Browsing by Author "Rastimo, Ilona"

Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces botulinum neurotoxin (BoNT), the most potent known natural toxin. Botulism is a potentially fatal disease caused by BoNT, mostly transmitted through contaminated food and leading to flaccid paralysis. In nature, C. botulinum can disseminate via oxygen-resistant spores. Understanding the genetic regulation of sporulation is crucial for controlling spores in food. In C. botulinum Group I ATCC 3502, it has been observed that the role of the sporulation regulator SigK is more diverse than that of other sporulation sigma factors, potentially regulating stress response in addition to sporulation. In the less studied strain C. botulinum Group II Beluga a binding site for SigK has been identified in the promoter of the BoNT encoding gene bontE, suggesting that SigK may regulate BoNT production. This licentiate thesis focuses on the role of SigK in the C. botulinum Group II strain Beluga. The aim of the study was to determine how deletion of sigK, the gene encoding SigK, would affect bacterial growth, toxin production, sporulation, and spore physiology. The study was conducted by comparing the wild-type strain Beluga PbontE-SNAP (WTPbontE-SNAP) with the sigK deletion mutant Beluga ΔsigK PbontE-SNAP (ΔsigKPbontE SNAP), both of which were tagged with the reporter snap gene under the control of the bontE promoter to monitor the expression of the bontE gene. Although ΔsigKPbontE-SNAP initiated sporulation normally, fewer mature spores were observed compared to WTPbontE-SNAP. Even though in ΔsigKPbontE-SNAP no heat resistant spores were detected, phase-bright spores were seen in phase contrast microscopy, indicating that the sporulation process was initiated successfully. Another important finding was that the spores produced by ΔsigKdid not germinate as frequently as those of WT, which could indicate structural problems of ΔsigK spores. However, some parts of the spore coat had likely been successfully formed, as the addition of lysozyme to the recovery medium did not result in the destruction of ΔsigK spores. In addition to sporulation, deletion of sigK also partially negatively impacted vegetative growth. The total amount of toxin produced was reduced in the ΔsigK strain only in the early time points, which could be explained by the slower growth of the strain. The findings suggest that in C. botulinum Group II Beluga, SigK is a late-stage sporulation factor that mainly regulates genes necessary for spore coat formation. Experiments regarding spore structure should be performed using electron microscopy. Also, since the conditions used in this study were optimal for bacteria, the experiments could be repeated under stressful conditions as well. This study provides a basis for elucidating the entire sporulation cascade in Beluga. This knowledge could contribute to control C. botulinum spores in food in the future