Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions Between GerD and Germinant Receptor Subunits in Spores

Spores of the bacterium can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores' extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control spores. In this study, we confirmed that the GerR germinant receptor's (GR) A, B, and C subunits and GerD co-localize in spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)-SGFP2 and GerD-mScarlet-I, but there was no FRET between GerD-mScarlet-I and either GerRA-SGFP2 or GerRC-SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)-SGFP2 and GerD-mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.