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
The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA and... Show moreThe maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA and modulate its conformation, and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of Clostridium difficile have been characterized to date. Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10-kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G + C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo. The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism. Show less
