Browsing by Subject "kaksikomponenttijärjestelmä"

Clostridium botulinum is an anaerobic, endospore-forming bacterium able to produce botulinum neurotoxin which causes life-threatening botulism to human and animals. The classical food botulism follows when food containing preformed neurotoxin is ingested. C. botulinum strains form four phenotypically distinct groups of organisms (I-V). The strains causing human botulism belong to groups I and II. They are a risk to the food industry since the spores of group I are higly resistant to heat and the strains of group II can grow at refrigeration temperatures. A profound understanding of the mechanisms behind cold and heat resistance of C. botulinum will minimize the risks posed by this pathogen in the food industry. Two-component systems utilizing a phospotransfer mechanism between a sensor kinase and a response regulator are known to be the mostly used mechanisms in adaptation to different environmental conditions. However, their role in the physiology and stress tolerance of C. botulinum is unclear. The purpose of this study was to find out the role of a CBO0366 gene, encoding a sensor kinase belonging to a two-component system, in the physiology and cold and heat resistance of C. botulinum. The novel ClosTron-system was used in this study to inactivate the CBO0366 gene of the ATCC 3502 strain of C. botulinum. The system is based on re-targeting the mobile group II intron of the pMTL007-plasmid by using Splicing by Overlap Extension PCR (SOE-PCR), resulting in the intron inserting into the CBO0366 gene of C. botulinum. The insertion of the intron interrupts the reading frame of the gene. The physiology and cold and heat resistance of the mutant strain were researched by using the wild type of ATCC 3502 as a control. The growth on blood and egg yolk agar, metabolic activity, and growth curves at 37 and 45 °C, of the mutant strain were identical to the wild type. By contrast, at 15 ° and 20 °C the mutant strain grew slower. The results indicate that the CBO0366 gene has a central role in the cold resistance of C. botulinum ATCC 3502. It thus can be expected that the sensor kinase encoded by CBO0366 is a part of a two-component system responsible for responses needed in adaptation to cold. Inactivation of the gene probably inhibits the cold signal and subsequently the specific response inside the cell. Thus adaptation to cold is slower. The mutant strain nevertheless grew at 15 °C so the gene is probably not essential in survival in lower temperatures.