Directory of Researchers

ISHIKAWA Shu
Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation
Associate Professor
Agriculture
Last Updated :2023/12/12

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Graduate School of Science, Technology and Innovation / Department of Science, Technology and Innovation
  • <Related Faculty / Graduate School / Others>

    Faculty of Agriculture / Department of Agrobioscience, Graduate School of Agricultural Science / Department of Agrobioscience, Engineering Biology Research Center

Teaching

Research Activities

Research Interests

  • 微生物学
  • 分子生物学
  • タンパク質複合体
  • リボゾーム
  • 遺伝子発現制御
  • 必須タンパク質
  • 転写制御
  • ゲノム
  • テイコ酸
  • GTP結合蛋白質
  • 大腸菌
  • ChIP-chip解析
  • タイリングチップ
  • 細胞骨格蛋白質
  • 細胞システム
  • システム生物学
  • 細菌細胞
  • 枯草菌
  • 生体分子
  • 細胞分裂
  • 細菌
  • 蛋白質

Research Areas

  • Life sciences / Genomics
  • Life sciences / Molecular biology
  • Life sciences / Applied microbiology

Committee Memberships

  • Apr. 2021 - Present, Journal of Bioscience and Bioengineering, Editor
  • 2015 - 2017, 日本ゲノム微生物学会, 評議員

Published Papers

  • Takahiko Kondo, Surachat Sibponkrung, Ken-yu Hironao, Panlada Tittabutr, Nantakorn Boonkerd, Shu Ishikawa, Hitoshi Ashida, Neung Teaumroong, Ken-ichi Yoshida

    Microbiology Research Foundation, 2023, The Journal of General and Applied Microbiology

    Scientific journal

  • Yuzheng Wu, Honami Kawabata, Kyosuke Kita, Shu Ishikawa, Kan Tanaka, Ken-ichi Yoshida

    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.

    Springer Science and Business Media LLC, 20 Dec. 2022, Microbial Cell Factories, 21 (1)

    Scientific journal

  • Kotaro Mori, Kaho Fukui, Ryotaro Amatsu, Shu Ishikawa, Valeria Verrone, Anil Wipat, Wilfried J. J. Meijer, Ken-ichi Yoshida

    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.

    Springer Science and Business Media LLC, Dec. 2022, Microbial Cell Factories, 21 (1)

    Scientific journal

  • Junya Yamamoto, Onuma Chumsakul, Yoshihiro Toya, Takuya Morimoto, Shenghao Liu, Kenta Masuda, Yasushi Kageyama, Takashi Hirasawa, Fumio Matsuda, Naotake Ogasawara, Hiroshi Shimizu, Ken-Ichi Yoshida, Taku Oshima, Shu Ishikawa

    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.

    27 May 2022, DNA research : an international journal for rapid publication of reports on genes and genomes, 29 (3), English, International magazine

    Scientific journal

  • Ryotaro Amatsu, Kotaro Mori, Shu Ishikawa, Wilfried Meijer, Ken-ichi Yoshida

    Geobacillus kaustophilus , a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus . The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus . Graphical abstract.

    Bio-Protocol, LLC, 2022, BIO-PROTOCOL, 12 (17), English, International magazine

    Scientific journal

  • Kotaro Mori, Valeria Verrone, Ryotaro Amatsu, Kaho Fukui, Wilfried J J Meijer, Shu Ishikawa, Anil Wipat, Ken-Ichi Yoshida

    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.

    10 Sep. 2021, Microorganisms, 9 (9), English, International magazine

    Scientific journal

  • Yuji Tsujikawa, Shu Ishikawa, Iwao Sakane, Ken-Ichi Yoshida, Ro Osawa

    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 cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. 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 that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to "monopolize" inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

    06 Aug. 2021, Scientific reports, 11 (1), 16007 - 16007, English, International magazine

    Scientific journal

  • Yayoi Gotoh, Kyosuke Kita, Kosei Tanaka, Shu Ishikawa, Toshio Suzuki, Ken-Ichi Yoshida

    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.

    30 Jul. 2021, The Journal of general and applied microbiology, English, Domestic magazine

    Scientific journal

  • Ken-ichi Yoshida, Yusuke Shirae, Ryo Nishimura, Kaho Fukui, Shu Ishikawa

    Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis . The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus . When iolQ was inactivated in G. kaustophilus , not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an 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 two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol 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 two iol operons responding to myo-inositol.

    Microbiology Society, 01 Jan. 2021, Microbiology, 167 (1), English, International magazine

    Scientific journal

  • Kyosuke Kita, Shu Ishikawa, Ken-ichi Yoshida

    ABSTRACT We report here the complete genome sequence of nitrogen-fixing Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.

    American Society for Microbiology, 03 Sep. 2020, Microbiology Resource Announcements, 9 (36)

    Scientific journal

  • Kyosuke Kita, Atsushi Ishida, Kosei Tanaka, Shu Ishikawa, Ken-ichi Yoshida

    Here, we report the complete genome sequence of 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.

    American Society for Microbiology, 23 Apr. 2020, Microbiology Resource Announcements, 9 (17)

    [Refereed]

    Scientific journal

  • Christophe Michon, Choong-Min Kang, Sophia Karpenko, Kosei Tanaka, Shu Ishikawa, Ken-Ichi Yoshida

    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.

    02 Mar. 2020, Communications biology, 3 (1), 93 - 93, English, International magazine

    [Refereed]

  • Yoshida, Ken-ichi, Ishikawa, Shu

    2019, J Nutr Sci Vitaminol, 65, S139 - S142, English

    [Refereed]

  • Nishihata Shogo, Kondo Takahiko, Tanaka Kosei, Ishikawa Shu, Takenaka Shinji, Kang Choong-Min, Yoshida Ken-ichi

    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 p

    BMC, Oct. 2018, BMC Microbiology, 18, English

    [Refereed]

    Scientific journal

  • Miyano Megumi, Tanaka Kosei, Ishikawa Shu, Mori Kotaro, Miguel-Arribas Andres, Meijer Wilfried J. J, Yoshida Ken-ichi

    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 Geobac

    BMC, Aug. 2018, Microbial Cell Factories, 17, English

    [Refereed]

    Scientific journal

  • Megumi Miyano, Kosei Tanaka, Shu Ishikawa, Shinji Takenaka, Andrés Miguel-Arribas, Wilfried J.J. Meijer, Ken-ichi Yoshida

    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., 27 Jan. 2018, Microbial Cell Factories, 17 (1), English

    [Refereed]

    Scientific journal

  • Megumi Miyano, Kosei Tanaka, Shu Ishikawa, Shinji Takenaka, Andrés Miguel-Arribas, Wilfried J.J. Meijer, Ken-ichi Yoshida

    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., 27 Jan. 2018, Microbial Cell Factories, 17 (1), English

    [Refereed]

    Scientific journal

  • Chumsakul O, Nakamura K, Ishikawa S, Oshima T

    2018, Methods in molecular biology (Clifton, N.J.), 1837, 33 - 47

    [Refereed]

  • Onuma Chumsakul, Divya P. Anantsri, Tai Quirke, Taku Oshima, Kensuke Nakamura, Shu Ishikawa, Michiko M. Nakano

    Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate ResD response regulator play primary roles in the transcriptional activation of genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR repressor controls transcription to support nitrate respiration. In addition, the ferric uptake repressor (Fur) can modulate transcription under anaerobic conditions. However, whether these controls are direct or indirect has been investigated only in a gene-specific manner. To gain a genomic view of anaerobic gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR, and Fur transcription factors (TFs) using in situ DNase I footprinting combined with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting by high-throughput sequencing [GeF-seq]). A significant number of sites were targets of ResD and NsrR, and a majority of them were also bound by Fur. The binding of multiple TFs to overlapping targets affected each individual TF's binding, which led to combinatorial transcriptional control. ResD bound to both the promoters and the coding regions of genes under its positive control. Other genes showing enrichment of ResD at only the promoter regions are targets of direct ResD-dependent repression or antirepression. The results support previous findings of ResD as an RNA polymerase (RNAP)binding protein and indicated that ResD can associate with the transcription elongation complex. The data set allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by an A- and T-rich sequence are often found at sites where all three TFs competitively bind. IMPORTANCE Bacteria encounter oxygen fluctuation in their natural environment as well as in host organisms. Hence, understanding how bacteria respond to oxygen limitation will impact environmental and human health. ResD, NsrR, and Fur control transcription under anaerobic conditions. This work using in situ DNase I footprinting uncovered the genome-wide binding profile of the three transcription factors (TFs). Binding of the TFs is often competitive or cooperative depending on the promoters and the presence of other TFs, indicating that transcriptional regulation by multiple TFs is much more complex than we originally thought. The results from this study provide a more complete picture of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to our further understanding of anaerobic physiology.

    AMER SOC MICROBIOLOGY, Jul. 2017, JOURNAL OF BACTERIOLOGY, 199 (13), English

    [Refereed]

    Scientific journal

  • Dong-Min Kang, Christophe Michon, Tetsuro Morinaga, Kosei Tanaka, Shinji Takenaka, Shu Ishikawa, Ken-ichi Yoshida

    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, English

    [Refereed]

    Scientific journal

  • Dong-Min Kang, Kosei Tanaka, Shinji Takenaka, Shu Ishikawa, Ken-ichi Yoshida

    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), 1026 - 1032, English

    [Refereed]

    Scientific journal

  • Kosei Tanaka, Ayane Natsume, Shu Ishikawa, Shinji Takenaka, Ken-ichi Yoshida

    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

  • Ayako Terakawa, Ayane Natsume, Atsushi Okada, Shogo Nishihata, Junko Kuse, Kosei Tanaka, Shinji Takenaka, Shu Ishikawa, Ken-ichi Yoshida

    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 - 13, English

    [Refereed]

    Scientific journal

  • Masahiro Tatsumi Akiyama, Taku Oshima, Onuma Chumsakul, Shu Ishikawa, Hisaji Maki

    Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two similar to 200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

    WILEY-BLACKWELL, Aug. 2016, GENES TO CELLS, 21 (8), 907 - 914, English

    [Refereed]

    Scientific journal

  • Koichi Higashi, Toru Tobe, Akinori Kanai, Ebru Uyar, Shu Ishikawa, Yutaka Suzuki, Naotake Ogasawara, Ken Kurokawa, Taku Oshima

    Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches.

    PUBLIC LIBRARY SCIENCE, Jan. 2016, PLOS GENETICS, 12 (1), English

    [Refereed]

    Scientific journal

  • Yoshihiro Toya, Takashi Hirasawa, Shu Ishikawa, Onuma Chumsakul, Takuya Morimoto, Shenghao Liu, Kenta Masuda, Yasushi Kageyama, Katsuya Ozaki, Naotake Ogasawara, Hiroshi Shimizu

    Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates.

    TAYLOR & FRANCIS LTD, Dec. 2015, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79 (12), 2073 - 2080, English

    [Refereed]

    Scientific journal

  • Yoshihiro Toya, Takashi Hirasawa, Shu Ishikawa, Onuma Chumsakul, Takuya Morimoto, Shenghao Liu, Kenta Masuda, Yasushi Kageyama, Katsuya Ozaki, Naotake Ogasawara, Hiroshi Shimizu

    Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates.

    TAYLOR & FRANCIS LTD, Dec. 2015, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79 (12), 2073 - 2080, English

    [Refereed]

    Scientific journal

  • Satohiko Murayama, Shu Ishikawa, Onuma Chumsakul, Naotake Ogasawara, Taku Oshima

    The amino acid sequence of the RNA polymerase (RNAP) alpha-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (alpha-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the alpha-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the alpha-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that alpha-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that alpha-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of alpha-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

    PUBLIC LIBRARY SCIENCE, Jul. 2015, PLOS ONE, 10 (7), English

    [Refereed]

    Scientific journal

  • Amarila Malik, Maria Tyas Hapsari, Iwao Ohtsu, Shu Ishikawa, Hiroshi Takagi

    Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce beta-fructans (inulin and levan), as well as alpha-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, May 2015, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 119 (5), 515 - 520, English

    [Refereed]

    Scientific journal

  • Amarila Malik, Maria Tyas Hapsari, Iwao Ohtsu, Shu Ishikawa, Hiroshi Takagi

    Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce beta-fructans (inulin and levan), as well as alpha-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

    SOC BIOSCIENCE BIOENGINEERING JAPAN, May 2015, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 119 (5), 515 - 520, English

    [Refereed]

    Scientific journal

  • Kosei Tanaka, Kana Iwasaki, Takuya Morimoto, Takatsugu Matsuse, Tomohisa Hasunuma, Shinji Takenaka, Onuma Chumsakul, Shu Ishikawa, Naotake Ogasawara, Ken-ichi Yoshida

    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), English

    [Refereed]

    Scientific journal

  • Bernadette Henares, Sushma Kommineni, Onuma Chumsakul, Naotake Ogasawara, Shu Ishikawa, Michiko M. Nakano

    The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes with no apparent relevance to NO metabolism and that the ResD response regulator and NsrR coordinately regulate transcription. To determine whether these genes are direct targets of NsrR and ResD, we used chromatin affinity precipitation coupled with tiling chip (ChAP-chip) and ChAP followed by quantitative PCR (ChAP-qPCR) analyses. The study showed that ResD and NsrR directly control transcription of the ykuNOP operon in the Fur regulon. ResD functions as an activator at the nasD and hmp promoters, whereas it functions at the ykuN promoter as an antirepressor of Fur and a corepressor for NsrR. This mechanism likely participates in fine-tuning of transcript levels in response to different sources of stress, such as oxygen limitation, iron limitation, and exposure to NO.

    AMER SOC MICROBIOLOGY, Jan. 2014, JOURNAL OF BACTERIOLOGY, 196 (2), 493 - 503, English

    [Refereed]

    Scientific journal

  • Bernadette Henares, Sushma Kommineni, Onuma Chumsakul, Naotake Ogasawara, Shu Ishikawa, Michiko M. Nakano

    The ResD response regulator activates transcription of diverse genes in Bacillus subtilis in response to oxygen limitation. ResD regulon genes that are the most highly induced during nitrate respiration include the nitrite reductase operon (nasDEF) and the flavohemoglobin gene (hmp), whose products function in nitric oxide (NO) metabolism. Transcription of these genes is also under the negative control of the NO-sensitive NsrR repressor. Recent studies showed that the NsrR regulon contains genes with no apparent relevance to NO metabolism and that the ResD response regulator and NsrR coordinately regulate transcription. To determine whether these genes are direct targets of NsrR and ResD, we used chromatin affinity precipitation coupled with tiling chip (ChAP-chip) and ChAP followed by quantitative PCR (ChAP-qPCR) analyses. The study showed that ResD and NsrR directly control transcription of the ykuNOP operon in the Fur regulon. ResD functions as an activator at the nasD and hmp promoters, whereas it functions at the ykuN promoter as an antirepressor of Fur and a corepressor for NsrR. This mechanism likely participates in fine-tuning of transcript levels in response to different sources of stress, such as oxygen limitation, iron limitation, and exposure to NO.

    AMER SOC MICROBIOLOGY, Jan. 2014, JOURNAL OF BACTERIOLOGY, 196 (2), 493 - 503, English

    [Refereed]

    Scientific journal

  • Ramona Duman, Shu Ishikawa, Ilkay Celik, Henrik Strahl, Naotake Ogasawara, Paulina Troc, Jan Loewe, Leendert W. Hamoen

    A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum.

    NATL ACAD SCIENCES, Nov. 2013, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110 (48), E4601 - E4610, English

    [Refereed]

    Scientific journal

  • Onuma Chumsakul, Kensuke Nakamura, Tetsuya Kurata, Tomoaki Sakamoto, Jon L. Hobman, Naotake Ogasawara, Taku Oshima, Shu Ishikawa

    Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using Ger-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.

    OXFORD UNIV PRESS, Aug. 2013, DNA RESEARCH, 20 (4), 325 - 337, English

    [Refereed]

    Scientific journal

  • Takeshi Ueda, Hiroki Takahashi, Ebru Uyar, Shu Ishikawa, Naotake Ogasawara, Taku Oshima

    The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex. © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

    Jun. 2013, DNA Research, 20 (3), 263 - 271, English

    [Refereed]

    Scientific journal

  • Ying Lei, Taku Oshima, Naotake Ogasawara, Shu Ishikawa

    Biofilm is a complex aggregate of cells that adhere to each other and produce an extracellular matrix. In Bacillus subtilis, an extracellular polysaccharide (EPS) and amyloid fiber (TasA), synthesized by the epsA-epsO and tapA-sipW-tasA operons, respectively, are the primary components of the extracellular matrix. In the current study, we investigated the functional role of the previously uncharacterized veg gene in B. subtilis. Overproduction of Veg, a small protein highly conserved among Gram-positive bacteria, stimulated biofilm formation via inducing transcription of the tapA-sipW-tasA operon. Moreover, overproduced Veg restored the impairment of biofilm formation in mutants carrying a deletion of of sinI, slrA, or slrR, encoding an antirepressor of SinR that acts as the master regulator of biofilm formation, while biofilm morphology in the absence of SinR was not affected by either additional veg deletion or overproduction, indicating that Veg negatively regulates SinR activity independently of the known antirepressors. Expression of sinR was not affected in Veg-overproducing cells, and amounts of SinR were similar in cells expressing different levels of Veg, strongly suggesting that Veg modulates the repressor activity of SinR. Interestingly, the results of in vivo pulldown assays of the SinR complex indicate that Veg inhibits the interactions between SinR and SlrR. Based on these findings, we propose that Veg or a Veg-induced protein acts as an antirepressor of SinR to regulate biofilm formation.

    AMER SOC MICROBIOLOGY, Apr. 2013, JOURNAL OF BACTERIOLOGY, 195 (8), 1697 - 1705, English

    [Refereed]

    Scientific journal

  • Ying Lei, Taku Oshima, Naotake Ogasawara, Shu Ishikawa

    Biofilm is a complex aggregate of cells that adhere to each other and produce an extracellular matrix. In Bacillus subtilis, an extracellular polysaccharide (EPS) and amyloid fiber (TasA), synthesized by the epsA-epsO and tapA-sipW-tasA operons, respectively, are the primary components of the extracellular matrix. In the current study, we investigated the functional role of the previously uncharacterized veg gene in B. subtilis. Overproduction of Veg, a small protein highly conserved among Gram-positive bacteria, stimulated biofilm formation via inducing transcription of the tapA-sipW-tasA operon. Moreover, overproduced Veg restored the impairment of biofilm formation in mutants carrying a deletion of of sinI, slrA, or slrR, encoding an antirepressor of SinR that acts as the master regulator of biofilm formation, while biofilm morphology in the absence of SinR was not affected by either additional veg deletion or overproduction, indicating that Veg negatively regulates SinR activity independently of the known antirepressors. Expression of sinR was not affected in Veg-overproducing cells, and amounts of SinR were similar in cells expressing different levels of Veg, strongly suggesting that Veg modulates the repressor activity of SinR. Interestingly, the results of in vivo pulldown assays of the SinR complex indicate that Veg inhibits the interactions between SinR and SlrR. Based on these findings, we propose that Veg or a Veg-induced protein acts as an antirepressor of SinR to regulate biofilm formation.

    AMER SOC MICROBIOLOGY, Apr. 2013, JOURNAL OF BACTERIOLOGY, 195 (8), 1697 - 1705, English

    [Refereed]

    Scientific journal

  • 大島拓, 石川周

    日本農芸化学会 ; 1962-, 2013, 化学と生物, 51 (10), 670 - 678, Japanese

    [Refereed]

  • Accurate regulation of chromosome initiation is essential for maintenance of genome integrity in Bacillus subtilis

    Shu Ishikawa, Hajime Okumura, Mika Yoshimura, Mikako Ueki, Taku Oshima, Naotake Ogasawara

    GENETICS SOC JAPAN, Dec. 2012, GENES & GENETIC SYSTEMS, 87 (6), 385 - 385, English

    [Refereed]

  • Hajime Okumura, Mika Yoshimura, Mikako Ueki, Taku Oshima, Naotake Ogasawara, Shu Ishikawa

    Bacterial chromosome replication is initiated by binding of DnaA to a DnaA-box cluster (DBC) within the replication origin (oriC). In Bacillus subtilis, six additional DBCs are found outside of oriC and some are known to be involved in transcriptional regulation of neighboring genes. A deletion mutant lacking the six DBCs (Delta 6) initiated replication early. Further, inactivation of spo0J in Delta 6 cells yielded a pleiotropic phenotype, accompanied by severe growth inhibition. However, a spontaneous suppressor in soj or a deletion of soj, which stimulates DnaA activity in the absence of Spo0J, counteracted these effects. Such abnormal pheno-typic features were not observed in a mutant background in which replication initiation was driven by a plasmid-derived replication origin. Moreover, introduction of a single DBC at various ectopic positions within the Delta 6 chromosome partly suppressed the early-initiation phenotype, but this was dependent on insertion location. We propose that DBCs negatively regulate replication initiation by interacting with DnaA molecules and play a major role, together with Spo0J/Soj, in regulating the activity of DnaA.

    OXFORD UNIV PRESS, Jan. 2012, NUCLEIC ACIDS RESEARCH, 40 (1), 220 - 234, English

    [Refereed]

    Scientific journal

  • Hajime Okumura, Mika Yoshimura, Mikako Ueki, Taku Oshima, Naotake Ogasawara, Shu Ishikawa

    Bacterial chromosome replication is initiated by binding of DnaA to a DnaA-box cluster (DBC) within the replication origin (oriC). In Bacillus subtilis, six additional DBCs are found outside of oriC and some are known to be involved in transcriptional regulation of neighboring genes. A deletion mutant lacking the six DBCs (Delta 6) initiated replication early. Further, inactivation of spo0J in Delta 6 cells yielded a pleiotropic phenotype, accompanied by severe growth inhibition. However, a spontaneous suppressor in soj or a deletion of soj, which stimulates DnaA activity in the absence of Spo0J, counteracted these effects. Such abnormal pheno-typic features were not observed in a mutant background in which replication initiation was driven by a plasmid-derived replication origin. Moreover, introduction of a single DBC at various ectopic positions within the Delta 6 chromosome partly suppressed the early-initiation phenotype, but this was dependent on insertion location. We propose that DBCs negatively regulate replication initiation by interacting with DnaA molecules and play a major role, together with Spo0J/Soj, in regulating the activity of DnaA.

    OXFORD UNIV PRESS, Jan. 2012, NUCLEIC ACIDS RESEARCH, 40 (1), 220 - 234, English

    [Refereed]

    Scientific journal

  • 2Ap03 Bacillus subtilis cell factory for production of scyllo-inositol promising for Alzheimer's disease (Bio-Based Production) :

    YOSHIDA Ken-ichi, ONUMA Chumsakul, ISHIKAWA Shu, OGASAWARA Naotake

    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, English

    Symposium

  • Yoshikazu Kawai, Jon Marles-Wright, Robert M. Cleverley, Robyn Emmins, Shu Ishikawa, Masayoshi Kuwano, Nadja Heinz, Nhat Khai Bui, Christopher N. Hoyland, Naotake Ogasawara, Richard J. Lewis, Waldemar Vollmer, Richard A. Daniel, Jeff Errington

    Teichoic acids and acidic capsular polysaccharides are major anionic cell wall polymers (APs) in many bacteria, with various critical cell functions, including maintenance of cell shape and structural integrity, charge and cation homeostasis, and multiple aspects of pathogenesis. We have identified the widespread LytR-Cps2A-Psr (LCP) protein family, of previously unknown function, as novel enzymes required for AP synthesis. Structural and biochemical analysis of several LCP proteins suggest that they carry out the final step of transferring APs from their lipid-linked precursor to cell wall peptidoglycan (PG). In Bacillus subtilis, LCP proteins are found in association with the MreB cytoskeleton, suggesting that MreB proteins coordinate the insertion of the major polymers, PG and AP, into the cell wall. The EMBO Journal (2011) 30, 4931-4941. doi: 10.1038/emboj.2011.358; Published online 30 September 2011

    NATURE PUBLISHING GROUP, Dec. 2011, EMBO JOURNAL, 30 (24), 4931 - 4941, English

    [Refereed]

    Scientific journal

  • Amarila Malik, Shu Ishikawa, Muhamad Sahlan, Naotake Ogasawara, Uyen Quynh Nguyen, Herman Suryadi

    Sucrose phosphorylase has been known for its activity to generate some intermediate drug precursors. In spite of the broad acceptor specificity of the enzyme, the difference in enzyme specificity between bacterial species was recognized. A number of lactic acid bacteria strains known as exopolysaccharide producer isolated previously from local foods, beverages and soil samples from Indonesia's biosphere were screened for gene encode sucrose phosphorylase using polymerase chain reaction (PCR). A 1476-bp gene fragment from Leuc mesenteroides MBFWRS-3(1) isolated from sugar containing-beverage from Solo, Central Java, called Wedang Ronde, was successfully cloned encoding 492 amino acid residues of sucrose phosphorylase SPaseWRS-3(1) (AN. HM536929). Its predicted molecular mass was found to be closed to the confirmed one by sodium dodecyl sulfate polyacrylamide gel electrophresis (SDS-PAGE) gel (approximately 57 kDa). Although, it revealed high similarity in amino acid sequences to the existing ones (99% identity to 1355SPase), this is the first report on SPase from Indonesia's biosphere. The recombinant SPaseWRS-3(1) expressed by Escherichia coli BL21 Star (TM) showed lower activity as compared to the reference SPase, which is 50%.

    ACADEMIC JOURNALS, Nov. 2011, AFRICAN JOURNAL OF BIOTECHNOLOGY, 10 (74), 16915 - 16923, English

    [Refereed]

    Scientific journal

  • Kensuke Nakamura, Taku Oshima, Takuya Morimoto, Shun Ikeda, Hirofumi Yoshikawa, Yuh Shiwa, Shu Ishikawa, Margaret C. Linak, Aki Hirai, Hiroki Takahashi, Md. Altaf-Ul-Amin, Naotake Ogasawara, Shigehiko Kanaya

    We identified the sequence-specific starting positions of consecutive miscalls in the mapping of reads obtained from the Illumina Genome Analyser (GA). Detailed analysis of the miscall pattern indicated that the underlying mechanism involves sequence-specific interference of the base elongation process during sequencing. The two major sequence patterns that trigger this sequence-specific error (SSE) are: (i) inverted repeats and (ii) GGC sequences. We speculate that these sequences favor dephasing by inhibiting single-base elongation, by: (i) folding single-stranded DNA and (ii) altering enzyme preference. This phenomenon is a major cause of sequence coverage variability and of the unfavorable bias observed for population-targeted methods such as RNA-seq and ChIP-seq. Moreover, SSE is a potential cause of false single-nucleotide polymorphism (SNP) calls and also significantly hinders de novo assembly. This article highlights the importance of recognizing SSE and its underlying mechanisms in the hope of enhancing the potential usefulness of the Illumina sequencers.

    OXFORD UNIV PRESS, Jul. 2011, NUCLEIC ACIDS RESEARCH, 39 (13), English

    [Refereed]

    Scientific journal

  • Yoko Kusuya, Ken Kurokawa, Shu Ishikawa, Naotake Ogasawara, Taku Oshima

    Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that Escherichia coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity precipitation coupled with DNA microarray) was conducted to visualize the distribution of Bacillus subtilis GreA on the chromosome and to determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.

    AMER SOC MICROBIOLOGY, Jun. 2011, JOURNAL OF BACTERIOLOGY, 193 (12), 3090 - 3099, English

    [Refereed]

    Scientific journal

  • Yoko Kusuya, Ken Kurokawa, Shu Ishikawa, Naotake Ogasawara, Taku Oshima

    Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that Escherichia coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity precipitation coupled with DNA microarray) was conducted to visualize the distribution of Bacillus subtilis GreA on the chromosome and to determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.

    AMER SOC MICROBIOLOGY, Jun. 2011, JOURNAL OF BACTERIOLOGY, 193 (12), 3090 - 3099, English

    [Refereed]

    Scientific journal

  • Onuma Chumsakul, Hiroki Takahashi, Taku Oshima, Takahiro Hishimoto, Shigehiko Kanaya, Naotake Ogasawara, Shu Ishikawa

    AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, delta abrB and delta abh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.

    OXFORD UNIV PRESS, Jan. 2011, NUCLEIC ACIDS RESEARCH, 39 (2), 414 - 428, English

    [Refereed]

    Scientific journal

  • Onuma Chumsakul, Hiroki Takahashi, Taku Oshima, Takahiro Hishimoto, Shigehiko Kanaya, Naotake Ogasawara, Shu Ishikawa

    AbrB is a global transcriptional regulator of Bacillus subtilis that represses the expression of many genes during exponential growth. Here, we demonstrate that AbrB and its homolog Abh bind to hundreds of sites throughout the entire B. subtilis genome during exponential growth. Comparison of regional binding of AbrB and Abh in wild-type, delta abrB and delta abh backgrounds revealed that they bind as homomer and/or heteromer forms with different specificities and affinities. We found four AbrB and Abh binding patterns were major. Three of these contain pairs of TGGNA motifs connected by A/T-rich sequences, differing in arrangement and spacing. We also assessed the direct involvement of these complexes in the control of gene expression. Our data indicate that AbrB usually acts as a repressor, and that the ability of Abh to act as a transcriptional regulator was limited. We found that changes to AbrB/Abh levels affect their binding at several promoters and consequently transcriptional regulation. Surprisingly, most AbrB/Abh binding events had no impact on transcription, suggesting an interesting possibility that AbrB/Abh binding is analogous to nucleoid-associated protein binding in Escherichia coli.

    OXFORD UNIV PRESS, Jan. 2011, NUCLEIC ACIDS RESEARCH, 39 (2), 414 - 428, English

    [Refereed]

    Scientific journal

  • Shu Ishikawa, Taku Oshima, Ken Kurokawa, Yoko Kusuya, Naotake Ogasawara

    To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the sigma(A) and beta' subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis.

    AMER SOC MICROBIOLOGY, Nov. 2010, JOURNAL OF BACTERIOLOGY, 192 (21), 5778 - 5787, English

    [Refereed]

    Scientific journal

  • Shu Ishikawa, Taku Oshima, Ken Kurokawa, Yoko Kusuya, Naotake Ogasawara

    To obtain insight into the in vivo dynamics of RNA polymerase (RNAP) on the Bacillus subtilis genome, we analyzed the distribution of the sigma(A) and beta' subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells using chromatin affinity precipitation coupled with gene chip mapping (ChAP-chip). In contrast to Escherichia coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences. This finding suggests that, in general, B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex and proceeds without intragenic transcription attenuation. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which can be identified as transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior between E. coli and B. subtilis during initiation and elongation steps might result in distinct strategies for postinitiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis.

    AMER SOC MICROBIOLOGY, Nov. 2010, JOURNAL OF BACTERIOLOGY, 192 (21), 5778 - 5787, English

    [Refereed]

    Scientific journal

  • Ling Juan Wu, Shu Ishikawa, Yoshikazu Kawai, Taku Oshima, Naotake Ogasawara, Jeff Errington

    Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division. The EMBO Journal (2009) 28, 1940-1952. doi: 10.1038/emboj.2009.144; Published online 4 June 2009

    NATURE PUBLISHING GROUP, Jul. 2009, EMBO JOURNAL, 28 (13), 1940 - 1952, English

    [Refereed]

    Scientific journal

  • Ebru Uyar, Ken Kurokawa, Mika Yoshimura, Shu Ishikawa, Naotake Ogasawara, Taku Oshima

    We performed chromatin immunoprecipitation-microarray analysis to investigate differences in function between StpA and H-NS in Escherichia coli cells. StpA binding regions essentially overlap those of H-NS in wild-type cells, while they are reduced to one-third in the hns mutant. The H-NS binding profile was unaffected by stpA inactivation.

    AMER SOC MICROBIOLOGY, Apr. 2009, JOURNAL OF BACTERIOLOGY, 191 (7), 2388 - 2391, English

    [Refereed]

    Scientific journal

  • Eunha Cho, Naotake Ogasawara, Shu Ishikawa

    The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChA-P-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaA(Y144C) mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.

    GENETICS SOC JAPAN, Apr. 2008, GENES & GENETIC SYSTEMS, 83 (2), 111 - 125, English

    [Refereed]

    Scientific journal

  • 細菌の細胞分裂の分子メカニズム (特集 原核細胞の細胞骨格)

    石川周, 小笠原直毅

    共立出版, 2008, 蛋白質核酸酵素, 53 (13), 1725 - 1731, Japanese

    [Refereed]

  • Shu Ishikawa, Yoshitoshi Ogura, Mika Yoshimura, Hajime Okumura, Eunha Cho, Yoshikazu Kawai, Ken Kurokawa, Taku Oshima, Naotake Ogasawara

    We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors-the local density of DnaA boxes and their affinities for DnaA-are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.

    OXFORD UNIV PRESS, Aug. 2007, DNA RESEARCH, 14 (4), 155 - 168, English

    [Refereed]

    Scientific journal

  • Shu Ishikawa, Yoshitoshi Ogura, Mika Yoshimura, Hajime Okumura, Eunha Cho, Yoshikazu Kawai, Ken Kurokawa, Taku Oshima, Naotake Ogasawara

    We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors-the local density of DnaA boxes and their affinities for DnaA-are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication.

    OXFORD UNIV PRESS, Aug. 2007, DNA RESEARCH, 14 (4), 155 - 168, English

    [Refereed]

    Scientific journal

  • Taku Oshima, Shu Ishikawa, Ken Kurokawa, Hirofumi Aiba, Naotake Ogasawara

    Heat-stable nucleoid-structuring protein (H-NS) is one of the main nucleoid proteins expressed in exponentially growing Escherichia coli cells. In addition to a role in nucleoid organization, H-NS functions as a pleiotropic regulator of gene expression. The genome-wide distribution of H-NS, compared with the distribution of RNA polymerase and transcriptionally active genes, was investigated using a high-density oligonucleotide chip. The new approach utilized in this study revealed that H-NS binds specifically to approximately 250 loci, covering >1000 genes, to maintain transcriptional inactivation. RNA polymerase was detected in >65% of H-NS binding sites with low or no transcriptional activity, indicating that the association of RNA polymerase to promoter regions is a general mode of transcription repression by H-NS. This study also revealed that most H-NS bound DNA have been horizontally acquired, which indicates that repression of inappropriate gene expression by H-NS plays an important role in the diversification of the E. coli genome. This study presents a comprehensive assessment of the distribution of H-NS within the E. coli genome, sheds light on the mechanism underlying the transcriptional regulation by H-NS, and provides new insight into bacterial genome evolution.

    OXFORD UNIV PRESS, Aug. 2006, DNA RESEARCH, 13 (4), 141 - 153, English

    [Refereed]

    Scientific journal

  • S Ishikawa, Y Kawai, K Hiramatsu, M Kuwano, N Ogasawara

    The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.

    BLACKWELL PUBLISHING, Jun. 2006, MOLECULAR MICROBIOLOGY, 60 (6), 1364 - 1380, English

    [Refereed]

    Scientific journal

  • S Ishikawa, Y Kawai, K Hiramatsu, M Kuwano, N Ogasawara

    The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.

    BLACKWELL PUBLISHING, Jun. 2006, MOLECULAR MICROBIOLOGY, 60 (6), 1364 - 1380, English

    [Refereed]

    Scientific journal

  • タイリングアレイを用いた細菌ゲノム機能研究の可能性

    小笠原直毅, 石川周, 黒川顕, 大島拓

    秀潤社, 2006, 細胞工学, 25 (10), 1155 - 1160, Japanese

    [Invited]

  • C Bongiorni, S Ishikawa, S Stephenson, N Ogasawara, M Perego

    The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Moll. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and Raff-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

    AMER SOC MICROBIOLOGY, Jul. 2005, JOURNAL OF BACTERIOLOGY, 187 (13), 4353 - 4361, English

    [Refereed]

    Scientific journal

  • C Bongiorni, S Ishikawa, S Stephenson, N Ogasawara, M Perego

    The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Moll. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and Raff-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

    AMER SOC MICROBIOLOGY, Jul. 2005, JOURNAL OF BACTERIOLOGY, 187 (13), 4353 - 4361, English

    [Refereed]

    Scientific journal

  • T Fukushima, S Ishikawa, H Yamamoto, N Ogasawara, J Sekiguchi

    A Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant. But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat. SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant. Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation. phases, and primer extension analysis indicated an identical transcriptional start point in both phases. The upstream sequence suggests that veg is transcribed by Esigma(A) RNA polymerase. Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase. Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.

    JAPANESE BIOCHEMICAL SOC, Apr. 2003, JOURNAL OF BIOCHEMISTRY, 133 (4), 475 - 483, English

    [Refereed]

    Scientific journal

  • K Kobayashi, SD Ehrlich, A Albertini, G Amati, KK Andersen, M Arnaud, K Asai, S Ashikaga, S Aymerich, P Bessieres, F Boland, SC Brignell, S Bron, K Bunai, J Chapuis, LC Christiansen, A Danchin, M Debarbouille, E Dervyn, E Deuerling, K Devine, SK Devine, O Dreesen, J Errington, S Fillinger, SJ Foster, Y Fujita, A Galizzi, R Gardan, C Eschevins, T Fukushima, K Haga, CR Harwood, M Hecker, D Hosoya, MF Hullo, H Kakeshita, D Karamata, Y Kasahara, F Kawamura, K Koga, P Koski, R Kuwana, D Imamura, M Ishimaru, S Ishikawa, Ishio, I, D Le Coq, A Masson, C Mauel, R Meima, RP Mellado, A Moir, S Moriya, E Nagakawa, H Nanamiya, S Nakai, P Nygaard, M Ogura, T Ohanan, M O'Reilly, M O'Rourke, Z Pragai, HM Pooley, G Rapoport, JP Rawlins, LA Rivas, C Rivolta, A Sadaie, Y Sadaie, M Sarvas, T Sato, HH Saxild, E Scanlan, W Schumann, JFML Seegers, J Sekiguchi, A Sekowska, SJ Seror, M Simon, P Stragier, R Studer, H Takamatsu, T Tanaka, M Takeuchi, HB Thomaides, Vagner, V, JM van Dijl, K Watabe, A Wipat, H Yamamoto, M Yamamoto, Y Yamamoto, K Yamane, K Yata, K Yoshida, H Yoshikawa, U Zuber, N Ogasawara

    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), 4678 - 4683, English

    [Refereed]

    Scientific journal

  • T Fukushima, S Ishikawa, H Yamamoto, N Ogasawara, J Sekiguchi

    A Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant. But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat. SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant. Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation. phases, and primer extension analysis indicated an identical transcriptional start point in both phases. The upstream sequence suggests that veg is transcribed by Esigma(A) RNA polymerase. Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase. Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.

    JAPANESE BIOCHEMICAL SOC, Apr. 2003, JOURNAL OF BIOCHEMISTRY, 133 (4), 475 - 483, English

    [Refereed]

    Scientific journal

  • T Fukushima, S Ishikawa, H Yamamoto, N Ogasawara, J Sekiguchi

    A Bacillus subtilis veg mutant exhibited a small reduction of absorbance, a large reduction of hexosamine release, and slow dipicolinic acid release from spores during germination with L-alanine as a germinant. But veg spores exhibited normal resistance to chloroform, 2-propanol, lysozyme, and heat. SDS-polyacrylamide gel electrophoresis of spore coat proteins revealed no difference in coat proteins between the wild type and the veg mutant. Northern and veg-lacZ fusion analyses indicated that the veg gene is transcribed in both the vegetative growth and sporulation. phases, and primer extension analysis indicated an identical transcriptional start point in both phases. The upstream sequence suggests that veg is transcribed by Esigma(A) RNA polymerase. Veg-GFP fusion protein was detected for vegetative cells and spores, but the fluorescence of mother cells disappeared completely in the late sporulation phase. Decoated spores containing Veg-GFP exhibited a strong green fluorescence in the core, but much weaker fluorescence in the cortex.

    JAPANESE BIOCHEMICAL SOC, Apr. 2003, JOURNAL OF BIOCHEMISTRY, 133 (4), 475 - 483, English

    [Refereed]

    Scientific journal

  • T Shida, K Mukaijo, S Ishikawa, H Yamamoto, J Sekiguchi

    A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm-r) chromosomal DNA, a Cm-r transformant 327UHSC (sacC::Cm-r degSU(Hy)) produced 3 times longer levan than that of the wild type.

    TAYLOR & FRANCIS LTD, Jul. 2002, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66 (7), 1555 - 1558, English

    [Refereed]

    Scientific journal

  • T Shida, K Mukaijo, S Ishikawa, H Yamamoto, J Sekiguchi

    A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm-r) chromosomal DNA, a Cm-r transformant 327UHSC (sacC::Cm-r degSU(Hy)) produced 3 times longer levan than that of the wild type.

    TAYLOR & FRANCIS LTD, Jul. 2002, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66 (7), 1555 - 1558, English

    [Refereed]

    Scientific journal

  • T Shida, K Mukaijo, S Ishikawa, H Yamamoto, J Sekiguchi

    A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm-r) chromosomal DNA, a Cm-r transformant 327UHSC (sacC::Cm-r degSU(Hy)) produced 3 times longer levan than that of the wild type.

    TAYLOR & FRANCIS LTD, Jul. 2002, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66 (7), 1555 - 1558, English

    [Refereed]

    Scientific journal

  • S Ishikawa, L Core, M Perego

    The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0Fsimilar toP intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The similar to48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0Fsimilar toP is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0Fsimilar toP or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0Fsimilar toP from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.

    AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jun. 2002, JOURNAL OF BIOLOGICAL CHEMISTRY, 277 (23), 20483 - 20489, English

    [Refereed]

    Scientific journal

  • LJ Core, S Ishikawa, M Perego

    In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators. The RapA phosphatase. is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity. PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide. Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid. A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1: 1 ratio and it is approximately 200 fold more active than a C-terminal arn ide peptide. Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA. Published by Elsevier Science Inc.

    ELSEVIER SCIENCE INC, Oct. 2001, PEPTIDES, 22 (10), 1549 - 1553, English

    [Refereed]

    Scientific journal

  • S Ishikawa, S Kawahara, J Sekiguchi

    The cwlV gene, which encodes Bacillus poly-myxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (M-r of 53,707 Dal. The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183 aa, M-r of 20,050 Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (V Delta S and U Delta S, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that V Delta S and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but U Delta S and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.

    SPRINGER VERLAG, Dec. 1999, MOLECULAR AND GENERAL GENETICS, 262 (4-5), 738 - 748, English

    [Refereed]

    Scientific journal

  • Peptidoglycan hydrolase LytF plays a role in cell separation with Cw1F during vegetative growth of Bacillus subtilis

    R Ohnishi, S Ishikawa, J Sekiguchi

    Peptidoglycan hydrolase, LytF (CwIE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwIF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-fagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by E sigma(D) RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.

    AMER SOC MICROBIOLOGY, May 1999, JOURNAL OF BACTERIOLOGY, 181 (10), 3178 - 3184, English

    [Refereed]

    Scientific journal

  • Peptidoglycan hydrolase LytF plays a role in cell separation with Cw1F during vegetative growth of Bacillus subtilis

    R Ohnishi, S Ishikawa, J Sekiguchi

    Peptidoglycan hydrolase, LytF (CwIE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwIF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-fagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by E sigma(D) RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.

    AMER SOC MICROBIOLOGY, May 1999, JOURNAL OF BACTERIOLOGY, 181 (10), 3178 - 3184, English

    [Refereed]

    Scientific journal

  • Genome analysis and germination related genes of Bacillus subtilis

    Fukushima Tatsuya, Ishikawa Shu, Yamamoto Hiroki, Sekiguchi Junichi

    日本生物工学会, 1999, 日本生物工学会大会講演要旨集, 11, 90 - 90, Japanese

    [Refereed]

    Symposium

  • Sekiguchi J, Sato T, Nanamiya H, Ohashi Y, Kawamura F, Takamatsu H, Kodama T, Watabe K, Ishikawa S

    1999, Tanpakushitsu Kakusan Koso, 44 (10), 1460 - 6

    [Refereed]

  • Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis

    S Ishikawa, Y Hara, R Ohnishi, J Sekiguchi

    Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the papQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical to cwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins of Escherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The P-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the crt IF gene is transcribed mainly by E sigma A RNA polymerase and weakly by E sigma H RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutations flaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

    AMER SOC MICROBIOLOGY, May 1998, JOURNAL OF BACTERIOLOGY, 180 (9), 2549 - 2555, English

    [Refereed]

    Scientific journal

  • Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis

    S Ishikawa, Y Hara, R Ohnishi, J Sekiguchi

    Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the papQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical to cwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins of Escherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The P-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the crt IF gene is transcribed mainly by E sigma A RNA polymerase and weakly by E sigma H RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutations flaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

    AMER SOC MICROBIOLOGY, May 1998, JOURNAL OF BACTERIOLOGY, 180 (9), 2549 - 2555, English

    [Refereed]

    Scientific journal

  • Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores

    S Ishikawa, K Yamane, J Sekiguchi

    The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis, We constructed a cwlJ::lacZ fusion in the B, subtilis chromosome, The beta-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by Eo(E) RNA polymerase, cwlJ-deficient spores responded to both L-alanine and AGFK, the A(580) values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination, However, the mutant spores released only slightly less hexosamine than did the wild-type spores, In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A(580), and slow release of dipicolinic acid, CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores, The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.

    AMER SOC MICROBIOLOGY, Mar. 1998, JOURNAL OF BACTERIOLOGY, 180 (6), 1375 - 1380, English

    [Refereed]

    Scientific journal

  • Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores

    S Ishikawa, K Yamane, J Sekiguchi

    The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis, We constructed a cwlJ::lacZ fusion in the B, subtilis chromosome, The beta-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by Eo(E) RNA polymerase, cwlJ-deficient spores responded to both L-alanine and AGFK, the A(580) values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination, However, the mutant spores released only slightly less hexosamine than did the wild-type spores, In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A(580), and slow release of dipicolinic acid, CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores, The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.

    AMER SOC MICROBIOLOGY, Mar. 1998, JOURNAL OF BACTERIOLOGY, 180 (6), 1375 - 1380, English

    [Refereed]

    Scientific journal

  • Localization of proteins onto the cell surface of Bacillus subtilis with a cell wall binding domain of autolysin

    Tsuchiya Atsushi, Ishikawa Shu, Sekiguchi Junichi

    日本生物工学会, 1998, 日本生物工学会大会講演要旨集, 10, 11 - 11, Japanese

    Symposium

  • S Kawahara, C Utsunomiya, S Ishikawa, J Sekiguchi

    Bacillus polymyxa var. colistinus autolysin was purified from a 1-l culture by ammonium sulfate fractionation, gel filtration, non-binding with a DEAE-Sepharose resin, and CM-Sepharose column chromatography. SDS-polyacrylamide gel (containing B. polymyxa var. colistinus cell mall) electrophoresis of the purified autolysin gave a single band at an M-r of 23 kDa exhibiting cell wall hydrolytic activity. Identification of the specific substrate bond cleaved by the autolysin indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase. The optimal temperature for the enzyme reaction was 40-50 degrees C, and the optimal pH was 4.0, which is extraordinarily unique for amidases.

    SOC FERMENTATION BIOENGINEERING, JAPAN, 1997, JOURNAL OF FERMENTATION AND BIOENGINEERING, 83 (5), 419 - 422, English

    [Refereed]

    Scientific journal

  • Study on a Bacillus subtilis autolysin, Cw1F, which appears during vegetative growth

    Hara Yoshiko, Ohnishi Ryo, Ishikawa Shu, Sekiguchi Junichi

    日本生物工学会, 1997, 日本生物工学会大会講演要旨集, 9, 22 - 22, Japanese

    Symposium

  • Study on a cw1J gene which affects germination of Bacillus subtilis spores

    Ishikawa Shu, Yamane Kunio, Sekiguchi Junichi

    日本生物工学会, 1997, 日本生物工学会大会講演要旨集, 9, 22 - 22, Japanese

    Symposium

MISC

  • H‐NSによる転写抑制を介した大腸菌ゲノムの多様性獲得機構

    東光一, 戸邉亨, 石川周, 鈴木穣, 小笠原直毅, 黒川顕, 黒川顕, 大島拓

    2016, 日本ゲノム微生物学会年会要旨集, 10th, 63, Japanese

  • 枯草菌ゲノム縮小株によるケミカル代替素材の生産

    増田健太, 森本拓也, 戸谷吉博, 平沢敬, 平沢敬, ONUMA Chumsakul, 石川周, 影山泰, 清水浩, 小笠原直毅, 尾崎克也

    2014, 日本農芸化学会大会講演要旨集(Web), 2014

  • 枯草菌ゲノム縮小株を用いた組換えタンパク質生産における転写,代謝フラックス解析

    戸谷吉博, 河崎優樹, 平沢敬, 増田健太, 森本拓也, ONUMA Chumsakul, 石川周, 大島拓, 影山泰, 尾崎克也, 小笠原直毅, 清水浩

    05 Mar. 2013, 日本農芸化学会大会講演要旨集(Web), 2013, 4B11A10 (WEB ONLY), Japanese

  • 大腸菌Hha,YdgTタンパク質による外来性遺伝子の転写抑制

    上田剛士, 高橋弘喜, 石川周, 小笠原直毅, 大島拓

    2013, 日本ゲノム微生物学会年会要旨集, 7th, 80, Japanese

  • 2Ap03 Bacillus subtilis cell factory for production of scyllo-inositol promising for Alzheimer's disease (Bio-Based Production)

    YOSHIDA Ken-ichi, ONUMA Chumsakul, ISHIKAWA Shu, OGASAWARA Naotake

    The Society for Biotechnology, Japan, Oct. 2012, 日本生物工学会大会講演要旨集, 64, 39 - 39, English

    Lecture materials

  • Functional analysis of the Veg protein that stimulates biofilm formation in Bacillus subtilis

    LEI YING, ISHIKAWA SHU, OGASAWARA NAOTAKE

    2012, 日本ゲノム微生物学会年会要旨集, 6th, 85, English

    Lecture materials

  • Yoko Kusuya, Ken Kurokawa, Shu Ishikawa, Naotake Ogasawara, Taku Oshima

    Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that Escherichia coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity precipitation coupled with DNA microarray) was conducted to visualize the distribution of Bacillus subtilis GreA on the chromosome and to determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells. © 2011, American Society for Microbiology. All Rights Reserved.

    Jun. 2011, Journal of Bacteriology, 193 (12), 3090 - 3099, English

    Lecture materials

  • Bacillus subtilis degU32(hy) proactively cancels catabolite repression

    IWASAKI KANA, ISHIKAWA SHU, OGASAWARA NAOTAKE, TAKENAKA SHINJI, YOSHIDA KEN-ICHI

    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), Japanese

    Lecture materials

  • Identification of Novel Nucleoid-associated Proteins in Bacillus subtilis

    LEI YING, ISHIKAWA SHU, OGASAWARA NAOTAKE

    2009, 日本分子生物学会年会講演要旨集, 32nd (Vol.1), 126, English

    Lecture materials

  • Studies on in vivo dynamics of Bacillus subtilis RNA polymerase through ChAP-chip analysis of RpoA

    MURAYAMA SATOHIKO, OSHIMA TAKU, ISHIKAWA SHU, OGASAWARA NAOTAKE

    2009, 日本分子生物学会年会講演要旨集, 32nd (Vol.4), 92, English

    Lecture materials

  • Search of nucleoid proteins in Bacillus subtilis: comparative analysis using ChAP(ChIP)-chip analysis between in B. subtilis and E. coli.

    CHUMSAKUL ONUMA, UYAL EBRU, ISHIKAWA SHU, OSHIMA TAKU, OGASAWARA NAOTAKE

    2009, 日本分子生物学会年会講演要旨集, 32nd (Vol.1), 27, English

    Lecture materials

  • 大腸菌のタイリングアレイ解析により明らかになったゲノム進化

    大島 拓, 小笠原 直毅, 石川 周, 黒川 顕, 饗場 浩文

    25 Feb. 2007, 日本細菌学雑誌, 62 (1), 46 - 46, Japanese

  • 1E12-1 Expression analysis of metal responsive genes in the moderately halophilic bacterium, Halomonas elongate OUT30018

    KOGA Ayumi, SUSUKI Koudai, OSHIMA Taku, ISHIKAWA Shu, KUROKAWA Ken, OGASAWARA Naotaka, SHINMYO Atsuhiko, YOSHIDA Kazuya, NAKAYAMA Hideki

    日本生物工学会, 2007, 日本生物工学会大会講演要旨集, 19, 116 - 116, Japanese

  • 枯草菌の胞子形成,胞子,発芽

    佐藤勉, 七宮英晃, 大橋由明, 河村富士夫, 高松宏治, 児玉武子, 渡部一仁, 石川周, 関口順一

    Aug. 1999, 蛋白質核酸酵素, 44 (10), 14 - 20, Japanese

    Introduction scientific journal

  • Formation, Structure and Germination of the Spore of Bacillus subtilis.

    SATO TSUTOMU, NANAMIYA HIDEAKI, OHASHI YOSHIAKI, KAWAMURA FUJIO, TAKAMATSU HIROMU, KODAMA TAKEKO, WATABE KAZUHITO, ISHIKAWA SHU, SEKIGUCHI JUN'ICHI

    共立出版, Aug. 1999, 蛋白質 核酸 酵素, 44 (10), 1460 - 1466, Japanese

    [Refereed]

    Introduction scientific journal

  • Localization of proteins onto the cell surface of Bacillus subtilis with a cell wall binding domain of autolysin.

    TSUCHIYA ATSUSHI, ISHIKAWA SHU, SEKIGUCHI JUN'ICHI

    日本生物工学会, 1998, 日本生物工学会大会講演要旨集, 1998, 11 - 11, Japanese

    Lecture materials

  • Study on a cwlJ gene which affects germination of Bacillus subtilis spores.

    ISHIKAWA SHU, YAMANE KUNIO, SEKIGUCHI JUN'ICHI

    日本生物工学会, 1997, 日本生物工学会大会講演要旨集, 1997, 22 - 22, Japanese

    Lecture materials

  • Study on a Bacillus subtilis autolysin, CwlF, which appears during vegetative growth.

    HARA YOSHIKO, ONISHI RYO, ISHIKAWA SHU, SEKIGUCHI JUN'ICHI

    日本生物工学会, 1997, 日本生物工学会大会講演要旨集, 1997, 22 - 22, Japanese

    Lecture materials

  • Bacillus colistinusの自己溶解酵素遺伝子cw1Vに関する研究 : 微生物

    石川 周, 宇都宮 千恵, 関口 順一, 河原 伸二

    社団法人日本農芸化学会, 05 Jul. 1995, 日本農藝化學會誌, 69, 228 - 228, Japanese

  • Study on a Bacillus colistinus autolysin gene, cwlU

    Ishikawa Shu, Kawahara Shinji, Rashid M. H., Sekiguchi Junichi

    日本生物工学会, 1995, 日本生物工学会大会講演要旨集, 7, 82 - 82, Japanese

Books etc

  • GeF-seq: A Simple Procedure for Base Pair Resolution ChIP-seq

    Onuma Chumsakul, Kensuke Nakamura, ISHIKAWA SHU, Taku Oshima

    Joint work, Humana Press, New York, NY, 2018, English

    Scholarly book

  • 高精度で結合領域を決定するGeF-seq

    大島 拓, 石川 周, Chumsakul Onuma, 中村 建介

    Joint work, 羊土社, Sep. 2014, Japanese

    Scholarly book

  • 細菌の発現制御機構をゲノムワイドに解析する :次世代シーケンサーを用いた高精度な網羅的解析の可能性

    大島 拓, 石川 周

    Single work, 日本農芸化学会, Oct. 2013, Japanese

    Scholarly book

  • 細菌の細胞分裂の分子メカニズム

    石川 周, 小笠原 直毅

    Joint work, 共立出版, Oct. 2008, Japanese

    Scholarly book

  • 枯草菌の胞子形成,胞子,発芽

    佐藤 勉, 七宮 英晃, 大橋 由明, 河村 富士夫, 高松 宏治, 児玉 武子, 渡部 一仁, 石川 周, 関口 順一

    Joint work, 共立出版, Aug. 1999, Japanese

    Scholarly book

Presentations

  • Study of the mechanism of bacterial growth inhibition by citrate

    Shu Ishikawa

    The;th Annual Meeting of the Society for Antibacterial and Antifungal Agents, Japan, 09 Sep. 2021, Japanese

    [Invited]

    Nominated symposium

Association Memberships

  • The Society for Biotechnology, Japan

    Apr. 2021 - Present
  • Society of Genome Microbiology, Japan

  • JAPAN SOCIETY FOR LACTIC ACID BACTERIA

  • グラム陽性菌ゲノム機能会議

Research Projects

  • 石川 周

    科学研究費補助金/挑戦的研究(開拓), Jun. 2018 - Mar. 2023, Principal investigator

    Competitive research funding

  • 吉田 健一

    科学研究費補助金/基盤研究(B), Apr. 2018 - Mar. 2022

    Competitive research funding

  • Shu ISHIKAWA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), 2015 - 2017, Principal investigator

    Competitive research funding

  • 汎用的高効率バイオプロセス細胞の創製

    Notake OGASAWARA

    Japan Science and Technology Agency (JST), Advanced Low Carbon Technology (ALCA), 2010 - 2016

    化学プロセスによる諸有用ケミカル素材の工業的生産を、バイオプロセスによる生産へ転換するために、革新的な「汎用的高効率バイオプロセス細胞」を創出します。具体的には、現在、諸有用分子の工業的合成等に用いられている枯草菌について、その増殖メカニズムの制御により細胞を素材生産期に導き、維持し、同時に、代謝フラックスの制御によって多様な産物を効率的に生産できる汎用性を備えたバイオプロセス技術を開発します。

    Competitive research funding

  • Shu ISHIKAWA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), 2012 - 2014, Principal investigator

    細菌の細胞分裂はFtsZが中心となり行われるが、FtsZは細胞膜に結合できないので、細胞膜に繋ぎとめる蛋白質が必須である。大腸菌ではFtsAが、シアノバクテリアではSepFがその役割を担うと考えられるが、増殖に必須である。枯草菌は両方の因子を備え、さらに同様の役割をすると考えられるEzrAも有するので、各々が破壊可能である。本研究では、酵母2ハブリッド解析、結晶構造、電子顕微鏡観察など多面的に調べ、SepFがFtsZを膜につなぎとめるメカニズムを解明した。また、EzrAがFtsZと直接結合せず、FtsAとの結合を介してZ-ringのダイナミズムを促進している、というモデルを提案した。

    Competitive research funding

  • Naotake OGASAWARA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(A)), 2011 - 2013

    我々は、1bpの解像度でDNA結合タンパク質とゲノムDNAとの結合領域を網羅的に解析可能なGeF-seq法を新たに開発し、進化的に大きく異なる細菌、大腸菌と枯草菌の核様体タンパク質について解析を行った。その結果、配列非特異的な結合様式を示すHUタンパク質は、枯草菌、大腸菌で同様の結合様式を示すものの、大腸菌では、DNAが屈曲あるいはヘアピン状の構造を取りうるDNA配列に、FisおよびIHFタンパク質が結合し、DNAと構造を取ることが示唆された。これらのタンパク質は枯草菌には存在しないことから、細菌種ごとに核様体構造が大きく異なる可能性が示された。

    Competitive research funding

  • Hiroki YAMAMOTO

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), 2011 - 2013

    枯草菌が桿状形態を維持して増殖するためには、細胞壁溶解酵素の活性が必要である。本研究では、これらの酵素の機能を制御する因子の解明を試みた。まず、LytEのシグナルペプチド(SPLytE)を用いてLytFを発現する株(SPLytE-LytF)では、分泌されたLytFは本来のセプタムと極だけでなく細胞側壁にも局在していた。また、lytEとcwlOの合成致死性についても、部分的ではあるものの相補できるようになっていた。以上の結果から、シグナルペプチドが細胞壁溶解酵素の局在性と機能を支配している可能性が示唆された。さらに詳細に調べた結果、SPLytEの疎水性領域の前半部分が重要であることがわかった。

    Competitive research funding

  • Hiroki YAMAMOTO

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), 2007 - 2009

    レクチンの一種であるConcanavalin Aの蛍光標識物(ConA-TMR)を用いることにより、枯草菌の細胞壁テイコ酸(WTA)を特異的に検出する方法を確立した。種々のテイコ酸修飾遺伝子変異株を用いて観察を行った結果、ConA-TMRはmajor WTAのグルコース修飾を効率よく検出し、minor WTAやリポテイコ酸は検出できないことが明らかになった。また、major WTAのグルコース修飾に関与するtagE条件変異株を用いて、細胞側壁におけるWTA修飾が螺旋状に行われていることを明らかにした。さらに側壁のWTA修飾制御には、細胞骨格蛋白質のひとつであるMreBが関与している可能性が示唆された。

    Competitive research funding

  • Naotake OGASAWARA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(若手研究(B)), 2005 - 2009

    FtsZは自己縮合してポリマー構造、さらにはリング構造を形成し、細菌の細胞分裂の基盤となる。この様な反応には、枯草菌ではFtsZと直接結合するFtsA、YlmF、ZapAとの結合が重要である。そこで、このような細胞分裂初期の分子メカニズムを解明するために、FtsZにランダムに変異を導入し、相互作用を失う変異FtsZを酵母2ハイブリッド解析により同定し、どのアミノ酸がFtsZ自身やFtsA、YlmF、ZapAとの結合に関わっているかを決定した。FtsZは、Plus End領域、T7 loop、Minus End領域、保存されていない領域、保存性の高いC末端配列からなる。Methanococcus JannaschiiのFtsZの結晶構造解析などから、FtsZポリマーは、異なるFtsZのPlusとMinus Endが結合して形成されると考えられていたが、本研究で得られた変異FtsZの結果はこれとよく一致しており、このようなモデルが枯草菌FtsZにも当てはまることがわかった。また、保存性の高いC末端配列に欠損、もしくは変異を持つ変異FtsZでは、FtsAとYlmFとの結合能を失うこと

    Competitive research funding

  • Shu ISHIKAWA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(特定領域研究), 2006 - 2007, Principal investigator

    細菌細胞の増殖を支える遺伝子・タンパク質のネットワークがどのように構築されているか、その基本原理の解明を目指して、2大モデル細菌である枯草菌・大腸菌を対象として、タイリングチップを用いたChIP-chip解析や質量分析法を用いた複合来解析等の新たな研究手法の導入を進め、新たな必須遺伝子機能、必須遺伝子産物間の機能ネットワーク、転写調節タンパク質と核様体タンパク質による転写制御ネットワーク、核様体タンパク質による細胞周期制御、代謝ネットワークのモデル化等について、多くの新たな知見を明らかにした。

    Competitive research funding

  • Naotake OGASAWARA

    Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(A)), 2005 - 2007

    枯草菌は、モデル生物として、ゲノム機能解析が進んでいる。その中で、ゲノム配列決定によって見出された機能未知、あるいは、未解析である遺伝子の破壊株ライブラリーの作成が、研究代表者を中心として行われた(Proc Natl Acad Sci USA,100,4678-4683,2003)。その結果、LB培地・37度という培養条件で、その破壊が致死となる必須遺伝子は271であるということが明らかとなった。その内、機能が不明確なものは22遺伝子であった。興味深いことに、その中の7遺伝子は、分子スイッチとして働くと考えられるGTP結合蛋白質をコードしていた(Microbiology,148,3539-3552,2002)。GTP結合蛋白質には、GTP結合型とGDP結合型の2つの状態があり、それぞれに特異的なeffectorに作用することにより分子スイッチとして働く。GTP-GDP型の変換は内在性のGTPase活性により行われるが、GTPase活性化因子、GTP-GDP交換促進因子等による調節を受けている。従って、GTP結合蛋白質の機能を解明するためには、そのGTPase活性を調節する因

    Competitive research funding

Industrial Property Rights

  • 枯草菌を用いた超高分子のL‐γ‐ポリグルタミン酸 の製造方法

    石川周, 山本純也, Chumsakul Onuma

    特願2022-159018, 20 Jul. 2022, 神戸大学

    Patent right

  • 枯草菌変異株及びその利用

    増田 健太, 劉 生浩, 森本 拓也, 小笠原 直毅, 石川 周, チュムサクル オンウマ

    特願2015-226957, 19 Nov. 2015, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-093321, 01 Jun. 2017

    Patent right

  • 組換え微生物及びその利用

    増田 健太, 劉 生浩, 森本 拓也, 小笠原 直毅, 石川 周, チュムサクル オンウマ

    特願2015-211548, 28 Oct. 2015, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-079640, 18 May 2017

    Patent right

  • 枯草菌変異株及びその利用

    増田 健太, 劉 生浩, 森本 拓也, 小笠原 直毅, 石川 周, チュムサクル オンウマ

    特願2015-211547, 28 Oct. 2015, 花王株式会社, 国立大学法人 奈良先端科学技術大学院大学, 特開2017-079639, 18 May 2017

    Patent right