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- Olsson, Jan
(författare)
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Control of Chromosome and Plasmid Replication in Escherichia coli
- 2003
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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.
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| 2. |
- Singh, Bhupender
(författare)
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Dynamic Organization of Molecular Machines in Bacteria
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism.In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phaseBacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase.In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.
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