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The spore-forming bacterium Clostridium botulinum is the causative agent of food- borne botulism, infant botulism and wound botulism. Botulinum neurotoxin (BoNT) is one of the most lethal substances known to man. Whilst the pathogenesis of C. botulinum is generally ascribed to BoNT, it is the capacity to produce endospores that lies at the heart of botulism. Spores enable the organism to persist in the environment and be transmitted from soil to foods and subsequently from person to person. Germination is the initial and perhaps most important step for the progression of disease. Understanding the molecular mechanisms of germination of C. botulinum spores is a major focus of research for the food industry due to C. botulinum group I proteolytic strain being a causative agent of a potentially fatal food poisoning. It occurs in low-acid canned foods of pH >4.6. Incidences of such food poisoning are rare due to adequate existing sterilisation and preservation systems. However, development of novel preservative systems and preventative strategies remains a target for the food industry with knowledge of the genetics of germination having direct applications in future disease prevention. This study encompassed four main areas of focus. These included (i) determination of appropriate germination conditions for spores of C. botulinum and evaluation of media suitable for the production of spores (ii) the use of reverse genetics and ClosTron technology followed by characterisation of three genes, sleB, cwlJ and ypeB thought to play a role in the critical stage of spore cortex lysis occurring during spore germination (iii) development of a system of forward genetics with the implementation of mariner-based random mutagenesis in C. botulinum and (iv) evaluation of the role of four small acid soluble protein (SASP) genes of the a/p- type in the protection of C. botulinum spores against heat and various chemicals. This study has culminated with the establishment of conditions specific for the germination of spores of C. botulinum ATCC 3502 strain. Combinations of nutrient germinants proved optimal with sodium bicarbonate playing a significant role functioning as a germinant itself or as a germinant co-factor. Evaluation of suitable sporulation media suggested the choice of medium for the production of spores to be a critical consideration for the performance of subsequent phenotyping and characterisation assays. Insertional mutagenesis using ClosTron technology uncovered a potential contributory role for SleB and YpeB in the spore cortex lysis stage of spore germination. A cell wall hydrolase, CwlJ is likely to be a minor cortex lytic enzyme during germination. The implementation of a system of random mutagenesis was successful in C. botulinum and future refinements of the protocol will be required to optimise all necessary requirements of a fully functional mariner-based mutagenesis system. The role of the four individual SASP genes in protection of spores against heat was difficult to ascertain but two genes, CBO1789 and CBO1790 have been implicated as having a role to play in the spore’s resistance to the DNA damaging chemical, nitrous acid. Collectively, this study has contributed and expanded our knowledge of current germination mechanisms of spores of the Group I proteolytic C. botulinum strain. It is hoped that continued study with this organism will lead to the design of either inhibitors of germination or artificial germinants that could allow spore killing under milder conditions. |
