Spore germinosome visualization and dynamics in Bacillus cereus

Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. B. cereus can survive for years in the form of spores posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. These dormant and resistant spores can germinate and grow when their surroundings become suitable, and the specific germination proteins of spores play a major role in the decision to initiate germination. Since germinated spores have lost the extreme resistance of the dormant spores, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. The thesis demonstrated that the germination proteins germinant receptor (GR) GerR sensing L-alanine and GerD acting as a scaffold for GRs’ assembly localize in the inner membrane (IM) of spores in germinosomes. Germination initiation involves, upon germinant binding, signal relay from germinosomes to the SpoVA channel thus facilitating the secretion of the spores’ dipicolinic acid, a compound key to thermal resistance. In our current study, we found that the SpoVAEa subunit of the channel could be visualized as fluorescent reporter proteins clustered in a number of foci in dormant spores of B. cereus. These foci move on the IM, but slower than in B. subtilis spores, and likely colocalize transiently with GerD-mScarlet-I in the germinosome. Furthermore, the thesis found interaction between the GerR B subunit and GerD using Förster Resonance Energy Transfer (FRET), but not with the GerR A or C subunits. The heterogeneity in sporulation and germination previously described in B. cereus ATCC 14579 strain was also seen reflected in this work in the behaviour of individual germinosome protein complexes. Finally, we attempted to address flexibility in the integration of genes into B. cereus by studying both genomic integration and inducible high-level overexpression from plasmid of various gene constructs made in vitro. These data will aid future fluorescent reporter protein usage.