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1992 Fiscal Year Final Research Report Summary

Analysis and application of an ice-nucleation plasmid from Pseudomonas viridiflava

Research Project

Project/Area Number 03650795
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 発酵工学
Research InstitutionKansai University

Principal Investigator

TOKUYAMA Tai  Kansai Univ., Faculty of Eng., Biotech., Prof, 工学部, 教授 (60067519)

Co-Investigator(Kenkyū-buntansha) HASEGAWA Yoshie  Kansai Univ., Faculty of Eng., Biotech., Associate Prof, 工学部, 助教授 (30156327)
OBATA Hitoshi  Kansai Univ., Faculty of Eng., Biotech., Prof, 工学部, 教授 (00067646)
Project Period (FY) 1991 – 1992
KeywordsIce-nucleation gene / Ice-nucleation plasmid / Shuttle vector / Cloning vector
Research Abstract

A cosmic library containing Pseudomonas viridiflava KUIN-2 DNA was constructed in the vector Homer III and used to transform E. coli HB101. Five colonies (INAC-1-INAC-5) with ice-nucleating activity (INA) were obtained and all these transformants had in common an approximately 13.2-kb Eco RI fragment (pNVR-1). Among them, the transformant INAC-2, which harbored a single plasmid (pCH-1) and had an excellent INA, was selected for studying the characteristics of the INA^+ transformant. The INA in culture was not detectable until the bacterial titer reached 10^5 to 10^6 cells/ml.
A novel plasmid pNVR-1, a self-circularized DNA segment which consists of 13.2 kb-Eco RI fragment, was introduced into both E. coli JM109 and P. aeruginosa PAO1161 by transformation. Both of the strains harboring the pNVR-1 plasmid showed good INA and resistance to ampicillin. In addition, the optimum growth temperatures for these strains shifted to temperatures lower than those of strains without pNVR-1.
P. viridif … More lava KUIN-2 originally had a single plasmid, called pINA, and three Eco RI fragments 13.2, 8.8, and 6.8 kb long were detected in pINA. The size of an EcoRI-digested fragment from pINA (13.2 kb) agreed with that of EcoRI-digested pNVR-1. From the results of Southern blotting with the probe pNVR-1, it was found that an EcoRI-digested fragment about 13.2 kb long from pINA hybridized with pNVR-1. Furthermore, E. coli JM109 harboring pINA had ice-nucleation activity, and the ice-nucleation temperatures, (T_<50>), of the transformations were -4.9 to -5.2. These results suggest that both the ampicillin-resistance gene and the ice-nucleation gene of P. viridiflava KUIN-2 were coded on pINA.
A mini-plasmid, pNVR-1012 (approximately 2.7 kb), was constructed from a novel plasmid pNVR-1 encoded ice-nicleation gene. This plasmid encoded an ampicillin resistance gene, and had unique restriction sites as EcoRI, Hind III, PstI, and SphI. To confirm the eligibility of pNVR-1012 as cloning vector, a 3.1 kb- Hind III fragment encoded for the salicylate hydroxylase (nahG) gene from NAH plasmid was ligated to pNVR-1012. The nahG gene was expressed in both E. coli and P. aeruginosa harboring this recombinant plasmid under the culture without salicylate. From the level of nahG expression in the recombinant strains, it is suggested that the origin of the expression of the nahG gene appear to derive from NAH plasmid promoter, and recombinant plasmid functions efficiently in P. aeruginosa rather than in E. coli. From these results, pNVR-1012 seems to be very useful for gene cloning vector in E. coli and P. aeruginosa. Less

  • Research Products

    (4 results)

All Other

All Publications (4 results)

  • [Publications] 大釜 英貴: "Expression of the ice-nucleation active gene of a novel plasmid pNVR-1 from Pseudomonas uiriditlaua in Escherichia coli and Pseudomonas aenu-ginosa." J.Ferment.Bioeng.74. 73-96 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 大釜 英貴: "Ice-nucleation gene of Pseudomonas uiridiflaua KUIN-2 coded on a plasmid" Biosci.Biotech.Biochem. 56. 1690-1691 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hideki Ohgama et al: "Expression of the ice nucleation active gene of a novel plasmid pNVR-1 from Pseudomonas viridiflava in Escherichia coli and Pseudomonas aeruginosa" J.Ferment. Bioeng. 74(2). 73-76 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hideki Ohgama et al: "Ice nucleation gene of Pseudomonas viridiflava KUIN-2 Coded on a plasmid" Biosci. Biotech. Biochem. 56(10). 1690-1691 (1992)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1994-03-24  

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