Flagella are complex structures with important roles in bacterial motility, virulence and protein secretion. As of yet, little work has been undertaken to characterise the function and regulation of flagella in Clostridium botulinum, an organism which produces an extremely potent neurotoxin which is the causative agent of botulism and is utilised for numerous pharmaceutical purposes. Genes encoding structural components of the flagellar basal body, hook and filament were inactivated using the ClosTron insertional mutation method, resulting in reduced swarming motility and reduced neurotoxin release from the cell into culture supernatant as determined by an ELISA method. To determine regulatory factors affecting flagellar assembly and secretion, sigD, the gene encoding δD, an alternative sigma factor that has been shown to be responsible for the expression of late flagellar genes in various bacterial species, was inactivated. The mutant strain was analysed by microarray and quantitative real-time reverse-transcription PCR methods. It was shown that δD positively regulates the expression of multiple genes including the late flagellar genes, the clostridiolysin S genes and an operon of secreted proteases. Phenotypic assays were performed on the sigD mutant and confirmed that it was null for motility and that proteolysis by secreted proteases was reduced. This reduced proteolysis phenotype was complemented back towards wild-type levels by the introduction of a plasmid containing the sigD gene from Clostridium sporogenes under the control of its native Pft9B promoter. To aid future work, a method for inducing and selecting for double homologous recombination events in C. botulinum was developed for the purpose of creating a strain in which the chromosomal botA gene is converted to a toxoid encoding sequence by allelic exchange. This work has therefore implicated flagella in the secretion of the neurotoxin, characterised the regulatory roles of δD and developed a novel method for allelic exchange in clostridia.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588079 |
| Date | January 2011 |
| Creators | Blount, Benjamin Andrew |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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