Visualization of SpoVAEa protein dynamics in dormant spores of Bacillus cereus and dynamic changes in their germinosomes and SpoVAEa during germination

Abstract

Bacillus cereus spores, like most Bacillus spores, can survive for years, germinate when their surroundings become suitable, and spore germination proteins play an important role in the initiation of germination. Because germinated spores lose dormant spores extreme resistance, information on the function of germination proteins could be useful in developing new strategies to control B. cereus spores. Prior work has shown that: i) the channel protein SpoVAEa exhibits high frequency movement in the outer leaflet of the inner membrane (IM) in dormant spores of B. subtilis; ii) the formation of the foci termed the germinosome between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing spores of B. cereus is slower than foci formation by GerR and GerD individually. However, the dynamics of movement of SpoVAEa in B. cereus spores, and the behaviour of the germinosome in B. cereus spore germination are unclear. In this study, we found that SpoVAEa fluorescent foci in dormant spores of B. cereus move on the IM, but slower than in B. subtilis spores, and likely colocalize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that: i) expression of GerR-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus; and ii) germinosome foci observed by Fluorescence Resonance Energy Transfer (FRET) between GerR-SGFP2 and GerD-mScarlet-I b can be lost soon after the spore phase transition. However this is not always the case, as some GerR-SGFP2 foci and GerD-mScarlet-I foci continued to exist, colocalize, and even show a weak FRET signal. These data highlight the heterogeneous behaviour of spore germination protein complexes and indicate that some complexes may persist well beyond the initiation of germination. IMPORTANCEBacillus cereus is commonly present in soil and does harm to humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, as well as the dynamics of a number of germination proteins in spore germination. Our results expand upon observations of the interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B and C subunits and GerD, as well as between SpoVAEa and GerD. The approaches used in this work could also be used to examine the interactions between GerD and SpoVAEa and other germination proteins in spores of other Bacillus species.