The studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to... Show moreThe studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to investigate C difficile were insufficient, a secondary goal was to develop novel methods for C. difficile studies. We developed a luciferase toolkit for use in C. difficile, not only for gene expression analysis, but also for protein-protein interaction studies in vivo, secretion of proteins or the analysis of complexes present at the cell surface. We investigated fluorescence microscopy for C. difficile, by comparing different fluorescent systems previously described, and introduced the use of the fluorophore HaloTag. Additionally, we analyzed C. difficile intrinsic fluorescence and report the potential limitations for live-cell microscopy. The development of the tools enabled the assessment of the localization of the TcdC C-terminus and the characterization of the HupA protein. Finally, we provide new insights into the chromosome remodelling and DNA replication by studying DnaA-dependent unwinding and characterizing the histone-like protein HupA. In the discussion, we present the implications of our findings and provide perspectives for further research on TcdC-mediated toxin regulation and chromosome dynamics, specifically during DNA replication. Show less
Over the last years major advances have been made in the field of C. difficile research. Despite the continuous progress of research in C. difficile epidemiology and molecular biology. This thesis... Show moreOver the last years major advances have been made in the field of C. difficile research. Despite the continuous progress of research in C. difficile epidemiology and molecular biology. This thesis shows that the development of molecular based techniques in detecetion and typing of C. difficile could be very valuable in combatting CDI. This also applies for the genetics and molecular biology of C. difficile in understanding the virulence of C. difficile. However, the continuing development of new techniques, both typing and genetics manipulation tools, has also led to the recognition of potential weaknesses of the currently frequent used techniques. Therefore, it is of utmost importance to keep up with the technical progress in the field of C. difficile research to enable further elucidation of the mechanisms that determine the virulence of C. difficile. Show less
