Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system

Mol Microbiol. 2003 Oct;50(1):291-301. doi: 10.1046/j.1365-2958.2003.03683.x.

Abstract

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Centrifugation, Density Gradient / methods
  • Chromosomes, Bacterial / metabolism*
  • DNA Replication* / genetics
  • DNA, Bacterial
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Mutation
  • Nitrogen Isotopes / metabolism
  • Nucleic Acid Hybridization / methods
  • R Factors / metabolism*
  • Replicon / physiology*
  • Temperature

Substances

  • DNA, Bacterial
  • Nitrogen Isotopes