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1999 Fiscal Year Final Research Report Summary

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
KeywordsBacleriophage / Bacillus / E. coli / Lysis / Endolysine / Holin / Disruption
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.

  • Research Products

    (10 results)

All Other

All Publications (10 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 2001-10-23  

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