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A key survival mechanism ofClostridium botulinum , the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organismBacillus subtilis , nothing is known about these mechanisms inC. botulinum. Using the ClosTron gene-knockout tool,sigK , encoding late-stage (stage IV) sporulation sigma factor K inB. subtilis , was disrupted inC. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast,sigK -complemented mutants sporulated successfully. Quantitative real-time PCR analysis ofsigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis ofspo0A , encoding the sporulation master switch, in thesigK mutant and the parent showed significantly reduced relative levels ofspo0A expression in thesigK mutant compared to the parent strain. Similarly,sigF showed significantly lower relative transcription levels in thesigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in thesigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation inC. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organismB. subtilis . Understanding the sporulation mechanism ofC. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation