Experiments on the bacterial nucleoid of Escherichia coli viewed as a physical entity

Experiments on the bacterial nucleoid of Escherichia coli viewed as a physical entity

Cunha, S.M.

Odijk, T. (promotor)

Applied Sciences

2004-10-04

In this thesis we focus on the compaction of DNA within Escherichia coli and aim to gain some understanding of the physical mechanism behind its spatial organization in the cell. Chapter I is a brief review of the current knowledge of nucleoid structure. We discuss a model for DNA compaction in the living Escherichia coli cell, for which this thesis has provided evidence. In this model the formation of the nucleoid is presented as the phase separation of supercoiled DNA in a suspension of proteins. Chapter II is a brief introduction to excluded-volume interactions and ends with an exposition of recent theoretical calculations that explain how the phase separation between DNA and proteins within E. coli may occur. In chapter III, a comparison between E. coli nucleoids isolated by the detergent and osmotic shock methods is presented. In chapter IV the presence of polyethylene glycol (PEG) was used to study the effect of polymeric depletion interactions on the dimensions of nucleoids isolated by osmotic shock. We derive the free energy of isolated nucleoids when compacted into the intracellular dimensions. A discussion of these results with reference to the phase separation theory developed by Odijk (1998) is given and we conclude that depletion can be the force behind the formation of the bacterial nucleoid. Based on dynamic light scattering (DLS) experiments we show that within isolated nucleoids the supercoil segments exhibit a fast dynamics. In chapter V the process of expansion of nucleoids released by osmotic shock is studied. In our experiments, nucleoids initially expanded quite fast, followed by a long intermediate period. The average exponent characterizing the dynamics of the intermediate phase is indicative of a process driven by excluded-volume. Nevertheless, there are substantial variations between the individual measurements, which are not fully understood. In Chapter VI we directly monitor the movement of DNA segments within isolated nucleoids. We conclude that the isolated nucleoid has a gel-like structure where supercoil segments show confined Brownian motion. The observed diffusion constant substantiates the DLS experiments preformed in Chapter IV.

dynamic light scattering
free energy
light microscopy
nucleoids

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Optima

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doctoral thesis

(c) 2004 S.M. Cunha