研究者データベース

横田 篤(ヨコタ アツシ)
総長、理事・副学長
理事・副学長

基本情報

所属

  • 総長、理事・副学長

職名

  • 理事・副学長

学位

  • 農学博士(北海道大学)

ホームページURL

J-Global ID

研究キーワード

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

研究分野

  • ライフサイエンス / 応用微生物学

職歴

  • 2000年 - 2006年 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年 - 2006年 Professor,Laboratory of Microbial Resources and Ecology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • 2006年 - 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年 - Professor,Laboratory of Microbial Physiology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Reseach Faculty of Agriculture, Hokkaido University
  • 1999年 - 2000年 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年 - 2000年 Associate Professor,Laboratory of Microbial Resources and Ecology, Research Group of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University
  • 1992年 - 1999年 北海道大学 農学部 農芸化学科 応用菌学講座 助教授
  • 1992年 - 1999年 Associate Professor,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1991年 - 1992年 北海道大学 農学部 農芸化学科 応用菌学講座 講師
  • 1991年 - 1992年 Lecturer,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1989年 - 1991年 北海道大学 農学部 農芸化学科 応用菌学講座 助手
  • 1989年 - 1991年 Research Associate,Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University
  • 1984年 - 1989年 味の素株式会社 中央研究所 微生物化学部 研究員
  • 1984年 - 1989年 Researcher,Central Research Laboratory of Ajinomoto Co., Inc., Kawasaki, Japan

学歴

  •         - 1984年   北海道大学   農学研究科   農芸化学専攻
  •         - 1984年   北海道大学
  •         - 1981年   北海道大学   農学研究科   農芸化学専攻
  •         - 1981年   北海道大学
  •         - 1979年   北海道大学   農学部   農芸化学科
  •         - 1979年   北海道大学

所属学協会

  • (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   

研究活動情報

論文

  • Siddabasave Gowda B. Gowda, Fengjue Hou, Divyavani Gowda, Hitoshi Chiba, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Shu-Ping Hui
    Analytica Chimica Acta 1288 342145 - 342145 2024年02月
  • Bifidobacterium longum subsp.longumにおける1-Kestose代謝メカニズムの解明
    難波 有希, 倉持 碧海, 山口 颯人, 吉田 佳鼓, 後藤 恭宏, 門田 吉弘, 栃尾 巧, 小椋 義俊, 林 哲也, 前田 智也, 横田 篤, 吹谷 智
    日本乳酸菌学会誌 33 2 120 - 120 日本乳酸菌学会 2022年06月
  • 腸内での二次胆汁酸生成と腸内細菌叢変化・疾病の誘導
    吹谷 智, 横田 篤, 石塚 敏
    機能性食品と薬理栄養 15 4 229 - 234 (株)インフォノーツパブリッシング 2022年02月 [査読無し][招待有り]
  • Miriam N Ojima, Yuya Asao, Aruto Nakajima, Toshihiko Katoh, Motomitsu Kitaoka, Aina Gotoh, Junko Hirose, Tadasu Urashima, Satoru Fukiya, Atsushi Yokota, Maher Abou Hachem, Mikiyasu Sakanaka, Takane Katayama
    Applied and environmental microbiology 88 2 e0143721  2022年01月25日 
    Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.
  • Isaiah Song, Yasuhiro Gotoh, Yoshitoshi Ogura, Tetsuya Hayashi, Satoru Fukiya, Atsushi Yokota
    Microorganisms 9 11 2021年10月29日 
    The human gut houses bile acid 7α-dehydroxylating bacteria that produce secondary bile acids such as deoxycholic acid (DCA) from host-derived bile acids through enzymes encoded by the bai operon. While recent metagenomic studies suggest that these bacteria are highly diverse and abundant, very few DCA producers have been identified. Here, we investigated the physiology and determined the complete genome sequence of Eubacterium sp. c-25, a DCA producer that was isolated from human feces in the 1980s. Culture experiments showed a preference for neutral to slightly alkaline pH in both growth and DCA production. Genomic analyses revealed that c-25 is phylogenetically distinct from known DCA producers and possesses a multi-cluster arrangement of predicted bile-acid inducible (bai) genes that is considerably different from the typical bai operon structure. This arrangement is also found in other intestinal bacterial species, possibly indicative of unconfirmed 7α-dehydroxylation capabilities. Functionality of the predicted bai genes was supported by the induced expression of baiB, baiCD, and baiH in the presence of cholic acid substrate. Taken together, Eubacterium sp. c-25 is an atypical DCA producer with a novel bai gene cluster structure that may represent an unexplored genotype of DCA producers in the human gut.
  • Bifidobacterium longum subsp.longumにおける1-Kestose代謝メカニズムの解明
    難波 有希, 山口 颯人, 吉田 佳鼓, 後藤 恭宏, 門田 吉弘, 栃尾 巧, 小椋 義俊, 林 哲也, 横田 篤, 吹谷 智
    日本生物工学会大会講演要旨集 2021年 230 - 230 (公社)日本生物工学会 2021年10月
  • Kenta Maegawa, Haruka Koyama, Satoru Fukiya, Atsushi Yokota, Koichiro Ueda, Satoshi Ishizuka
    The British journal of nutrition 127 11 1 - 10 2021年07月14日 
    Enterohepatic circulation of 12α-hydroxylated (12αOH) bile acid (BA) is enhanced depending on the energy intake in high-fat diet-fed rats. Such BA metabolism can be reproduced using a diet supplemented with cholic acid (CA), which also induces simple steatosis, without inflammation and fibrosis, accompanied by some other symptoms that are frequently observed in the condition of non-alcoholic fatty liver in rats. We investigated whether supplementation of the diet with raffinose (Raf) improves hepatic lipid accumulation induced by the CA-fed condition in rats. After acclimation to the AIN-93-based control diet, male Wistar rats were fed diets supplemented with a combination of Raf (30 g/kg diet) and/or CA (0·5 g/kg diet) for 4 weeks. Dietary Raf normalised hepatic TAG levels (two-way ANOVA P < 0·001 for CA, P = 0·02 for Raf and P = 0·004 for interaction) in the CA-supplemented diet-fed rats. Dietary Raf supplementation reduced hepatic 12αOH BA concentration (two-way ANOVA P < 0·001 for CA, P = 0·003 for Raf and P = 0·03 for interaction). The concentration of 12αOH BA was reduced in the aortic and portal plasma. Raf supplementation increased acetic acid concentration in the caecal contents (two-way ANOVA P = 0·001 as a main effect). Multiple regression analysis revealed that concentrations of aortic 12αOH BA and caecal acetic acid could serve as predictors of hepatic TAG concentration (R2 = 0·55, P < 0·001). However, Raf did not decrease the secondary 12αOH BA concentration in the caecal contents as well as the transaminase activity in the CA diet-fed rats. These results imply that dietary Raf normalises hepatic lipid accumulation via suppression of enterohepatic 12αOH BA circulation.
  • Shota Hori, Minako Satake, Ohji Kohmoto, Ryo Takagi, Kazufumi Okada, Satoru Fukiya, Atsushi Yokota, Satoshi Ishizuka
    The Journal of nutrition 151 3 523 - 530 2021年03月11日 
    BACKGROUND: Primary 12α-hydroxylated bile acids (12αOH BAs) enhance intestinal iron uptake due to their ability ex vivo to chelate iron. However, no information is available on their role in vivo, especially in the liver. OBJECTIVES: To investigate the effects and mechanisms of primary 12αOH BAs on hepatic iron concentration in vivo. METHODS: Male Wistar King A Hokkaido male rats (WKAH/HkmSlc) rats aged 4-5 weeks were fed a control diet or a diet with cholic acid (CA; 0.5 g/kg diet), the primary 12αOH BA, for 2 weeks (Study 1) or 13 weeks (Study 2). In Study 3, rats fed the same diets were given drinking water either alone or containing vancomycin (200 mg/L) for 6 weeks. The variables measured included food intake (Studies 1-3), bile acid profiles (Studies 1 and 3), hepatic iron concentration (Studies 1-3), fecal iron excretion (Studies 1 and 2), iron-related liver gene expression (Studies 2 and 3), and plasma iron-related factors (Studies 2 and 3). RESULTS: In Study 1, CA feed reduced the hepatic iron concentration (-16%; P = 0.005) without changing food intake or fecal iron excretion. In Study 2, we found a significant increase in the aortic plasma concentration of lipocalin 2 (LCN2; +65%; P < 0.001), an iron-trafficking protein. In Study 3, we observed no effect of vancomycin treatment on the CA-induced reduction of hepatic iron concentration (-32%; P < 0.001), accompanied by increased plasma LCN2 concentration (+72%; P = 0.003), in the CA-fed rats despite a drastic reduction in the secondary 12αOH BA concentration (-94%; P < 0.001) in the aortic plasma. CONCLUSIONS: Primary 12αOH BAs reduced the hepatic iron concentration in rats. LCN2 may be responsible for the hepatic iron-lowering effect of primary 12αOH BAs by transporting iron out of the liver.
  • Rika Hirano, Mikiyasu Sakanaka, Kazuto Yoshimi, Naohisa Sugimoto, Syogo Eguchi, Yuko Yamauchi, Misaki Nara, Shingo Maeda, Yuta Ami, Aina Gotoh, Takane Katayama, Noriho Iida, Tamotsu Kato, Hiroshi Ohno, Satoru Fukiya, Atsushi Yokota, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki Nakai, Shin Kurihara
    Gut Microbes 13 1 2021年01月01日
  • Masaru Wada, Satoru Fukiya, Azusa Suzuki, Nanae Matsumoto, Miki Matsuo, Atsushi Yokota
    Bioscience of microbiota, food and health 40 1 80 - 83 2021年 
    Although bifidobacteria are already widely used as beneficial microbes with health-promoting effects, their amino acid utilization and metabolism are not yet fully understood. Knowledge about the metabolism of sulfur-containing amino acids in bifidobacteria is especially limited. In this study, we tested the methionine utilization ability of several bifidobacterial strains when it was the sole available sulfur source. Although bifidobacteria have long been predominantly considered to be cysteine auxotrophs, we showed that this is not necessarily the case.
  • Ja Young Lee, Hidehisa Shimizu, Masahito Hagio, Satoru Fukiya, Masamichi Watanabe, Yasutake Tanaka, Ga Hyun Joe, Hitoshi Iwaya, Reika Yoshitsugu, Keidai Kikuchi, Misaki Tsuji, Nanako Baba, Takuma Nose, Koji Tada, Taketo Hanai, Shota Hori, Akari Takeuchi, Yumiko Furukawa, Bungo Shirouchi, Masao Sato, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Satoshi Ishizuka
    Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1865 12 2020年12月 
    © 2020 The Authors There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.
  • Siddabasave Gowda B. Gowda, Divyavani Gowda, Chongsheng Liang, Yonghan Li, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
    Metabolites 10 10 398 - 398 2020年10月08日 
    Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are novel endogenous lipids with important physiological functions in mammals. We previously identified a new type of FAHFAs, named short-chain fatty acid esterified hydroxy fatty acids (SFAHFAs), with acetyl or propyl esters of hydroxy fatty acids of carbon chains, C ≥ 20. However, sensitive determination of SFAHFAs is still a challenge, due to their high structural similarity and low abundance in biological samples. This study employs one-step chemical derivatization following total lipid extraction using 2-dimethylaminoethylamine (DMED) for enhanced detection of SFAHFAs. The labeled extracts were subjected to ultrahigh performance liquid chromatography coupled to linear ion trap quadrupole-Orbitrap mass spectrometry (UHPLC/LTQ-Orbitrap MS). Our results demonstrated that the detection sensitivities of SFAHFAs increased after DMED labeling, and is highly helpful in discovering six additional novel SFAHFAs in the cecum and colon contents of WKAH/HKmSlc rats fed with normal and high-fat diet (HFD). The identified DMED labeled SFAHFAs were characterized by their detailed MS/MS analysis, and their plausible fragmentation patterns were proposed. The concentrations of SFAHFAs were significantly reduced in the cecum of HFD group compared to the control. Hence, the proposed method could be a promising tool to apply for the enhanced detection of SFAHFAs in various biological matrices, which in turn facilitate the understanding of their sources, and physiological functions of these novel lipids.
  • Siddabasave Gowda B. Gowda, Chongsheng Liang, Divyavani Gowda, Fengjue Hou, Kentaro Kawakami, Satoru Fukiya, Atsushi Yokota, Hitoshi Chiba, Shu‐Ping Hui
    Rapid Communications in Mass Spectrometry 34 17 2020年09月15日
  • Shota Hori, Takayuki Abe, Dong Geun Lee, Satoru Fukiya, Atsushi Yokota, Nao Aso, Bungo Shirouchi, Masao Sato, Satoshi Ishizuka
    The Journal of nutritional biochemistry 83 108412 - 108412 2020年05月20日 [査読有り][通常論文]
     
    High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.
  • Hiroka Koguchi, Natsumi Ishigami, Mikiyasu Sakanaka, Kako Yoshida, Sayaka Hiratou, Mina Shimada, Satoru Fukiya, Kei Sonoyama, Atsushi Yokota
    Microorganisms 8 3 2020年03月13日 [査読有り][通常論文]
     
    Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.
  • Siddabasave Gowda B Gowda, Zi-Jun Gao, Zhen Chen, Takayuki Abe, Shota Hori, Satoru Fukiya, Satoshi Ishizuka, Atsushi Yokota, Hitoshi Chiba, Shu-Ping Hui
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 2020年01月17日 [査読有り][通常論文]
  • Hiroko Yoshioka, Masamichi Watanabe, Fumio Nanba, Toshio Suzuki, Satoru Fukiya, Atsushi Yokota, Toshiya Toda
    Journal of nutritional science and vitaminology 66 6 571 - 576 2020年 
    Equol (Eq) is a metabolite of soy isoflavone daidzein (De) produced by the intestinal microbiota. The clinical effectiveness of soy isoflavone is considered to depend on the individual ability of Eq production. Previous studies have demonstrated that habitual dietary patterns may influence the production of Eq. For example, high Eq producers consumed less fat as a percentage of energy than low Eq producers. However, the inhibitory factors of Eq production are unknown. Recently, it was reported that bile acids induced by high-fat diet consumption may be a host-related factor controlling the composition of the intestinal microbiota. In this study, we investigated the effect of cholic acid (CA) administration, a mimic of the microbiota altered by a high-fat diet, on Eq production in mice. CA administration significantly decreased the levels of the De metabolites Eq, dihydrodaidzein, and O-desmethylangolensin in the serum of mice. However, CA administration did not affect the total molar concentration of De and its metabolites. Moreover, CA administration increased the levels of secondary bile acids, particularly deoxycholic acid (DCA), which has strong antibacterial activity in the cecum contents of mice. Thus, CA administration may increase the levels of DCA, a secondary bile acid, resulting in inhibition of Eq production. These findings may help to reveal the factors inhibiting Eq production and enhance the clinical effectiveness of isoflavone intake.
  • 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年08月 [査読有り][通常論文]
     
    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 2019年03月 [査読有り][通常論文]
     
    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.
  • Lee DG, Hori S, Kohmoto O, Kitta S, Yoshida R, Tanaka Y, Shimizu H, Takahashi K, Nagura T, Uchino H, Fukiya S, Yokota A, Ishizuka S
    Bioscience, Biotechnology, and Biochemistry 83 7 1329 - 1335 2019年03月 [査読有り][通常論文]
     
    Difructose anhydride III (DFAIII) is a prebiotic involved in the reduction of secondary bile acids (BAs). We investigated whether DFAIII modulates BA metabolism, including enterohepatic circulation, in the rats fed with a diet supplemented with cholic acid (CA), one of the 12α-hydroxylated BAs. After acclimation, the rats were fed with a control diet or a diet supplemented with DFAIII. After 2 weeks, each group was further divided into two groups and was fed diet with or without CA supplementation at 0.5 g/kg diet. BA levels were analyzed in aortic and portal plasma, liver, intestinal content, and feces. As a result, DFAIII ingestion reduced the fecal deoxycholic acid level via the partial suppression of deconjugation and 7α-dehydroxylation of BAs following CA supplementation. These results suggest that DFAIII suppresses production of deoxycholic acid in conditions of high concentrations of 12α-hydroxylated BAs in enterohepatic circulation, such as obesity or excess energy intake. Abbreviation: BA: bile acid; BSH: bile salt hydrolase; CA: cholic acid; DCA: deoxycholic acid; DFAIII: difructose anhydride III; MCA: muricholic acid; MS: mass spectrometry; NCDs: non-communicable diseases; LC: liquid chromatography; SCFA: short-chain fatty acid; TCA: taurocholic acid; TCDCA: taurochenodeoxycholic acid; TDCA: taurodeoxycholic acid; TUDCA: tauroursodeoxychlic acid; TαMCA: tauro-α-muricholic acid; TβMCA: tauro-β-muricholic acid; TωMCA: tauro-ω-muricholic acid.
  • Kataoka N, Vangnai AS, Pongtharangkul T, Yakushi T, Wada M, Yokota A, Matsushita K
    Bioscience, Biotechnology, and Biochemistry 83 2 372 - 380 2019年02月 [査読有り][通常論文]
     
    To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).
  • Mikiyasu Sakanaka, Shingo Nakakawaji, Shin Nakajima, Satoru Fukiya, Arisa Abe, Wataru Saburi, Haruhide Mori, Atsushi Yokota
    Applied and environmental microbiology 84 17 2018年09月01日 [査読有り][通常論文]
     
    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.
  • 乳酸桿菌の対数期に膜小胞上に局在するリポテイコ酸
    白石 宗, 横田 伸一, 佐藤 耶舞羽, 伊藤 利章, 吹谷 智, 山本 聡, 佐藤 豊孝, 横田 篤
    日本乳酸菌学会誌 29 2 116 - 116 日本乳酸菌学会 2018年07月
  • T. Shiraishi, S. Yokota, Y. Sato, T. Ito, S. Fukiya, S. Yamamoto, T. Sato, A. Yokota
    Beneficial Microbes 9 4 653 - 662 2018年 [査読有り][通常論文]
     
    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 2017年11月 [査読有り][通常論文]
     
    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 2017年09月 [査読有り][通常論文]
     
    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 2017年06月 [査読有り][通常論文]
     
    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.-
  • Lactobacillus gasseri種に共通する種特異的なリポテイコ酸の化学構造
    久富 亮佑, 白石 宗, 佐藤 耶舞羽, 森田 直樹, 吹谷 智, 佐藤 豊孝, 横田 篤, 横田 伸一
    日本乳酸菌学会誌 28 2 123 - 123 日本乳酸菌学会 2017年06月
  • 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 2017年04月 [査読有り][通常論文]
     
    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.
  • Lactobacillus gasseri種に共通するリポテイコ酸の特異的構造
    久富 亮佑, 白石 宗, 佐藤 耶舞羽, 森田 直樹, 吹谷 智, 佐藤 豊孝, 横田 篤, 横田 伸一
    日本細菌学雑誌 72 1 86 - 86 日本細菌学会 2017年02月
  • Hiroyuki Nagano, Kana Shibano, Yu Matsumoto, Atsushi Yokota, Masaru Wada
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 6 1156 - 1164 2017年 [査読有り][通常論文]
     
    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 4.3.1.16) 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 2017年 [査読有り][通常論文]
     
    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
  • Atsushi Yokota, Masato Ikeda
    AMINO ACID FERMENTATION 159 V - VI 2017年 [査読有り][通常論文]
  • 構造情報の蓄積から見えてきたリポテイコ酸の細菌種による構造多様性.
    白石宗, 横田伸一, 吹谷智, 横田篤
    日本乳酸菌学会誌 28 3 177 - 177 2017年 [査読無し][招待有り]
  • 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 2016年08月 [査読有り][通常論文]
     
    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 Yokota
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 121 2 172 - 177 2016年02月 [査読有り][通常論文]
     
    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 2016年 [査読有り][通常論文]
     
    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.
  • Kazunori Sawada, Masaru Wada, Takuya Hagiwara, Susumu Zen-in, Keita Imai, Atsushi Yokota
    Metabolic Engineering Communications 2 67 - 75 2015年12月01日 [査読有り][通常論文]
     
    The effect of pyruvate kinase gene (pyk) deletion on the physiology of Corynebacterium glutamicum ATCC13032 was investigated under biotin-sufficient, non-glutamate-producing conditions. In a complex medium containing 100. g/L glucose, a defined pyk deletion mutant, strain D1, exhibited 35% enhancement in glucose consumption rate, 37% increased growth and a 57% reduction in respiration rate compared to the wild-type parent. Significant upregulation of phosphoenolpyruvate (PEP) carboxylase and downregulation of PEP carboxykinase activities were observed in the D1 mutant, which may have prevented over-accumulation of PEP caused by the pyk deletion. Moreover, we found a dramatic 63% reduction in the activity of malate:quinone oxidoreductase (MQO) in the D1 mutant. MQO, a TCA cycle enzyme that converts malate to oxaloacetate (OAA), constitutes a major primary gate to the respiratory chain in C. glutamicum, thus explaining the reduced respiration rate in the mutant. Additionally, pyruvate carboxylase gene expression was downregulated in the mutant. These changes seemed to prevent OAA over-accumulation caused by the activity changes of PEP carboxylase/PEP carboxykinase. Intrinsically the same alterations were observed in the cultures conducted in a minimal medium containing 20. g/L glucose. Despite these responses in the mutant, metabolic distortion caused by pyk deletion under non-glutamate-producing conditions required amelioration by increased biomass production, as metabolome analysis revealed increased intracellular concentrations of several precursor metabolites for building block formation associated with pyk deletion. These fermentation profiles and metabolic alterations observed in the mutant reverted completely to the wild-type phenotypes in the pyk-complemented strain, suggesting the observed metabolic changes were caused by the pyk deletion. These results demonstrated multilateral strategies to overcome metabolic disturbance caused by pyk deletion in this bacterium.
  • Yu Matsumoto, Yoshiaki Yasutake, Yuki Takeda, Tomohiro Tamura, Atsushi Yokota, Masaru Wada
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 99 17 7137 - 7150 2015年09月 [査読有り][通常論文]
     
    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 2015年06月 [査読有り][通常論文]
     
    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 2015年04月 [査読有り][通常論文]
     
    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.
  • Hagio M, Shimizu H, Joe GH, Takatsuki M, Shiwaku M, Xu H, Lee JY, Fujii N, Fukiya S, Hara H, Yokota A, Ishizuka S
    Toxicology Letters 232 1 246 - 252 2015年01月 [査読有り][通常論文]
     
    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年12月18日 [査読有り][通常論文]
     
    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 2014年12月 [査読有り][通常論文]
     
    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.
  • Sakanaka M, Tamai S, Hirayama Y, Onodera A, Koguchi H, Kano Y, Yokota A, Fukiya S
    Journal of Bioscience and Bioengineering 118 5 489 - 495 2014年11月 [査読有り][通常論文]
     
    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 2014年07月 [査読有り][通常論文]
     
    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 2014年05月 [査読有り][通常論文]
     
    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.
  • Lee JY, Arai H, Nakamura Y, Fukiya S, Wada M, Yokota A
    Journal of Lipid Research 54 11 3062 - 3069 2013年11月 [査読有り][通常論文]
     
    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
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 69 Pt 10 1131 - 1134 2013年10月 [査読有り][通常論文]
     
    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 2013年08月 [査読有り][通常論文]
     
    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 2013年05月 [査読有り][通常論文]
     
    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.
  • Hirayama Y, Sakanaka M, Fukuma H, Murayama H, Kano Y, Fukiya S, Yokota A
    Applied and Environmental Microbiology 78 14 4984 - 4994 2012年07月 [査読有り][通常論文]
     
    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 2012年04月 [査読有り][通常論文]
     
    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 2012年04月 [査読無し][通常論文]
     
    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 2012年 [査読有り][通常論文]
     
    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 2012年 [査読有り][通常論文]
     
    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 2011年11月 [査読無し][通常論文]
     
    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 2010年12月 [査読無し][通常論文]
     
    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 2010年10月 [査読無し][通常論文]
     
    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 2010年08月 [査読無し][通常論文]
     
    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 2010年07月 [査読無し][通常論文]
     
    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 2010年03月 [査読無し][通常論文]
     
    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.
  • Suzuki T, Nishimukai M, Takechi M, Taguchi H, Hamada S, Yokota A, Ito S, Hara H, Matsui H
    Journal of agricultural and food chemistry 58 3 1927 - 1932 2010年02月 [査読有り][通常論文]
  • Satoshi Ishizuka, Ami Iwama, Achmad Dinoto, Akarat Suksomcheep, Kohshi Maeta, Takanori Kasai, Hiroshi Hara, Atsushi Yokota
    MOLECULAR NUTRITION & FOOD RESEARCH 53 S62 - S67 2009年05月 [査読無し][通常論文]
     
    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 2009年05月 [査読無し][通常論文]
     
    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 2009年05月 [査読無し][通常論文]
     
    L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16), 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 2009年04月 [査読無し][通常論文]
     
    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 2009年03月 [査読無し][通常論文]
     
    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 2009年02月 [査読無し][通常論文]
     
    Cellobiose 2-epimerase (CE, EC 5.1.3.11) 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 2008年11月 [査読無し][通常論文]
     
    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.
  • Fukiya S, Yokota A
    Seikagaku. The Journal of Japanese Biochemical Society 80 5 421 - 425 5 2008年05月25日 [査読有り][通常論文]
  • Eriko Yahata, Yuki Sawatari, Hisashi Sugiyama, Akihiro Hanaoka, Atsushi Yokota, Hamo Saruyama
    Food Science and Technology Research 14 3 285 - 292 2008年05月 [査読有り][通常論文]
     
    The lactic acid fermentation of instant Chinese noodle sheet by Lactobacillus 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 nonfermented 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.
  • Eriko Yahata, Yuki Sawatari, Hisashi Sugiyama, Akihiro Hanaoka, Atsushi Yokota, Haruo Saruyama
    FOOD SCIENCE AND TECHNOLOGY RESEARCH 14 3 285 - 292 2008年05月 [査読無し][通常論文]
     
    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 2008年02月 [査読無し][通常論文]
     
    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 2007年12月 [査読無し][通常論文]
     
    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 2007年09月 [査読無し][通常論文]
     
    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 2007年09月 [査読無し][通常論文]
     
    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 16 5385  2007年08月 [査読有り][通常論文]
  • Yuki Sawatari, Atsushi Yokota
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 73 12 3909 - 3915 2007年06月 [査読無し][通常論文]
     
    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 2007年05月 [査読無し][通常論文]
     
    Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+-ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+-ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P <= 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.
  • ディノト アハマド, 吹谷 智, 横田 篤
    日本乳酸菌学会誌 = Journal of Japan Society for Lactic Acid Bacteria 17 2 110 - 117 2006年12月01日 [査読無し][通常論文]
  • Yuki Sawatari, Tomomi Hirano, Atsushi Yokota
    JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY 52 6 349 - 356 2006年12月 [査読無し][通常論文]
     
    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 2006年12月 [査読無し][通常論文]
     
    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 2006年10月 [査読無し][通常論文]
     
    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 2006年03月 [査読無し][通常論文]
     
    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 2006年01月 [査読無し][通常論文]
     
    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 2005年12月 [査読無し][通常論文]
     
    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 2005年08月 [査読無し][通常論文]
     
    A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345(T) and Lactobacillus fermentum JCM 1173(T), and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 X 10(3) cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10(4) cells/g feces on day 4 and 10(5) cells/g feces on days 11 to 18. However, cell populations of 10(6) to 10(7) cells/g feces were detected in the second trial. The total bacterial count, measured by 4',6-diamidino-2-phenylindole (DAPI) staining, was approximately 10(11) cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.
  • R Aoki, M Wada, N Takesue, K Tanaka, A Yokota
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 8 1466 - 1472 2005年08月 [査読無し][通常論文]
     
    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.
  • Kimiko Minamida, Maki Kaneko, Midori Ohashi, I Nengah Sujaya, Teruo Sone, Masaru Wada, Atsushi Yokota, Hiroshi Hara, Kozo Asano, Fusao Tomita
    Journal of Bioscience and Bioengineering 99 6 548 - 554 The Society for Biotechnology, Japan 2005年06月 [査読有り][通常論文]
     
    The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 108–109 cells/g dry feces within a week from 106 cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.
  • Kimiko Minamida, Kazuki Shiga, I Nengah Sujaya, Teruo Sone, Atsushi Yokota, Hiroshi Hara, Kozo Asano, Fusao Tomita
    Journal of Bioscience and Bioengineering 99 3 230 - 236 The Society for Biotechnology, Japan 2005年03月 [査読有り][通常論文]
     
    The effects of difructose anhydride III (di-D-fructofuranose-1,2′:2,3′-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.
  • 澤渡 優喜, 横田 篤
    日本乳酸菌学会誌 16 2 75 - 83 日本乳酸菌学会誌 2005年 [査読有り][通常論文]
     
    我々は小麦粉を乳酸菌で発酵させた新規発酵食品として発酵即席麺の開発研究を行っている。乳酸菌発酵を利用した小麦粉発酵食品はきわあて少ないが,代表例としてサワーブレッドがあげられる。中華麺製造にはかんすいが用いられ,発酵環境はアルカリ性(pH約8.5)であるため,アルカリ耐性乳酸菌が必要となる。植物性乳酸菌を主体とする12種(亜種を含む)46菌株を対象にスクリーニングを行った結果,<I>Lactobacillus</I>(以下,Lb.)<I>pentosus</I>および<I>Lb.plantarum</I>の2菌種にのみ麺発酵能が認められた。最適株として選択した<I>Lb.plantarum</I> NRIC 0380を用いて調製した発酵即席麺は,麺物性が顕著に向上し,軽い酸味が付与されたこれまでにない良好な特性を示した。発酵培地としての麺生地の食品化学的特徴と乳酸菌の生理的特性の関連性についても考察した。
  • 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 2004年10月 [査読無し][通常論文]
     
    Di-D-fructofuranose-1,2':2,3'-dianhydride (DFA III) was shown to enhance Ca absorption in rat and human intestine. The effects of DFA III administration (9 g per day for 4 weeks that corresponded to 3-fold the optimal dosage of DFA III) on human intestinal microbiota were studied using denaturing gradient gel electrophoresis (DGGE). The major groups of human intestinal microbiota reported previously: the Bacteroides, the Clostridium coccoides group (Clostridium cluster XIVa), the Clostridium leptum group (Clostridium cluster IV), and the Bifidobacterium group were detected. The similarity of 30 DGGE profiles based on the V3 region (before and after administration to the 15 subjects) of the 16S rDNA were calculated using Pearson's correlation based on numbers, positions and intensity of bands, and then a dendrogram of DGGE profiles was constructed by the unweighted pair group method using arithmetic average (UPGMA) clustering method. By these analyses, no difference in DGGE profiles after DFA III administration was observed in healthy subjects, while two subjects with chronic constipation showed different profiles, namely on numbers, positions and the intensity of some bands. Their stools were softer and stool frequencies increased and they obtained relief from constipation.
  • S Ren, F Tomita, A Yokota, K Asano
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 8 1627 - 1633 2004年08月 [査読有り][通常論文]
     
    Microorganisms were screened for their ability to release cadmium from scallop hepatopancreas, which is the main residue after removing of the edible parts of scallop. The isolated strain, 23-0-11, identified as Arthrobacter nicotinovorans, secreted a protease which released cadmium from scallop hepatopancreas into the liquid medium. The molecular mass of the enzyme was estimated to be 27 kDa. The sequence of the 15 N-terminal amino acids of the protease showed no close similarity with any other protein. Compared with a commercial enzyme, the purified protease had greater ability to release cadmium. The enzyme activity was greatest at 50degreesC and pH 7.0, and was enhanced in the presence of Ca2+, Mg2+ and Mn2+, while being strongly inhibited by Co2+. The inhibition profile by the serine protease inhibitor, phenylmethylsulphonyl fluoride (PMSF), confirmed that the protease belonged to the serine protease family.
  • NS Antara, IN Sujaya, A Yokota, K Asano, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 98 2 92 - 98 2004年08月 [査読有り][通常論文]
     
    Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25degreesC at night to 50degreesC during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.
  • IN Sujaya, NS Antara, T Sone, Y Tamura, WR Aryanta, A Yokota, K Asano, F Tomita
    WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 20 2 143 - 150 2004年03月 [査読無し][通常論文]
     
    Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer ( ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae ( 35 strains), Candida glabrata ( six strains), Pichia anomala ( three strains) and Issatchenkia orientalis ( seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.
  • KIKUCHI H.
    Journal of Applied Glycoscience 51 4 291 - 296 2004年 [査読無し][通常論文]
  • 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 2003年11月 [査読無し][通常論文]
     
    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 2003年08月 [査読無し][通常論文]
     
    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 2003年05月 [査読無し][通常論文]
     
    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 2.4.1.93) 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 2.4.1.200) 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 2003年02月 [査読無し][通常論文]
     
    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 2003年01月 [査読無し][通常論文]
     
    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 2003年01月 [査読無し][通常論文]
     
    The gene encoding a 2,6-beta-D-fructan 6-levanbiohydrolase (LF2ase) (EC 3.2.1.64) 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 2002年06月 [査読無し][通常論文]
     
    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 2002年04月 [査読無し][通常論文]
     
    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 2002年04月 [査読無し][通常論文]
     
    '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.
  • M Suto, M Takebayashi, K Saito, M Tanaka, A Yokota, F Tomita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 93 1 88 - 90 2002年01月 [査読有り][通常論文]
     
    One hundred and sixty-nine endophytic fungi and 81 endophytic bacteria were isolated from 14 plants in total. Among them, 155 fungi (91.7%) and 52 bacteria (64%) were found to produce xylanase. The inside part of plants is a novel and good source for isolating xylanase producers in comparison with soil.
  • H Sekine, T Shimada, C Hayashi, A Ishiguro, F Tomita, A Yokota
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 57 4 534 - 540 2001年11月 [査読無し][通常論文]
     
    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 2001年06月 [査読無し][通常論文]
     
    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 2000年11月 [査読無し][通常論文]
     
    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 2000年11月 [査読無し][通常論文]
     
    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 2000年09月 [査読無し][通常論文]
     
    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 2000年08月 [査読無し][通常論文]
     
    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 2000年01月 [査読無し][通常論文]
     
    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 3.1.1.64).
  • N Phay, T Higashiyama, M Tsuji, H Matsuura, Y Fukushi, A Yokota, F Tomita
    PHYTOCHEMISTRY 52 2 271 - 274 1999年09月 [査読無し][通常論文]
     
    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 1999年05月 [査読無し][通常論文]
  • K Saito, T Hira, T Suzuki, H Hara, A Yokota, F Tomita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 4 655 - 661 1999年04月 [査読無し][通常論文]
     
    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.
  • NAOWARAT C.
    Thai Journal of Biotechnology 1 1 37 - 45 1999年 [査読無し][通常論文]
  • Y Nagasaka, K Kurosawa, A Yokota, F Tomita
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 50 3 323 - 330 1998年09月 [査読無し][通常論文]
     
    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 1998年08月 [査読無し][通常論文]
     
    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 1997年12月 [査読無し][通常論文]
     
    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 1997年10月 [査読無し][通常論文]
     
    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 1997年10月 [査読無し][通常論文]
     
    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 1997年06月 [査読無し][通常論文]
     
    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.
  • Atsushi Yokota
    Nippon Nogeikagaku Kaishi 71 1 9 - 14 1997年 [査読有り][通常論文]
  • A Yokota, M Henmi, N Takaoka, C Hayashi, Y Takezawa, Y Fukumori, F Tomita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 83 2 132 - 138 1997年 [査読無し][通常論文]
     
    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 1997年01月 [査読無し][通常論文]
     
    The gene encoding an extracellular inulin fructotransferase (depolymerizing) (inulase ZI) (EC 2.4.1.93), 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 2.4.1.200) 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 1996年07月 [査読無し][通常論文]
  • K Kawasaki, A Yokota, F Tomita
    JOURNAL OF FERMENTATION AND BIOENGINEERING 82 6 604 - 606 1996年 [査読無し][通常論文]
     
    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 1995年12月 [査読無し][通常論文]
     
    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.
  • A YOKOTA, O YAMAUCHI, F TOMITA
    LETTERS IN APPLIED MICROBIOLOGY 21 5 330 - 333 1995年11月 [査読無し][通常論文]
     
    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.
  • A YOKOTA, S AMACHI, S ISHII, F TOMITA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 10 2004 - 2007 1995年10月 [査読無し][通常論文]
     
    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.
  • K KAWASAKI, A YOKOTA, F TOMITA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 59 10 1938 - 1943 1995年10月 [査読無し][通常論文]
     
    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.
  • Toshiyuki Obara, Atsushi Yokota, Fusao Tomita
    日本エネルギー学会誌 74 4 228 - 232 1995年 [査読無し][通常論文]
     
    For the purpose of removing nitrogen atoms from heavy oil by microbial procedures, the first approach was made to obtain microorganisms capable of degrading quinoline as the N-containing model compound by enrichment culture. One isolate, identified as Pseudomonas putida Q26, was chosen for further study. The degradation of quinoline by this strain yielded initially 2-hydroxyquinoline and then 8-hydroxycoumarin, resulting finally in the complete degradation. This strain was able to degrade quinoline and a few methylquinoline in a complex basic fraction of coal-liquids. It was observed that microbial treatment decreased nitrogen content of the basic fraction of hydrogenated recycle solvent derived from Taiheiyo coal. © 1995, The Japan Institute of Energy. All rights reserved.
  • A YOKOTA, Y TERASAWA, N TAKAOKA, H SHIMIZU, F TOMITA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 58 12 2164 - 2167 1994年12月 [査読無し][通常論文]
     
    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.
  • A YOKOTA, H SHIMIZU, Y TERASAWA, N TAKAOKA, F TOMITA
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 41 6 638 - 643 1994年08月 [査読無し][通常論文]
     
    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.
  • K KAWASAKI, A YOKOTA, S OITA, C KOBAYASHI, S YOSHIKAWA, SI KAWAMOTO, S TAKAO, F TOMITA
    JOURNAL OF GENERAL MICROBIOLOGY 139 12 3275 - 3281 1993年12月 [査読無し][通常論文]
     
    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.
  • A YOKOTA, K KONDO, M NAKAGAWA, KOJIMA, I, F TOMITA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 57 5 745 - 749 1993年05月 [査読無し][通常論文]
     
    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.
  • F TOMITA, A YOKOTA
    NIPPON NOGEIKAGAKU KAISHI-JOURNAL OF THE JAPAN SOCIETY FOR BIOSCIENCE BIOTECHNOLOGY AND AGROCHEMISTRY 65 6 998 - 1002 1991年06月 [査読有り][通常論文]
  • A YOKOTA, S HIRAYAMA, K ENOMOTO, Y MIURA, S TAKAO, F TOMITA
    JOURNAL OF FERMENTATION AND BIOENGINEERING 72 4 258 - 261 1991年 [査読無し][通常論文]
     
    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.
  • A YOKOTA, K ENOMOTO, F TOMITA
    JOURNAL OF FERMENTATION AND BIOENGINEERING 72 4 262 - 265 1991年 [査読無し][通常論文]
     
    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.
  • J HARIANTONO, A YOKOTA, S TAKAO, F TOMITA
    JOURNAL OF FERMENTATION AND BIOENGINEERING 71 5 367 - 369 1991年 [査読無し][通常論文]
     
    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.
  • A YOKOTA, SHIIO, I
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 54 2 547 - 548 1990年02月 [査読無し][通常論文]
  • S OITA, A YOKOTA, S TAKAO
    JOURNAL OF FERMENTATION AND BIOENGINEERING 69 4 256 - 258 1990年 [査読無し][通常論文]
  • SHIIO, I, A YOKOTA, K KAWAMURA
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 8 2169 - 2175 1989年08月 [査読無し][通常論文]
  • A YOKOTA, S OITA, S TAKAO
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 8 2037 - 2044 1989年08月 [査読無し][通常論文]
  • A YOKOTA, S TAKAO
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 3 705 - 711 1989年03月 [査読無し][通常論文]
  • SHIIO, I, A YOKOTA, Y TORIDE, SI SUGIMOTO
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 53 1 41 - 48 1989年01月 [査読無し][通常論文]
  • A YOKOTA, SHIIO, I
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 52 2 455 - 463 1988年02月 [査読無し][通常論文]
  • SHIIO, I, A YOKOTA, S SUGIMOTO
    AGRICULTURAL AND BIOLOGICAL CHEMISTRY 51 9 2485 - 2493 1987年09月 [査読無し][通常論文]
  • S TAKAO, A YOKOTA, M TANIDA
    JOURNAL OF FERMENTATION TECHNOLOGY 62 4 329 - 334 1984年 [査読無し][通常論文]
  • S TAKAO, A YOKOTA, M TANIDA
    JOURNAL OF FERMENTATION TECHNOLOGY 61 6 643 - 645 1983年 [査読無し][通常論文]

書籍

  • Atsushi Yokota, Masato Ikeda (担当:編者(編著者))
    Springer 2017年 (ISBN: 9784431565185)
  • 横田篤, 大西康夫, 小川順 (担当:編者(編著者))
    文永堂出版 2016年07月 (ISBN: 4830041315) 327
  • Kenji Sonomoto, Atsushi Yokota (担当:編者(編著者))
    Caister Academic Press, Norfolk, UK 2011年 (ISBN: 9781904455820)
  • 乳酸菌とビフィズス菌のサイエンス
    (担当:共編者(共編著者))
    京都大学出版会 2010年
  • Handbook of Corynebacterium glutamicum (eds. L. Eggeling and M. Bott)
    (担当:分担執筆)
    CRC Press 2005年
  • 「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年
  • 「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年
  • 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年

その他活動・業績

特許

  • 新規二次胆汁酸生成抑制剤(もしくは方法)
    2010-048241
  • 乳酸菌利用による発酵即席麺の物性改良法
    3873129
  • 乳酸菌利用による発酵即席麺
    3769616

受賞

  • 2019年09月 公益財団法人 秋山記念生命科学振興財団 2019年度秋山財団賞
     腸内細菌叢の制御における胆汁酸の役割に関する微生物生理学的研究 
    受賞者: 横田 篤
  • 2019年07月 日本乳酸菌学会 日本乳酸菌学会賞
     乳酸菌・ビフィズス菌・腸内細菌と胆汁酸の相互作用に関する総合的研究 
    受賞者: 横田 篤
  • 2012年10月 公益社団法人 日本生物工学会 生物工学功績賞
     有用物質生産菌の中枢代謝強化に関する基盤研究 
    受賞者: 横田 篤
  • 1996年03月 公益社団法人 日本農芸化学会 農芸化学奨励賞
     エネルギー代謝変異による有用微生物の育種に関する研究 
    受賞者: 横田 篤

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 前田 智也, 横田 篤
     
    本研究では、発酵産業に重要な大腸菌やコリネ型細菌の発酵生産効率の向上を目的として、中枢代謝が活性化している様々な呼吸鎖酵素欠損株の適応的実験室進化を行うことで、産業微生物の細胞増殖と目的物質生産のバランスを最適化させる方法を明らかにすることを最終目的としている。 大腸菌やコリネ型細菌において、酸化的リン酸化の阻害によるエネルギー欠乏の誘導が糖代謝などの中枢代謝を活性化させる一方、著しい増殖悪化を引き起こすことが先行研究から明らかにされている。申請者は、本研究において、エネルギー欠乏株の多くが酢酸を単一炭素源として生育できない、または著しく生育が阻害されることを見出した。酢酸を単一炭素源とした場合、取り込んだ酢酸をまず活性化してアセチルリン酸へ変換する過程で1分子のATPを消費し、その後基質レベルのリン酸化では1分子のATPしか合成できないため、ATP合成は酸化的リン酸化に依存していると考えられる。そのため、酢酸を単一炭素源とする選択圧をかける実験室進化を行うことで、酸化的リン酸化が阻害されているエネルギー欠乏株において、エネルギー欠乏への潜在的な適応能力が活性化された進化株が出現し得るのではないかと考えた。そこで今年度は、まず大腸菌の様々な呼吸鎖酵素を欠損したエネルギー欠乏株6株を親株として、酢酸最少培地を用いた適応的実験室進化を行った。適応的実験室進化は、コントロールとして呼吸鎖を欠損していない野生株を用い、また同一培養条件における反復数4として合計28系列行った。このような適応的実験室進化により、酢酸最少培地における生育が回復または、増殖速度が向上した進化株を取得することに成功した。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 横田 篤, 前田 智也, 片岡 尚也, 和田 大
     
    本研究では、野生株W1485から取得したΔΔ株(NDH-IとCytbo3を共に欠損)が糖消費活性と呼吸活性の著しい上昇、酢酸の生成、NADHの滞留、グルタミン酸(Glu)の著量蓄積などの異常な糖代謝を示すことを発見した。さらにメタボローム解析、RNA-Seqにより、ペントースリン酸経路、酢酸生成経路に加えて、Gluをγ-アミノ酪酸(GABA)に変換するグルタミン酸脱炭酸酵素遺伝子gadABなどの発現上昇が認められた。また、ΔΔ株を用いた発酵生産の効率化について、1,3-ブタンジオールとGABAの高生産化を目指し、どちらの物質に対してもΔΔ変異が生産性向上に寄与することを実証した。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2013年04月 -2014年03月 
    代表者 : 横田 篤
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2011年 -2012年 
    代表者 : 横田 篤, 石塚 敏
     
    大腸癌のリスク因子として大腸内に生成される発がん性二次胆汁酸が知られている.本研究では,腸内細菌による胆汁酸の還元的代謝により生成される発がん性二次胆汁酸を低減させる新しい手法として,腸内細菌の嫌気呼吸を促進させ,胆汁酸の酸化的代謝を活性化させることを試みた.そこでラットにフマル酸を添加した飼料を摂取させ,盲腸内の嫌気呼吸の誘導を試みたところ,代表的な発がん性二次胆汁酸であるデオキシコール酸の低減可能性は示された.しかし,5~10%と高濃度のフマル酸添加必要であり,この濃度ではラットが下痢を起こすことから、実用的な抑制にはフマル酸のカプセル化等,添加方法の検討が必要である.
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2009年 -2011年 
    代表者 : 横田 篤, 吹谷 智, 森田 直樹, 横田 伸一
     
    遊離胆汁酸はその細胞膜損傷作用により腸内細菌にとって最も過酷な環境ストレス因子になっている.これまでの研究で,研究代表者らはLactobacillus gasseri JCM1131Tにヒトの主要な胆汁酸であるコール酸に対する適応現象を見出している.これは,対数増殖期の細胞を非致死濃度である4mMのコール酸に30分間暴露した後,致死濃度の15mMのコール酸で処理したところ,7時間後でも生残率がほぼ100%であったのに対し,何も処理しない細胞は生残率が千分の一あるいは一万分の一に急速に低下したというものである.この際の適応細胞では,致死濃度のコール酸による細胞膜の損傷が軽減されていて,適応に伴う細胞膜脂質組成の変化が示唆された. そこで適応細胞と非適応細胞から全脂質を抽出し,それらを中性脂質,糖脂質,リン脂質の3区分に分画した.それぞれの画分の比率は適応前後で変化なく,それぞれの画分の脂肪酸組成についても変化がなかった.しかし,糖脂質とリン脂質の組成に有意な変動があった.糖脂質では糖鎖の鎖長が長くなった.またリン脂質ではカルジオリピンの比率が有意に増大した. コール酸適応におけるカルジオリピンの役割について調べるため,ベシクル実験を行った.その結果,カルジオリピンはコール酸による攻撃に対してベシクルに抵抗性を付与することが分かり,カルジオリピンが適応機構に深く関わっていることが示唆された. 本菌のゲノム上には,カルジオリピンの合成に作用すると考えられる2つのカルジオリピン合成酵素遺伝子が存在する.この遺伝子を単独又は両方欠損させた変異株を構築し,それらの株のコール酸適応能を調べた.単独欠損株ではいずれの株でもリン脂質画分におけるカルジオリピンの存在比が低下したが,コール酸適応により野生株並みに増大し,野生株と変わらないコール酸適応を示した.二重変異株ではカルジオリピンが殆ど検出されなかったが,それにも拘わらず,コール酸適応を示した.したがって,カルジオリピンはコール酸適応に必須ではないことが分かった.しかしこの二重変異株では,糖脂質含量の有意な減少とリン脂質含量の有意な増大が見られ,その変化は適応後により顕著になった.このことはカルジオリピンが細胞膜の脂質組成を決定する重要な役割を担っていることを示している.
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2004年 -2006年 
    代表者 : 横田 篤, 吹谷 智, 湯本 勳, 和田 大
     
    糞便サンプルから,一次胆汁酸であるコール酸(CA)とケノデオキシコール酸(CDCA)をそれぞれ大腸ガンのプロモーター活性を持つ二次胆汁酸,デオキシコール酸(DCA)とリトコール酸(LCA),に変換する7α-脱水酸化活性を持つ未知の腸内細菌の分離を目的として研究を行った. 嫌気チャンバー内で糞便サンプルから合計619株の腸内細菌を分離し,これらのCAあるいはCDCAからの二次胆汁酸生成活性をTLC,HPLC,GC-MS分析により検出した.その結果,8株の二次胆汁酸生成菌を取得した.16SrRNA遺伝子の塩基配列の決定により,DCAやLCA生成菌株としてClostridum scindensとC.leptumを得たが,これらは既知の変換菌であった。他の6株はCDCAからLCAとは異なる代謝産物として7-オキソリトコール酸(3α-hydroxy-7-oxo-5β-cholanoic acid,7-keto-LCA)を生成していた.これら6株は7-keto-LCA生成菌としては既知のEscherichia coli(3株)やBacteroides fragilis(2株)に加えて,今まで報告のなかったBeacteroides intestinalsを含むことが判明した.本株をB.intestinalis AM-1と命名した.AM-1株はCAからは7-keto-DCAを生成した.基準株JCM13265^Tでも同様の活性が確認された. 変換反応を既知の変換株E.coli HB101およびB.fragilis JCM11019^Tと比較したところ,AM-1株は変換反応培養時の生育量は他株に比べて低いが,変換率は90%以上であり,菌体当たりの変換活性が格段に高いことが分かった.本反応を触媒する酵素7α-hydroxysteroid dehydrogenase活性はこれら3株間に大差を認めなかった.
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2001年 -2002年 
    代表者 : 横田 篤, 冨田 房男
     
    酸化的リン酸化欠損変異(F_1-ATPase欠損変異)に伴うエネルギー代謝の撹乱が大腸菌細胞におよぼす影響を,プロテオーム変動解析とDNAマクロアレイ法によるトランスクリプトーム解析により検討した。 [方法] (1)サンプル調製:大腸菌K-12野生株W1485と,そのF_1-ATPase欠損変異株をグルコース最少培地でケモスタット培養し,解析の試料とした. (2)プロテオーム解析:両株の全発現タンパク質を2次元電気泳動によって分離し,両株で差異の認められるタンパク質のスポットを飛行時間型質量分析計を用いて同定した. (3)DNAアレイ法による遺伝子発現解析:両株の細胞より全RNAを抽出し,33PラベルcDNAを合成した.これをDNAマクロアレイとハイブリダイゼーションさせ,アレイ画像読み取りと画像解析を行い,野生株と変異株の遺伝子発現の差を解析した. [結果] 解糖系諸酵素ではプロテオーム解析と活性測定により変異株での発現上昇が認められたものがいくつかあったが(phosphoglycerate kinase, enolase, pyruvate kinase I, pyruvate dehydrogenase), DNAアレイでの発現上昇はPyruvate dehydrogenaseのみであった.TCAサイクルの多くの酵素では発現低下が認められ,上記3項目全てにおいて一致したのはcitrate synthase, succinyl-coA syntehtase, malate dehydrogenase, isocitrate lyase, malate synthaseであった.一方succinate dehydrogenase, fumarate hydrataseでは活性とプロテオームにおいてのみ発現低下が確認された.呼吸鎖ではNADH dehydrogenase-2とcytochrome bdの活性上昇がDNAアレイにおいても確認された.
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1998年 -2000年 
    代表者 : 横田 篤, 富田 房男
     
    産業上重要な次の3種類の菌株について,エネルギー代謝に関わるH^+-ATPaseに着目した微生物機能の解析と応用について検討し,以下の結果を得た.以下の一連の検討は,エネルギー代謝の操作が微生物機能の新しい改変を可能にすることを示している. 1.大腸菌:野生株から取得したF_1-ATPase活性欠損株を最少培地で連続培養して調製した生理学的に均一な細胞について,中枢代謝を検討した.欠損株では解糖系酵素活性は上昇,TCAサイクル関連酵素活性は低下し,呼吸鎖の成分であるNADHデヒドロゲナーゼの活性は増大した.大腸菌のゲノム情報に基づき,2次元電気泳動により発現タンパク質のプロテオーム変動解析を行い,F_1-ATPase欠損変異株においては細胞成分が質的に作り変えられていることを明らかにした. 2.コリネ型グルタミン酸生産菌:Corynebacterium glutamicumのH^+-ATPase活性低下変異株の発酵経過を調べた.変異株では菌体当たりの糖消費量が増大していること,また,グルタミン酸はほとんど生産されなくなり,代わりにピルビン酸,アラニン,乳酸の生成量が増加することを見出した.H^+-ATPase遺伝子のクローニングを行い,完全長の遺伝子を得た.本遣伝子を大腸菌-コリネバクテリウムシャトルベクターに連結し,C.glutamicum野生株の形質転換を行った.その結果,形質転換体のH^+-ATPase活性は野生株の約2.7倍に上昇した. 3.乳酸菌:チーズスターター乳酸菌Lactococcus lactisにおいて,H^+-ATPase活性の低下は酸性感受性を与え,細胞内pHの恒常性維持にH^+-ATPaseが深く関わることが明らかになった.本菌のH^+-ATPaseオペロン全遺伝子をクローニングした.
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1998年 -1999年 
    代表者 : 冨田 房男, 横田 篤
     
    先に得られた物質AM-1及びNCA-D6関連物質の単離精製、を行った。 AM-1関連物質と想定されるAM-2を単離した。 AM-2はキチン合成酵素の一部を欠損した株にのみ生育阻害をもたらし、同時に行った野生株には生育阻害は見られなかった。また、その他の糸状菌Aiternaria sp.、Fusarium oxysporum、細菌Salmonella typhymurium、Bacillus subtillisにも生育阻害は見られなかった。AM-2の機器分析の結果分子量が303、分子式がC_<21>H_<37>Nであることがわかった。AM-2は、野生株の粗精製キチン合成酵素反応を阻害することが確認された。また、^3H-uracil、^3H-thymidine、^3H-leucineを用いた取り込み実験の結果、AM-2が3H-uracilと^3H-leucineの取り組みを阻害しており、AM-2によるRNAおよびタンパク合成が影響を受けていると示唆された。 NCA-D6関連の少なくとも4つの物質が確認された。NCA-D6も、キチン合成酵素の一部欠損株にのみ生育阻害をもたらし、同時に行った野生株には生育阻害は見られなかった。また、その他の糸状菌Aiternaria sp.、Fusarium oxysporum、細菌Salmomella typhymurium、Bacillus subtillisにも生育阻害は見られなかった。分子量646、分子式C_<37>H_<58>O_9であり、その構造を明らかにした。NCA-D6は野生株の粗精製キチン合成酵素反応を阻害し また、^3H-uracil、^3H-thymidine、^3H-leucineを用いた取り込み実験の結果、3H-leucineの取り込みを阻害しており、タンパク合成が影響を受けていると示唆された。既知の物質ではあったものの、キチン合成を阻害するという報告は本研究が初めてである。関連物質について単離精製を進め、機器分析にかける段階である。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1995年 -1997年 
    代表者 : 冨田 房男, 佐山 晃司, 原 博, 葛西 隆則, 横田 篤
     
    1.製造原料の供給:チコリよりイヌリンを抽出精製するプロセスをビ-トからの蔗糖精製と同様の工程で検討し、イヌリン調製法を確立した。レバンについてもレバン生産菌のジャー培養法の確立を行い、その培養液からの回収、精製プロセスを確立した。また、粗抽出液や培養液中にオリゴ糖生産酵素を添加するなどの方法で原料精製プロセスの省略化を図ったオリゴ糖生産についても検討している。2.オリゴ糖生産菌の探索、生産菌の育種改良:オリゴ糖生産菌の探索を土壌微生物に加え植物内生菌より行い、その中からオリゴ糖生産株としてキシロビオースを生産する糸状菌を新たに単離した。また、DFAIII、DFAIVの生成酵素遺伝子(ift、lft)のクローニングと塩基配列の決定を行い、大腸菌での高発現系を構築し、それぞれ共に元株より高活性のオリゴ糖生成酵素の調製に成功した。3.オリゴ糖生産プロセスの構築:オリゴ糖生産菌株の培養法またはクローン化し高発現し生産された酵素の諸性質をそれぞれ決定し、DFAIII、DFAIV,イヌロトリオース、レバンビオースの生成酵素生産条件、酵素反応条件を確立した。DFAIII、DFAIVについては酵素反応液中からの精製方法を確立し、酵母による発酵、イオン交換カラム、結晶化することにより精製オリゴ糖標品を大量に調製した。4.オリゴ糖の栄養・機能性評価:3により大量調製したDFAIIIの加工特性や腸内細菌による利用性ラットや人での栄養評価を行った。そのうちラットを用いたカルシウム吸収促進効果を調べたところ、DFAIIIはラフィノースやフラクトオリゴ糖以上のカルシウム吸収促進を有することがわかり、その促進部位・作用は大腸部位での腸内細菌による発酵基質としての他、小腸部位でも濃度依存的にカルシウム吸収を促進することがわかった。DFAIIIについてもDFAIIIと同様の評価を検討している。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1996年 -1996年 
    代表者 : 横田 篤, 須藤 学, 冨田 房男
     
    大腸菌及びコリネ型細菌のH^+-ATPアーゼの活性低下変異株について研究を進め下に示す結果を得た。 1)これまでに当研究室で得られていた大腸菌のH^+-ATPアーゼ欠損株の炭素の流れを解糖系の酸素及び糖の取込み系について解析した。ピルビン酸キナーゼ-1とホスホグリセリン酸キナーゼの活性が元株の約1.5倍、グルコース取込み系(ホスホトランスフェラーゼ系)が約2.1倍、チトクロームb含有量が約1.4倍(呼吸が約1.7倍)に上昇しており、これらの共同作用で炭素の流れ(フラックス)に変化がでたものと結論した。中でもグルコース取込み系の増大は、ピルビン酸の生産向上と協同してフラックスの変化に最も大きな寄与があるものと考えられる。 2)コリネ型細菌のH^+-ATPアーゼの活性が元株の約30%にまで低下した変異株を得てその解析を試みた。本変異株は生育初期には元株よりも生育が遅いが最終的な生育量はほとんど同じであった。本菌においても大腸菌と同様にピルビン酸の生産が増大し、オキソグルタール酸の生産はなくなり、クエン酸サイクルが動いていないことが示された。その結果グルタミン酸の生産はほとんどなくなり、アラニンの生産が増大した。また本菌でも糖消費が増大していたがこれもグルコース取込み系(ホスホトランスフェラーゼ系)の増大が大きく寄与していることが示された。
  • 2. 腸内細菌と胆汁酸の相互作用に関する総合的研究
    研究期間 : 1996年
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1995年 -1995年 
    代表者 : 横田 篤, 冨田 房男
     
    大腸菌K-12株由来のリボ酸要求性ピルビン酸生産菌を新株として、形質導入法によりF_1-ATPaseの欠損変異遺伝子atpA401を導入して得られたTBLA-1株では、生育は親株の70%に低下したが、菌体当りのグルコース消費速度及びピルビン酸生産速度が親株の2倍及び2.5倍に増大し、その結果ピルビン酸の生産効率が大幅に向上した。平成7年度はTBLA-1株の性質解明とともに、新たに大腸菌K-12株野生株からも同様なF_1-ATPaseの欠損変異株を導き、この株についての性質も検討し、以下の結果を得た。 1.TBLA-1株における解糖系諸酵素活性の変化:グルコースの取込みにかかわるホスホトランスフェラーゼ系を除く解糖系の全ての酵素活性の測定を行い、ホスホグリセリン酸キナーゼ及びピルビン酸キナーゼIの活性が対数増殖期において親株の最大1.5倍に増大していることを認めた。この二つの酵素は共に基質レベルのリン酸化を触媒するものであり、F_1-ATPaseの欠損変異におけるエネルギー(ATP)欠乏を補うレスポンスとして合目的的と考えられた。その他の酵素については両株間で有意な差が認められなかった。 2.大腸菌K-12株野生株からのF_1-ATPase欠損変異株の取得:大腸菌K-12株野生株W1485に、形質導入法によりF_1-ATPaseの欠損変異遺伝子atpA401を導入しHBA-1株を取得した。 3.培養条件の検討:TBLA-1、HBA-1両株の培養条件を検討し、鉄イオンの添加と、有機窒素源としてポリペプトンの添加が有効であることを認めた。 4.HBA-1の糖代謝:3の条件でTBLA-1、HBA-1両株を培養した結果、HBA-1株の培養では著量のピルビン酸が生成した。ピルビン酸生産株でない野生株由来のF_1-ATPaseの欠損株でもピルビン酸が蓄積することから、F_1-ATPaseの欠損株では解糖系が強く活性化されていることが示唆された。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1994年 -1994年 
    代表者 : 横田 篤, 富田 房男
     
    大腸菌K-12株由来のリポ酸要求性ピルビン酸生産菌を親株として、形質導入法によりF_1-ATPaseの欠損変異遺伝子atpA401を導入して得られたTBLA-1株では、生育は親株の70%に低下したが、菌体当たりのグルコース消費速度およびピルビン酸生産速度が親株の2倍および2.5倍に増大し、その結果ピルビン酸生産効率が大幅に向上した。平成6年度はTBLA-1株の性質を解明することを目的として検討し、次に示す成果を得た。 1.F_1-ATPaseレベルとピルビン酸生産効率との関連性の検討:TBLA-1株から親株の10%の活性を持つF_1-ATPaseの部分復帰変異株を取得した。この株のピルビン酸生産能は親株と同じであった。従って、ピルビン酸生産の効率化のためには、F_1-ATPaseがさらに低下する必要があることが分かった。 2.TBLA-1株における酸素消費量の変化:ピルビン酸生産培養時のTBLA-1株の酸素消費量は親株の1.7倍に増大していた。 3.電子伝達系のチトクロムb含有量の測定:分光学的な測定の結果、TBLA-1株では含有量が親株の3倍に増大していた。2.の結果と合わせて考えると、TBLA-1株では糖消費の増大の結果過剰に生成されるNADHを再酸化するために、これらの変化が起こったと考察された。 4.TBLA-1株における解糖系諸酵素活性の変化:glucose phosphate isomerase,phosphofructokinase,fructose bisphosphate aldolase,phosphoglycerate mutase,pyruvate kinaseの5種類の酵素活性について測定したところ、pyruvate kinaseの活性がTBLA-1において約50%増大しており、解糖系が活性化されていることが見出された。
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 1992年 -1994年 
    代表者 : 富田 房男, 須藤 学, 横田 篤
     
    オリゴ糖は抗う蝕作用、便秘改善、血清脂質改善などの生理作用や、低カロリー甘味物質として注目を集めている。またオリゴ糖は植物や微生物の健康に対しても有効であることが示されてきた。このようにオリゴ糖の重要性が認識されるにつれて、その効率的な生産手段が求められるようになってきた。そこで我々はイヌリンやレバンなどの未利用多糖類から微生物によってオリゴ糖を生産することとし、土壌サンプルからこれらの多糖分解菌を分離し、オリゴ糖生産菌をスクリーニングした。 1.イヌリンからのオリゴ糖生産:イヌリンからDFAIIIを生産するArthrobacter sp.H65‐7は菌体内DFAIII分解酵素によってDFAIIIをイヌロビオースに変換することが分かった。菌体反応をにより、DFAIIIからイヌロビオース生産が可能であることを示した。Streptomyces rocheiE87はイヌリンからイヌロトリオースを生産するエキソ型酵素を生産することが分かった。本酵素の精製を行い、酵素化学的性質を解明した。 2.レバンからのオリゴ糖生産:土壌から分離されたレバン分解菌Streptomyces exfoliatus F3‐2とArthrobacter nicotinovorans SG9は、レバンからレバンビオースとDFAIVをそれぞれ効率よく生産することが分かった。レバンビオース生成酵素を精製し、性質を解明した。 3.糖転移反応を用いるオリゴ糖生産:Arthrobacter sp.MS9と同定された土壌分離菌のレバンビオース生成酵素を高濃度のイヌロビオースに作用させたところ、糖転移生成物と思われる3糖が生成された。構造解析の結果、このオリゴ糖はイヌロビオースの還元末端側のフラクトース残基にβ2‐6結合でフラクトースが1残基結合した新規3糖であった。
  • 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
    研究期間 : 1988年

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  • 2019年 - 現在   日本生物工学会   監事
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  • 2008年   日本生物工学会   活性化WG委員   日本生物工学会
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  • 1999年 - 2001年   日本生物工学会   英文誌 (J. Biosci. Bioeng.) 編集委員   日本生物工学会
  • 1996年 - 2000年   日本生物工学会   北日本支部庶務幹事   日本生物工学会
  • 2000年   日本農芸化学会   北海道支部評議員   日本農芸化学会
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  • 1995年 - 1999年   日本生物工学会   活動強化委員   日本生物工学会
  • 1995年 - 1996年   日本農芸化学会   北海道支部幹事   日本農芸化学会
  • 1991年 - 1992年   日本農芸化学会   北海道支部幹事   日本農芸化学会


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