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Browsing by Subject "geenitekniikka"

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  • Joutsen, Suvi (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2007)
    Clostridium botulinum is an obligatory anaerobic, gram-positive and spore-forming food-borne pathogen. It produces one of the most toxic substances known, botulinum neurotoxin. Botulinum toxin causes a rare but life-threatening illness called botulism. The significance of botulism may grow in the future with the development of the modern food industry. C. botulinum strains form four physiologically distinct groups (I-IV). The four groups are diverse, as for example the group I spores have a high heat resistance, while group II strains are capable of growing in refridgerated temperatures. Toxins from the groups I and II are human pathogens, while toxins produced by group III strains are pathogenic only to animals. There are seven distinct serotypes of botulinum toxins, and these are marked with letters A-G. Inhibition of neurotransmitter acetylcholine release caused by botulinum toxin causes paralysis. All bacteria have sigma factors, which are an important part of transcription initiation mechanism. Sigma factors are components of the RNA polymerase holoenzymes. These are involved in recognizing specific promoters, and by binding to different promoters they enable the initiation of transcription. Bacteria have many different sigma factors that are divided into two classes: in first class there are house-keeping σ70-factors and stress-activated alternative sigma factors and in the second class σ54-factors, which are involved in nitrogen metabolism and stress tolerance. Only little research has been done on C. botulinum sigma factors and there is, for example, no knowledge of their heat and cold resistance. In this research we studied the importance of σ54-factor in the colony morphology, metabolism and heat and cold resistance of C. botulinum ATCC 3502. A deletion mutant for the gene CBO0224 of C. botulinum was constructed with ClosTron technique, in which a target-specified group II intron is inserted into the genome. After activation, the intron interrupts the gene and inhibits transcription. The mutant strain was compared to the wild type by growing it on blood and egg yolk agar plates, on which it grew slower and the colonies were smaller. The metabolism of the mutant was studied with API-test, in which the mutant did not show any sign of fermentation of glucose, maltose, salicin or trehalose, or hydrolysis of esculin or utilization of urea. The mutant was also compared to the wild type by growing both strains at different temperatures, at 15°C and 20°C, at the optimum temperature of 37°C and at 45°C. The mutant grew slower than the wild type at all studied temperatures. The σ54-factor of C. botulinum strain ATCC 3502 has a clearly influence on the bacterium's capability of growing both in an optimal and a stressful environment. In this research the first genetically modified strain of the σ54-factor of C. botulinum ATCC 3502 strain was constructed to produce information on the sigma factor's importance to the metabolism and stress resistance of the bacterium.