Determination of nucleotide sequence related to the plasmid replication region in Enterococcus faecalis and its application to a new shuttle vector

Kotaro Kirimura, Kiyotake Kamigaki, Tadashi Ebihara, Yoshitaka Ishii, Shinji Kanayama, Shoji Usami

    Research output: Contribution to journalArticle

    Abstract

    The 5.1-kb plasmid pAMα1Δ2, a derivative of the 9.6-kb plasmid pAMα1 which is harbored by Enterococcus faecalis ATCC 14508, has a region necessary for replication in E. faecalis. The nucleotide sequence related to the replication region in pAMα1Δ2 was determined and found to contain an open reading frame of 720-bp encoding a replication protein. The sequence showed 54.5 and 48.5% homology to those encoding the RepAs of plasmids pLA103 from Lactobacillus acidophilus and pFA3 from Neisseria gonorrhoeae, respectively. A recombinant 5.8-kb plasmid, pEFX6, which can be used as a shuttle vector between Escherichia coli and some strains of E. faecalis, was constructed by combining the tetracycline resistance gene of pAMα1 and the replication regions of pAMα1Δ2 and pUC18 for E. faecalis and E. coli, respectively. This shuttle vector was successfully used to clone and express the gelatinase gene from E. faecalis subsp. zymogenes IFO 3989 in E. faecalis C57, a strain showing no gelatinase activity.

    Original languageEnglish
    Pages (from-to)566-571
    Number of pages6
    JournalJournal of Bioscience and Bioengineering
    Volume87
    Issue number5
    DOIs
    Publication statusPublished - 1999

    Fingerprint

    Genetic Vectors
    Enterococcus faecalis
    Nucleotides
    Escherichia coli
    Plasmids
    Genes
    Gelatinases
    Derivatives
    Proteins
    Tetracycline
    Lactobacillus acidophilus
    Tetracycline Resistance
    Neisseria gonorrhoeae
    Open Reading Frames
    Clone Cells

    Keywords

    • Electroporation
    • Enterococcus faecalis
    • Gelatinase
    • Replication protein
    • Shuttle vector

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering

    Cite this

    Determination of nucleotide sequence related to the plasmid replication region in Enterococcus faecalis and its application to a new shuttle vector. / Kirimura, Kotaro; Kamigaki, Kiyotake; Ebihara, Tadashi; Ishii, Yoshitaka; Kanayama, Shinji; Usami, Shoji.

    In: Journal of Bioscience and Bioengineering, Vol. 87, No. 5, 1999, p. 566-571.

    Research output: Contribution to journalArticle

    Kirimura, Kotaro ; Kamigaki, Kiyotake ; Ebihara, Tadashi ; Ishii, Yoshitaka ; Kanayama, Shinji ; Usami, Shoji. / Determination of nucleotide sequence related to the plasmid replication region in Enterococcus faecalis and its application to a new shuttle vector. In: Journal of Bioscience and Bioengineering. 1999 ; Vol. 87, No. 5. pp. 566-571.
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    abstract = "The 5.1-kb plasmid pAMα1Δ2, a derivative of the 9.6-kb plasmid pAMα1 which is harbored by Enterococcus faecalis ATCC 14508, has a region necessary for replication in E. faecalis. The nucleotide sequence related to the replication region in pAMα1Δ2 was determined and found to contain an open reading frame of 720-bp encoding a replication protein. The sequence showed 54.5 and 48.5{\%} homology to those encoding the RepAs of plasmids pLA103 from Lactobacillus acidophilus and pFA3 from Neisseria gonorrhoeae, respectively. A recombinant 5.8-kb plasmid, pEFX6, which can be used as a shuttle vector between Escherichia coli and some strains of E. faecalis, was constructed by combining the tetracycline resistance gene of pAMα1 and the replication regions of pAMα1Δ2 and pUC18 for E. faecalis and E. coli, respectively. This shuttle vector was successfully used to clone and express the gelatinase gene from E. faecalis subsp. zymogenes IFO 3989 in E. faecalis C57, a strain showing no gelatinase activity.",
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