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Eclipse period during replication of plasmid R1: Contributions from Structural events and from Copy-Number-control system
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Evolutionary Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2003 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 50, no 1, 291-301 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2003. Vol. 50, no 1, 291-301 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90567DOI: 10.1046/j.1365-2958.2003.03683.xPubMedID: 14507381OAI: oai:DiVA.org:uu-90567DiVA: diva2:162961
Available from: 2003-04-29 Created: 2003-04-29 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Control of Chromosome and Plasmid Replication in Escherichia coli
Open this publication in new window or tab >>Control of Chromosome and Plasmid Replication in Escherichia coli
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Life is cellular. Cells grow and divide to give two new cells; this process is called the cell cycle. The chromosome in a bacterium is replicated into two identical copies before the cell divides. DNA replication is a fundamental process common to all forms of life.

In my thesis, I have studied control of chromosome and plasmid replication in Escherichia coli, a rod-shaped bacterium. Plasmids are extrachromosomal autonomously replicating DNA molecules.

I have combined the classical Meselson-Stahl density-shift and DNA hybridisation with theoretical analysis of DNA replication. The minimal time between two successive replications of the same molecule, the eclipse, was determined for both plasmid and chromosome.

The aim was to investigate the processes ensuring the precise timing of chromosome replication in the cell cycle. In wild-type strains, the chromosomal eclipse was long. Mutations affecting the so-called sequestration process, the superhelicity of the DNA, and the initiation protein, DnaA, reduced the eclipse.

Fast-growing E. coli has overlapping replicative phases with synchronous initiation from multiple initiation sites, oriC. I have investigated the complex interplay between different control processes by measuring the length of the eclipse and the degree of asynchronous initiation in various mutants.

I have measured the eclipse period of plasmid R1 during up- and down-shifts in plasmid copy number. The length of the eclipse was found to be determined by structural events as well as by the properties of the copy-number-control system.

During downshift from very high copy numbers, the rate of plasmid replication started very slowly and gradually increased until the normal copy number was achieved, in accordance with the +n model.

The CopB system of plasmid R1 was shown to be a rescue system preventing cells with few plasmid copies from losing the plasmid in some of the daughter cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 65 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 838
Keyword
Microbiology, Mikrobiologi
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-3471 (URN)91-554-5627-8 (ISBN)
Public defence
2003-05-22, B:10, Biomedicinskt Centrum BMC, Uppsala, 14:00
Opponent
Supervisors
Available from: 2003-04-29 Created: 2003-04-29Bibliographically approved

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