| 1. |
|
|
| 2. |
- Olsson, Jan A, et al.
(författare)
-
Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome.
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 334:5, s. 919-31
-
Tidskriftsartikel (refereegranskat)abstract
- Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin. During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC. Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth. Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication. Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse. In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively. The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations. The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse. An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.
|
|
| 3. |
- Olsson, Jan
(författare)
-
Control of Chromosome and Plasmid Replication in Escherichia coli
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 4. |
- Olsson, Jan, et al.
(författare)
-
Eclipse period during replication of plasmid R1 : Contributions from Structural events and from Copy-Number-control system
- 2003
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 50:1, s. 291-301
-
Tidskriftsartikel (refereegranskat)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.
|
|
