Bacillus cereus spore formation, structure and germination

Bacterial spores arespecializeddifferentiated celltypes fortypes,specificallydesigned for thesurvival of adverse conditions. Their structure is highly unique and very different from the structure of normal vegetative bacterial cells. Spores cause massive problems in the food industry, because their remarkable resistance allows them to survive food processing and conservation methods. The spore-forming Bacillus cereus is an important food-borne pathogen, is famousfor its ability to causefood poisoning, andisan importantspoilage organism inpasteurizeddairy products. The work presented in this thesis has focused on the formation, structure and germination of B. cereus spores. An easy and efficient way of producing synchronized and homogeneous B. cereus spore batches was developed, using a chemically defined medium in combination with an airlift fermenter system. This setup allowed precise monitoring and manipulation of key growth- and sporulation parameters. The conditions employed resulted in synchronous growth and sporulation, which facilitated gene-expression studies. The kinetics of expression of sigA , sigB , sigF and sigG followed the model developed for Bacillus subtilis , underscoring the conservation of sporulation mechanisms among bacilli. B. cereus was able to form spores on the chemically defined medium without glucose but with lactate as a main carbon source. Sporulation was not induced by nutrient limitation, because significant amounts of carbon and nitrogen sources were still present when the cells started to sporulate. The presence of glutamate delayed the final stages of sporulation, but not the moment of sporulation initiation. Clearly, the concentration of glutamate influenced key spore properties such as heat resistance and germination. The alternative sigma factor σ B , encoded by the sigB gene, is an important stress response regulator of B. cereus . An increase in sigB transcription was observed upon glucose depletion, coinciding with the transition from exponential growth to the stationary phase. This increase was specifically associated with the depletion of glucose. Deletion of sigB had a significant impact on spore heat resistance and spore germination properties. Spore heat resistance is caused by the physicochemical structure of the spore, which protects vital spore components such as membranes, proteins, and the DNA. A spin-probe-based Electron Spin Resonance (ESR) method for measuring the internal structure of intact bacterial spores was developed and applied, and provided the first direct data on the aqueous environment in the various compartments of B. subtilis and B. cereus spores. From the results obtained, it was concluded that the core cytoplasm is not in a glassy state. Instead, a three-dimensional molecular matrix incorporating free but highly viscous water exists in the core. Notably, neither heat activation nor partial germination (the excretion of DPA but not full rehydration and enzyme activity) altered the structural properties of the core matrix significantly. Complete germination resulted in the disappearance of the structure in the core, and a decrease of the micro viscosity in the core cytoplasm to levels encountered in normal vegetative cells. For a quantitative analysis of the behavior of individual spores in a large, germinating spore-population, a flow cytometry (FCM) method was developed and applied. By using several different fluorescent dyes, distinct germination parameters such as DNA accessibility and esterase activity were quantified. Finally, spore properties from a large number of B. cereus strains, including the B. cereus laboratory model strain ATCC14579 and a number of recent isolates from environmental and industrial settings were analyzed. The strains tested showed a large variation in heat resistance, and the majority had a higher heat resistance than the laboratory model strain. With respect to germination, many of the strains were less sensitive to the nutrients tested as compared to the laboratory model strain. Heat activation and ageing enhanced germination in response to several nutrients in various isolates. The knowledge that was gained and the methods that were developed in this study are expected to contribute to progress in the spore research field, and to enhanced spore control in the food industry.