The effect of segregational drift on multicopy plasmid evolution

Plasmids are extra-chromosomal genetic elements commonly found in prokaryotic cells. The multicopy nature of plasmids enables the emergence of intracellular genetic diversity or heterogeneity. Nonetheless, until recent years plasmid evolution and allele dynamics studies have yielded limited empirical data of the subpopulation structure generated by multicopy plasmids. In this thesis we studied the evolutionary forces at play during plasmid evolution. Plasmid allele inheritance contains a component of random genetic drift, segregational drift. To characterize its properties, we established an experimental system to introduce and trace single plasmid alleles at the resolution of individual cells in evolving populations. We employed natural transformation to create a new plasmid allele at a specific frequency in the population and then followed the vertically inherited plasmid alleles during an experimental evolution experiment. The evolution experiments were combined with mathematical modelling to validate and extend some of our experimental results. We demonstrate that segregational drift has an effect on the evolution of multicopy plasmids by hindering the fixation of novel alleles. Both neutral and beneficial alleles on multicopy plasmids are more prone to extinction than alleles present in single copy chromosomes. We proved that factors that govern the plasmid life cycle, like the mode of plasmid inheritance and the mode of plasmid replication influence plasmid allele dynamics. Our study further reveals that the establishment probability of novel alleles depends on the strength of selection and the replicon copy number.This thesis provides a valuable insight to plasmid allele dynamics at the intracellular and population levels. Multicopy genetic element evolution is governed by an interplay of evolutionary forces acting in multiple levels, where the copy number of the replicon has special importance