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1989 Fiscal Year Final Research Report Summary

Molecular genetic studies on pathogenicity of genus Bacteroides.

Research Project

Project/Area Number 62480154
Research Category

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

Allocation TypeSingle-year Grants
Research Field 細菌学
Research InstitutionThe University of Tokushima

Principal Investigator

OHNISHI Yoshinari  The University of Tokushima, Dept.of Bacteriol., Professor, 医学部, 教授 (10037400)

Co-Investigator(Kenkyū-buntansha) KINOUCHI Takemi  The University of Tokushima, Dept.of Bacteriol., Lecturer, 医学部, 講師 (80136217)
ONO Tsuneko  The University of Tokushima, Dept.of Bacteriol., Assistant Professor, 医学部, 助手 (40035514)
AKIMOTO Shigeru  The University of Tokushima, Dept.of Bacteriol., Lecturer, 医学部, 講師 (10159337)
Project Period (FY) 1987 – 1989
KeywordsBacteroides / pathogenicity / gene cloning / neuraminidase / DNA sequence / mobilization / electroporation / positive-selection vector
Research Abstract

1. A gene encoding neuraminidase of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The neuraminidase gene nanH was subcloned from the cosmid and was located within a 1.8-kilobase-pair XhoI-KpnI restriction endbnuclease fragment. DNA sequencing of the insert revealed an open reading frame which encodes 296 amino acids. The predicted protein sequence had no marked similarity to known bacterial neuraminidase sequences. A 5.4-kb BamHI-SaII fragment containing the nanH gene was cloned into the Escherichia coli-Bacteroides shuttle vector pVAL-1. The resulting plasmid, pKK105, was mobilized from E. coli strain HB101 to Bacteroides uniformis strain BU1001 by the conjugative plasmid R751.
The neuraminidase activity in crude cell extracts from strain BU1001 carrying pKK105 was found to be 1600-fold greater than that found in strain HB101 carrying the lame plasmid.
2. High voltage electroporation was applied to transformation of B. thetaiotaomicron strain 5482 using the shuttle plasmid pVAL-1. With the optimized procedure, B. thetaiotaomicorn was transformed with an efficiency up to 3xlO^5 transformants per mug pVAL-1 plasmid DNA.
3. A plasmid vector pUCST5 carrying a lethal gene pnd was constructed for the use of positive-selection of recombinant plasmids.

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Sakikawa,T.: "The pnd gene in plasmid R16:Nucleotide sequence and gene expression leading to cell Mg^<2+> release and stable RNA degradation." Biochim.Biophys.Acta. 1007. 158-166 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Akimoto.S.: "Transcriptional regalation of F plasmid gene srnB:rifampicin promoted in vitro reathrough of a terminator in the leader region." Mol.Microbiol.3. 787-796 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ono,T.: "Cloning and expression of the Bacteroides fragilis neuraminidase gene nanH in Escherichia coli." Microbial Pathogenesis. (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Akimoto,S.: "Transformation of Bacteroides thetaiotaomicron by electroporation." Microbiol.Immunol.(1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 小野恒子: "Bacteroides fragilisのノイラミニダ-ゼ遺伝子のクロ-ニングと大腸菌からBacteroidesへの伝達" 嫌気性菌感染症研究. 20. (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakikawa, T., et al.: "The pnd gene in plasmid R16: Nucleotide sequence and gene expression leading to cell Mg release and stable RNA degradation." Biochim. Biophys. Acta 1007, 158-166, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akimoto, S., et al.: "Transcriptional regulation of F plasmid gene srnB: rifampicin promoted in vitro readthrough of a terminator in the leader region." Mol. Microbiol. 3, 787-796, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ono, T., et al.: "Cloning and expression of the Bacteroides fragilis neuraminidase gene nanH in Escherichia coli." Microbial Pathogenesis 1990.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akimoto, S., et al.: "Transformation of Bacteroides thetaiotaomicron by electroporation." Microbiol. Immunol. 1990.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ono, T., et al.: "Cloning of Bacteroide fragilis neuraminidase gene in Escherichia coli and transferring the gene to Bacteroides.(in Japanese)" Proceedings of 20th meeting of the Japanese association for anaerobic infection research. 1990.

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

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Published: 1993-03-26  

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