Molecular characterisation of industrially relevant clostridium botulinum strains
Publication Date
July 31, 2026
Creator
Rawson, Alexander
Abstract
Clostridium botulinum is an organism of importance due to the production of an extremely potent neurotoxin, which is the causative agent of botulism, a potentially fatal paralytic condition or exploited for its therapeutic and cosmetic applications. However, limited research has been carried out on the bacterium due safety reasons and the requirement for costly specialist high containment laboratory facilities providing significant obstacles. The aim of this project was to deliver a suite of non toxigenic strains which would be fit for widening the field of C. botulinum research, and then applying such strains for further characterisation of this industrially significant pathogen.
Employing CRISPR/Cas9 technology, a stable isolate of the model C. botulinum Group I strain ATCC 3502 was genetically manipulated to generate non-toxigenic variants. Three ‘safe’ strain alternatives were created and subsequently phenotypically characterised to ensure suitability as safe surrogate strains for research applications.
To add to the strain toolbox, neurotoxin negative reporter strains were generated and characterised. This allows for the safe study of the regulatory changes altering toxin expression in the native bacterium. To identify previously unknown toxin regulators a reporter strain was subjected to a transposon mutagenesis experiment, however this approach did not yield any definitive candidates.
Subsequently, this strain toolbox was applied to study the means by which the botulinum neurotoxin exits the bacterial cell, a fundamental question of C. botulinum biology that remained unknown. Through targeted gene deletions of identified putative toxin export systems, extensive phenotypic characterisation of each set of mutants was completed and the titre of neurotoxin released from the mutant cells was determined. Only deletion of structural and regulatory components of the flagellar, which houses a flagella export apparatus homologous to the Gram-negative type III secretion system, caused a reduction in extracellular neurotoxin.
To determine if global regulatory changes in these mutant strains were responsible for the decrease in toxin release, RNA-Sequencing was performed on the sigD and flgB deletion mutant strains. It was shown that SigD, an alternative sigma factor, is responsible for the expression of a wide range of genes, including late stage flagellar genes, and even influencing differential expression of the neurotoxin. Identification of the upregulated expression of neurotoxin cluster genes in the flgB mutant cannot account for the decrease in extracellular toxin. The data presented in this project has strongly implicated the flagella export apparatus as having a role in neurotoxin export in C. botulinum.
Item Type
ethesis
Thesis Type
PhD
Supervisors
University Of Nottingham
Subjects (LC)
Associated Schools / Departments
UoN Repository URI
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