Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4

Leo Eberl, Claus Sternberg, Michael Christian Givskov, E. Grohmann, M. Gerlitz, H. Schwab

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.
    Original languageEnglish
    JournalMolecular Microbiology
    Volume12
    Issue number1
    Pages (from-to)131-141
    ISSN0950-382X
    Publication statusPublished - 1994

    Cite this

    Eberl, L., Sternberg, C., Givskov, M. C., Grohmann, E., Gerlitz, M., & Schwab, H. (1994). Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. Molecular Microbiology, 12(1), 131-141.
    Eberl, Leo ; Sternberg, Claus ; Givskov, Michael Christian ; Grohmann, E. ; Gerlitz, M. ; Schwab, H. / Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. In: Molecular Microbiology. 1994 ; Vol. 12, No. 1. pp. 131-141.
    @article{62553fa442bc4297ac1123bfe1c923f3,
    title = "Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4",
    abstract = "The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.",
    author = "Leo Eberl and Claus Sternberg and Givskov, {Michael Christian} and E. Grohmann and M. Gerlitz and H. Schwab",
    year = "1994",
    language = "English",
    volume = "12",
    pages = "131--141",
    journal = "Molecular Microbiology",
    issn = "0950-382X",
    publisher = "Wiley-Blackwell",
    number = "1",

    }

    Eberl, L, Sternberg, C, Givskov, MC, Grohmann, E, Gerlitz, M & Schwab, H 1994, 'Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4', Molecular Microbiology, vol. 12, no. 1, pp. 131-141.

    Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. / Eberl, Leo; Sternberg, Claus; Givskov, Michael Christian; Grohmann, E.; Gerlitz, M.; Schwab, H.

    In: Molecular Microbiology, Vol. 12, No. 1, 1994, p. 131-141.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4

    AU - Eberl, Leo

    AU - Sternberg, Claus

    AU - Givskov, Michael Christian

    AU - Grohmann, E.

    AU - Gerlitz, M.

    AU - Schwab, H.

    PY - 1994

    Y1 - 1994

    N2 - The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.

    AB - The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.

    M3 - Journal article

    VL - 12

    SP - 131

    EP - 141

    JO - Molecular Microbiology

    JF - Molecular Microbiology

    SN - 0950-382X

    IS - 1

    ER -