The role of CwlJ and SleB in germination of Bacillus licheniformis spores
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Bacterial spores are a major issue for the health sector and the food industry. These spores are made inside a cell when nutrients become scares in a process called sporulation, and can survive very harsh conditions for a very long time. When conditions return to normal, they undergo germination where they return to vegetative growth. Bacillus Licheniformis is one such spore forming bacterium. It causes food spoilage and may cause food poisoning. Because of reported problems with B.licheniformis in production of cooked ham where the bacteria survived sterilization methods that usually worked, a project studying germination in B.licheniformis was started at the Institute of Food Safety and Infection Biology, NMBU. An important part of the sporulation is the breakdown of a very thick modified peptidoglycan layer called the cortex. Studies done on the closely related species Bacillus subtilis have shown that there are two enzymes responsible for this breakdown of the cortex. These are CwlJ and SleB, and their homologues in B. licheniformis will be investigated in this thesis. Three mutants were created to study the role of CwlJ and SleB. The single mutants ΔcwlJ and ΔsleB were made previously by the research group, but the double mutant ΔcwlJ,ΔsleBwas made from ΔcwlJ in this thesis using a “markerless gene replacement” method. Spores of the mutants and the wild type were made, and germination measured by three different assays. In two of the assays germination is induced by L-alanine. In the third assay, germination is induced by exogenous Ca2+-Dipicolinic acid (CaDPA) which activates CwlJ in B.subtilis. Successful germination was observed in both the single mutants (ΔcwlJ and ΔsleB) and the wild type (MW3), but only partly germination, and no out-growth was observed in the double mutant (ΔcwlJ,ΔsleB). These observations are in agreement to what is seen in B.subtilis.