.
  • H Sakurai, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61 (1) 87 - 92 0916-8451 1997/01 [Not refereed][Not invited]
    The gene encoding an extracellular inulin fructotransferase (depolymerizing) (inulase ZI) (EC, designated ift gene, was cloned from the genomic DNA of Arthrobacter sp. H65-7, and expressed in Escherichia coli for the first time. Sequence analysis showed a single open reading frame consisting of 1314 base pairs that encoded a signal peptide of 32 amino acids and a mature protein of 405 amino acids, The primary structure showed a homology of 49.8% with that of the inulin fructotransferase (DFA I-producing) (EC from Arthrobacter globiformis S14-3. E. coli cells carrying the ift gene produced the active enzyme under control of the lac promoter, The expression of the ift gene was improved by a plasmid, pIFT-B, in which the ift gene was immediately downstream from the lac promoter. An E. coli transformant carrying pIFT-B expressed twice as much activity of inulase II as that of the original strain, Arthrobacter sp, H65-7. Most of the enzyme activity existed within the cells.
  • N Phay, H Yada, T Higashiyama, A Yokota, A Ichihara, F Tomita
    JOURNAL OF ANTIBIOTICS 49 (7) 703 - 705 0021-8820 1996/07 [Not refereed][Not invited]
  • K Kawasaki, A Yokota, F Tomita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 82 (6) 604 - 606 0922-338X 1996 [Not refereed][Not invited]
    Pyruvic acid-producing Escherichia coli W1485lip2 was transformed with the plasmid of KT901EA that carries the tryptophanase structural gene from Enterobactera aerogenes SM-18 downstream of the tac promoter. In the course of culture of the transformant in a pyruvic acid fermentation medium, isopropyl-beta-D-thiogalactopyranoside and other nutrients were supplied to the medium to induce tryptophanase expression. After 32-h culture, 28.6 g/l of pyruvic acid had been produced, and a large amount of tryptophanase had been accumulated in the cells. By addition of ammonia and indole to the culture broth, the enzymatic synthesis of L-tryptophan was then started. Our results showed that 23.7 g/l of L-tryptophan had been produced after reaction for 36 h (total 68 h).
  • Y Nagasaka, N Muraki, A Kimura, M Suto, A Yokota, F Tomita
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 44 (3-4) 451 - 458 0175-7598 1995/12 [Not refereed][Not invited]
    A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35% - 56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.
    LETTERS IN APPLIED MICROBIOLOGY 21 (5) 330 - 333 0266-8254 1995/11 [Not refereed][Not invited]
    A Strain of streptomycetes producing an extracellular inulin-degrading enzyme was isolated from a soil sample. This strain was identified as Streptomyces rochei E87. The inulin-degrading enzyme was found to degrade inulin into inulotriose as the main end product. This enzyme was induced by inulin. Under favourable culture conditions, the enzyme activity in the culture supernatant fluid reached 1.0 U ml(-1) after incubation for 3 d. Using this enzyme, inulotriose was produced in a weight yield of over 70% from an initial 10-50 g l(-1) of inulin.
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 (10) 2004 - 2007 0916-8451 1995/10 [Not refereed][Not invited]
    Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp, lactis C2 as spontaneous neomycin-resistant mutants, Characteristics of the representative mutant, No, 1016-51, were compared with the parental strain in cultures using a jar fermenter with the pH controlled at various values, At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical, At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24h decreased to 60, 36, and 60% of the parental strain, respectively, During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80h, On the other hand, at pH 4.0, the viable count of the strain No. 1016-51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability, Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 (10) 1938 - 1943 0916-8451 1995/10 [Not refereed][Not invited]
    We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Entepobacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E, coli JM109 transformed with pKT901EA (JM109/pKT901EA) was inducible with isopropyl-beta-D-thiogalactopyranoside, and 3.6 times higher than that of E. aerogenes SM-18, Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated, The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.
  • 日本エネルギー学会誌 74 (4) 228 - 232 1995 [Not refereed][Not invited]
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 58 (12) 2164 - 2167 0916-8451 1994/12 [Not refereed][Not invited]
    An F-1-ATPase-defective mutant, TBLA-1, was constructed by the transduction of a defective gene for the alpha subunit of F-1-ATPase, atpA401, into Escherichia coli W1485lip2, a lipoic acid-requiring pyruvic acid producer. The pyruvic acid production of the strain TBLA-1 was found to be improved markedly compared with that of strain W1485lip2. In cultures using a jar fermenter, the strain W1485lip2 consumed 50 g/liter of glucose and produced 25 g/liter of pyruvic acid after culture for 32 h, while strain TBLA-1 consumed the same amount of glucose, and produced more than 30 g/liter of pyruvic acid in a 24-h culture. A revertant, No. 63-1, derived from the strain TBLA-1, had a normal level of F-1-ATPase activity, and showed a similar pattern of pyruvic acid production to that of strain W1485lip2.
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 41 (6) 638 - 643 0175-7598 1994/08 [Not refereed][Not invited]
    A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid as the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32-40 h at pH 6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermenter culture was discussed.
    JOURNAL OF GENERAL MICROBIOLOGY 139 (12) 3275 - 3281 0022-1287 1993/12 [Not refereed][Not invited]
    A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory element for transcription. A partial open reading frame homologous to the 5' end of E. coli tnaB was observed at the 3'-flanking region of tnaA. These genes may thus constitute an operon as in E. coli.
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 57 (5) 745 - 749 0916-8451 1993/05 [Not refereed][Not invited]
    Levan-assimilating microorganisms obtained from soil samples were screened for levan-degrading enzyme production. Among them, the strain F3-2 was found to produce an extracellular levan-degrading enzyme that hydrolyzed levan to a disaccharide identified as levanbiose. The strain F3-2 was identified as Streptomyces exfoliatus F3-2. This levan-degrading enzyme was specifically induced by levan, and the activity was increased to 2.58 units/ml by improvement of the culture conditions. The optimum temperature and pH for the enzyme reaction were 60-degrees-C and 5.5, respectively. The crude levan-degrading enzyme was stable within a pH range of 3.0 to 9.0 and at up to 50-degrees-C. Using this crude levan-degrading enzyme, 50 mg/ml of levan was converted to levanbiose in amounts of 42 mg/ml.
    JOURNAL OF FERMENTATION AND BIOENGINEERING 72 (4) 258 - 261 0922-338X 1991 [Not refereed][Not invited]
    A bacterial strain H65-7 isolated from soil as an inulin-assimilating microorganism produces inulin fructotransferase (inulase II) which converts inulin into di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III). This strain was classified as Arthrobacter sp. The inulase II production was induced by inulin, and markedly enhanced by the addition of yeast extract. Under optimal conditions, the enzyme activity in the culture supernatant reached 90 units/ml after cultivation for 18 h. The optimum pH and temperature for the enzyme reaction were 5.5 and 60-degrees-C, respectively. The enzyme was stable within a pH range of 4.5 to 9.0 and at up to 75-degrees-C. Using this crude enzyme, 300 mg/ml of inulin was converted into 237 mg/ml of DFA III after incubation for 4 h.
    JOURNAL OF FERMENTATION AND BIOENGINEERING 72 (4) 262 - 265 0922-338X 1991 [Not refereed][Not invited]
    Arthrobacter sp. H65-7 produced inulin fructotransferase (depolymerizing) which converts inulin into di-D-fructofuranose 1, 2':2, 3' dianhydride (DFA III) and small amounts of oligosaccharides. The enzyme was purified 8-fold with a yield of 13% from a culture supernatant by ammonium sulfate precipitation followed by DEAE-Toyopearl 650 M column chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 49,000 by SDS-polyacrylamide gel electrophoresis, and 100,000 by gel filtration. The isoelectric point of the enzyme was determined to be pH 4.7. The optimal pH and temperature for the enzyme reaction were 5.5, and 60-degrees-C, respectively. The enzyme was stable with a pH range of 4.5 to 9.0, and up to 70-degrees-C. After exhaustive digestion of inulin by this enzyme, nystose and 1-F-fructofuranosyl-nystose were produced in addition to DFA III.
    JOURNAL OF FERMENTATION AND BIOENGINEERING 71 (5) 367 - 369 0922-338X 1991 [Not refereed][Not invited]
    Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27-degrees-C and under the yeast cell concentration of 2.7 x 10(9) cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15-degrees-C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 54 (2) 547 - 548 0002-1369 1990/02 [Not refereed][Not invited]
    JOURNAL OF FERMENTATION AND BIOENGINEERING 69 (4) 256 - 258 0922-338X 1990 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 (8) 2169 - 2175 0002-1369 1989/08 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 (8) 2037 - 2044 0002-1369 1989/08 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 (3) 705 - 711 0002-1369 1989/03 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 (1) 41 - 48 0002-1369 1989/01 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 52 (2) 455 - 463 0002-1369 1988/02 [Not refereed][Not invited]
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 51 (9) 2485 - 2493 0002-1369 1987/09 [Not refereed][Not invited]
    JOURNAL OF FERMENTATION TECHNOLOGY 62 (4) 329 - 334 0385-6380 1984 [Not refereed][Not invited]
    JOURNAL OF FERMENTATION TECHNOLOGY 61 (6) 643 - 645 0385-6380 1983 [Not refereed][Not invited]

Books etc

  • Atsushi Yokota, Masato Ikeda (Editor)
    Springer 2017 (ISBN: 9784431565185)
  • 横田篤, 大西康夫, 小川順 (Editor)
    文永堂出版 2016/07 (ISBN: 4830041315) 327
  • Kenji Sonomoto, Atsushi Yokota (Editor)
    Caister Academic Press 2011 (ISBN: 9781904455820)
  • 乳酸菌とビフィズス菌のサイエンス
    (Joint editor)
    京都大学出版会 2010
  • Handbook of Corynebacterium glutamicum (eds. L. Eggeling and M. Bott)
    CRC Press 2005
  • 「微生物利用の大展開」今中忠行 監修
    株式会社エヌ・ティー・エス 2002
  • 「食による生体機能調節の新展開」栄養・食糧科学セレクション3 <安本教傳,葛西隆則 編集>
    日本食品出版 2002
  • Food for Health in the Pacific Rim (eds. J. R. Whitaker, N. F. Haard, C. F. Shoemaker and R. P. Singh)
    Food & Nutrition Press, Inc. 1999


  • Defective F1-ATPase
    John Wiley & Sons, Inc. USA  2009  [Not refereed][Not invited]
  • The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation (eds. M. C. Flickinger and S. W. Drew)
    John Wiley & Sons  1999  [Not refereed][Not invited]

Industrial Property Rights

  • 新規二次胆汁酸生成抑制剤(もしくは方法)
  • 乳酸菌利用による発酵即席麺の物性改良法
  • 乳酸菌利用による発酵即席麺

Awards & Honors

  • 2019/09 The Akiyama Life Science Foundation The Akiyama Life Science Foundation Award 2019
    受賞者: YOKOTA Atsushi
  • 2019/07 Japan Society for Lactic Acid Bacteria Japan Society for Lactic Acid Bacteria (JSLAB) Award
     Comprehensive studies on the interactions between bile acid and lactic acid bacteria/bifidobactria/gut microbes 
    受賞者: YOKOTA Atsushi
  • 2012/10 The Society for Biotechnology, Japan Achievement Award of the Society for Biotechnology, Japan
     Basic research on the enhancement of central metabolism in bacteria producing useful metabolites 
    受賞者: YOKOTA Atsushi
  • 1996/03 The Japan Society for Bioscience, Biotechnology, and Agrochemistry The Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for Encouragement of Young Scientists
    受賞者: YOKOTA Atsushi

Research Grants & Projects

  • 2. 腸内細菌と胆汁酸の相互作用に関する総合的研究
    Date (from‐to) : 1996
  • 1. エネルギー代謝に着目した有用微生物の機能解析と産業利用
    Date (from‐to) : 1988

Educational Activities

Teaching Experience

  • バイオ産業創成学
    開講年度 : 2019
    課程区分 : 修士課程
    開講学部 : 農学院

Campus Position History

  • 2013年4月1日 
  • 2013年4月1日 
  • 2013年4月1日 
  • 2015年4月1日 
  • 2015年4月1日 
  • 2015年4月1日 
  • 2017年4月1日 
  • 2017年4月1日 
  • 2017年4月1日 
  • 2020年10月1日 
  • 2013年4月1日 
  • 2015年4月1日 
  • 2017年4月1日 
  • 2020年10月1日 

Position History

  • 2013年4月1日 
  • 2013年4月1日 
  • 2013年4月1日 
  • 2015年4月1日 
  • 2015年4月1日 
  • 2015年4月1日 
  • 2017年4月1日 
  • 2017年4月1日 
  • 2017年4月1日 
  • 2020年10月1日 
  • 2013年4月1日 
  • 2015年4月1日 
  • 2017年4月1日 
  • 2020年10月1日 

Committee Membership

  • 1999 -2001   The Society for Biotechnology, Japan   Editor, J. Biosci. Bioeng.   The Society for Biotechnology, Japan

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