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YOSHIDA Ken-ichiGraduate School of Science, Technology and Innovation / Department of Science, Technology and InnovationProfessor
Research activity information
■ Award- Nov. 2023 兵庫県, 兵庫県科学賞
- Jan. 2019 FEMS, Honorary FEMS Ambassador
- Mar. 2014 日本農芸化学会, 日本農芸化学会論文賞2013, Award for Excellence to Authors Publishing in Bioscience, Biotechnology, and Biochemistry in 2013Japan society
- Mar. 2009 日本農芸化学会, 日本農芸化学会論文賞2008, Award for Excellence to Authors Publishing in Bioscience, Biotechnology, and Biochemistry in 2008Japan society
- Mar. 2009 日本農芸化学会, 日本農芸化学会英文誌Bioscience, Biotechnology, and Biochemistry論文賞, Identification of Two Major Ammonia-Releasing Reactions Involved in Secondary Natto FermentationOfficial journal
- Mar. 2003 日本農芸化学会, 農芸化学奨励賞, ゲノム情報に基づく枯草菌の逆遺伝学的研究
- Abstract Bacillus velezensis S141 helps soybean establish specific symbiosis with strains of Bradyrhizobium diazoefficiens to form larger nodules and improve nitrogen fixation efficiency. In this study, we found that the dry weight of soybean roots increased significantly in the presence of S141 alone under drought conditions. Hence, S141 improved the root growth of soybean under limited water supply conditions. S141 can produce some auxin, which might be involved in the improved nodulation. Inactivating IPyAD of S141, which is required for auxin biosynthesis, did not alter the beneficial effects of S141, suggesting that the root growth was independent of auxin produced by S141. Under drought conditions, soybean exhibited some responses to resist osmotic and oxidative stresses; however, S141 was relevant to none of these responses. Although the mechanism remains unclear, S141 might produce some substances that stimulate the root growth of soybean under drought conditions.Corresponding, Oxford University Press (OUP), Nov. 2024, Bioscience, Biotechnology, and Biochemistry, English[Refereed]Scientific journal
- ABSTRACT Here we present a “breathing” vessel consisting of expanded polytetrafluoroethylene, which allows gas exchange but no liquid permeation. The bacterial culture inside needs only agitation to promote air supply. Using this setup, a Bacillus subtilis cell factory for scyllo-inositol production grew to produce scyllo-inositol efficiently. The results indicate that our approach represents a sustainable “greener” approach for the cell factory.Lead, Oxford University Press (OUP), Sep. 2024, Bioscience, Biotechnology, and Biochemistry, English[Refereed]Scientific journal
- ABSTRACT Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli–Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo -inositol into scyllo -inositol at 30°C. In a scaled-up reaction, 10 g of myo -inositol was converted to 1.8 g of scyllo -inositol, which was further purified to yield 970 mg of pure powder. Notably, myo -inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo -inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo -inositol, holding potential pharmaceutical significance as scyllo -inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.American Society for Microbiology, Jul. 2024, Applied and Environmental Microbiology, 90(7) (7), English[Refereed]Scientific journal
- American Chemical Society (ACS), Jul. 2024, ACS Synthetic Biology, 13(7) (7), 2199 - 2214, English[Refereed]Scientific journal
- Elsevier BV, Jun. 2024, Current Opinion in Biotechnology, 87, 103114 - 103114, English[Refereed][Invited]Scientific journal
- Abstract Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.Oxford University Press (OUP), Jan. 2024, Biology Methods and Protocols, 9(1) (1), English[Refereed]Scientific journal
- Abstract Aim Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. Methods and results We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. Conclusion Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.Oxford University Press (OUP), Dec. 2023, Journal of Applied Microbiology, 134(12) (12)[Refereed]Scientific journal
- Elsevier BV, Dec. 2023, Biofilm, 6, 100152 - 100152[Refereed]Scientific journal
- Abstract Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.Oxford University Press (OUP), Mar. 2023, Nucleic Acids Research, 51(6) (6), 2974 - 2992, English[Refereed]Scientific journal
- Corresponding, Microbiology Research Foundation, 2023, The Journal of General and Applied Microbiology, English[Refereed]Scientific journal
- Abstract Background Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer’s disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. Results The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. Conclusion The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.Corresponding, Springer Science and Business Media LLC, Dec. 2022, Microbial Cell Factories, 21(1) (1), English[Refereed]Scientific journal
- Abstract Background Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. Results In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. Conclusions This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.Corresponding, Springer Science and Business Media LLC, Dec. 2022, Microbial Cell Factories, 21(1) (1), English[Refereed]Scientific journal
- Corresponding, Bio-Protocol, LLC, Sep. 2022, BIO-PROTOCOL, 12(17) (17), English[Refereed][Invited]Scientific journal
- Abstract Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate. Here, we show that this growth defect could be restored by silencing redundant or less important genes affecting exponential growth by manipulating the global transcription factor AbrB. Comparative transcriptome analysis revealed that AbrB-regulated genes were upregulated and those involved in central metabolic pathway and synthetic pathways of amino acids and purine/pyrimidine nucleotides were downregulated in MGB874 compared with the wild-type strain, which we speculated were the cause of the growth defects. By constitutively expressing high levels of AbrB, AbrB regulon genes were repressed, while glycolytic flux increased, thereby restoring the growth rate to wild-type levels. This manipulation also enhanced the productivity of metabolites including γ-polyglutamic acid. This study provides the first evidence that undesired features induced by genome reduction can be relieved, at least partly, by manipulating a global transcription regulation system. A similar strategy could be applied to other genome engineering-based challenges aiming toward efficient material production in bacteria.Oxford University Press (OUP), May 2022, DNA Research, 29(3) (3)[Refereed]Scientific journal
- Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators inside the conjugation operon to be bypassed, but also the differential expression of a subset of genes. Here, we show that some conjugation operons on plasmids belonging to the Inc18 family of Gram-positive broad host-range plasmids do not possess an antitermination system, suggesting that the absence of an antitermination system may have advantages. The possible (dis)advantages of conjugation operons possessing (or not) an antitermination system are discussed.MDPI AG, Mar. 2022, Microorganisms, 10(3) (3), 587 - 587, English[Refereed]Scientific journal
- Corresponding, Microbiology Research Foundation, 2022, The Journal of General and Applied Microbiology, 68(2) (2), 87 - 94, English[Refereed]Scientific journal
- Springer Science and Business Media LLC, Dec. 2021, Scientific Reports, 11(1) (1), 16007 - 16007, English, International magazine
Abstract Lactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene clusterinuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth geneinuD (Ldb1384) was up-regulated most prominently. AlthoughBacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression ofinuABCDEF . When freshly cultured cells of the recombinantB. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply thatinuABCDEF might encode a novel ABC transporter inL. delbrueckii to “monopolize” inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.[Refereed]Scientific journal - Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when rappLS20 was overexpressed.Corresponding, MDPI AG, Sep. 2021, Microorganisms, 9(9) (9), 1931 - 1931, English, International magazine[Refereed][Invited]Scientific journal
- Strains of Lactococcus lactis subsp. cremoris are used to produce yogurt containing exopolysaccharides with a sticky texture. When strain G3-2 producing exopolysaccharides was grown at elevated temperatures, a spontaneous mutant EPSC, which had lost exopolysaccharides biosynthesis, was isolated. Genomes of the two strains were determined to be composed of a 2.4-Mb chromosome and up to eleven plasmids, and it was revealed that one of the plasmids encoding the gene cluster for exopolysaccharides biosynthesis was lost selectively in EPSC.Corresponding, Microbiology Research Foundation, 2021, The Journal of General and Applied Microbiology, English, Domestic magazine[Refereed]Scientific journal
- Corresponding, Microbiology Society, Jan. 2021, Microbiology, 167(1) (1), English, International magazine
Geobacillus kaustophilus iol gene cluster required for inositol catabolism has been postulated with reference to theiol genes inBacillus subtilis iol gene cluster ofG. kaustophilus B. subtilis iolQ is known to be involved in the regulation ofiolX encodingscyllo -inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899 ) of theiol gene cluster and was termediolQ inG. kaustophilus iolQ was inactivated inG. kaustophilus myo -inositol dehydrogenase activity due togk1899 expression but also the transcription of the twoiol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein inEscherichia coli in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the twoiol promoter regions and that DNA binding was antagonized bymyo -inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of theiol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the twoiol operons responding tomyo -inositol.[Refereed]Scientific journal - Corresponding, Springer Science and Business Media LLC, Dec. 2020, Biotechnology and Bioprocess Engineering, 25(6) (6), 872 - 885, English[Refereed][Invited]Scientific journal
- Corresponding, American Society for Microbiology, Sep. 2020, Microbiology Resource Announcements, 9(36) (36), English
ABSTRACT We report here the complete genome sequence of nitrogen-fixingPaenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related toPaenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.[Refereed]Scientific journal - The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus velezensis S141 with USDA110 into soybean resulted in enhanced nodulation and N2-fixing efficiency by producing larger nodules. To understand the role of S141 on soybean and USDA110 symbiosis, putative genes related to IAA biosynthesis were disrupted, suggesting that co-inoculation of USDA110 with S141ΔyhcX reduces the number of large size nodules. It was revealed that yhcX may play a major role in IAA biosynthesis in S141 as well as provide a major impact on soybean growth promotion. The disruption of genes related to cytokinin biosynthesis and co-inoculation of USDA110 with S141ΔIPI reduced the number of very large size nodules, and it appears that IPI might play an important role in nodule size of soybean–Bradyrhizobium symbiosis. However, it was possible that not only IAA and cytokinin but also some other substances secreted from S141 facilitate Bradyrhizobium to trigger bigger nodule formation, resulting in enhanced N2-fixation. Therefore, the ability of S141 with Bradyrhizobium co-inoculation to enhance soybean N2-fixation strategy could be further developed for supreme soybean inoculants.Corresponding, MDPI AG, May 2020, Microorganisms, 8(5) (5), 678 - 678, English[Refereed]Scientific journal
- Here, we report the complete genome sequence ofLead, American Society for Microbiology, Apr. 2020, Microbiology Resource Announcements, 9(17) (17)
Aeribacillus pallidus PI8, a thermophilic bacterium, isolated from soybean stem extract. The sequence was determined using Illumina and Nanopore sequencers. Bioinformatic analyses of the genome sequence revealed the presence of possible bacteriocin gene clusters.[Refereed]Scientific journal - A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.Mar. 2020, Communications biology, 3(1) (1), 93 - 93, English, International magazine[Refereed]
- Elsevier BV, Apr. 2019, Process Biochemistry, 79, 74 - 80[Refereed]Scientific journal
- 2019, J Nutr Sci Vitaminol, 65, S139 - S142, English[Refereed]
- Elsevier BV, 2019, Process Biochem, 79, 74 - 80, English[Refereed]Scientific journal
- Background Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin pBMC, Oct. 2018, BMC Microbiology, 18, English[Refereed]Scientific journal
- Background: Bacterial strains of the genus Geobacillus grow at high temperatures of 50-75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming GeobacBMC, Aug. 2018, Microbial Cell Factories, 17, English[Refereed]Scientific journal
- Background: The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. Results: In this study, the oriT sequence of pLS20cat (oriT LS20) was eliminated to obtain the plasmid, pLS20catoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catoriT was able to mobilize longer DNA segments, up to 113kb of chromosomal DNA containing oriT LS20, after mixing the liquid cultures of the donor and recipient for only 15min. Conclusions: The chromosomal DNA mobilization mediated by pLS20catoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.BioMed Central Ltd., Jan. 2018, Microbial Cell Factories, 17(1) (1), English[Refereed]Scientific journal
- A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential.Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions.Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.Last, Springer Science and Business Media LLC, Jan. 2018, Biotechnology Letters, 40(1) (1), 189 - 196, English[Refereed]Scientific journal
- Our previous report demonstrated that epigallocatechin gallate (EGCg) promotes translocation of glucose transporter 4 (GLUT4) in skeletal muscle. In this study, we investigated the molecular mechanism of GLUT4 translocation by EGCg at the physiological concentration range. In L6 cells, EGCg induced phosphorylation of phosphatidylinositide 3'-kinase (PI3K) and downstream protein kinase C (PKC) λ/ξ without affecting the phosphorylation of insulin receptor and Akt. EGCg-induced GLUT4 translocation was suppressed by RNA interference-mediated knockdown of PI3K and treatment with PKC inhibitor Go6983. Moreover, EGCg increased Rac1 activity and actin remodelling as downstream events of PKCλ/ξ. These results indicate that EGCg induced GLUT4 translocation through a PI3K-dependent pathway, but its mode of action differed from that of insulin. EGCg also induced GLUT4 translocation through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent pathway. 67 kDa laminin receptor, which is a target molecule of EGCg, was not involved in EGCg-induced glucose uptake in L6 cells. The oral administration of EGCg suppressed postprandial hyperglycaemia accompanied by GLUT4 translocation through both PI3K- and AMPK-dependent pathways, and promoted glycogen accumulation in skeletal muscle of ICR mice. EGCg promotes GLUT4 translocation through both PI3K- and AMPK-dependent pathways and glycogen accumulation in skeletal muscle.2018, Food and Function, 9(8) (8), 4223 - 4233, English, International magazine[Refereed]Scientific journal
- Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-β-oxidative, CoA-dependent or (II) β-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.Dec. 2017, AMB Express, 7(1) (1), 130 - 130, English, International magazine[Refereed]Scientific journal
- To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. (126)Leu, (132)Leu, and (135)Lys were implicated in the binding of phosphopantothenic acid, and (100)Tyr and (131)Lys in that of adenosyl biphosphate. The data supported the participation of (83)Glu and (133)Tyr in catalyzing acetyl-group transfer to l-2-phenylglycine. 5-3B NatA catalyzes the enantioselective N-acetylation of l-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.SPRINGER, Nov. 2017, BIOTECHNOLOGY LETTERS, 39(11) (11), 1699 - 1707, English[Refereed]Scientific journal
- Background: Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD(+)-dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. Results: Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. Conclusions: IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.BIOMED CENTRAL LTD, Jul. 2017, BMC MICROBIOLOGY, 17(1) (1), 154, English[Refereed]Scientific journal
- Bacillus subtilis genes iolG, iolW, iolX, ntdC, yfiI, yrbE, yteT, and yulF belong to the Gfo/Idh/MocA family. The functions of iolG, iolW, iolX, and ntdC are known; however, the functions of the others are unknown. We previously reported the B. subtilis cell factory simultaneously overexpressing iolG and iolW to achieve bioconversion of myo-inositol (MI) into scyllo-inositol (SI). YulF shares a significant similarity with IolW, the NADP(+)-dependent SI dehydrogenase. Transcriptional abundance of yulF did not correlate to that of iol genes involved in inositol metabolism. However, when yulF was overexpressed instead of iolW in the B. subtilis cell factory, SI was produced from MI, suggesting a similar function to iolW. In addition, we demonstrated that recombinant His(6)-tagged YulF converted scyllo-inosose into SI in an NADPH-dependent manner. We have thus identified yulF encoding an additional NADP(+)-dependent SI dehydrogenase, which we propose to rename iolU.TAYLOR & FRANCIS LTD, May 2017, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81(5) (5), 1026 - 1032, English[Refereed]Scientific journal
- May 2017, Genome Announc., 5(48) (48), EnglishGenome Sequence of Bacillus velezensis S141, a New Strain of Plant Growth-Promoting Rhizobacterium Isolated from Soybean Rhizosphere.[Refereed]
- Background: A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. Results: When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. Conclusions: The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.BIOMED CENTRAL LTD, Apr. 2017, MICROBIAL CELL FACTORIES, 16, English[Refereed]Scientific journal
- BACKGROUNDAspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. RESULTSThe genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repenseMK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. CONCLUSIONGiven its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. (c) 2016 Society of Chemical IndustryWILEY-BLACKWELL, Jan. 2017, JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, 97(1) (1), 95 - 101, English[Refereed]Scientific journal
- Accumulating evidence suggests that dietary taurine (2-aminoethanesulfonic acid) exerts beneficial anti-inflammatory effects in the large intestine. In this study, we investigated the possible impact of taurine on human colonic microbiota using our single-batch fermentation system (Kobe University Human Intestinal Microbiota Model; KUHIMM). Fecal samples from eight humans were individually cultivated with and without taurine in the KUHIMM. The results showed that taurine remained largely undegraded after 30 h of culturing in the absence of oxygen, although some 83% of the taurine was degraded after 30 h of culturing under aerobic conditions. Diversity in bacterial species in the cultures was analyzed by 16S rRNA gene sequencing, revealing that taurine caused no significant change in the diversity of the microbiota; both operational taxonomic unit and Shannon-Wiener index of the cultures were comparable to those of the respective source fecal samples. In addition, principal coordinate analysis indicated that taurine did not alter the composition of bacterial species, since the 16S rRNA gene profile of bacterial species in the original fecal sample was maintained in each of the cultures with and without taurine. Furthermore, metabolomic analysis revealed that taurine did not affect the composition of short-chain fatty acids produced in the cultures. These results, under these controlled but artificial conditions, suggested that the beneficial anti-inflammatory effects of dietary taurine in the large intestine are independent of the intestinal microbiota. We infer that dietary taurine may act directly in the large intestine to exert anti-inflammatory effects.2017, PloS one, 12(7) (7), e0180991, English, International magazine[Refereed]Scientific journal
- Background: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified. Results: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His(6)-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not. Conclusions: Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.BIOMED CENTRAL LTD, Oct. 2016, BMC MICROBIOLOGY, 16(1) (1), 1 - 13, English[Refereed]Scientific journal
- We devised a single-batch fermentation system to simulate human colonic microbiota from fecal samples, enabling the complex mixture of microorganisms to achieve densities of up to 1011 cells/mL in 24 h. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that representatives of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. On the earlier stages of fermentation (up to 9 h), trace mixtures of acetate, lactate, and succinate were detectable; on the later stages (after 24 h), larger amounts of acetate accumulated along with some of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human colonic microbiota. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) resulted in an increased population in genus Bifidobacterium, concomitant with significant increases in acetate production. The results suggested that this fermentation system may be useful for in vitro, preclinical evaluation of the effects of prebiotics prior to testing in humans.PUBLIC LIBRARY SCIENCE, Aug. 2016, PLOS ONE, 11(8) (8), English[Refereed]Scientific journal
- We modified GntR regulation in Bacillus subtilis to devise transient induction systems. GntR is the repressor antagonized by gluconate to induce transcription of the gntRKPZ operon for gluconate catabolism. On the other hand, the gnt operon is repressed by glucose via carbon catabolite repression involving CcpA/P-ser-HPr, which binds to two cre sites: one located in the gnt promoter region and the other within the gntR coding region. We initiated gntKPZ encoding of enzymes for gluconate catabolism expressed independently from the operon; this allowed constitutive degradation of gluconate. Both cre sites were mutated to abolish catabolite repression. The mutated gnt promoter was set up to drive the expression of the lacZ reporter under the control of GntR. Even in the presence of glucose, lacZ was induced upon the addition of gluconate and shut down again as gluconate was consumed. Thus, modified GntR regulation enables artificial transient induction. This will allow us to design a flexible metabolic engineering system with genes expressed only temporarily as desired.MICROBIOL RES FOUNDATION, 2016, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 62(6) (6), 277 - 285, English[Refereed]Scientific journal
- Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis -amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance.WILEY-BLACKWELL, Jun. 2015, JOURNAL OF BASIC MICROBIOLOGY, 55(6) (6), 780 - 789, English[Refereed]Scientific journal
- Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.INFORMA HEALTHCARE, May 2015, INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 66(3) (3), 300 - 307, English[Refereed]Scientific journal
- Microbes employ cell membranes for reducing exogenous stresses. Polyamino acid display on microbial cell surfaces and their effects on microbial chemical stress tolerance were examined. Growth analysis revealed that displays of polyarginine, polyaspartate and polytryptophan substantially enhanced tolerance of Escherichia coli to NaCl. A titration assay indicated that polyarginine and polyaspartate altered cell surface charges, implying tolerance enhancement via ion atmosphere and/or ionic bond network formations for electrostatic ion repulsion. The enhancement by polytryptophan may have arisen from surface hydrophobicity increase for hydrophobic ion exclusion, because of a strong correlation between hydrophobic characters of amino acids and their effects on tolerance enhancement. The display also enhanced tolerance to other salts and/or alcohols in E. coli and to NaCl in Saccharomyces cerevisiae. Thus polyamino acid display has the potential as an approach for conferring chemical stress tolerance on various microbes.SPRINGER, Feb. 2015, BIOTECHNOLOGY LETTERS, 37(2) (2), 429 - 435, English[Refereed]Scientific journal
- Background: The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P). Results: Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase. Conclusions: Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.BIOMED CENTRAL LTD, Feb. 2015, BMC MICROBIOLOGY, 15(1) (1), English[Refereed]Scientific journal
- 2015, Austin J Clin Neurol, 2(4) (4), 1040, Englishscyllo-Inositol, a Therapeutic Agent for Alzheimer’s Disease[Refereed]Scientific journal
- 2015, International Journal of Food Sciences and Nutrition, 66, 300 - 307, English[Refereed]Scientific journal
- Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.TAYLOR & FRANCIS LTD, 2015, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79(11) (11), 1906 - 1914, English[Refereed]Scientific journal
- Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis cell factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the cell factory all "useless" genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the cell factory and its possible applications.LANDES BIOSCIENCE, Sep. 2014, BIOENGINEERED, 5(5) (5), 331 - 334, English[Refereed]Scientific journal
- Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis cell factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the cell factory all “useless” genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the cell factory and its possible applications.Landes Bioscience, Jul. 2014, Bioengineered Bugs, 5(5) (5), 331 - 334, English[Refereed]Scientific journal
- Wageningen Academic Publishers, May 2014, Industrial, medical and environmental applications of microorganisms. Current status and trends, 470 - 475, EnglishPoly-β-hydroxybutyrate accumulation in Bradyrhizobium japonicum depends on proteins referred to as phasins[Refereed][Invited]International conference proceedings
- Wageningen Academic Publishers, May 2014, Industrial, medical and environmental applications of microorganisms. Current status and trends, 636 - 640, EnglishA Bacillus subtilis cell factory for producing scyllo-inositol[Refereed][Invited]International conference proceedings
- Background: Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. Results: The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917(T), respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The K-m values of the enzymes on each galloyl ester were comparable; however, the k(cat)/K-m values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. Conclusion: Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.BIOMED CENTRAL LTD, Apr. 2014, BMC MICROBIOLOGY, 14(1) (1), English[Refereed]Scientific journal
- Background: Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. Results: The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917(T), respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The K-m values of the enzymes on each galloyl ester were comparable; however, the k(cat)/K-m values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. Conclusion: Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.BIOMED CENTRAL LTD, Apr. 2014, BMC MICROBIOLOGY, 14, 87, English[Refereed]Scientific journal
- N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of L-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for L-2-phenylglycine and its chloro-and hydroxyl-derivatives. The K-m and V-max values for L-2-phenylglycine were 0.145 +/- 0.026 mM and 43.6 +/- 2.39 mu mol . min(-1) . mg protein(-1), respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to L-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).AMER SOC MICROBIOLOGY, Mar. 2014, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 80(5) (5), 1770 - 1776, English[Refereed]Scientific journal
- N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of L-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for L-2-phenylglycine and its chloro-and hydroxyl-derivatives. The K-m and V-max values for L-2-phenylglycine were 0.145 +/- 0.026 mM and 43.6 +/- 2.39 mu mol . min(-1) . mg protein(-1), respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to L-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).AMER SOC MICROBIOLOGY, Mar. 2014, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 80(5) (5), 1770 - 1776, English[Refereed]Scientific journal
- Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.MICROBIOL RES FOUNDATION, 2014, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 60(5) (5), 175 - 182, English[Refereed]Scientific journal
- Genomes of two nondomesticated strains of Bacillus subtilis subspecies subtilis, NDmed and NDfood, have been sequenced. Both strains form very thick and spatially complex biofilms on submerged surfaces. Moreover, biofilms of the NDmed isolate were shown to be highly resistant to antimicrobials action.American Society for Microbiology, 2014, Genome Announcements, 2(5) (5), English[Refereed]Scientific journal
- Background: Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-beta-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression. Results: Quantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB. Conclusions: These results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.BIOMED CENTRAL LTD, Dec. 2013, BMC MICROBIOLOGY, 13(1) (1), English[Refereed]Scientific journal
- Background: Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. Results: All "useless" genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. Conclusions: The B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.BIOMED CENTRAL LTD, Dec. 2013, MICROBIAL CELL FACTORIES, 12(1) (1), English[Refereed]Scientific journal
- Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using beta-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and alpha-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.AMER SOC MICROBIOLOGY, Sep. 2013, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 79(17) (17), 5151 - 5158, English[Refereed]Scientific journal
- The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of > 99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.SPRINGER, Jul. 2013, BIOTECHNOLOGY LETTERS, 35(7) (7), 1053 - 1059, English[Refereed]Scientific journal
- Simple pharmacological studies on inositol stereoisomers are presented in this study. Male ICR mice were orally administered 1 g/kg BW of three inositol stereoisomers, myo-inositol (MI), d-chiro-inositol (DCI), and scyllo-inositol (SI), and blood plasma samples and skeletal muscle fractions were prepared after an hour. The plasma samples were subjected to gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) analysis. None of the three stereoisomers was seen in untreated samples, but substantial amounts ranging from 2.5 to 6.5 mM were detected only after administration, indicating that orally administered inositol stereoisomers were readily absorbed and their levels elevated in the bloodstream. In addition, plasma of SI-administered animals contained substantial MI, suggesting a possible metabolic conversion of SI to MI. In the skeletal muscle fractions, glucose transporter type 4 (GLUT4) content in the plasma membrane increased, indicating that inositol stereoisomers stimulated GLUT4 translocation. © 2013 American Chemical Society.May 2013, Journal of Agricultural and Food Chemistry, 61(20) (20), 4850 - 4854, English[Refereed]Scientific journal
- Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-gamma-glutamate (gamma-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in gamma-PGA production and in the total extracellular protease activity. The defect in gamma-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline senile protease encoded by aprE plays an indispensable role in supplying materials to produce gamma-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either gamma-PGA production or total protease activity.TAYLOR & FRANCIS LTD, Apr. 2013, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77(4) (4), 802 - 809, English[Refereed]Scientific journal
- Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β- glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts. © 2012 Springer-Verlag.Apr. 2013, Applied Microbiology and Biotechnology, 97(7) (7), 2929 - 2938, English[Refereed]Scientific journal
- Background: The agrichemical 4-aminopyridine is used as a bird repellent in crop fields and has an epileptogenic action in a variety of animals, including man and mouse. 4-Aminopyridine is biodegraded in the environment through an unknown mechanism. Results: A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-amino-3-hydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. 4-Amino-3-hydroxypyridine could not be further metabolized and probably accumulated as a dead-end product in the culture. Biodegradability tests and partial sequence analysis of the enrichment culture indicated that 4-aminopyridine was mainly degraded via 3,4-dihydroxypyridine and that the metabolite is probably cleaved by 3-hydroxy-4-pyridone dioxygenase. Seven culturable predominant bacterial strains (strains 4AP-A to 4AP-G) were isolated on nutrient agar plates. Changes in the bacterial populations of 4-aminopyridine, 3,4-dihydroxypyridine, or formate/ammonium chloride enrichment cultures were monitored by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Sequence analysis of the 16S rRNA gene fragments derived from predominant DGGE bands indicated that Pseudomonas nitroreducens 4AP-A and Enterobacter sp. 4AP-G were predominant in the three tested enrichment cultures and that the unculturable strains Hyphomicrobium sp. 4AP-Y and Elizabethkingia sp. 4AP-Z were predominant in 4-aminopyridine and formate/ammonium chloride enrichment cultures and in the 3,4-dihydroxypyridine enrichment culture, respectively. Among the culturable strains, strain 4AP-A could utilize 3,4-dihydroxypyridine as a growth substrate. Although we could not isolate strain 4AP-Y on several media, PCR-DGGE analysis and microscopy indicated that the unique bi-polar filamentous bacterial cells gradually became more dominant with increasing 4-aminopyridine concentration in the medium. Conclusions: Hyphomicrobium sp. 4AP-Y, P. nitroreducens 4AP-A, and Elizabethkingia sp. 4AP-Z probably play important roles in 4-aminopyridine degradation in crop fields. In the enrichment culture, 3,4-dihydroxypyridine and its metabolites including formate might be shared as growth substrates and maintain the enrichment culture, including these indispensable strains.BIOMED CENTRAL LTD, Mar. 2013, BMC MICROBIOLOGY, 13(1) (1), English[Refereed]Scientific journal
- 2013, Organohalogen Compounds, 75, 625 - 628, EnglishAryl hydrocarbon receptor enhances the expression of multidrug-resistant mdr1b through p53 in mouse hepatoma cellsScientific journal
- Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 pnitrophenyl- glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.2013, Bioscience, Biotechnology and Biochemistry, 77(8) (8), 1709 - 1714, English[Refereed]Scientific journal
- Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (Delta pyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (Delta pyrF Delta pyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous beta-galactosidase and a-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.AMER SOC MICROBIOLOGY, Oct. 2012, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 78(20) (20), 7376 - 7383, English[Refereed]Scientific journal
- We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY, Sep. 2012, JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 22(9) (9), 1279 - 1287, English[Refereed]Scientific journal
- Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, D-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His(6)-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.SOC GENERAL MICROBIOLOGY, Aug. 2012, MICROBIOLOGY-SGM, 158(8) (8), 1942 - 1952, English[Refereed]Scientific journal
- Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in . The recombinant protease, over-expressed in , was inactive but addition of acetone to crude cell extracts restored the activity and removed many proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, alpha-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.SPRINGER, May 2012, BIOTECHNOLOGY LETTERS, 34(5) (5), 949 - 955, English[Refereed]Scientific journal
- Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P (alsSD) promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic-anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P (alsSD) promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.SPRINGER, May 2012, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 94(3) (3), 651 - 658, English[Refereed]Scientific journal
- Transformation of an aryl hydrocarbon receptor (AhR) is the initial step to express the multiple toxicity of halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs) including dioxins. Therefore, it has been suggested that suppression of the transformation induced by HAHs and PAHs leads to reduce their toxicological effects. In this study, the antagonistic effect of 110 indigenous plants (192 plant parts) used as medicine and/or food by the Ainu on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation was investigated. Of these, a stalk of Aralia elata (Miq.) Seemann and a bark of Fraxinus mandshurica Rupr. var. japonica Maxim. exhibited the strong antagonistic effect in a dose-dependent manner. An antioxidative activity and polyphenol content were also measured, and the strong correlation (r= 0.96) between these two parameters could be confirmed. However, correlation coefficients of the antagonistic effect of 192 extracts compared to their antioxidative activity and polyphenol content were 0.17 and 0.20, respectively. These results suggest that the Ainu-selected traditional beneficial plants are useful source for findings of novel AhR antagonists, and the antagonistic activity of these plants may be independent on their antioxidative activity and polyphenol content.WILEY-BLACKWELL, Apr. 2012, JOURNAL OF FOOD SCIENCE, 77(4) (4), C420 - C429, English[Refereed]Scientific journal
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 39 - 39, English2Ap03 Bacillus subtilis cell factory for production of scyllo-inositol promising for Alzheimer's disease (Bio-Based Production) :Symposium
- Background: A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available. Results: Bacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium. Conclusions: The engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol.BIOMED CENTRAL LTD, Sep. 2011, MICROBIAL CELL FACTORIES, 10, English[Refereed]Scientific journal
- DNA methylation in Streptomyces griseus IFO 13350 was analyzed by high-performance liquid chromatographic analysis and bisulfite-based analysis to reveal two methylation sites, 5'-GC(5m)CGGC-3' and 5'-GAG(5m)CTC-3'. The methylation was reconstituted in Escherichia coli by simultaneous expression of S. griseus SGR4675 and S. achromogenes M.SacI. The E. coli cells produced plasmids that mimicked the methylation profile of S. griseus DNA, which was readily introduced into S. griseus. The results of this study raise the possibility of a promising approach to establish efficient transformation in several streptomycetes.KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY, Jul. 2011, JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 21(7) (7), 675 - 678, English[Refereed]Scientific journal
- We cloned the genes encoding the two distinct extracellular halotolerant proteases of Bacillus subtilis FP-133 Expro-I and Expro-II, which were classified as alkaline serine and neutral proteases respectively. Three-dimensional modeling suggested that acidic and polar amino acid residues located on the surface stabilize protein structure in the presence of relatively high NaCl concentrations.TAYLOR & FRANCIS LTD, Jan. 2011, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75(1) (1), 148 - 151, English[Refereed]Scientific journal
- To investigate the preventive effects of tea on hyperglycemia and insulin resistance, male C57BL/6J mice were given a high-fat diet containing 29% lard and also green or black tea ad libitum for 14 weeks. Both teas suppressed body weight gain and deposition of white adipose tissue caused by the diet. In addition, they improved hyperglycemia and glucose intolerance by stimulating glucose uptake activity accompanied by the translocation of glucose transporter (GLUT) 4 to the plasma membrane in muscle. Long-term consumption of the high-fat diet reduced levels of insulin receptor beta-subunit, GLUT4 and AMP-activated protein kinase a in muscle, and green and black tea suppressed these decreases. The results strongly suggest that green and black tea suppress high-fat diet-evoked hyperglycemia and insulin resistance by retaining the level of GLUT4 and increasing the level of GLUT4 on the plasma membrane in muscle.AMER CHEMICAL SOC, Dec. 2010, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 58(24) (24), 12916 - 12923, English[Refereed]Scientific journal
- In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 mu M catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 mu M catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKC lambda/zeta without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKC lambda/zeta in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.ROYAL SOC CHEMISTRY, Nov. 2010, FOOD & FUNCTION, 1(2) (2), 167 - 173, English[Refereed]Scientific journal
- In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 mu M catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 mu M catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKC lambda/zeta without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKC lambda/zeta in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.ROYAL SOC CHEMISTRY, Nov. 2010, FOOD & FUNCTION, 1(2) (2), 167 - 173, English[Refereed]Scientific journal
- The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological and toxicological effects by binding to its agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Previously we demonstrated that flavonoids suppressed the TCDD-induced DNA-binding activity of the AhR in a structure-dependent manner. In this study, we investigated the mechanisms by which flavonoids suppressed the AhR-mediated signal transduction in mouse hepatoma Hepa-1c1c7 cells. Flavones and flavonols suppressed the TCDD-induced nuclear translocation of the AhR and dissociation of its partner proteins, heat shock protein 90 and X-associated protein 2, whereas flavanones and catechins did not. Flavonoids of all these four subclasses suppressed the phosphorylation of both AhR and Arnt and the formation of a heterodimer consisting of these proteins. Since certain flavonoids are known to inhibit mitogen-activated protein kinases (MAPKs), we confirmed the contribution of MAPK/ERK kinase (MEK) to the AhR-mediated signal transduction by using U0126, an inhibitor of MEK1/2. U0126 suppressed TCDD-induced phosphorylation of the AhR and Arnt followed by the DNA-binding activity of the AhR. Flavanones and catechins suppressed the TCDD-induced phosphorylation of ERK1/2. The inhibition of MEK/ERK phosphorylation is one of the mechanisms by which flavanones and catechins suppress the AhR-mediated signal transduction in Hepa-1c1c7 cells. (C) 2010 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Sep. 2010, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 501(1) (1), 134 - 141, English[Refereed]Scientific journal
- In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5'-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated. Second, two genes for purine nucleoside phosphorylase, punA and deoD, were inactivated. Third, to enhance purine nucleotide biosynthesis, the pur operon repressor PurR and the 5'-UTR of the operon, containing the guanine riboswitch, were disrupted. Finally, the -10 sequence of the pur promoter was optimized to elevate its transcription level. The resulting mutant was capable of producing 6 g/L inosine from 30 g/L glucose in culture broth without the detectable by-production of hypoxanthine. This indicates the validity of this approach for the breeding of the next generation of B. subtilis strains for industrial nucleoside production.SPRINGER, Aug. 2010, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 87(6) (6), 2195 - 2207, English[Refereed]Scientific journal
- Bacillus subtilis IolT is the major myo-inositol transporter for growth, while IolF is a minor one unable to support growth. We found that either loIT or IolF was sufficient for moderate growth using D-chiro-inositol. Conversely to IolT, IolF transported n-chiro-inositol more preferentially than myo-inositol. These results indicate that IolT and IolF are different in substrate specificity.TAYLOR & FRANCIS LTD, Jun. 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(6) (6), 1312 - 1314, English[Refereed]Scientific journal
- 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes a wasting syndrome characterized by a loss of body weight accompanied by a decrease in adipose tissue weight, i.e., insulin resistance-like symptoms. Therefore, the effects of TCDD on an insulin signaling pathway in mature 3T3-L1 adipocytes were investigated to obtain insight into the underlying mechanisms. TCDD downregulated expression of insulin receptor beta-subunit (IR beta), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (GLUT4) and decreased insulin-stimulated glucose uptake activity. TCDD also upregulated expression of TNF-alpha, one of insulin resistance-inducing factors. Anti-TNF-alpha neutralization antibody and silencing of TNF-alpha receptor 1 (TNFR1) diminished the TCDD-induced downregulation of IR beta, IRS1, and GLUT4. Moreover, the experiments using small interfering RNA for an aryl hydrocarbon receptor (AhR) revealed that the TCDD-evoked changes of IR beta, IRS1, GLUT4, and TNF-alpha were dependent on AhR. TCDD also stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), and their inhibitors abrogated the TCDD-induced downregulation of IR beta, IRS1, and GLUT4; upregulation of TNF-alpha; and activation of NF-kappa B. Taken together, TCDD stimulates expression and secretion of TNF-alpha in adipocytes through activation of AhR, ERK1/2, and JNK, and the secreted TNF-alpha causes the downregulation of IR beta, IRS1, and GLUT4 through TNFR1, resulting in insulin resistance.OXFORD UNIV PRESS, Jun. 2010, TOXICOLOGICAL SCIENCES, 115(2) (2), 482 - 491, English[Refereed]Scientific journal
- The Bacillus subtilis asnH operon, comprising yxbB, yxbA, yxnB, asnH and yxaM, is induced dramatically in the transition between exponential growth and stationary phase in rich sporulation medium. The asnH operon is transcribed to produce an unstable long transcript covering the entire operon as well as a short one corresponding to the first three genes. Northern blot analysis revealed that the discrete band corresponding to the short transcript was detectable even 1 h after the addition of excess rifampicin, suggesting its unusual stability. The transcription start site of the operon was determined; its corresponding promoter was most likely sigma-A dependent and under tight control of AbrB and CodY. Within the 5'-proximal region of the transcript preceding yxbB, there is a mysterious long sequence triplication (LST) segment, consisting of a tandem repeat of two highly conserved 118 bp units and a less conserved 129 bp unit. This LST segment was not involved in regulation by AbrB and CodY. Transcriptional fusion of the 5'-region containing the LST segment to lacZ resulted in a significant increase in beta-galactosidase synthesis in cells; the LST segment was thought to prevent degradation of the 5'-region-lacZ fusion transcript. These results suggest that the 5'-region containing the LST segment could function as an mRNA stabilizer that prolongs the lifetime of the transcript to which it is fused.SOC GENERAL MICROBIOLOGY, Jun. 2010, MICROBIOLOGY-SGM, 156(6) (6), 1632 - 1641, English[Refereed]Scientific journal
- 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes a wasting syndrome characterized by a loss of body weight accompanied by a decrease in adipose tissue weight, i.e., insulin resistance-like symptoms. Therefore, the effects of TCDD on an insulin signaling pathway in mature 3T3-L1 adipocytes were investigated to obtain insight into the underlying mechanisms. TCDD downregulated expression of insulin receptor beta-subunit (IR beta), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (GLUT4) and decreased insulin-stimulated glucose uptake activity. TCDD also upregulated expression of TNF-alpha, one of insulin resistance-inducing factors. Anti-TNF-alpha neutralization antibody and silencing of TNF-alpha receptor 1 (TNFR1) diminished the TCDD-induced downregulation of IR beta, IRS1, and GLUT4. Moreover, the experiments using small interfering RNA for an aryl hydrocarbon receptor (AhR) revealed that the TCDD-evoked changes of IR beta, IRS1, GLUT4, and TNF-alpha were dependent on AhR. TCDD also stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), and their inhibitors abrogated the TCDD-induced downregulation of IR beta, IRS1, and GLUT4; upregulation of TNF-alpha; and activation of NF-kappa B. Taken together, TCDD stimulates expression and secretion of TNF-alpha in adipocytes through activation of AhR, ERK1/2, and JNK, and the secreted TNF-alpha causes the downregulation of IR beta, IRS1, and GLUT4 through TNFR1, resulting in insulin resistance.OXFORD UNIV PRESS, Jun. 2010, TOXICOLOGICAL SCIENCES, 115(2) (2), 482 - 491, English[Refereed]Scientific journal
- Diabetes mellitus is a complex disease that is characterized by the defection of insulin sensitivity in such peripheral tissues as skeletal muscle, adipose tissue and liver. We have previously demonstrated that certain inositol derivatives stimulated glucose uptake accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane in L6 myotubes. We investigated in this present study whether an oral intake of D-pinitol (PI) and myo-inositol (MI) would affect GLUT4 translocation in the skeletal muscle of mice. PI or MI at 1 g/kg BW administered orally to mice 30 min before a post-oral injection of glucose at 2 g/kg BW resulted in both PI and MI increasing GLUT4 translocation in the skeletal muscle and lowering the plasma glucose and insulin levels. PI and MI, therefore, have the potential to prevent diabetes mellitus by reducing the postprandial blood glucose level and stimulating GLUT4 translocation in the skeletal muscle.TAYLOR & FRANCIS LTD, May 2010, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 74(5) (5), 1062 - 1067, English[Refereed]Scientific journal
- scyllo-Inositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; another inositol isomer, which is abundant in nature). Previous studies elucidated the MI catabolic pathway in B. subtilis as comprising multiple stepwise reactions catalysed by a series of lol enzymes. The first step of the pathway converts MI to scyllo-inosose (SIS) and involves the MI dehydrogenase lolG. Since lolG does not act on SI, we suspected that there could be another enzyme converting SI into SIS, namely an SI dehydrogenase. Within the whole genome, seven genes paralogous to iolG have been identified and two of these, iolX and iolW (formerly known as yisS and yvaA, respectively), were selected as candidate genes for the putative SI dehydrogenase since they were both prominently expressed when B. subtilis was grown on medium containing SI. iolX and iolW were cloned in Eschefichia colt and both were shown to encode a functional enzyme, revealing the two distinct SI dehydrogenases in B. subtilis. Since inactivation of iolX impaired growth with SI as the carbon source, lolX was identified as a catabolic enzyme required for SI catabolism and it was shown to be NAD(+) dependent. The physiological role of lolW remains unclear, but it may be capable of producing SI from SIS with NADPH oxidation.SOC GENERAL MICROBIOLOGY, May 2010, MICROBIOLOGY-SGM, 156(5) (5), 1538 - 1546, English[Refereed]Scientific journal
- SPRINGER, 2010, ANIMAL CELL TECHNOLOGY: BASICS & APPLIED ASPECTS, 16, 327 - 331, English[Refereed]International conference proceedings
- Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt). This complex binds to DNA. It has been shown that the AhR bonds to a DNA sequence called the dioxin response element (DRE), which controls the expression of battery genes. It is reported that TCDD releases arachidonic acid from membrane phospholipids via activation of phospholipase A(2)s (PLA(2)s) in various cell types. Recently, we demonstrated that the TCDD-activated AhR binds to the second intron of the Pla2g4a gene, which encodes cytosolic phospholipase A(2)alpha (cPLA(2)alpha), in mouse hepatoma Hepa-1c1c7 cells. This result suggests that Pla2g4a appears to be a target gene of the AhR. In the present study, we investigated whether the transcriptional regulation of Pla2g4a is dependent on the AhR in Hepa-1c1c7 cells. Treatment of the cells with TCDD increased mRNA expression of Pla2g4a and enzymatic activity of PLA(2). while this increased expression was not observed in AhR-defective c12 cells. After transient transfection of an Ahr gene-expressing plasmid into the 02 cells, expression of Pla2g4a was increased by TCDD. These results indicate that Pla2g4a may be a novel target gene of the AhR, and its transcriptional induction is mediated through binding of the AhR to the second intron of Pla2g4a, although this target site does not have a typical DRE sequence. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Oct. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108(4) (4), 277 - 281, English[Refereed]Scientific journal
- Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt). This complex binds to DNA. It has been shown that the AhR bonds to a DNA sequence called the dioxin response element (DRE), which controls the expression of battery genes. It is reported that TCDD releases arachidonic acid from membrane phospholipids via activation of phospholipase A(2)s (PLA(2)s) in various cell types. Recently, we demonstrated that the TCDD-activated AhR binds to the second intron of the Pla2g4a gene, which encodes cytosolic phospholipase A(2)alpha (cPLA(2)alpha), in mouse hepatoma Hepa-1c1c7 cells. This result suggests that Pla2g4a appears to be a target gene of the AhR. In the present study, we investigated whether the transcriptional regulation of Pla2g4a is dependent on the AhR in Hepa-1c1c7 cells. Treatment of the cells with TCDD increased mRNA expression of Pla2g4a and enzymatic activity of PLA(2). while this increased expression was not observed in AhR-defective c12 cells. After transient transfection of an Ahr gene-expressing plasmid into the 02 cells, expression of Pla2g4a was increased by TCDD. These results indicate that Pla2g4a may be a novel target gene of the AhR, and its transcriptional induction is mediated through binding of the AhR to the second intron of Pla2g4a, although this target site does not have a typical DRE sequence. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Oct. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 108(4) (4), 277 - 281, English[Refereed]Scientific journal
- Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515-535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.SPRINGER, Apr. 2009, CYTOTECHNOLOGY, 59(3) (3), 177 - 182, English[Refereed]Scientific journal
- Inositol derivatives are natural ingredients found in plants and animals. Some of them have been reported to help the action of insulin, which induces glucose uptake involving the translocation of glucose transporter 4 (GLUT4) to the plasma membrane particularly in muscle tissues. Here, we investigated the effect of eight inositol derivatives on glucose uptake in L6 muscle myotubes, and on GLUT 4 translocation to plasma membrane in ex vivo assay Using murine skeletal muscle. At 1 mM, seven inositol derivatives other than myo-inositol stimulated glucose uptake in the absence Of insulin. However, at 100 mu M, only L-chiro-inositol, muco-inositol and epi-inositol increased the glucose uptake compared to control. The ex vivo assay confirmed that D-pinitol, D-chiro-inositol, L-chiro-inositol, muco-inositol and epi-inositol stimulated GLUT4 translocation. These results suggest that inositol derivatives can exert insulin-like effect in muscle cells.SPRINGER, 2009, ANIMAL CELL TECHNOLOGY: BASIC AND APPLIED ASPECTS, VOL 15, 15, 217 - 222, English[Refereed]International conference proceedings
- A number of 2',3',4'-trihydroxy-2-phenylacetophenone derivatives were synthesized and examined for growth inhibition of several kinds of bacteria. 2',3',4'-Trihydroxy-2-phenylacetophenone itself exhibited no antibacterial activity, but some of its derivatives showed various antibacterial activities depending on functional groups introduced on the 2-phenyl ring. Eighteen out of 24 compounds synthesized in this study appeared to possess antibacterial activities against at least two Gram-positive strains of Bacillus subtilis and Staphylococcus aureus, 2-(biphenyl-4-yl)-2',3',4'-trihydroxy-acetophenone being the most active with LC50 of 5.8 mu M and 5.6 mu M respectively. However, none of the synthesized compounds exhibited inhibitory effects on Gram-negative strains, such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica, suggesting that anti-Gram-positive specificity of the antibacterial compounds.TAYLOR & FRANCIS LTD, Jan. 2009, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(1) (1), 124 - 128, English[Refereed]Scientific journal
- Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1,700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.GENETICS SOC JAPAN, Dec. 2008, GENES & GENETIC SYSTEMS, 83(6) (6), 455 - 468, English[Refereed]Scientific journal
- In this study, We investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation Of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGC-increasecl uptake. Therefore, ECCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin. (C) 2008 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2008, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 377(1) (1), 286 - 290, English[Refereed]Scientific journal
- Dioxins enter the body through the diet and cause various toxicological effects through transformation of an aryl hydrocarbon receptor (AhR). Plant extracts and phytochemicals including flavonoids are reported to suppress this transformation. This paper investigates the suppression by a cacao polyphenol extract (CPE) of AhR transformation in vivo. The CPE was administered orally to C57BL/6 mice at 100 mg/kg of body weight, followed 1 h later by 3-methylcholanthrene (MC), an AhR agonist, injected intraperitoneally at 10 mg/kg of body weight. CPE suppressed the MC-induced transformation to the control level by inhibiting the formation of a heterodimer between AhR and an aryl hydrocarbon receptor nuclear translocator in the liver at 3 h postadministration. It also suppressed MC-induced cytochrome P4501A1 expression and NAD(P)H:quinone-oxidoreductase activity, whereas it increased glutathione S-transferase activity at 25 h. CPE constituents and their metabolites might contribute, at least in part, to the suppression of AhR transformation. The results indicate that the intake of CPE suppressed the toxicological effects of dioxins in the body.AMER CHEMICAL SOC, Nov. 2008, JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 56(21) (21), 10399 - 10405, English[Refereed]Scientific journal
- Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammoniareleasing reaction.TAYLOR & FRANCIS LTD, Jul. 2008, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 72(7) (7), 1869 - 1876, English[Refereed]Scientific journal
- The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for myo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane1,2- dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione catalyzed by IolD to yield 5-deoxy-D-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the iol operon. In this reaction 2-deoxy-5-keto-D-gluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO2 by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO2.AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Apr. 2008, JOURNAL OF BIOLOGICAL CHEMISTRY, 283(16) (16), 10415 - 10424, English[Refereed]Scientific journal
- Halogenated and polycyclic aromatic hydrocarbons, exogenous ligands of the aryl hydrocarbon receptor (AhR), cause various toxicological effects through the transformation of the AhR. In this study, we investigated the antagonistic effects of indigoids on the transformation in addition to their agonistic ones. In a cell-free system, indigoids induced the transformation dose-dependently, but suppressed the transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the binding of 3-methyleholanthrene to the AhR. In mouse hepatoma Hepa-1clc7 cells, indigoids, especially indirubin, suppressed the transformation and expression of CYPIAI by inhibiting the translocation of AhR into the nucleus. When orally administered to mice at 10 mg/kg BW/day for three successive days, indigoids did not induce AhR transformation and expression of the CYPIA subfamily in the liver, while indirubin and indigo upregulated quinone reductase activity. These results indicate that indigoids are able to bind to the AhR as ligands and exhibit antagonistic effects at lower concentrations in mammalian cells. (c) 2007 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Feb. 2008, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 470(2) (2), 187 - 199, English[Refereed]Scientific journal
- 2008, Journal of Clinical Biochemistry and Nutrition, 43(Suppl.1), 460 - 463, EnglishSuppressive effects of propolis extract on cytochrome P4501A1 expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin[Refereed]Scientific journal
- Some of inositol derivatives have been reported to help the action of insulin stimulating glucose uptake in skeletal muscle cells. Rat L6 myotubes were employed in an attempt to develop an in vitro model system for investigation of the possible insulin-like effect of eight inositol derivatives, namely allo-inositol, D-chiro-inositol L-chiro-inositol, epi-inositol, muco-inositol, myo-inositol, scyllo-inositol and D-pinitol. At a higher concentration of 1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1mM only D-chiro-inositol, L-chiro-inositol, epi-inositol and muco-inositol could induce glucose uptake, indicating their significant insulin-mimetic activity. Immunoblot analyses revealed that at least D-chiro-inositol, L-chiro-inositol, epi-inositol, muco-inositol and D-pinitol were able to induce translocation of glucose transporter 4 (GLUT4) to plasma membrane not only in L6 myotubes but also in skeletal muscles of rats ex vivo. These results demonstrated that L6 myotubes appeared efficient as an in vitro system to identify inositol derivatives exerting an insulin-like effect on muscle cells depending on the induced translocation of GLUT4.SPRINGER, Dec. 2007, CYTOTECHNOLOGY, 55(2-3) (2-3), 103 - 108, English[Refereed]Scientific journal
- Halogenated and polycyclic aromatic hydrocarbons induce diverse biochemical responses through the transformation of a cytosolic aryl hydrocarbon receptor (AhR). In mouse hepatoma Hepa-1c1c7 cells, curcumin, a yellow pigment of Curcuma longa, did not inhibit the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced translocation of the AhR into the nucleus, but rather accelerated it. In the nucleus, curcumin inhibited the TCDD-induced heterodimerization of the AhR with an AhR nuclear translocator (Arnt), an essential partner for the transformation, and also dose-dependently inhibited the TCDD-evoked phosphorylation of both the AhR and Arnt. Moreover, curcumin significantly inhibited the TCDD-induced activation of protein kinase C (PKC), which is involved in the transformation, decreased the TCDD-induced DNA-binding activity of the AhR/Arnt heterodimer, and downregulated CYP1A1 expression. In a cell-free system, curcumin inhibited the binding of 3-methylcholanthrene, an AhR agonist, to the receptor. These results indicate that curcumin is able to bind to the AhR as a ligand, but suppresses its transformation by inhibiting the phosphorylation of AhR and Arm, probably by PKC. (c) 2007 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Oct. 2007, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 466(2) (2), 267 - 273, English[Refereed]Scientific journal
- Bacillus subtilis LmrA is known to be a repressor that regulates the hnrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the hnrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the 1mrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter P-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the hnrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.AMER SOC MICROBIOLOGY, Jul. 2007, JOURNAL OF BACTERIOLOGY, 189(14) (14), 5170 - 5182, English[Refereed]Scientific journal
- Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently WE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An WE ortholog was cloned from USDA191. USDA191 WE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 WE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis WE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myoinositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and WE.TAYLOR & FRANCIS LTD, Dec. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(12) (12), 2957 - 2964, English[Refereed]Scientific journal
- Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in ΔaseR and ΔczrA cells. Candidate (i) partly thiolate, α3-helix (for AseR) and (ii) tetrahedral, non-thiolate, α5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (α3) and zinc (α5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting α3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA. © 2006 Blackwell Publishing Ltd.Feb. 2006, Molecular Microbiology, 59(4) (4), 1341 - 1356, English[Refereed]Scientific journal
- Horseradish peroxidase isozyme C1a (HRP C1a) is widely used as a reporter enzyme in a variety of detection procedures such as enzyme-linked immunosorbent assay (ELISA) and western blotting. We previously isolated the gene encoding HRP C1a and showed that HRP C1a is at first translated as a preproprotein containing propeptides at its N- and C-termini (N-terminal secretion signal peptide and C-terminal propeptide; CTPP). The signal peptide (sp) is necessary for endoplasmic reticulum (ER) translocation and the CTPP acts as a vacuolar sorting determinant. Furthermore, HRP C1a was secreted into the culture medium from cells expressing the HRP C1a gene without the CTPP region. We optimized the secretory production system of HRP C1a in tobacco plants. To determine a suitable signal peptide for high-efficient secretion of proteins, three types of sp derived from HRP C1a (C1Psp), beta-D-glucan exohydrolase (GEsp) and 38 kDa peroxidase (38Psp) were compared. GE and 38P are secretory proteins highly accumulated in the culture medium of BY2 cells. The secretion efficiency was increased by 34% and 53% when GEsp and 38Psp was used, respectively. Next, we used a translational enhancer, the 5'-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5'-UTR). The production of HRP C1a was increased by placing NtADH 5'UTR in front of the ORF in BY2 cells. These results indicate that the localization and expression level of recombinant proteins can be controlled by the use of propeptides and 5'UTR, respectively. Finally, high-efficiency secretory production of the HRP C1a was also achieved in transgenic tobacco.SOC BIOSCIENCE BIOENGINEERING JAPAN, Aug. 2006, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 102(2) (2), 102 - 109, English[Refereed]Scientific journal
- Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiroinositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.TAYLOR & FRANCIS LTD, Aug. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(8) (8), 1913 - 1920, English[Refereed]Scientific journal
- D-chiro-Inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABC-DEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IoIG and then to 3(D)-(3,5/4)-trihydroxycyclohexane-1,2-dione by IoIE. In this study, we identified ioll encoding inosose isomerase, which converts 2KMI to 1-keto-(D)-chiro-inositol (1KDCI), and found that IoIG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of loll and IoIG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.AMER SOC MICROBIOLOGY, Feb. 2006, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72(2) (2), 1310 - 1315, English[Refereed]Scientific journal
- Screening of indigenous plants from Japan for modulating effects on transformation of the aryl hydrocarbon receptorThe aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with which halogenated and polycyclic aromatic hydrocarbons such as dioxins and benzo[a]pyrene interact as ligands. Since such compounds cause various toxicological effects, including cancer, through the transformation of AhR, it is important to determine influence of modulating factors. It has been reported that certain plant components such as flavonoids and indoles can affect AhR transformation. In this study, to obtain clues to novel ligands of AhR, 191 species of indigenous plants were collected in Japan, and their 50% methanolic extracts (total 368 plant parts) were tested for modulating effects on AhR transformation in a cell-free system using a rat hepatic cytosolic fraction. Among tested extracts at a concentration of 1 mg dry weight of plant/mL, 174 of 368 extracts suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD)-induced AhR transformation to 50% or less, while 9 extracts per se induced AhR transformation equivalent to more than 20% of that induced by 1 nM TCDD. Mallotus japonicus (Thunb.) Muell. (leaf) and Trichosanthes rostrata Kitamura (fruit and fruit skin) strongly suppressed 1 nM TCDD-induced AhR transformation, while Phellodendron amurense Ruprecht (seed) per se strongly induced AhR transformation. These results suggest that a large variety of plants in Japan contain various compounds modulating, mainly suppressing, AhR transformation.Asian Pacific Organization for Cancer Prevention, 2006, Asian Pacific Journal of Cancer Prevention, 7(2) (2), 208 - 220, English[Refereed]Scientific journal
- Suppressive effects of ethanolic extracts prepared from propolis group 12 and its main botanical origin (leaf bud of Baccharis dracunculifolia) on transformation of the aryl hydrocarbon receptor (AhR), the initial action of dioxin toxicity, were investigated. It was found that suppressive effects of propolis on AhR transformation were relatively higher than those of resins of its botanical origin in cell-free system and in Hepa-1c1c7 cells. When the composition of chemical ingredients was measured, propolis contained slightly higher amounts of flavonoid aglycones as compared with its botanical origin with the same characteristics. Moreover, antiradical activity, one of the typical biological activities of flavonoids, in propolis was also slightly higher than that in its botanical origin. These results indicate that not only propolis but also its botanical origin contains high amounts of flavonoid aglycones and that both of them are useful dietary sources for flavonoids with a potency to prevent dioxin toxicity. © 2005 American Chemical Society.Dec. 2005, Journal of Agricultural and Food Chemistry, 53(26) (26), 10306 - 10309, English[Refereed]Scientific journal
- Mar. 2005, Proceedings of 2004 International Conference on O-CHA(tea) Culture and Science, 547-548, EnglishSuppressive effects of catechins on differentiation of 3T3-L1 preadipocytesInternational conference proceedings
- Mar. 2005, Proceedings of 2004 International Conference on O-CHA (tea) Culture and Science, 561-562, EnglishBlack tea (Camellia sinensis) suppresses hyperglycemia in STZ-induced diabetic ratsInternational conference proceedings
- 2005, Proceedings of 2004 International Conference on O-CHA(tea) Culture and Science, 594-595, EnglishTea has the potential to reduce the dioxin riskInternational conference proceedings
- In plants, the plasma membrane Na+/H+ antiporter is the only key enzyme that extrudes cytosolic Na+ and contributes to salt tolerance. But in fungi, the plasma membrane Na+/H+ antiporter and Na+-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na+ and Li+ efflux across the plasma membrane during salt stress and for K+ efflux at high pH and high K+. To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na+ and Li+ than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K+ than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na+, Li+, and K+) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K+-ATPase activity, Na+-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress. (C) 2004 Wiley Periodicals, Inc.WILEY-BLACKWELL, Mar. 2004, BIOTECHNOLOGY AND BIOENGINEERING, 85(7) (7), 776 - 789, English[Refereed]Scientific journal
- Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.SPRINGER-VERLAG, Oct. 2003, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 62(5-6) (5-6), 517 - 522, English[Refereed]Scientific journal
- JAPANESE BIOCHEMICAL SOC, May 2003, SEIKAGAKU, 75(5) (5), 407 - 410, JapaneseDNA microarray analysis of Bacillus subtilis[Refereed]Scientific journal
- To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.NATL ACAD SCIENCES, Apr. 2003, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(8) (8), 4678 - 4683, EnglishScientific journal
- Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the Bacillus subtilis genome project, we found that a mutant with inactivation of iolT (ydjK) exhibited a growth defect on myo-inositol as the sole carbon source. The putative product of iolT exhibits significant similarity with many bacterial sugar transporters in the databases. In B. subtilis, the iolABCDEFGHIJ and iolRS operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the iol genes, iolF was predicted to encode an inositol transporter. Inactivation of iolF alone did not cause such an obvious growth defect on inositol as the iolT inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single iolT inactivation. Determination of inositol uptake by the mutants revealed that iolT inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the K(m) and V(max) values for the IolT and IolF inositol transporters, indicated that iolT and iolF encode major and minor inositol transporters, respectively. Northern and primer extension analyses of iolT transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by final sigma(A) RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the iolT promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the iol divergon.Feb. 2002, Journal of bacteriology, 184(4) (4), 983 - 91, English, International magazine[Refereed]Scientific journal
- It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B. subtilis two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (http://www.genome.ad.jp/kegg/expression).AMER SOC MICROBIOLOGY, Dec. 2001, JOURNAL OF BACTERIOLOGY, 183(24) (24), 7365 - 7370, English[Refereed]Scientific journal
- We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-beta -D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.OXFORD UNIV PRESS, Sep. 2001, NUCLEIC ACIDS RESEARCH, 29(18) (18), 3804 - 3813, English[Refereed]Scientific journal
- We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in Bacillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.OXFORD UNIV PRESS, Feb. 2001, NUCLEIC ACIDS RESEARCH, 29(3) (3), 683 - 692, English[Refereed]Scientific journal
- Cytochrome bd biosynthesis in Bacillus subtilis: Characterization of the cydABCD operonUnder aerobic conditions Bacllus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa(3)-, an aa(3)-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.AMER SOC MICROBIOLOGY, Dec. 1998, JOURNAL OF BACTERIOLOGY, 180(24) (24), 6571 - 6580, English[Refereed]Scientific journal
- Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilisPrevious determination of the nucleotide sequence of the ioi region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iolJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh). Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5' region of iolA, which is probably recognized by sigma(A). SB nuclease analysis implied that a rho-independent terminator like structure in the 3' region of iolJ might be a terminator for iol transcription. Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes. Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found. Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcription-negative regulator (presumably a repressor) for the iol operon. Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by sigma(A). The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus.AMER SOC MICROBIOLOGY, Jul. 1997, JOURNAL OF BACTERIOLOGY, 179(14) (14), 4591 - 4598, English[Refereed]Scientific journal
- A 20-kDa protein of Bacillus thuringiensis var. israelensis (Bti) has been shown to be necessary for the efficient expression of the 27-kDa mosquitocidal protein gene in Escherichia coli. We have investigated the effects of this 20-kDa protein on the expression of two 130-kDa mosquitocidal protein genes (cryIV A, cryIV B) in E.coli by supplying the 20-kDa protein gene in trans. When a recombinant plasmid, pLH4BX, which was constructed to express cryIV A under the E.coli lac promoter on pUC19,coexisted with the 20-kDa protein gene, a striking increase in production of the fused CryIV A was detected. This was not accompanied by an increase in the amount of intracellular mRNA, suggesting that 20-kDa protein exerts a posttranscriptional effect. We conclude that the 20-kDa protein also stimulates the production of 130-kDa protein in E.Coli.Japan Society for Bioscience, Biotechnology, and Agrochemistry, Sep. 1992, Bioscience, biotechnology, and biochemistry, 56(9) (9), 1429 - 1433, English
- Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1990, Nippon Nōgeikagaku Kaishi, 64(10) (10), 1612 - 1615, Japanese
- Lead, Nov. 2019, 生物工学会誌, 97(11) (11), 692 - 693, JapaneseFEMS2019報告[Refereed][Invited]Meeting report
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 185 - 185, Japanese2P-043 Characterization of aspartic proteases from Aspergillus repens and Aspertic glaucus
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 185 - 185, Japanese2P-041 Characterization of internal substrate and acetyl-CoA binding sites in Nacetyltransferase from Chryseobacterium sp. 5-3B
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 185 - 185, Japanese2P-042 Cloning, heterologous expression, and enzymatic characterization of bacterial diamine transaminases
- 日本生物工学会, 2015, 日本生物工学会大会講演要旨集, 67, 178 - 178, Japanese2P-013 Phasin genes involved in PHB accumulation in Bradyrhizobium japonicum
- 日本工業出版, 2015, 環境浄化技術, 14(7-8) (7-8), 92 - 97, Japanese微生物のゲノム（前編）[Invited]Introduction scientific journal
- 日本工業出版, 2015, 環境浄化技術, 14(9-10) (9-10), Japanese微生物のゲノム（後編）[Invited]Introduction scientific journal
- 2015, バイオサイエンスとインダストリー, 73(4) (4), 284 - 287, Japaneseアルツハイマー病治療薬として期待されるシロイノシトールの微生物生産[Invited]Introduction scientific journal
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 113 - 113, Japanese2P-025 Optimizing secretion of phytase in Bacillus subtilis
- 日本生物工学会, 2014, 日本生物工学会大会講演要旨集, 66, 32 - 32, Japanese1P-061 Characterization of N-acetyltransferase from Chryseobacterium sp.
- 日本生物工学会, Nov. 2013, 生物工学会誌, 91(11) (11), 625 - 628, Japanese枯草菌を活用する生理活性イノシトールの開発[Invited]Introduction scientific journal
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 28 - 28, Japanese1P-043 Cloning, expression, and characterization of N-acetyltransferase from Chryseobacterium sp. 5-3B
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 135 - 135, Japanese2P-122 Optimizing bioconversion from myo-inositol to scyllo-inositol in Bacillus subtilis
- 日本生物工学会, 2013, 日本生物工学会大会講演要旨集, 65, 228 - 228, Japanese3P-159 Optimizing secretion of phytase from Bacillus subtilis
- 日本農芸化学会, Oct. 2012, 化学と生物, 55(10) (10), 704 - 705, Japanese[Invited]Introduction scientific journal
- 日本生物工学会, Oct. 2012, 生物工学会誌, 90(10) (10), 623 - 624, Japanese代謝アドオンシステムと物質生産[Invited]Introduction scientific journal
- The Society for Biotechnology, Japan, Oct. 2012, 日本生物工学会大会講演要旨集, 64, 39 - 39, English2Ap03 Bacillus subtilis cell factory for production of scyllo-inositol promising for Alzheimer's disease (Bio-Based Production)Lecture materials
- シーエムシー出版, Feb. 2012, 月刊バイオインダストリー, 29(2) (2), 15 - 20, Japanese大豆（ダイズ）の新規機能性成分開発[Invited]Introduction scientific journal
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 36 - 36, Japanese2Ca09 Purification and characterization of halotolerant amylases from Bacillus subtilis FP-133
- 日本生物工学会, 2012, 日本生物工学会大会講演要旨集, 64, 36 - 36, Japanese2Ca08 Gene cloning and expression of eggshell membrane degrading enzyme from Pseudomonas aeruginosa ME-4
- 日本生物工学会, Oct. 2011, 生物工学会誌, 89(10) (10), 585 - 588, Japanese有用希少イノシトールのバイオアベイラビリティーとバイオコンバージョン生産[Refereed][Invited]Introduction scientific journal
- 日本生物工学会, 2011, 日本生物工学会大会講演要旨集, 63, 70 - 70, Japanese1Kp15 Inositol dehydrogenase genes of Geobacillus kaustophilus HTA426
- 日本生物工学会, 2011, 日本生物工学会大会講演要旨集, 63, 134 - 134, Japanese2Da11 Purification and characterization of aspartic protease II from Aspergillus repens MK82
- 2011, 日本分子生物学会年会プログラム・要旨集(Web), 34th, 2P - 0003 (WEB ONLY), JapaneseBacillus subtilis degU32(hy) proactively cancels catabolite repressionLecture materials
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 76 - 76, Japanese3P-1076 Characterization of N-acetyltransferase from Microbacterium paraoxydans FK-2-1
- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 229 - 229, Japanese2S-Dp02 Production and bioavailability of rare inositols
- Flavonoids have been reported to be dietary antagonists of an aryl hydrocarbon receptor (AhR). However, little is known about the molecular mechanism on their antagonistic effects. In this study, the inhibitory effect of flavonoids on ligand binding to the AhR and interaction between flavonoids and the AhR complex (AhRc) were investigated in each flavonoid subclass. Flavone, flavonol, and flavanone but not catechin inhibited the specific binding between the AhR and 3-methylcholanthrene dose-dependently, indicating that the former three subclasses possibly act as competitive antagonists of the AhR. However, catechin in addition to the former three subclasses directly interacted with the AhRc by surface plasmon resonance analysis. The dissociation constant values showed an inverse correlation with the suppressive effect on the DNA binding activity. These results suggest that flavone, flavonol, and flavanone act as competitive antagonists of the AhR, while catechin associates with the AhRc and indirectly exhibits its antagonistic effects. (c) 2007 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2007, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 359(3) (3), 822 - 827, English
- Jan. 2007, Global Regulatory Networks in Bacillus subtilis, 75-90, EnglishA holistic view of inositol catabolism in Bacillus subtilisIntroduction scientific journal
- Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently WE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An WE ortholog was cloned from USDA191. USDA191 WE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 WE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis WE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myoinositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and WE.TAYLOR & FRANCIS LTD, Dec. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(12) (12), 2957 - 2964, English
- Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiroinositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.TAYLOR & FRANCIS LTD, Aug. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(8) (8), 1913 - 1920, English
- (公社)日本栄養・食糧学会, Apr. 2006, 日本栄養・食糧学会大会講演要旨集, 60回, 206 - 206, Japanese筋肉細胞における(-)-エピガロカテキンガレートによるグルコースの取り込み亢進作用機構について
- (公社)日本栄養・食糧学会, Apr. 2006, 日本栄養・食糧学会大会講演要旨集, 60回, 235 - 235, Japanese芳香族炭化水素により誘導されるアリール炭化水素受容体の形質転換に対するカカオポリフェノールの抑制効果
- D-chiro-Inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABC-DEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiro-inositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI. Copyright © 2006, American Society for Microbiology. All Rights Reserved.Feb. 2006, Applied and Environmental Microbiology, 72(2) (2), 1310 - 1315, English
- Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in Delta aseR and Delta czrA cells. Candidate (i) partly thiolate, alpha 3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha 5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha 3) and zinc (alpha 5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha 3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.BLACKWELL PUBLISHING, Feb. 2006, MOLECULAR MICROBIOLOGY, 59(4) (4), 1341 - 1356, English
- Dioxins enter the body mainly through diet and cause the various toxicological effects by binding to the cytosolic aryl hydrocarbon receptor (AhR) followed by its transformation. In recent reports, it has been shown that certain natural compounds suppress AhR transformation in vitro. In this study, we demonstrated that ethanolic extract from molokhia, known as Egyptian spinach, showed the strongest suppressive effect on AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cell-free system using rat hepatic cytosol among 41 kinds of extracts from vegetables and fruits. The molokhia extract also suppressed TCDD-induced AhR transformation in mouse hepatoma Hepa-1c1c7 cells and in intestinal permeability system constructed with human colon adenocarcinoma Caco-2 cells and human hepatoma HepG2 cells. Moreover, oral administration of the molokhia extract (100 mg/kg body weight) decreased 3-methylcholanthrene-induced AhR transformation to the control level by inhibiting translocation of the AhR from cytosol into the nucleus in the liver of rats. The molokhia extract-administered rat liver showed a tolerance to TCDD-induced AhR transformation by ex vivo experiment. These results indicate that molokhia is an attractive food for isolation and identification of a natural antagonist for the AhR. (c) 2005 Elsevier Ltd. All rights reserved.PERGAMON-ELSEVIER SCIENCE LTD, Feb. 2006, FOOD AND CHEMICAL TOXICOLOGY, 44(2) (2), 250 - 260, English
- Sep. 2005, 化学と生物, 43 9 566-568, Japanese枯草菌のイノシトール分解系―機能解明とその応用の可能性―Introduction scientific journal
- 日本農芸化学会, 2005, 化学と生物, 43 9 566-568(9) (9), 566 - 568, Japanese今日の話題「枯草菌のイノシトール分解系－機能解明とその応用の可能性－[Refereed]Others
- The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine). The two- to threefold repression of expression of the ilv-leu operon during logarithmic-phase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments. A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses. Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending. The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two- to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.AMER SOC MICROBIOLOGY, Dec. 2004, JOURNAL OF BACTERIOLOGY, 186(23) (23), 7971 - 7979, English
- The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATA WT, which could play an important role in LmrA binding.AMER SOC MICROBIOLOGY, Sep. 2004, JOURNAL OF BACTERIOLOGY, 186(17) (17), 5640 - 5648, English
- The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IoIG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.SOC GENERAL MICROBIOLOGY, Mar. 2004, MICROBIOLOGY-SGM, 150(3) (3), 571 - 580, English
- The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.Mar. 2004, Microbiology (Reading, England), 150(Pt 3) (Pt 3), 571 - 580, English, International magazine[Refereed]
- 2004, 月刊海洋, 36(8) 579-587, Japaneseモデル微生物としての枯草菌Others
- 2004, Proceedings of 2004 International Conference on O-CHA (tea) Culture and Science, 561-562Black tea (Camellia sinensis) suppresses hyperglycemia in STZ-induced diabetic rats
- We investigated the organization and expression of the Bacillus subtilis sigY operon, the first gene of which codes for sigma(Y), a member of the extracytoplasmic function (ECF) family of sigma factors. The sigY operon, comprising six genes (sigY, yxlC, D, E, F, and G), was induced upon nitrogen starvation; it was continuously transcribed from the 31st base upstream of sigY to a neighboring convergent gene, yxlH, resulting in a 4.2-kb mRNA. The expression of the sigY operon was also positively autoregulated through sigma(Y), suggesting that its transcription is likely to be directed by sigma(Y). Deletion analysis of the sigY promoter, which was localized by primer extension, revealed the promoter region of sigY with the "-10" and "-35" sequences of CGTC and TGAACG, respectively. The latter sequence was distinct from those recognized by sigma(W), sigma(X), and sigma(M). The ay-directed transcription of sigY was under negative regulation involving Yx1D. sigY disruption affected sporulation induced by nitrogen starvation, but sigY induction upon nitrogen starvation was not associated with the sporulation process. The organization and function of the sigY operon are significantly conserved in several microorganisms living in adverse living environments.JAPANESE BIOCHEMICAL SOC, Dec. 2003, JOURNAL OF BIOCHEMISTRY, 134(6) (6), 935 - 946, English
- A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic, enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k(cat)/K-m 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.SOC GENERAL MICROBIOLOGY, Sep. 2003, MICROBIOLOGY-SGM, 149(9) (9), 2331 - 2343, English
- Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF ) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB , pucJKLM , glnQHMP , nasDEF , oppABCDF , nasA , nasBCDEF and ywrD for positive regulation, and gltAB , pel , ywdIJK , yycCB , yttA , yxkC , ywlFG , yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.BLACKWELL PUBLISHING LTD, Jul. 2003, MOLECULAR MICROBIOLOGY, 49(1) (1), 157 - 165, English
- To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.NATL ACAD SCIENCES, Apr. 2003, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(8) (8), 4678 - 4683, English
- Target gene candidates of the seven extracytoplasmic function (ECF) sigma factors of Bacillus subtilis have been surveyed using DNA microarray analysis of mRNA extracted from cells grown in Luria-Bertani broth, in which an ECF sigma factor gene was placed under the control of the spac promoter on multicopy plasmid pDG148 and overexpressed. The number of target candidates for each of the sigma factors varied greatly. and a total of 278 genes were selected. Interestingly, the above target gene candidates shared only one gene out of 94 target genes of the general stress sigma B that have been reported in the literature thus far. Furthermore, lacZ-fusion experiments based on the results of DNA microarray analysis indicated that each ECF sigma factor directs transcription of its own operon, with the exception of sigZ. The DNA microarray data collected in this study are available at the KEGG Expression Database web site (http://www.genome.ad.jp/kegg/expression/). (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Mar. 2003, FEMS MICROBIOLOGY LETTERS, 220(1) (1), 155 - 160, English
- 2003, 生化学, 75(5) 407-410, Japanese枯草菌のDNA マイクロアレイ解析Others
- Japan Society for Bioscience Biotechnology and Agrochemistry, 2003, Nippon Nogeikagaku Kaishi, 77(1) (1), 12 - 17, Japanese
- 05 Mar. 2001, 日本農芸化学会誌, 75, 105, Japanese枯草菌イノシトール分解系遺伝子の機能解析
- With the completion of the determination of its entire genome sequence, one of the next major targets of Bacillus subtilis genomics is to clarify the whole gene regulatory network. To this end, the results of systematic experiments should be compared with the rich source of individual experimental results accumulated so far. Thus, we constructed a database of the upstream regulatory information of B. subtilis (DBTBS). The current version was constructed by surveying 291 references and contains information on 90 binding factors and 403 promoters. For each promoter, all of its known cis-elements are listed according to their positions, while these cis-elements are aligned to illustrate their consensus sequence for each transcription factor. All probable transcription factors coded in the genome were classified with the Pfam motifs. Using this database, We compared the character of B. subtilis promoters with that of Escherichia coli promoters. Our database is accessible at http://elmo.ims.u-tokyo.ac.jp/dbtbs/.OXFORD UNIV PRESS, Jan. 2001, NUCLEIC ACIDS RESEARCH, 29(1) (1), 278 - 280, English[Refereed]
- 25 Nov. 2000, 日本分子生物学会年会プログラム・講演要旨集, 23rd, 450, Japanese枯草菌イノシトール分解系遺伝子の機能解析
- The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system, YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six ytr genes form a single operon, transcribed from a putative sigma(A)-dependent promoter present upstream of ytrA. YtrA, which possesses a helix-turn-helix. motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation. It is known that B. subtilis produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation, When either the entire ytr operon or its last gene, ytrF, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient. We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation.AMER SOC MICROBIOLOGY, Oct. 2000, JOURNAL OF BACTERIOLOGY, 182(19) (19), 5454 - 5461, English[Refereed]
- SOC GENERAL MICROBIOLOGY, Sep. 2000, MICROBIOLOGY-UK, 146, 2091 - 2092, EnglishThe 409 bp tandem repeat spanning genes yxaK and yxaL is absent from the Bacillus subtilis chromosome[Refereed]Report scientific journal
- Systematic study of gene expression and transcription organization in the gntZ-ywaA region of the Bacillus subtilis genomeWithin the framework of the international project 'The functional analysis of the Bacillus subtilis genome' in Japan and Europe, the gene expression and transcription organization of the gntZ-ywaA region (160 kb) of the B. subtilis genome has been systematically analysed. First, all unanalysed genes comprising more than 80 amino acids (125 genes) in this region were inactivated through integration of plasmid pMUTIN. No essential gene was found which could not be inactivated. All the integrants grew normally in both nutrient sporulation medium and glucose minimal medium. But an integrant in the yxbG gene exhibited an oligosporogenic phenotype in the nutrient sporulation medium. The synthesis of beta-galactosidase was examined, as a reporter for expression of the inactivated genes, during growth and sporulation in the two media. The results indicated that 36% of the promoters were inactive when cells were grown in at least one of these two media. Furthermore, the transcription of the 119 genes in this region was analysed by Northern blotting, resulting in a transcription map. The results indicate that the gntZ-ywaA region contains at least 24 polycistronic operons, including several published ones. The operons newly found in this work are yxaAB, yxaGH, yxaJKL, yxbBA-yxnB-asnH-yxaM. yxbCD, yxcED, yxdJK, yxeFGH, yxeKLMNOPQ, yxeR-yxxB, hutPHUIGM, bgIPH-yxiE, wapA-yxxG, yxiM-deaD, katB-yxiS, yxjCDEF, yxjJI and yxkF-mmsX.SOC GENERAL MICROBIOLOGY, Mar. 2000, MICROBIOLOGY-UK, 146(3) (3), 573 - 579, English[Refereed]
- Three asparagine synthetase genes of Bacillus subtilisThree asparagine synthetase genes, asnB, asnH, and asnO (yisQ), were predicted from the sequence of the Bacillus subtilis genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of asnB was detected only during exponential growth, that of asnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnO was seen only later in sporulation. In a minimal medium, both asnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of asnH was partially repressed. Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprising yxbB, yxbA, yxnB, asnH, and yxaM. The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an Escherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In B. subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of asnB led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B. subtilis might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation.AMER SOC MICROBIOLOGY, Oct. 1999, JOURNAL OF BACTERIOLOGY, 181(19) (19), 6081 - 6091, English[Refereed]
- Transcription of the Bacillus subtilis iol divergon is negatively regulated by a repressor encoded by iolR, which belongs to the DeoR family of bacterial regulators. Gel retardation analysis involving the IolR protein synthesized in Escherichia coli revealed that IolR bound specifically and independently to each of the iol and iolRS promoter regions, with higher affinity to iol. DNase I footprinting revealed that IolR affected DNase I sensitivity either in the iol promoter region between nucleotides -46 and +51 or in iolRS between -79 and -2 (+1 is the transcription initiation nucleotide of both iol and iolRS), indicating its interaction with the extended regions of the iol and iolRS promoters. Deletion analysis indicated that the iol region between -23 and +21 is involved mainly in IolR binding and negative regulation, while the iolRS region between -70 and -44 comprises at least part of the cis-acting sequences for IolR binding and negative regulation. Sequence examination of the extended regions revealed that a tandem direct repeat consisting of two relatively conserved Il-mer sequences, WRAYCAADARD (where D is A, G or T; R is A or G; W is A or T; and Y is C or T), found in each of the iol and, iolRS regions might be a determinant sequence for the IolR-DNA interaction. Actual involvement of the direct repeats in the IolR-DNA interaction was shown by the deficiency of IolR-binding and negative regulation that was caused by substitution of the conserved bases within the conserved sequences. These results imply a unique mode of interaction of IolR with the target DNA.(C) 1999 Academic Press.ACADEMIC PRESS LTD, Jan. 1999, JOURNAL OF MOLECULAR BIOLOGY, 285(3) (3), 917 - 929, English[Refereed]
- 1999, 蛋白質 核酸 酵素, 44, 1449 - 1459
- 01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 247 - 247, JapaneseFunction analysis of the gntZ-ywaA region(150kb)of the Bacillus subtilis genome
- Identification and expression of the Bacillus subtilis fructose-1,6-bisphosphatase gene (fbp)The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as yydE. The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis,fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly, B. subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.AMER SOC MICROBIOLOGY, Aug. 1998, JOURNAL OF BACTERIOLOGY, 180(16) (16), 4309 - 4313, English[Refereed]
- Bacillus subtilis Is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, Including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage Infection has played an important evolutionary role in horizontal gene transfer, in particular In the propagation of bacterial pathogenesis.MACMILLAN MAGAZINES LTD, Nov. 1997, NATURE, 390(6657) (6657), 249 - 256, English
- Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY regionWithin the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from got to sacXY. This 65 kb region contains 64 ORFs (62 complete and two partial genes). The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, beta-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th-48th, 51st and 58th genes are designated deaD, pepT, galE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose 4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, sigma-factor of RNA polymerase and catalase, respectively. The 60th-64th genes are celRABCD, which are probably involved in cellobiose utilization. Gene organization and gene features in the gnt-sacXY region are discussed.SOC GENERAL MICROBIOLOGY, Nov. 1996, MICROBIOLOGY-UK, 142(11) (11), 3113 - 3123, English[Refereed]
- 01 Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 750 - 750, Japanese枯草菌ゲノムsacXY-gnt領域(177kb)の遺伝子解析の終了と今後の機能未知遺伝子の機能解析のストラテジーの模索
- 社団法人日本農芸化学会, 05 Mar. 1996, 日本農藝化學會誌, 70, 253 - 253, Japanese枯草菌sacS-gnt領域(333°-344°)のゲノム解析 : 微生物
- 社団法人日本農芸化学会, 05 Mar. 1996, 日本農藝化學會誌, 70, 255 - 255, Japanese枯草菌イノシトール資化遺伝子の機能解析 : 微生物
- The Bacillus subtilis gnt operon is negatively regulated via interaction of the gnt repressor (GntR) with an operator upstream of gntR, which is antagonized by gluconate. An 8 bp insertional operator mutation (gntOi) of the gnt operon was constructed which affected the expression level of this operon. Two suppressors of this gnt0i mutation, exhibiting normal expression, were also isolated; one involved a threonine substitution for the Ala-48 residue (gntR48T) within the helix-turn-helix DNA-binding motif of GntR, and the other an adenine substitution for the guanine at nucleotide -4 within the gnt0i operator (gntOiM4A) (+ 1 is the transcription initiation site). The gntR48T mutation by itself rendered the gnt operon partially constitutive. When the gntR43L mutation, which renders the gnt operon fully constitutive, was introduced into the gntOi or gntOiM4A mutant, the operator mutations were found not to affect the promoter activity of the gnt operon. These in vivo results indicate that the gnt0i mutation affects the operator interaction with GntR, causing a low expression level even in the presence of gluconate. In vitro gel retardation and DNase I footprint analyses demonstrated that even when gluconate was present, GntR still bound to the gnt0i operator region.SPRINGER VERLAG, Sep. 1995, MOLECULAR & GENERAL GENETICS, 248(5) (5), 583 - 591, English[Refereed]
- BACILLUS-SUBTILIS GNT REPRESSOR MUTANTS THAT DIMINISH GLUCONATE-BINDING ABILITYThe Bacillus subtilis gnt operon is negatively regulated by GntR, which is antagonized by gluconate. Three GntR mutants with diminished gluconate-binding ability were obtained. Two were missense mutants (Met-209 to Ile and Ser-230 to Leu), whereas the third had a deletion of the C-terminal 23 amino acids. The mutant GntR proteins were unable to become properly detached from the gnt operator even in the presence of gluconate.AMER SOC MICROBIOLOGY, Aug. 1995, JOURNAL OF BACTERIOLOGY, 177(16) (16), 4813 - 4816, English[Refereed]Introduction scientific journal
- 社団法人日本農芸化学会, 05 Jul. 1995, 日本農藝化學會誌, 69, 286 - 286, Japanese枯草菌イノシトールオペロンの遺伝子構造と転写制御 : 微生物
- 社団法人日本農芸化学会, 05 Jul. 1995, 日本農藝化學會誌, 69, 227 - 227, English枯草菌sacS-gnt領域(333゜-344゜)のゲノム解析 : 微生物
- CLONING AND SEQUENCING OF A 29-KB REGION OF THE BACILLUS-SUBTILIS GENOME CONTAINING THE HUT AND WAPA LOCIWithin the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 29 kb chromosome segment, which contains the hut operon (335 degrees) and the wapA gene, has been cloned and sequenced, This region (28954 bp) contains 21 complete ORFs and one partial one. The 5th, 6th and 17th genes correspond to hutH encoding histidase, hutP encoding the positive regulator for the hut operon and wapA encoding a precursor of three major wall-associated proteins, respectively. A homology search for their products deduced from the 21 complete ORFs revealed that nine of them exhibit significant homology to known proteins such as urocanase (Pseudomonas putida), a protein involved in clavulanic acid biosynthesis (Streptomyces griseus), amino acid permeases (lysine, Escherichia coli; histidine, Saccharomyces cerevisiae; and others), beta-glucoside-specific phosphotransferases (E. coli and Erwinia chrysanthemi) and 6-phospho-beta-glucosidases (E. coli and Erw. chrysanthemi). Based on the features of the determined sequence and the results of the homology search, as well as on genetic data and sequence of the hut genes reported by other groups, it is predicted that the B. subtilis hut operon may consist of the following six genes (6th-1st), the last of which is followed by a typical p-independent transcription terminator: hutP, hutH, EE57A (hutU) encoding urocanase, EE57B (hutI) encoding imidazolone-5-propionate hydrolase, EE57C (hutG) encoding formiminoglutamate hydrolase and EE57D (tentatively designated as hutM) possibly encoding histidine permease. Interestingly, the direction of transcription of these hut genes is opposite to that of the movement of the replication fork.SOC GENERAL MICROBIOLOGY, Feb. 1995, MICROBIOLOGY-UK, 141(2) (2), 337 - 343, English[Refereed]
- Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 36-kb chromosome segment, which covers the region between the gnt and iol operons, has been cloned and sequenced. This region (36447 bp) contains 33 complete open reading frames (ORFs genes) including the four gnt genes and one partial gene. A homology search for the products of the 33 complete ORFs revealed significant homology to known proteins in 16 of them such as tetracycline resistance protein (Clostridium perfringens), asparagine synthetase (Arabidopsis thaliana), aldehyde dehydrogenase (Pseudomonas oleovorans), 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (P. paucimobilis), heat shock protein HtpG (Escherichia coli), galactose-proton symporter (E. coli), auxin-induced protein (common tobacco), glucitol operon repressor (E. coli) and methylmalonate-semialdehyde dehydrogenase (P. aeruginosa). Unlike the regions we sequenced so far, this region contained two short sequence multiplications: one was a tandem sequence duplication (409 and 410 bp), and the other a triplication consisting of two highly conserved 118-bp tandem sequences preceded by a less conserved similar sequence (129 bp). The reasons for the presence of these sequence multiplications in the gnt to iol region were deduced.Universal Academy Press Inc., 1995, DNA Research, 2(2) (2), 61 - 69, English[Refereed]
- Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus. This region (23351 bp) contains 25 complete open reading frames (ORFs genes) including deoR, dra, nupC and pdp and two partial ones. The region (5140 bp) containing these four genes, being also sequenced by H. H. Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19. However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method. A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them. It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E. coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.Universal Academy Press Inc., 1995, DNA Research, 2(6) (6), 295 - 301, English[Refereed]
- CLONING AND NUCLEOTIDE SEQUENCING OF A 15 KB REGION OF THE BACILLUS-SUBTILIS GENOME CONTAINING THE IOL OPERONWithin the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced. This region (14974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase. All the genes identified are transcribed in the same direction as that of the movement of the replication fork. A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B. subtilis). and PhoB and FtsE proteins (Escherichia coli). It also implied that two genes (B65D and B65E) might encode a set of two-component regulatory proteins and that the BGSF gene might encode a protein belonging to the ATP-binding cassette (ABC) family. Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted.SOC GENERAL MICROBIOLOGY, Sep. 1994, MICROBIOLOGY-UK, 140(19) (19), 2289 - 2298, English[Refereed]
- Bacillus licheniformis was able to utilize gluconate as the sole carbon source as efficiently as Bacillus subtilis did. Southern analysis indicated that B. licheniformis likely possesses only one gnt determinant. The nucleotide sequence (6278 bp) of the B. licheniformis DNA containing the gnt operon was determined, revealing the five complete open reading frames (ORF genes). The putative product of the first gene, oug, did not show any significant homology to known proteins, but those of the second to fifth genes exhibited striking homology to the gntRKPZ genes of B. subtilis, respectively, indicating that they are the corresponding gnt genes of B. licheniformis. Not only is the organization of the gnt genes of these two Bacilli highly conserved, but so are the cis regulatory elements of their gnt operon. Sequence analysis of the upstream regions of these two gnt operons implied that a chromosome rearrangement in B. subtilis might have occurred immediately upstream of the gnt operon during evolution, causing it to diverge from a common ancestor into B. licheniformis and B. subtilis.Universal Academy Press Inc., 1994, DNA Research, 1(4) (4), 157 - 162, English[Refereed]
- TAYLOR & FRANCIS LTD, Jul. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(7) (7), 1200 - 1201, English[Refereed]Introduction scientific journal
- ACADEMIC PRESS LTD, May 1993, JOURNAL OF MOLECULAR BIOLOGY, 231(2) (2), 167 - 174, English[Refereed]Introduction scientific journal
- To delineate the mosquitocidal regions of the ISRH3 (CryIVB) and ISRH4 (CryIVA) proteins, which are two of the mosquitocidal 130-kDa proteins contained in the crystalline protein bodies (CPBs) of Bacillus thuringiensis var. israelensis (BTI), a deletion analysis of these protein genes has been done. Based on the evidence that each 130-kDa protein had two mosquitocidal regions, N-terminal and C-terminal ones, and these two regions shared a common part in the center of the 130-kDa proteins, deleted genes on this region were constructed. As the protein products which lacked the central region had reduced activities, the central region could be important for the mosquitocidal activity. The mosquitocidal and non-mosquitocidal truncated gene products of 130-kDa protein genes were also applied to a cultured lepidopteran cell line, TN-368. The mosquitocidal proteins caused the swelling and disruption of the cells in spite of the insecticidal specificity of CPBs of BTI, but the non-mosquitocidal proteins did not. Therefore, TN-368 cells were sensitive to the mosquitocidal fragments of 130-kDa proteins of BTI under the assay conditions used.TAYLOR & FRANCIS LTD, Apr. 1993, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 57(4) (4), 584 - 590, English[Refereed]
- The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho-11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an M(r) of 38 351, was determined. E. coli, bearing pIOL05d15, in which expression of the idh gene is under the control of the lac promoter of pUC118, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-beta-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same M(r) and substrate specificity as those of the B. subtilis enzyme.ELSEVIER SCIENCE BV, Dec. 1991, GENE, 108(1) (1), 121 - 125, English[Refereed]
- Joint work, Research Signpost, Oct. 2012, EnglishEscherichia coli and Bacillus subtilis; the frontiers of molecular microbiology revisitedTextbook
- Joint work, 化学同人, Mar. 2012, Japanese遺伝子工学と未来社会 「遺伝子工学」Textbook
- Joint work, 三共出版, Mar. 2012, Japaneseバイオコンバージョンによって効率的にビタミンを作る－イノシトール 「微生物機能学」Textbook
- Joint work, Springer, 2011, EnglishInositol derivatives stimulate glucose transport in muscle cells, in “Animal Cell Technology: Basic & Applied Aspects, Vol. 15, Eds. by, Koji Ikura, Masaya Nagao, Akira Ichikawa, Kiichiro Teruya and Sanetaka Shirahata, pp. 225-231.Scholarly book
- Joint work, Springer, 2010, Englishδin L6 myotubes, in "Animal Cell Technology: Basic & Applied Aspects", Vol. 16, Eds. by, Masamichi Kamihira, Yoshinori Katakura, and Akira Ito, pp.327-331, 2010.Scholarly book
- Joint work, 地人書館, Jan. 2008, Japanese微生物増殖学の現在・未来Textbook
- 日本農芸化学会 2021年度西日本・中四国・関西支部 合同大会, Sep. 2021, Japanese枯草菌接合伝達プラスミドpLS20の伝達効率の再評価Oral presentation
- Metabolic Engineering 14, Jun. 2021, EnglishMonitoring NADPH Levels by Luciferase Luminescence in Bacillus subtilis
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- 第13回日本ゲノム微生物学会年会, 2019, Japanese, Domestic conference有用希少イノシトールを生産する枯草菌細胞工場[Invited]Nominated symposium
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- 10th Conference on Recombinant Protein Production, 2019, English, International conferencemyo-Inositol-1-phosphate synthase “restored” in Bacillus subtilis to produce scyllo-inositol, a therapeutic agent for Alzheimer's disease, from glucoseOral presentation
- BACELL2019, 2019, English, International conferenceMonitoring NADPH regeneration by luciferase luminescence in Bacillus subtilisOral presentation
- The 10th International Symposium of Innovative BioProduction Kobe, 2019, English, International conferenceEngineering conjugation in Gram-positive bacteria[Invited]Nominated symposium
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- 2018年度度グラム陽性菌ゲノム機能会議, 2018, Japanese, Domestic conference枯草菌における人工的NADPH再生供給系の駆動とその効果Oral presentation
- 日本ゲノム微生物学会2017年度大会, 2018, Japanese, Domestic conferenceダイズ根粒菌のPHB蓄積制御を司るPhaRの多面的遺伝子発現調節Oral presentation
- 2018年度度グラム陽性菌ゲノム機能会議, 2018, Japanese, Domestic conferenceダイズ-ダイズ根粒菌の共生窒素固定を促進するBacillus velezensis S141のゲノム解析Oral presentation
- BACELL2018, 2018, English, International conferenceRapid conjugative mobilization of a 100 kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for selftransferOral presentation
- The Third International Symposium on Rice Science in Global Health, 2018, English, International conferenceProduction of scyllo-inositol: conversion of rice bran into a promising disease-modifying therapeutic agent for Alzheimer's diseasePoster presentation
- 2018年度度グラム陽性菌ゲノム機能会議, 2018, Japanese, Domestic conferenceGeobacillus kaustophilusの新規形質転換法の開発Poster presentation
- 9th International Symposium of Innovative BioProduction Kobe (iBioK), 2018, English, International conferenceEnzyme secretion in Bacillus subtilis enhanced by customization of signal peptides[Invited]Invited oral presentation
- Metabolic engineering 12, 2018, English, International conferenceNADPH to Improve Enzyme Performance in Bacillus SubtilisPoster presentation
- 13th European Nitrogen Fixation Conference, 2018, English, International conferenceBradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulationOral presentation
- ASBA2018, 2018, English, International conferenceBacillus subtilis engineered for production of scyllo-inositol from glucosePoster presentation
- Second Interdisciplinary and Research Alumni Symposium iJaDe2018, 2018, English, International conferenceBacillus subtilis cell factory converting agricultural wastes into rare inositol[Invited]Invited oral presentation
- Metabolic engineering 12, 2018, English, International conferenceBacillus Subtilis Cell Factories for Production of Two Inositol-Stereoisomers from GlucoseOral presentation
- 2018 iBioN, The Symposium on Biorefinery and Biprocess Topics, 2018 - Innovative Bio-production of Fuels and Chemicals, 2018, English, International conferenceBacillus subtilis cell factories converting agricultural wastes into scyllo-inositol[Invited]Invited oral presentation
- 第70回日本生物工学会大会, 2018, Japanese, Domestic conference100 kbを超える枯草菌染色体DNAセグメントの簡便迅速な接合伝達Oral presentation
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- 日本農芸化学会2017年度大会, 2017, Japanese, Domestic conference枯草菌を宿主としたL-gluconate生産系の開発Oral presentation
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- 日本農芸化学会2017年度大会, 2017, Japanese, Domestic conferenceコリネ型細菌を用いたフェルラ酸からプロトカテク酸の生産Oral presentation
- National University of Singapore, 2017, English, International conferenceRecombinant enzyme secretion in Bacillus subtilis enhanced by customization of signal peptides[Invited]Invited oral presentation
- Institut für Systembiotechnologie, Universität des Saarlandes, 2017, English, International conferenceRecombinant enzyme secretion in Bacillus subtilis enhanced by customization of signal peptides[Invited]Invited oral presentation
- 9th Conference on Recombinant Protein Production, 2017, English, International conferenceRecombinant enzyme secretion in Bacillus subtilis enhanced by customization of signal peptides[Invited]Invited oral presentation
- IUMS2017, Singapore, 2017, English, International conferenceProduction of rare inositols: conversion of agricultural wastes into value added productsOral presentation
- The 8th International Symposium of Innovative BioProduction Kobe, 2017, English, International conferenceProduction of rare inositols: conversion of agricultural wastes into value added productsOral presentation
- 2017年年度度グラム陽性菌ゲノム機能会議, 2017, Japanese, Domestic conferenceProduction of myo-inositol from glucose in Bacillus subtilisOral presentation
- 第11回ゲノム微生物学会, 2017, Japanese, Domestic conferenceLactococcus lactis subsp. cremoris FC 株のポリサッカライド合成遺伝子群の同定Poster presentation
- 2017年年度度グラム陽性菌ゲノム機能会議, 2017, Japanese, Domestic conferenceGeobacillus kaustophilus の pLS20 系プラスミド形質転換とその増殖フェーズ依存性Oral presentation
- 19th International Conference on Bacilli & Gram-Positive Bacteria, 2017, English, International conferenceGeobacillus kaustophilus Crh is independent of glucose catabolite repression but represses inositol catabolic genesOral presentation
- 日本農芸化学会2017年度大会, 2017, Japanese, Domestic conferenceChryseobacterium sp. 5-3B由来N-アセチルトランスフェラーゼの基質認識部位の探索Oral presentation
- Rector’s Lecture, Uniwersytet Mikołaja Kopernika, 2017, English, International conferenceBio-production in Kobe and a new generation of genome editing[Invited]Invited oral presentation
- Enzyme Engineering XXIV, 2017, English, International conferenceBacillus subtilis cell factory converting phytic acid into scyllo-inositol, a therapeutic agent for Alzheimer's diseaseOral presentation
- Micalis, INRA Jouy-en-Josas, 2017, English, International conferenceBacillus subtilis cell factory converting agricultural wastes into rare inositol isomer[Invited]Invited oral presentation
- 2016年年度度グラム陽性菌ゲノム機能会議, 2016, Japanese, Domestic conference納豆菌由来接合伝達プラスミドを用いたGeobacillus kaustophilusの形質転換Oral presentation
- 第68回 日本生物工学会大会, 2016, Japanese, Domestic conference納豆菌由来pLS20cat を用いた新規プラスミドベクターシステムの開発Poster presentation
- 第68回 日本生物工学会大会, 2016, Japanese, Domestic conference枯草菌のiolH がイノシトール代謝において果たす生理的意義Poster presentation
- 2016年年度度グラム陽性菌ゲノム機能会議, 2016, Japanese, Domestic conference枯草菌によるミニセルロソームの構築Poster presentation
- 第68回 日本生物工学会大会, 2016, Japanese, Domestic conferenceハイマンノース型糖鎖を有するAspergillus glaucus MA0196 由来アスパルティックプロテアーゼの特性解析Poster presentation
- 平成28年度 神戸大学大学院農学研究科 公開講座, 2016, Japanese, Domestic conferenceダイズ由来の新規健康増進化合物ピニトールの開発Public discourse
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- BACELL2016, 2016, English, International conferencePossible common regulators of ytsJ for malic enzyme and gndA for 6-phosphogluconate dehydrogenase in Bacillus subtilisOral presentation
- 2nd International Conference on Post-Translational Modifications in Bacteria, 2016, English, International conferenceInositol catabolism in Geobacillus kaustophilus is repressed by Crh phosphorylated independently of glucose catabolite repressionOral presentation
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- Metabolic Engineering 11, 2016, English, International conferenceFurther improvement of Bacillus subtilis cell factory producing scyllo-inositol, a promising therapeutic agent for Alzheimer’s diseaseOral presentation
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- 日本農芸化学会関西支部例会（第492回講演会）, Dec. 2015, Japanese, Domestic conferenceダイズ根粒菌のphaR欠損変異は多面的な表現型変化を引き起こすOral presentation
- 第67回日本生物工学会大会（2015）, Oct. 2015, Japanese, Domestic conference細菌由来ジアミントランスアミナーゼ遺伝子のクローニングと発現酵素の特性解析Poster presentation
- 第67回日本生物工学会大会（2015）, Oct. 2015, Japanese, Domestic conferenceかつお節のかび付けに使用されるAspergillus 属糸状菌由来アスパルティックプロテアーゼの特性解析Poster presentation
- 第67回日本生物工学会大会（2015）, Oct. 2015, Japanese, Domestic conferenceChryseobacterium sp. 5-3B 株由来N-アセチルトランスフェラーゼの部位特異的アミノ酸置換と機能解析Poster presentation
- 第67回日本生物工学会大会（2015）, Oct. 2015, Japanese, Domestic conferenceBradyrhizobium japonicum のPHB 蓄積に関わるファジン遺伝子の解析Poster presentation
- 2015 年度グラム陽性菌ゲノム機能会議, Aug. 2015, Japanese, Domestic conference枯草菌を用いたL-glucose 生産系の開発Oral presentation
- 2015 年度グラム陽性菌ゲノム機能会議, Aug. 2015, Japanese, Domestic conference枯草菌Rok のytsJ およびgndA プロモーターへの結合Oral presentation
- 2015 年度グラム陽性菌ゲノム機能会議, Aug. 2015, Japanese, Domestic conferenceGeobacillus kaustophilus HTA426 のIolE が転写制御に関与する可能性Oral presentation
- 8th International Conference on Gram-Positive Microorganisms, Jun. 2015, English, International conferenceEnhanced production of bio-based chemicals by using Bacillus subtilis genome reduced strain as a platform cell factoryPoster presentation
- 8th International Conference on Gram-Positive Microorganisms, Jun. 2015, English, International conferenceA third generation of Bacillus subtilis cell factory for producing scyllo-inositolPoster presentation
- 8th International Conference on Gram-Positive Microorganisms, Jun. 2015, English, International conferenceA Hyperphosphorylation of DegU interferes CcpA-dependent catabolite repression of rocG in Bacillus subtilisOral presentation
- BACELL2015, Apr. 2015, English, International conferenceHyperphosphorylation of DegU cancels CcpA-dependent catabolite repression of rocG in Bacillus subtilis[Invited]Invited oral presentation
- International Conference on Metabolic Engineering in Bacteria, Apr. 2015, English, International conferenceBacillus subtilis cell factory engineered for efficient bioconversion of myo-inositol into scyllo-inisitol, a therapeutic agent for Alzheimer's disease[Invited]Invited oral presentation
- 第9回日本ゲノム微生物学会年会, Mar. 2015, Japanese, Domestic conference枯草菌DegUの過剰なリン酸化昂進はカタボライト抑制を早期に解除するOral presentation
- 2015年度日本農芸化学会大会, Mar. 2015, Japanese, Domestic conferenceGeobacillus kaustphillusのイノシトール代謝系遺伝子群の発現制御Oral presentation
- 2015年度日本農芸化学会大会, Mar. 2015, Japanese, Domestic conferenceChryseobacterium sp. 5-3B由来N-アセチルトランスフェラーゼの発現と特性解析Oral presentation
- 日本農芸化学会関西支部例会（第487回講演会）, Dec. 2014, Japanese, Domestic conferenceアルファルファ根粒菌に見出されたイノシトール合成系に関する研究Oral presentation
- 第66回 日本生物工学会大会, Sep. 2014, Japanese, Domestic conference枯草菌フィターゼ分泌の高度効率化Poster presentation
- 2014年度グラム陽性菌ゲノム機能会議, Sep. 2014, Japanese, Domestic conference枯草菌フィターゼ分泌の高度効率化Oral presentation
- 第66回 日本生物工学会大会, Sep. 2014, Japanese, Domestic conferenceハイマンノース型糖鎖を有する Aspergillus glaucus MA0196 由来アスパルティックプロテアーゼの特性解析Poster presentation
- 2014年度グラム陽性菌ゲノム機能会議, Sep. 2014, Japanese, Domestic conferenceグルコン酸代謝システムを用いた、標的遺伝子の一時的な誘導（パルス=インダクションシステム）の構築Poster presentation
- 2014年度グラム陽性菌ゲノム機能会議, Sep. 2014, Japanese, Domestic conferenceイノシトール資化性微生物の検索と分離菌におけるiol遺伝子群の解析Poster presentation
- The 11th European Nitrogen Fixation Conference, Sep. 2014, English, Tenerife, Spain, International conferenceTo be, or not to be: that is the question. - myo-inositol in Sinorhizobium melilotiPoster presentation
- 2014年度グラム陽性菌ゲノム機能会議, Sep. 2014, Japanese, Domestic conferenceGeobacillus kaustophilus HTA426のiol遺伝子群の発現調節Oral presentation
- 第66回 日本生物工学会大会, Sep. 2014, Japanese, Domestic conferenceChryseobacterium sp. 5-3B 由来 N - アセチルトランスフェラーゼの特製解析Poster presentation
- IUMS2014, Jul. 2014, English, Montreal, Canada, International conferenceA Bacillus subtilis cell factory efficiently converting myo-inositol into scyllo-inositol, a potential therapeutic agent for Alzheimer's diseasePublic symposium
- Metabolic Engineering X, Jun. 2014, English, Cancouver, Canada, International conferenceBacterial cell factory for production of scyllo-inositol, a potential therapeutic agent for Alzheimer’s diseaseOral presentation
- BACELL2014, Apr. 2014, English, Bratislava, Slovakia, International conferenceInositol dehydrogenases in Geobacillus kaustophilus are regulated by a Crh homolog but not by glucose[Invited]Invited oral presentation
- 第8回日本ゲノム微生物学会年会, Mar. 2014, Japanese, Domestic conference枯草菌を用いたシロ-イノシトールの高効率バイオコンバージョン法の確立Oral presentation
- 2014年度日本農芸化学会大会, Mar. 2014, Japanese, Domestic conferenceアルツハイマー病への適応が期待されるシロ－イノシトールを生産する枯草菌細胞工場[Invited]Nominated symposium
- 2014年度日本農芸化学会大会, Mar. 2014, Japanese, 東京, Domestic conferenceBacillus subtilisFP-133由来耐塩性アミラーゼの特性解析Oral presentation
- One day workshop in "Lessons from interaction between plant and microbe: strategies in symbiosis and competition", Nov. 2013, English, Kobe University Brussels European Centre, International conferencePossible inositol synthesis in rhizobia with physiological significance?Nominated symposium
- BioMicroWorld 2013, Oct. 2013, English, Universidad Complutense de Madrid (Complutense University of Madrid), International conferenceTakenaka. Poly-β-hydroxybutyrate (PHB) accumulation in Bradyrhizobium japonicum depends on proteins referred to as phasinsOral presentation
- 18th International Congress on Nitrogen Fixation, Oct. 2013, English, Phoenix Seagaia Resort, Miyazaki, Japan, International conferencePhysiological significance of possible myo-inositol synthesis in Sinorhizobium melilotiOral presentation
- BioMicroWorld 2013, Oct. 2013, English, Universidad Complutense de Madrid (Complutense University of Madrid), International conferenceA Bacillus subtilis cell factory to produce syllo-inositol, a disease-modifying therapeutic agent for Alzheimer's diseaseOral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島国際会議場, Domestic conference枯草菌フィターゼ分泌の高度効率化Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conference枯草菌フィターゼ分泌の高度効率化Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conference枯草菌のNADPH再生機構．2013年度グラム陽性菌ゲノム機能会議Oral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島国際会議場, Domestic conference枯草菌によるシローイノシトール生産の効率化Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conference枯草菌アセトイン／2，3－ブタンジオール発酵の効率化Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conferenceシローイノシトールの高効率バイオコンバージョン法の確立Oral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島国際会議場, Domestic conferenceかつお節のかび付けに使用されるAspergillus glaucus MA0196由来アスパルティックプロテアーゼの特性解析Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conferenceイノシトール-1-リン酸合成酵素とイノシトールモノフォスファターゼの融合Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, English, 筑波山ホテル江戸屋, Domestic conferenceRational strategies to switch cofactor specificity of inositol dehydrogenases of Bacillus subtilisPoster presentation
- 日本農芸化学会関西・中四国・西日本支部 日本ビタミン学会近畿・中国四国・九州沖縄地区 2013年度 合同広島大会, Sep. 2013, Japanese, 広島県立大学, Domestic conferencePseudomonas aeruginosa ME-4由来エステラーゼを用いた卵殻膜の可溶化と生理活性ペプチドの探索Oral presentation
- 日本農芸化学会関西・中四国・西日本支部 日本ビタミン学会近畿・中国四国・九州沖縄地区 2013年度 合同広島大会, Sep. 2013, English, 広島県立大学, Domestic conferenceOptimizing secretion of heterologous thermostable cellulases in Bacillus subtilisOral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島国際会議場, Domestic conferenceChryseobacterium sp. 5-3由来N-アセチルトランスフェラーゼの発現と特性解析Poster presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波山ホテル江戸屋, Domestic conferenceBacillus subtilis FP-133由来C末端領域欠損アミラーゼの特性解析Poster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, International conferenceIdentification, characterization, and comparative analysis of tannnase from Lactobacillus plantarum, L. paraplantarum, and L. pentosusPoster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, Montecatini Terme, Tuscany, Italy, International conferenceEfficient bioconversion of myo-inositol to scyllo-inositol by genetically modified Bacillus suntilis requires global metabolic change to improve NADPH regenerationPoster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, Montecatini Terme, Tuscany, Italy, International conferenceCharacterization of a helotolerant extracellular amylase from Bacillus subtilis FP-133Poster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, Montecatini Terme, Tuscany, Italy, International conferenceBacillus subtilis iolH encodes an additional triosephosphate isomerasePoster presentation
- BACELL 2013, Apr. 2013, English, Newcastle University, UK, International conferenceBacillus subtilis possesses two distinct triosephosphate isomerases[Invited]Invited oral presentation
- 日本農芸化学会2013年度大会, Mar. 2013, Japanese, 東北大学, Domestic conferenceアルファルファ根粒菌のイノシトール合成系はストレスで誘導されるOral presentation
- 日本農芸化学会2013年度大会, Mar. 2013, Japanese, 東北大学, Domestic conferenceChryseobacterium sp. 5-3由来N-アセチルトランスフェラーゼの遺伝子クローニングと発現Oral presentation
- 第7回日本ゲノム微生物学会年会, Mar. 2013, Japanese, 長浜バイオ大学, Domestic conferenceBradyrhizobium japonicumのPHB蓄積に関わるパラログ遺伝子の機能解析Oral presentation
- ＪＢＡ発酵と代謝研究会講演会美味しい健康生活は微生物が作る, Feb. 2013, Japanese, 京都大学, Domestic conference枯草菌を活用する生理活性イノシトールの開発[Invited]Invited oral presentation
- 動物細胞工学会JAACT2013, 2013, Japanese, 福井, Domestic conference芳香族炭化水素受容体依存的な多剤耐性遺伝子Mdr1の転写調節機構Poster presentation
- 日本農芸化学会関西・中四国・西日本支部および日本ビタミン学会近畿・中国四国・九州沖縄地区合同大会, 2013, Japanese, 広島, Domestic conferencePseudomonas aeruginosa ME-4由来エラスターゼを用いた卵殻膜の可溶化と生理活性ペプチドの探索Oral presentation
- The 33rd International Symposium on Halogenated Persistent Organic Pollutants and POPs (DIOXIN2013), 2013, English, Daegu, Korea, International conferenceAryl hydrocarbon receptor enhances the expression of multidrug-registant Mdr1b through p53 in mouse hepatoma cellsPoster presentation
- The 4th International Symposium of Innovative Bioproduction Kobe (iBioK), Jan. 2013, English, Kobe University, International conferenceBio-production of biologically active substances: achievements and perspectives of iBioK[Invited]Nominated symposium
- The 2nd Asian Conference on Plant-Microbe Symbiosis and Nitrogen Fixation, Oct. 2012, English, Hilton Phuket Arcadia Resort & Spa, Thailand, International conferencePossible myo-inositol synthesis in Sinorhizobium melilotiPoster presentation
- The 90th Anniversary Meeting, The Society for Biotechnology, Japan, Oct. 2012, English, 神戸国際会議場, International conferenceBacillus subtilis cell factory for production of scyllo-inositol promising for Alzheimer’s disease[Invited]Nominated symposium
- 植物微生物研究会第22回研究交流会, Sep. 2012, Japanese, 神戸大学, Domestic conferenceアルファルファ根粒菌におけるイノシトール合成系の存在と意義Oral presentation
- 10th European Nitrogen Fixation Conference, Sep. 2012, English, Munich University, International conferencePhaP phasins of Bradyrhizobium japonicum playing an important role in poly--hydroxybutyrate synthesisPoster presentation
- 10th European Nitrogen Fixation Conference, Sep. 2012, English, Munich University, International conferencePhaP phasins of Bradyrhizobium japonicum playing an important role in poly-beta-hydroxybutyrate synthesisPoster presentation
- 平成24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conference乳酸菌由来タンナーゼの配列多様性と酵素活性に関する研究Poster presentation
- 24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conference新規イノシトール合成酵素の特性解析と応用Poster presentation
- 24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conference枯草菌におけるNADPH再生バランス機構の理解とその応用平成Poster presentation
- 24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conference枯草菌iolHの生理学的及び酵素学的機能の解明Poster presentation
- 平成24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conferenceGeobacillus kaustophilus HTA426が有する3種イノシトール脱水素酵素の生理的意義Oral presentation
- 24年度グラム陽性菌のゲノム生物学研究会, Aug. 2012, Japanese, 焼津グランドホテル, Domestic conferenceBacillus subtilis FP-133由来耐塩性アミラーゼの特性解析Poster presentation
- Metabolic Engineering IX: Metabolic Engineering and Synthetic Biology, Jun. 2012, English, Biarritz, France, International conferenceA cell factory of Bacillus subtilis engineered for the simple bioconversion of myo-inositol to scyllo-inositol, a potential therapeutic agent for Alzheimer's diseasePoster presentation
- BACELL 2012, Apr. 2012, English, Trinity College, Dublin, Ireland, International conferenceBacillus subtilis cell factory producing scyllo-inositol, a potential therapeutic agent for Alzheimer’s disease[Invited]Invited oral presentation
- 第6回日本ゲノム微生物学会年会, Mar. 2012, Japanese, 立教大学, Domestic conference人為的なイノシトール異性体バイオコンバージョンが誘発するトランスクリプトーム変動Oral presentation
- 日本農芸化学会2012年度(平成24年度)大会, Mar. 2012, Japanese, 京都女子大学, Domestic conferenceバクテリア細胞内でのイノシトール立体異性体相互変換の可能性Oral presentation
- 日本農芸化学会2012年度(平成24年度)大会, Mar. 2012, Japanese, 京都女子大学, Domestic conferenceChryseobacterium so. 5-3B由来N-アセチルトランスフェラーゼの特性解析Oral presentation
- 日本農芸化学会2012年度(平成24年度)大会, Mar. 2012, Japanese, 京都女子大学, Domestic conference4－アミノピリジンの分解に関わる微生物群集の組成と分解経路の推定Oral presentation
- 2011年度日本農芸化学会関西支部第472回講演会, Dec. 2011, Japanese, 神戸大学, Domestic conference根粒菌に見出されたミオ-イノシトール-1-リン酸合成酵素Oral presentation
- 日本生物工学会 第1回代謝工学研究部会シンポジウム, Nov. 2011, Japanese, 大阪大学, Domestic conference枯草菌の代謝改変による有用希少イノシトールの生産[Invited]Nominated symposium
- 2011年度日本農化会関西・中部支部合同大会, Oct. 2011, Japanese, 京都大学, Domestic conference枯草菌によるイノ シトール異性体変換の効率化Oral presentation
- 2011年度日本農化会関西・中部支部合同大会, Oct. 2011, Japanese, 京都大学, Domestic conferenceかつお節のかび付けに使用されるAspergillus glaucus MA0196由来アスパルティックプロテアーゼの精製と特性解析Oral presentation
- 灘酒研究会講演会, Sep. 2011, Japanese, 灘酒研究会, Domestic conference納豆菌を使った健康増進成分の生産についてPublic discourse
- 第63回日本生物工学会大会, Sep. 2011, Japanese, 東京農業工業大学, Domestic conferenceかつお節のかび付けに用いられるAspergillus repens MK82 由来アスパルティックプロテアーゼII の精製と特性解析Oral presentation
- 植微微生物研究会 研究交流会, Sep. 2011, Japanese, 岡山大学, Domestic conferenceSinorhizobium melilotiのミオ-イノシトール-1-リン酸合成酵素Poster presentation
- 日本ゲノム微生物学会「ゲノム微生物学会ワークショップ -ゲノムで繋がる微生物研究の新展開-」, Sep. 2011, Japanese, Domestic conferenceGeobacillus kaustophilus HTA426 の３種のイノシトール脱水素酵素パラログPoster presentation
- 第63回日本生物工学会大会, Sep. 2011, Japanese, 東京農業工業大学, Domestic conferenceGeobacillus kaustophilus HTA426 のイノシトール脱水素酵素遺伝子の解析Oral presentation
- IUMS2011, Sep. 2011, English, Sapporo COnvention Center, International conferenceGene cloning and expression of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4Poster presentation
- IUMS2011, Sep. 2011, Japanese, Sapporo Convention Center, International conferenceBacillus subtilis cell factory for production of rare inositols[Invited]Nominated symposium
- 平成23年度グラム陽性菌ゲノム機能会議, Aug. 2011, Japanese, 福山大学, Domestic conference枯草菌によるイノシトール異性体変換の精密測定と効率化条件の検討Oral presentation
- 平成23年度グラム陽性菌ゲノム機能会議, Aug. 2011, Japanese, 福山大学, Domestic conference枯草菌iolH遺伝子の酵素学的及び生理学的機能の解明Poster presentation
- 平成23年度グラム陽性菌ゲノム機能会議, Aug. 2011, Japanese, 福山大学, Domestic conference枯草菌degU32変異によるカタボライト抑制の積極的解除Oral presentation
- 平成23年度グラム陽性菌ゲノム機能会議, Aug. 2011, Japanese, 福山大学, Domestic conferenceGeobacillus kaustophilus HTA426の3種iolGパラログが同時発現する意義の考察Poster presentation
- 平成23年度グラム陽性菌ゲノム機能会議, Aug. 2011, Japanese, 福山大学, Domestic conferenceBacillus subtilis FP-133由来耐塩性酵素の特性解析Oral presentation
- 6th International Conference on Gram-positive Microorganisms, Jun. 2011, English, Montecatini Terme, Tuscany, Italy, International conferenceThree functional inositol dehydrogenases of Geobacillus kaustophilus HTA426 encoded by a single operonPoster presentation
- 6th International Conference on Gram-positive Microorganisms, Jun. 2011, English, Montecatini Terme, Tuscany, Italy, International conferenceCharacterization of halotolerant extracellular enzymes from Bacillus subtilis FP-133Poster presentation
- 6th International Conference on Gram-positive Microorganisms, Jun. 2011, English, Montecatini Terme, Tuscany, Italy, International conferenceBacillus subtilis cell factory for bio-productionPoster presentation
- 日本農芸化学会大会, Mar. 2011, Japanese, 京都大学, Domestic conferenceかつお節のかび付けに使用されるAspergillus glaucus MA0196由来アスパルティックプロテアーゼの精製と特性解析Oral presentation
- 第62回日本生物工学会大会, Oct. 2010, Japanese, (社)日本生物工学会, 宮崎市, Domestic conference有用希少イノシトールのバイオアベイラビリティーとバイオコンバージョン生産Oral presentation
- 第33回日本分子生物学会年会・第83回日本生化学会大会合同大会（BMB2010）, 2010, Japanese, Domestic conferenceマウス肝腫瘍由来Hepa-1c1c7細胞におけるアリール炭化水素受容体 (AhR) を介した多剤耐性遺伝子mdr1bの発現誘導Poster presentation
- 日本農芸化学会2010年度大会, 2010, Japanese, Domestic conferenceパスウエイデザイン枯草菌による2種の有用希少イノシトールの選択生産Oral presentation
- 日本農芸化学会2009年度大会, 2009, Japanese, Domestic conference納豆発酵におけるγ-PGA生産に重要な役割を果たす納豆菌菌体外プロテアーゼの解析Oral presentation
- 2009年度グラム陽性細菌ゲノム機能会議, 2009, Japanese, Domestic conferencePoly-γ-glutamate production during natto fermentation requires the functional extracellular alkaline protease AprEOral presentation
- 第32回日本分子生物学会年会, 2009, Japanese, Domestic conference枯草菌転写因子DegUによるグルタミン酸脱水素酵素遺伝子rocGの転写制御Oral presentation
- 第3回日本ゲノム微生物学会年会, 2009, Japanese, Domestic conference枯草菌をモデルとした新規抗菌薬剤の作用メカニズム解析Oral presentation
- 第32回日本分子生物学会年会, 2009, Japanese, Domestic conference枯草菌のscyllo-inositol脱水素酵素遺伝子とその転写調節因子の同定Poster presentation
- 日本食品学工学会第56回大会, 2009, Japanese, Domestic conference筋肉組織における糖輸送担体GLUT4の細胞膜移行に及ぼすプロポリス抽出物の影響についてOral presentation
- 第14回日本フードファクター学会, 2009, Japanese, Domestic conference筋肉細胞におけるアシル化カテキンによるGLUT4膜移行促進効果とその作用機構についてPoster presentation
- 日本農芸化学会2009年度大会, 2009, Japanese, Domestic conferenceヨモギ抽出物によるGLUT4膜移行促進作用機構の解明Oral presentation
- 日本農芸化学開関西支部第462回講演会, 2009, Japanese, Domestic conferenceダイズ由来ピニトールの血糖値降下作用と肥満抑制Oral presentation
- BACELL2009, 2009, English, International conferencescyllo-Inositol metabolism in Bacillus subtilisOral presentation
- 日本農芸化学会2009年度大会, 2009, Japanese, Domestic conferenceL6筋管細胞におけるアシルカテキンによるインスリン応答性糖輸送担体(GLUT4)の細胞膜移行促進効果Oral presentation
- 日本農芸化学会2009年度大会, 2009, Japanese, Domestic conferenceGeobacillus kaustophilusのイノシトール資化不全変異株取得Oral presentation
- 2009年度グラム陽性細菌ゲノム機能会議, 2009, Japanese, Domestic conferenceThree functional inositol dehydrogenase paralogs encoded by a single operon of Geobacillus kaustophilus HTA426Oral presentation
- 第32回日本分子生物学会年会, 2009, Japanese, Domestic conferenceGeobacillus kaustophilus HTA426の3種のイノシトール脱水素酵素をコードするオペロンPoster presentation
- The 4th International Conference on Polyphenols and Health, 2009, English, International conferenceEpigallocatechin-3-gallate regulate glucose metabolism in skeletal muscle cellsPoster presentation
- 5th International Conference on Gram-positive Microorganisms, 2009, English, International conferenceA bioconversion process to produce scyllo-inositol, a promising drug candidate for Alzheimer's diseasePoster presentation
- 5th International Conference on Gram-positive Microorganisms, 2009, English, International conference2’,3’,4’-Trihydroxy-2- phenylacetophenone derivatives, a novel and potent class of anti-Gram-positive antibacterial agentsOral presentation
- 7th European Nitrogen Fixation Conference, Jul. 2006, English, 7th European Nitrogen Fixation Conference, Aarhus Denmark, International conferenceFunctional analysis of NodD transcription factor paralogs of Sinorhizobium fredii USDA191 involved in regulation of the nodulation genesPoster presentation
- 2005 International chemical congress of pacific basin societies (PACIFICHEM 2005) December 15th-20th, Programp.4TECH, #162, Abstract is available on CD., Dec. 2005, English, 未記入, Honolulu, Hawaii, International conferencePrevention of dioxin toxicity by food factoresOral presentation
- Rikkyo International Symposium ' From bacteria to organelle', Aug. 2005, English, 立教大学理学部, 東京, Domestic conferenceFunctional analysis of NodD transcription factor paralogs of Shinorhizobium fredii USDA191 involved in regulation of the nodulation genes.Oral presentation
- 2005年度日本農芸化学会大会講演要旨集, Mar. 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference茶の飲用はアリール炭化水素受容体の活性化を抑制するOral presentation
- 日本農芸化学会第438回講演会 講演要旨集p.7, 2005, Japanese, 日本農芸化学会, 京都, Domestic conference紅茶の飲用がラットのインスリン感受性組織の脂質代謝に及ぼす影響Oral presentation
- 日本農芸化学会2005年度大会 大会講演要旨集p.65, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference枯草草の薬剤耐性に関与する転写因子のレギュロン機能解析Oral presentation
- 日本農芸化学会2005年度大会 大会講演要旨集p.224, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference枯草菌イノシトール分解系を応用したD-chiro-inositol の発酵生産Oral presentation
- 日本農芸化学会2005年度関西・中四国・西日本支部合同大会 講演要旨集p.82, 2005, Japanese, 日本農芸化学会, 大阪, Domestic conference枯草菌イノシトール分解系に関与するiolG とiolI の新規機能Oral presentation
- 第28回日本分子生物学会年会 講演要旨集p.415, 2005, Japanese, 日本分子生物学会, 博多, Domestic conference枯草菌HTH蛋白質の機能解析－脂肪酸分解に関わるHTH転写制御因子の解析Poster presentation
- 日本動物細胞工学会2005年度大会 講演要旨集p.58, 2005, Japanese, 日本動物細胞工学会, 東京, Domestic conferenceモロヘイヤはアリール炭化水素受容体の形質転換を抑制するOral presentation
- 日本農芸化学会2005年度関西・中四国・西日本支部合同大会 講演要旨集p.75, 2005, Japanese, 日本農芸化学会, 大阪, Domestic conferenceフラボノイド類とアリール炭化水素受容体との相互作用についてOral presentation
- 第28回日本分子生物学会年会 講演要旨集p.439, 2005, Japanese, 日本分子生物学会, 博多, Domestic conferenceダイオキシン受容体AhRの大腸菌内での発現精製とその機能解析Poster presentation
- 第20回香辛料研究会 講演要旨集p.25, 2005, Japanese, 日本香辛料研究会, 京都, Domestic conferenceクルクミンのダイオキシン毒性抑制効果についてOral presentation
- 日本農芸化学会2005年度大会 大会講演要旨集p.285, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conferenceカテキンがインスリン応答性糖輸送活性に及ぼす影響Oral presentation
- 第10回日本フードファクター学会（JSoFF） 講演要旨集p.61, 2005, Japanese, 日本フードファクター学会, 岡山, Domestic conferenceインジゴイドがアリール炭化水素受容体の形質転換に及ぼす影響についてOral presentation
- 第10回日本フードファクター学会（JSoFF） 講演要旨集p.53, 2005, Japanese, 日本フードファクター学会, 岡山, Domestic conferenceイノシトール分解系を応用したピニトール強化納豆の作製Oral presentation
- 日本農芸化学会第438回講演会 講演要旨集p.8, 2005, Japanese, 日本農芸化学会, 京都, Domestic conferenceアントラキノン類が示す新規生理活性：グリコース輸送担体の機能変調Oral presentation
- 第28回日本分子生物学会年会 講演要旨集p.479, 2005, Japanese, 日本分子生物学会, 博多, Domestic conferenceアリール炭化水素受容体複合体に対するフラボノイド類の作用機序の解明Poster presentation
- 日本農芸化学会2005年度大会 大会講演要旨集p.118, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conferenceアリール炭化水素受容体の形質転換に影響をおよぼす植物の検索Oral presentation
- 13th International Conference in Bacilli, Abstract book T79, 2005, English, 未記入, San Diego, California, International conferenceTranscription of Bacillus subtilis asnH operon under the dual control of AbrB abd CodY is stabilized by the 5'-untranslated region of its transcript containing a long sequence triplicationOral presentation
- 第15回植物微生物研究会, 2005, Japanese, 植物微生物研究会, 高松, Domestic conferenceSinorhizobium fredii USDA191のnodD1/nodD2パラログの機能解析Oral presentation
- 第15回植物微生物研究会, 2005, Japanese, 植物微生物研究会, 高松, Domestic conferenceSinorhizobium fredii USDA191 NodD1 の大腸菌内での発現精製Oral presentation
- 日本農芸化学会2005年度大会 大会講演要旨集p.99, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference(-)-エピガロカテキンガレートとアリール炭化水素受容体との相互作用についてOral presentation
- 第1回機能性食品開発研究会, Nov. 2004, Japanese, 未記入, 大阪商工会議所, Domestic conference納豆菌（枯草菌）のイノシトール分解系の全貌解明Oral presentation
- 近畿地域アグリビジネス創出フェア, 2004, Japanese, 未記入, 未記入, Domestic conference神戸大学農学部生物機能化学科生物機能開発化学教育研究分野Oral presentation
- 2004年度日本農芸化学会大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference枯草菌窒素代謝制御因子TnrAによるilv-leuオペロンの制御Oral presentation
- 第2回情報生命学研究交流会, 2004, Japanese, 未記入, 未記入, Domestic conference枯草菌機能未知HTH制御因子のレギュロン解析Oral presentation
- グラム陽性菌のゲノム生物学研究会T02, 2004, Japanese, 未記入, 未記入, Domestic conference枯草菌の薬剤耐性に関与する転写制御因子の探索とそのレギュロンの解析Oral presentation
- 2004年度日本農芸化学会大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference枯草菌の薬剤耐性に関与する可能性のある転写制御因子の制御ターゲットの探索Oral presentation
- 岡山・島根・鳥取大学交流会, 2004, Japanese, 未記入, 未記入, Domestic conference枯草菌のグルタミン依存性アスパラギン合成酵素パラログの発現制御と機能の解析Oral presentation
- グラム陽性菌のゲノム生物学研究会T21, 2004, Japanese, 未記入, 未記入, Domestic conference枯草菌のグルタミン依存性アスパラギン合成酵素パラログの発現制御と機能の解析Oral presentation
- 日本農芸化学会第437回講演会, 2004, Japanese, 日本農芸化学会, 神戸大学, Domestic conference枯草菌のグルタミン依存性アスパラギン合成酵素パラログの機能と発現制御の解析Oral presentation
- 2004年度日本農芸化学会大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference枯草菌のクエン酸サイクルに関与する遺伝子発現制御因子のマイクロアレイ解析Oral presentation
- はりま産学交流会・拡大一日神戸大学神戸大学の知的資産の競演、あなたが選ぶ!!シーズコンペ!!, 2004, Japanese, 未記入, 未記入, Domestic conference枯草菌のイノシトール分解系の全貌解明とその応用Oral presentation
- 2004年度日本農芸化学会大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference枯草菌のイノシトール分解系の逆遺伝学～遺伝子機能の同定と転写制御機構の解明～Oral presentation
- 2004年度日本農芸化学会大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference枯草菌asnHオペロンの発現調節Oral presentation
- 第9回日本フードファクター学会, 2004, Japanese, 日本フードファクター学会, 淡路夢舞台国際会議場, Domestic conference筋肉細胞のグルコース取り込み活性に及ぼすアントラキノン類の影響Oral presentation
- 奈良先端大Bio-COEセミナー, 2004, Japanese, 未記入, 未記入, Domestic conference逆遺伝学的アプローチによる枯草菌イノシトール分解系の解明Oral presentation
- ミニ国際シンポジウム：マリンゲノムの新展開「深海微生物のゲノム生物学」, 2004, Japanese, 未記入, 未記入, Domestic conferenceモデル微生物としての枯草菌～ポストゲノム時代の逆遺伝学研究～Oral presentation
- International Conference of O-CHA(tea) Culture and Science., 2004, English, 未記入, 未記入, International conferenceTea has the potential to reduce the dioxin risk.Oral presentation
- 2004 International Conference On O-CHA(tea) Culture And Science, 2004, English, 未記入, 未記入, International conferenceTea has the potential to reduce the dioxin risk.Oral presentation
- International Conference of O-CHA(tea) Culture and Science., 2004, English, 未記入, 未記入, International conferenceSuppressive effects of catechins on differentiation of 3T3-L1 preadipocytes.Oral presentation
- 2004 International Conference On O-CHA(tea) Culture And Science, 2004, English, 未記入, 未記入, International conferenceSuppressive effects of catechins on differentiation of 3T3-L1 preadipocyte.Oral presentation
- 奈良女子大学理学部外来セミナー, 2004, English, 未記入, 未記入, Domestic conferenceReverse genetics of myo-inositol catabolism in bacteria.Oral presentation
- 第27回日本分子生物学会, 2004, Japanese, 日本分子生物学会, 神戸, Domestic conferenceIdentification of a 2-keto-myo-inositol dehydratase gene of Shinorhizobium fredii USDA191Oral presentation
- International Conference of O-CHA(tea) Culture and Science., 2004, English, 未記入, 未記入, International conferenceBlack tea (Cameria sinensis) suppresses hyperglycemia in STZ-induced diabetic rats.Oral presentation
- 科学研究費補助金／基盤研究(B), Apr. 2018 - Mar. 2022, Principal investigatorCompetitive research funding
- JSPS, bilateral programs joint projects/seminars, Kobe University, Apr. 2019 - Mar. 2021, Principal investigatorTransforming functional bacterial communities: how does horizontal genetic transfer impact the functional society of bacteria?
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), University of Tsukuba, 01 Apr. 2016 - 31 Mar. 2020"A Sweet and Calorie-free Bacillus!?": Production of L-glucose in B. subtilisIn this study we aimed to construct an L-glucose production system in Bacillus subtilis by heterologous expression of iolM, iolN and lgnH genes to produce L-gluconate from myo-inositol. By using synthetic genes optimized for the codon usages of B. subtilis and a strong promoter, these genes can be expressed in B. subtilis at some extent. We introduced these three genes as an operon on a plasmid to B. subtilis strain deficient for intrinsic inositol catabolic genes, however, conversion of myo-inositol to L-gluconate was not observed. It may be due to low expression of these genes. On the other hand, to obtain a mutant protein of LgdA (L-glucose dehydrogenase) having reverse reaction activity, we have determined its three-dimensional structure. We identified several residues for substrate binding, of which R178 was proven to be essential for inositol dehydrogenation activity, but not for L-glucose dehydrogenation.
- 学術研究助成基金助成金／挑戦的研究（萌芽）, Apr. 2017 - Mar. 2019, Principal investigatorCompetitive research funding
- 学術研究助成基金助成金／挑戦的萌芽研究, Apr. 2014 - Mar. 2016, Principal investigatorCompetitive research funding
- 独立行政法人日本学術振興会, 二国間交流事業（オープンパートナーシップ共同研究）, 2016, Principal investigator好熱バチルス細胞工場を実現する新規遺伝子操作技術の開発Competitive research funding
- 日本学術振興会, 二国間交流事業（オープンパートナーシップ共同研究）, 2015, Principal investigator二国間交流「好熱バチルス細胞工場を実現する新規遺伝子操作技術の開発」Competitive research funding
- 二国間交流事業（オープンパートナーシップ共同研究）, 2014, Principal investigator二国間交流「好熱バチルス細胞工場を実現する新規遺伝子操作技術の開発」Competitive research funding
- 科学研究費補助金／基盤研究(B), 2010, Principal investigatorCompetitive research funding
- 科学研究費補助金／萌芽研究, 2008, Principal investigatorCompetitive research funding
- 科学研究費補助金／特定領域研究, 2008, Principal investigatorCompetitive research funding
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Fukuyama University, 2005 - 2007Strategic disinterment of bacterial potential drug-resistance genesAs part of the comprehensive function analysis of the HTH-regulatory proteins in the framework of Bacillus subtilis post-genome project, we conducted the function analysis of the regulons governed by the MarR, MerR and TetR families of the HTH-regulatory proteins, the targets of which tend to be drug-resistance genes. Out of them, we list those which are the prominent achievements from their analysis. (i) The YsiA regulon was found to be involved in fatty acid β-oxidation, whose members are five operons (ysiAB-etfBA, ykuFG, yhfL, yusLKJ, and ywjF-acdA-rpoE). Each of these operons carry a YsiA-box in its promoter region to which YsiA binds. The in vivo and in vitro analyses revealed that long chain fatty acyl-CoAs with 14 to 20 carbon atoms antagonized the YsiA protein. (ii) The LmrA/YxaF regulon comprises lmrAB, yxaGH, and yxaF was found to be involved in flavonoid degradation as well as lincomycin resistance, which were dually regulated by the LmrA and YxaF proteins. It was considered that lincomycin resistance is not induced by lincomycin, but the lmrB gene responsible to lincomycin exhaust is likely related to flavonoid degradation by chance. (iii) The YkvE regulon comprising ykcABC, ydfNOP, yodED, and yvaB is likely induced by chlorohydroquinone. (iv) The yhblJ-yhcAB operon was induced by indole acetic acid, which is repressed by YhbI. This operon is likely involved in indole acetic acid secretion. (v) The analysis of multi-drug resistant operons revealed that YcnC, YusO, and YwoH negatively regulate ycnCB, yusOP, and ywoHG, respectively.
- 日本学術振興会, 科学研究費助成事業, 若手研究(A), 2002 - 2004枯草菌のアスパラギン生合成の逆遺伝学的研究枯草菌等グラム陽性菌のアスパラギン合成は研究の例がなく、その機構と生理的意義の解明が待たれる。本研究課題は、枯草菌ゲノムに見出された3種のアスパラギン合成酵素様遺伝子asnB, asnH, asnOの発現調節と生理的意義の解明を目指す。本年度はasnHの発現調節機構の解析とasnOの機能解析をさらに推進した。 asnHを含むオペロンは、細胞内の栄養状態を感知するCodYと遷移期の(胞子形成開始期を含む)遺伝子発現を司るArbBによって転写調節される。このオペロンの転写産物の5'-UTRに存在する3回リピート配列(各約120bp)が、上記の調節に関与しているかどうか検討した。その結果、3回リピート配列はCodY, AbrBどちらの調節にも関係しておらず、むしろ転写産物を安定化させる機能を担うことが示唆されたので、この点の詳細を検討した。 昨年度、放射ラベルされたアスパラギン酸を基質として大腸菌や枯草菌の細胞抽出物を作用させ、生産されるアスパラギンを定量するアッセイ系を確立した。本年度はこのアッセイ系を利用して、asnOの担う胞子形成期のアスパラギン合成活性を評価した結果、asnOが実際にアスパラギン合成活性を担うことが強く示唆された。また、大腸菌のasnBをasnOに代えて発現させた場合にはアスパラギン合成の増強が見られたが、この発現によってasnO欠損による胞子形成不全は補れなかった。即ち、asnOの胞子形成への寄与はアスパラギン合成以外の何らかの機能によるものであると示唆された。詳細な顕微鏡観察の結果、asnO変異株の胞子は一旦脱水したのちに速やかに吸水し、そのことが耐熱性の消失につながる可能性が考えられた。そこで胞子の吸水を阻止することが知られるpdaA変異を追加導入したところ、asnO変異株の胞子の吸水が見られなくなった。しかし、この2重変異株の胞子はpdaAの単一変異のそれよりも耐熱性が低く、即ち胞子の吸水だけではasnO変異の影響を説明できないことがわかった。
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research on Priority Areas, Fukuyama University, 2000 - 2004Investigation of Comprehensive Vision of Transcriptional regulation in Bacillus subtilisMore than 500 Bacillus subtilis DNA microarray data were obtained, using the cells grown in various cultivation conditions and exposed to numerous environmental stresses, to perform gene clustering of altered genes, and using the transcriptional regulatory mutants, to extract the target gene candidates of the transcriptional regulatory factors. A fundamental network of metabolic regulation concerned with the biosynthesis of branched-chain amino acids involving carbon and nitrogen regulations by CcpA, CodY and TnrA and stringent control conducted by RelA, was revealed from the data of gene clustering and identification of target gene candidates. On the base of the DNA microarray data of DNA-binding transcriptional regulatory factors, we completed comprehensive molecular genetic analysis of ECF-sigma factors, and almost completed that of 34 two-component regulatory systems. Consequently, the function of only several two-component systems remained to be unknown. Moreover, we performed the molecular genetic analysis of the MarR, TetR and MerR families of HTH-DNA binding regulatory proteins among more than 200 proteins, for which the DNA maicroarray analysis of were almost completed, identifying at least one target gene of almost all proteins of the three families. Especially, it is notable that we found that YsiA protein was involved in global fatty acid degradation regulation. These results of the five-year project, unveiling a broad outline of the transcriptional network of B. subtilis, was published in 39 scientific papers listed below together with more than 100 of presentations at scientific meetings.
- 日本学術振興会, 科学研究費助成事業, 奨励研究(A), 福山大学, 2000 - 2001枯草菌のイノシトール分解系の解明枯草菌はイノシトールを炭素源として利用するための特殊な分解系を持つ。その分解系遺伝子はiol operonを構成しており、イノシトールの存在下で誘導される。筆者は枯草菌イノシトール分解系の分子生物学的解明を目指した。iolGが分解系の初発反応を担うinositol dehydrogenaseを、iolRが転写制御を担うrepressorをコードすることは既知であり、イノシトールの分解過程で生成する誘導物質がIolRをオペレーターから解離させるものと想定される。そこでまず誘導物質生成までの過程とそれ以降に関わる遺伝子を区別する実験系を確立した結果、誘導物質生成に不可欠なのはiolBCDEGの5遺伝子であることがわかった。そしてこれらの各遺伝子を大腸菌内で発現させ産物が示す酵素活性を検討し、iolEが2段階目の反応を担うinosose dehydrataseを、iolDが3段階目のdiketodeoxyinositol hydrolaseをコードすることを明らかにした。本年度はこの第3段階目の反応産物deoxyketohexonate(DKH)を精製することに成功し、その化学構造を詳細にする研究に着手することができ今後の結果が期待される。残るiolBCが関与する反応により誘導物質が生成されるものと考えらたので、これら2つの遺伝子をそれぞれ、また同時に発現させて酵素活性を調べたところ、これらの遺伝子産物は同時発現したときにのみ,複合体を形成し第4段階のDKH kinaseとしての酵素活性を発揮することがわかった。さらにこの反応産物(おそらくDKHP)はIolRとオペレーターDNAとの特異的な結合を阻害することを見出し、DKHPが細胞内の誘導物質として機能することを明らかにした。以上、本研究を通じ枯草菌のイノシトール分解系の細胞内誘導物質の生成までの反応系を分子生物学的に詳細に解明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, 奨励研究(A), 福山大学, 1998 - 1999枯草菌のイノシトール分解素の解明枯草菌等の微生物は特殊なイノシトール分解系を発達させている。枯草菌のイノシトール分解系遺伝子はiol divergon(iolABCDEFGHIJ&iolRSoperons)を構成し、イノシトールの存在下で誘導される。筆者は枯草菌イノシトール分解系の分子生物学的解明を目指した。iolGが分解系の初発反応を担うinositol dehydrogenaseを、iolRが転写制御を担うrepressorをコードすることは既知であり、イノシトールの分解過程で生成する誘導物質がIolRをオペレーターから解離させるものと想定される。そこでまず誘導物質生成までの過程とそれ以降に関わる遺伝子を区別する実験系を確立した結果、誘導物質生成に不可欠なのはiolBCDEGの5遺伝子であることがわかった。そしてこれらの各遺伝子を大腸菌内で発現させ、その産物の示す酵素活性を詳細に検討したところ、iolEが2段階目の反応を担うinosose dehydrataseを、iolDが3段階目のdiketodeoxyinositol hydrolaseをコードすることが明らかとなった。従って、残るiolBCが関与する反応により誘導物質が生成されるものと考えらる。一方、本研究の遂行過程でIolRの制御下にある新規遺伝子iolTを染色体上のiol divergonとはかけ離れた位置に発見した。この発見はIolR傘下の遺伝子発現制御ネットワーク"iol regulon"の存在を示唆した。iolTを破壊するとイノシトールの取り込み能力が著しく低下したことから、iolTはイノシトールの主たる取り込みを担う輸送蛋白をコードすると考えられた。またiol divergonに含まれるiolFもこの取り込みに部分的に関与することもわかり、iol regulonには少なくとも2つの輸送系が含まれ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, 奨励研究(A), 福山大学, 1995 - 1995枯草菌イノシトールオペロンの遺伝子構造と発現調節機構の解明枯草菌イノシトールオペロンの遺伝子構造と発現制御機構を明らかとするため以下の研究を行った。本オペロンはiolABCDEFGHIJの10個の遺伝子より構成されるものと推定され、またその上流に逆向きに存在する遺伝子がリプレッサー遺伝子iolRであることが分かっていた。 1.イノシトールオペロンのプロモーターの解析 イノシトールの添加により転写誘導される本オペロンのプロモーターとiolRのプロモーターを同定した。iolRはその下流のiolSと共に約2kbの転写産物として発現していた。 2.オペロンの転写終結点の同定 転写終結点をiolJ遺伝子の直後に同定した。この位置で終結する転写は本オペロンプロモーターからの転写であり、従って本オペロンは10個のiol遺伝子をコードする約11.5kbの非常に長い転写単位として発現していることが分かった。 (平成8年3月現在、以上2点に関する論文を執筆中である。) 3.IolRの認識するオペレーターの解析 大腸菌内でIolRを生産しDNase I foot printでプロモーターDNAとの結合を調べた結果、IolRは特異的にプロモーター領域に結合し約100bpもの長い領域をカバーする事が分かった。またDNA二重螺旋のほぼ1周期ごとに切断が増強された。IolRを精製し詳細な解析を試みている。 4.イノシトールオペロン遺伝子の機能解析 枯草菌のイノシトール分解系は未だ不明である。各遺伝子の機能を同定し分解系の詳細を明らかにする第一歩として10種の変異株の変異点を同定し、変異株の取れない遺伝子は遺伝子破壊を行った。これら変異株、破壊株を利用して各遺伝子の機能解析を進めている。
- 日本学術振興会, 科学研究費助成事業, 重点領域研究, 福山大学, 1990 - 1992枯草薗グルコン酸オペロンのリプレッサー蛋白質の構造と機能GntR蛋白の機能構造を明らかにするために主として次の2つの解析方法で研究した。1つは、分子遺伝学的な方法でのGntR蛋白の機能構造の解析であり、もう一つの方法は、Oregon Health Sciences UniversityのBrennen博士との共同研究であるGntRの結晶化とそのX線解析である。 分子遺伝学的方法でGntR蛋白の機能ドメインの解析では、前年度に4種のDNA結合能に影響する点変異の同定に成功している。Leu-43変異はGntRのDNAへの結合能を完全に欠失させ、他の点変異(Thr-66,Lys-74とGln-75)は、DNA結合能を野生型に比して低下させた。これらの変異はすべてGntRファミリーの保存領域に存在する事が明らかになった。さらにエフェクターであるグルコン酸の結合ドメインを同定するため、レポーターであるクロラムフェニコール耐性をグルコン酸で誘導可能とした系で、ヒドロキシルアミン処理により多数の誘導不能GntR変異を得た。これらの変異GntR蛋白質遺伝子の塩基配列を決定し、2種類の点変異(Ile-209とLeu-230)とC末端から23アミノ酸の欠失変異の同定した。これらの変異はすべてGntRファミリーの非保存領域にあたるC末端側に存在するので、この領域にGntRファミリーの制御蛋白質のエフエクターとの結合ドメインが存在すると考えられるた。 Brennan博士との共同研究のGntR蛋白の結晶化では、最近PEG溶液からかなリ大型の結晶(約100-クロン)が得られたが、さらにX線解析に耐える結晶を得るベく努力している。
- 日本学術振興会, 科学研究費助成事業, 重点領域研究, 福山大学, 1990 - 1991枯草菌グルコン酸オペロンのリプレッサ-蛋白質の構造と機能本課題遂行のため、グルコン酸オペロンのリプレッサ-(GntR)蛋白の結晶化、分子遺伝学詣なGntR蛋白の機能ドメインの解析、およびGntR蛋白のDNA結合のキネテックスの解析の3手法を採用している。各々の手法で得られた成果を箇条書にする。 1.Brennan博士との共同研究のGntR蛋白の結晶化では、最近PEG溶液からかなりの良好な結晶が得られたが尚小型である。X線解析に耐える大きさの結晶を得るべく努力している。 2.分子遺伝学的方法でGntR蛋白のDNA結合ドメインを解析した成果を述べる。欠失解析の結果DNA結合能にはN末側の71アミノ酸が必要であることが明らかになった。つぎに、クロラムフエニコ-ル耐性をグルコ-ン酸で誘導可能として系で、ヒドロキシルアミン処理により多数のGntR方異を得た。この中でGntR給白の安定性に影響しない変異をSDSーPAGEにより選抜し、その変異gntRの塩基配列を決定することにより方異蛋白の置換アミノ酸を同定した。その結果、4種のDNA結合能に影響する点変異の同定に成功はた。Leuー43変異はGntRのDNAへの結合能を完全に欠失させ、他の点変異(Thrー66,Lysー74とGlnー75)は、DNA結合能を野生型に比して低下させた。これらの方異はすべてGntRフアミリ-の保存領域に存在した。従って、GntRのDNA結合ドメインはN末端側に存在し、特にこのフアミリ-の保存領域がDNA結合能に重要な役割を果していることが類推できた。 3.GntR蛋白のDNA結合のキネテックスの解析により、この蛋白のオペレ-タDNAへの結合能は誘導物質たるグルコン酸あるいはグルコノーδーラクトンが存在すると特異的に阻害されるが、逆にオペレ-タ-配列を持たないDNAへの結合能が誘起されることが判った。
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