In this thesis, a general approach to complete de novo sequencing, annotation and bioinformatic analyses of bacterial genomes and pan-genomes was devised and implemented. The tools developed and implemented were used to process and analyse genome- and transcriptome-based data that allowed a reconstruction on the phylogeny of the genus Listeria and the evolution of adaptation traits enabling its survival in the intracellular environment.An efficient finishing pipeline was constructed using a combination of commercial and open source components connected by self-written software that was successfully employed for the assembly of one bacterial chromosome and five plasmids. In order to achieve a congruent and exact annotation for 12 chromosomes and 18 plasmids, a modular comparative annotation system was created including rapid primary automatic annotation followed by manual corrections that can be easily used to predict functions for a large amount of prokaryotic replicons simultaneously.Considerable extensions to the author s previously published GECO comparative analysis suite were implemented to assess relations of prokaryotic strains inside pan-genomes. These can be employed to (i) predict the total size of a pan-genome, as well as the distribution of conservation to identify indels supporting taxonomic or phenotypic divisions, (ii) rapidly export data necessary for the construction of phylogenomic and phenotypical trees to delineate between minor mutations and horizontal gene transfer, (iii) to identify and visualize diverging regions inside related replicons to recognize hyperdynamic hotspots, mobile elements and prophages, (iv) and to comparatively visualize limited regions for detailed assessment. A public GECO-LisDB comparative analysis server was set up bearing the largest number of completely sequenced listeriae available to date [97].In order to allow comparative identification of small non-coding RNAs, a client/server software called sRNAdb was devised, which can rapidly detect the presence of sRNAs inside complete replicons.These pipelines were employed for evolutionary analysis of genus Listeria focusing on pathogenic species L. monocytogenes. All compared chromosomes of the species displayed extensive similarities indicative of a highly conserved functionality. Observed differences between phylogenetic trees indicate that minor SNP variations may have a profound impact on adaptation and thus available niches, while gene-scale indels often associated with horizontal gene transfer are rarely conserved. Most duplications found inside the genus are ancient and do not contribute to current evolutionary adaptation of listeriae.Differentially distributed genes were predominantly found in nine highly variable regions or as a part of rare mobile genetic elements or prophages, highlighting the stability of the backbone of the species. Interestingly, only a subset of strains displayed putatively functional CRISPR adaptive immune systems of different types, or other identifiable defences against aggressive foreign DNA, indicating other factors to sustain the observed chromosomal stability, which now deserves further attention.A further factor expanding the genomes in a subset of strains is represented by plasmids, which putatively descended from a common ancestor. Plasmid-encoded genes with identifiable functions frequently belonged to the category of stress response, which may be most beneficial in defence against disinfectants applied in food processing environments.New candidate genes that may influence virulence of Listeria were predicted based on their phylogenetic distribution, functional domains and intracellular expression. Prophage-related genes had a major effect on intracellular survival of listeriae as recognized by attenuated virulence of deletion mutants.Loss of virulence was previously identified as a recurring pattern of listerial evolution and could be seen in genomes, intracellular expression and phenotypes of several compared strains of all lineages, especially lineage III. This indicates a drift towards an obligate saprophytic or commensal lifestyle within the eukaryotic host, which may support exchange of genetic information in the nutrient-rich gastrointestinal tract and serve as a vector for transportation to a new location.In conclusion, these studies aimed at the development of pipelines intended for the identification of evolutionary patterns within the pan-genome of Listeria monocytogenes and related species and uncovered new candidate genes valuable for diagnostics and virulence research.
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