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Title: Whole-genome analysis of sporulation and germination in Clostridium difficile
Author: Dembek, Marcin
ISNI:       0000 0004 5918 0229
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2014
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Clostridium difficile is a Gram-positive, obligate anaerobe and a leading cause of hospital-acquired diarrhoea in humans. Under conditions that are not favourable for growth, C. difficile triggers sporulation, producing metabolically dormant endospores through asymmetric cell division. These are regarded as the principal infective stage in the C. difficile life cycle, but must germinate to allow for vegetative cell growth and toxin production, representing an attractive target for intervention. A detailed understanding of sporulation and germination could thus have direct applications for disease prevention. While sporulation and germination in the model sporeformer Bacillus subtilis is well understood, little is known about these events in C. difficile. Therefore, the primary aim of this project was to provide a genome-wide overview of sporulation and germination in C. difficile and to identify genes that are essential in these processes using a combination of molecular microbiology, microscopy, transcriptomics and next-generation sequencing. The first part of this thesis is devoted to the characterisation of C. difficile sporulation and germination dynamics, and is followed by a transcriptional analysis of temporal gene expression in germinating spores. A functional analysis of the 511 genes identified as differentially regulated during germination is provided and the results validated for a number of selected genes. One gene in particular, encoding a membrane associated cell wall protein Cwp7, is examined in more detail. In the second part of this thesis I describe the construction of the first comprehensive transposon mutant library which is then used to identify genes that are essential for sporulation and/or germination using Transposon-Directed Insertion Site Sequncing (TraDIS). The results of this study are validated by constructing in-frame deletions in four selected genes followed by a thorough analysis of the resulting phenotypes. Two genes: CD0125 encoding a homologue of B. subtilis SpoIIQ and CD3567 encoding a putative cell wall hydrolase are shown to be involved in spore formation while CD0106 encoding a homologues of B. subtilis CwlD is shown to be involved in germination.
Supervisor: Fairweather, Neil Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral