| Bacterial spores are a metabolically ?sleeping state? of the organisms and can survive extreme environmental stress for prolonged periods. Such survival is a major problem to the food industry as spores are commonly present in the ingredients of many manufactured food products. ?Awakening? of spores progresses through germination and outgrowth followed by vegetative cell division. The molecular mechanisms that awake these dormant structures and ensure their recovery from possibly endured injury have recently been addressed with genomics studies. These tools have allowed for an analysis of the germination receptors and outgrowth-specific gene-expression program in aerobic spore-formers of the genus Bacillus. In wild-type B. subtilis cells grown under standard conditions information on the molecular processes involved in germination/outgrowth progression shows that among the germination specific genes also some stress specific genes are, transiently, expressed. What a possible functional role of this could be during germination/outgrowth is unclear. In the current STW proposal we will use targeted mutant analysis to study in detail the contribution of individual genes and regulatory units to the germination and outgrowth progression of Bacillus subtilis spores. As an initial positive control we will knock-out two genes of which our laboratory has meanwhile shown that they are important for the stress response of vegetative cells against sorbic acid, the most used food preservative. In order to know which phase the spores have passed, germination/outgrowth stage specific GFP-reporters will be used. To assess the behaviour of the internal pH, a very important physiological parameter, we will use a pH sensitive GFP expressed in spores. Preliminary experiments to make such reporters and the pH sensor have been successfully performed in our laboratory. Finally, where we will have identified candidate germination control genes we will verify their role in germination and outgrowth of spores after a thermal treatment as well as their presence and function in real food spoilage isolates. |
