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Related Experiment Videos

Trans-complementable copy-number mutants of plasmid ColE1

A J Twigg, D Sherratt

    Nature
    |January 10, 1980
    PubMed
    Summary

    Researchers identified a non-essential region in ColE1-like plasmids that, when deleted, increases copy number. This suggests a plasmid-encoded protein regulates plasmid levels in Escherichia coli.

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    Area of Science:

    • Molecular Biology
    • Microbiology
    • Genetics

    Background:

    • Plasmid ColE1, a high-copy-number plasmid in Escherichia coli, exhibits relaxed replication control.
    • Replication of ColE1 plasmids does not require plasmid-specified proteins for extensive DNA synthesis.
    • Evidence suggests a plasmid-specified protein may regulate ColE1 replication in viable cells.

    Purpose of the Study:

    • To investigate the role of plasmid-specified proteins in the replication control of ColE1-like plasmids.
    • To identify specific regions within ColE1-like plasmids that influence copy number.
    • To elucidate the mechanism of plasmid copy number regulation.

    Main Methods:

    • Deletion mutagenesis of non-essential regions in ColE1-like plasmids and the pBR322 cloning vector.
    • Analysis of copy number in modified plasmids within Escherichia coli.
    • Complementation studies using a co-resident plasmid to assess diffusible gene products.

    Main Results:

    • Deletion of a specific non-essential region from ColE1-like plasmids resulted in a significantly increased copy number.
    • These high-copy-number deletion mutants were stably maintained in Escherichia coli.
    • Presence of a complementing plasmid restored the normal copy number, indicating a diffusible regulatory factor.
    • Deletion of the equivalent region in pBR322 also increased copy number and abolished conjugal transfer ability.

    Conclusions:

    • A plasmid-specified diffusible gene product regulates the copy number of ColE1-like plasmids.
    • The identified non-essential region encodes a negative regulator of plasmid replication or maintenance.
    • This regulatory mechanism is crucial for maintaining stable plasmid levels in host cells.

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