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Related Experiment Videos

Multicopy plasmids affect replisome positioning in Bacillus subtilis.

Jue D Wang1, Megan E Rokop, Melanie M Barker

  • 1Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Journal of Bacteriology
|October 19, 2004
PubMed
Summary

In Bacillus subtilis, multicopy plasmids and their associated DNA replication machinery (replisomes) can exhibit dynamic, non-random subcellular positioning. This suggests that plasmid location influences replisome localization within bacterial cells.

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Area of Science:

  • Bacterial cell biology
  • Molecular microbiology
  • Genetics

Background:

  • Subcellular localization of cellular components is crucial for bacterial function.
  • The spatial organization of DNA replication machinery and genetic elements like plasmids is not fully understood in bacteria.
  • Previous studies suggest characteristic positions for DNA replication machinery, chromosomes, and plasmids.

Purpose of the Study:

  • To visualize the subcellular localization of a multicopy plasmid (pHP13) in living Bacillus subtilis cells.
  • To investigate the effect of multicopy plasmids on the localization of the DNA replication machinery (replisome).
  • To understand the relationship between plasmid positioning and replisome dynamics.

Main Methods:

  • Utilized a live-cell imaging approach in Bacillus subtilis.

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  • Employed a fusion of lac operators and LacI-green fluorescent protein (GFP) to track plasmid pHP13.
  • Visualized DNA polymerase localization using a DNA polymerase subunit-GFP fusion protein.

    • Main Results:

    • In most cells, plasmids were mobile and randomly distributed; however, some cells showed plasmid clusters at cell poles.
    • Multicopy plasmids induced additional, mobile replisome foci in uncharacteristic cellular locations.
    • Replisome foci in plasmid-free cells were relatively stationary, unlike those in plasmid-containing cells.
    • Plasmid-associated replisomes were recruited to the plasmid's subcellular position.

    Conclusions:

    • Multicopy plasmids can influence the subcellular localization and dynamics of the bacterial DNA replication machinery.
    • The positioning of plasmid-associated replisomes appears to be dictated by the plasmid's location.
    • Hypothesized that chromosomal replication origin (oriC) location establishes the subcellular position of the chromosomal replisome.