Organisation of the Mycobacterium smegmatis chromosome and its role in cell division

Abstract

Tuberculosis remains a global health problem, exacerbated by the increasing emergence of multi‐drug resistant strains. The identification of new drug targets and the discovery of new anti‐tuberculosis drugs is therefore a high priority. Although little is currently known about mycobacterial cell division, the process is essential for the survival and expansion of all bacterial species so may involve proteins that represent excellent drug targets. In this thesis, proven tools for the study of bacterial cell division such as live‐cell time‐lapse imaging and Fluorescent Repressor Operator System (FROS) were adapted for use in mycobacteria. Application of such techniques, fluorescent tagging of cell division proteins and deletion of parA in M. smegmatis helped to elucidate some interesting characteristics of mycobacterial cell division. In contrast to model organisms, live cell imaging and septal staining indicated that M. smegmatis can grow and divide asymmetrically and divides at a range of lengths suggesting a fundamentally different mechanism of division regulation. The chromosome was hypothesised to play a key role in cell division so was investigated further by labelling a specific chromosomal loci. The key finding was that M. smegmatis cells only contain 1 or 2 chromosomal copies and that regardless of cell length, the nucleoid occupies almost the entire intracellular space. To examine if the nucleoid organisation is important for cell division, a putative chromosome segregation gene parA was disrupted. The ΔparA mutant displayed a classic cell division phenotype characterised by the production of anuclear mini‐cells. The mechanism responsible for the ΔparA mutant phenotype was studied further by applying live cell imaging, FROS and expressing a ParA‐mCherry fusion protein. The data obtained from all work presented was collated and used to propose a novel model of bacterial cell division regulation applicable to mycobacteria where the nucleoid plays a central role and ParA is required to ensure correct nucleoid placement.