Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.
Clostridium botulinum is a dangerous pathogen that forms the deadly botulinum neurotoxin. Strains of C. botulinum are present in the environment as spores. Under suitable conditions, the dormancy of the bacterial spore is broken, and germination occurs. Germination is initiated following the recognition of small molecules by a specific germinant receptor (GR) located within spores. Currently, the identification and characterisation of these GRs remains unknown, but is critical if strategies are to be developed to either prevent spore germination altogether, or to germinate all the spores and then inactivate the emergent sensitive vegetative cells. The present study has characterised two functionally active GRs in C. botulinum which act in synergy and cannot function individually, and a related functionally active GR in C. sporogenes. These GRs respond to amino acids. Other GRs appear to form part of a complex involved in controlling the speed of germination, or are not functionally active. This study provides new insights into the mechanisms involved in germination and will allow us to develop new strategies to control this deadly pathogen.