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Development of self-disruptive E. coli cells by using phage encoded lysis genes

Research Project

Project/Area Number 09555250
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field 生物・生体工学
Research InstitutionTokyo Institute of Technology

Principal Investigator

TANJI Yasunori  Tokyo Institute of Technology, Assistant Prof., 生命理工学部, 助教授 (00282848)

Co-Investigator(Kenkyū-buntansha) UNNO Hajime  Tokyo Institute of Technology, Prof., 生命理工学部, 教授 (10087471)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥10,100,000 (Direct Cost: ¥10,100,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥4,700,000 (Direct Cost: ¥4,700,000)
KeywordsBacleriophage / Bacillus / E. coli / Lysis / Endolysine / Holin / Disruption / T4ファージ
Research Abstract

Bacteriophages specific to Bacillus amyloliqueficiens, a gram-positive bacterium, were isolated from a local sewage treatment center. Using a lysis assay, a gene was isolated and its nucleotide sequence revealed one open reading frame of 375 bp. Over expression of the cloned gene yielded a 13 kDa lysis protein. Two additional proteins, endolysine and holin, responsible for the lysis of B. amyloliqueficiens were also identified from the same phage. Endolysine degrades peptidoglycan of the cell. Holin degrades the cytoplasmic membrane thus allowing endolysine to reach the periplasm and gain access to the peptidoglycan layer. Right after the induction of the holin expression, growth of E. coli cells was halted. On the other hand, expression of endolysine did not give any detectable change on E. coli cells. Lysis genes cloned in pET26b(+) vector were expressed in BL21(DE3) host cell. Expressed protern is expected to be translocated into the periplasmic space driven by signal peptide fused in frame at N-terminal end of lysis protein. Immediate cell disruption was observed when the holin was expressed in the logarithmic growth phase. The production of endolysine with the N-terninal fusion of signal peptide did not cause immediate cell lysis but caused morphological change of the BL21 cells. Resuspension of this endolysine producing E. coli cell pellet with pure water caused cell lysis followed by β-galactosidase release to the medium. Furthermore, exogenous effects of the purified endolysinon growth of B. amyloliqueficiens and B. subtiils were observed.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] A. Muyombwe et al: "Cloning and expression of a gene encoding the lytic ..."J. Biosci, Bioeng.. 88. 221-225 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Suda et al: "Evidence for a novel Chlorella virus-encoded aliginate ..."FEMS Microbiology Letters. 180. 140-143 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.Soejima et al: "Purfication of phase lysozymes and their ..."化学工学シンポジウムシリーズ. 70. 140-143 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M. Morita et al: "Dual expressoon system for the production of ..."化学工学シンポジウムシリーズ. 70. 144-147 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Asami et al: "Characterization of oxygen-dependent lysis of ..."J. biosci. Bioeng.. (in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Muyombwe, Y. Tanji, H. Unno: "Cloning and expression of a gene encoding the lytic function of Bacillus amyloliquefaciens phage. Evidence of and auxiliary lysis"J. Biosci. Bioeng..

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Suda, Y. Tanji, K. Hori, and H. Unno: "Evidence for a novel Chlorella virus-encoded aliginate lyase"FEMS Microbiology Letters. 180. 45-53 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Soejima, A. Muyombwe, Y. Tanji, K. Hori, S. yamamoto, and H. Unno: "Purification of phage lysozymes and their exogenous effects on bacterial growth"Symposium series of Chemical Engineering. Vol. 70, Biochemical Engineering and Bioseparation Engineering. 140-143 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M. Morita, K. Asami, Y. Tanji, S. Yamamoto, K. Hori, and H. Unno: "Dual expression system for the production of β-glucuronidase in Escherichia coli and its release into the medium"Symposium series of Chemical Engineering. Vol. 70, Biochemical Engineering and Bioseparation Engineering. 144-147 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Asami, Y. Tanji & H. Unno: "Characterization of oxygen-dependent lysis of Escherichia coli cells infected by bacteriophage T4"J. Biosci. Bioeng.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.Muyombwe et al.: "Cloning and expression of a gene encodeng the lyti --"J. Biosci. Bioeng. 88(2). 221-225 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Suda et al: "Evidence for a novel Chlorella virus-encoded aliginate --"FEMS Microbiology Letters. 180. 45-53 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] A.Soejima et al.: "Puriification of phage lysozymes and their --"化学工学シンポジウムシリーズ. 70. 140-143 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] M. Morita et al.: "Dual expression system for the production of --"化学工学シンポジウムシリーズ. 70. 144-147 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K. Asami et al.: "Charactrization of oxygen-dependent lysis of --"J. Biosci. Bioeng. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 丹治保典: "T4ファージの溶菌酵素を用いた自己破砕大腸菌の作成" 酵素工学ニュース. 39. 10-13 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kazuhiro Asami et al: "Recovery of in Tracellular metabolits by phage lysis" 化学工学シンポジウムシリーズ. 65. 43-47 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kazuhiro Asami et al: "Influence of dereased oxygen Tension on Premature lysis" Phage Biology Meeting. 46 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 森田 昌知: "T4ファージ由来リゾチームが大腸菌の溶菌に及ぼす影響" 化学工学会第31回秋季大会要旨集. 第2分冊. 6 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Yasunori Tanji: "Characterization of E.ivli by producing phage encoded." 化学工学シンポジウムシリーズ. 61. 131-134 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 丹治, 保典: "自己溶菌性大腸菌の開発" 化学工学会群馬大会研究発表講演要旨集. 109 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kazuhiro Asami: "Synchronized Disruption of Escherichia coli Cells by T4 Phage lnfection" J.Ferment.Bioeng.83・6. 511-516 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Yasunori Tanji: "Charac Terization of Escherichia coli lysis by producing phage encoded lysis protein" 化学工学会第30回秋季大会 研究発表講演要旨集. 3分冊. 297 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Kazuhiro Asami: "Disruption of Escherichia coli by lnducible Phage Lysis" Droc.Asia-Pac.Biochem.Eng.Conf. (Beijing). 853-856 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Yasunori Tanji: "Use of Cloned T4-Phage Gene to Disrupt Escherichia coli" Proc.Asia-Pac.Biochem. Eng.Conf.(Beijing). 861-864 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Yasunori Tanji: "Controlled expression of T4 phage encoded lysis genes for the gentle" J.Ferment.Bioeng.85・1. 74-78 (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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