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

Abstract

Spores of the bacterium Bacillus cereus 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 B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus 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.

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

“…Considering initiation of spore nutrient germination, a recent study suggested that in B. subtilis, GerAB is responsible for sensing L-alanine, indicating that the function of GerA's B subunit is to detect nutrient germinants [27]. Our recent studies also suggest that there is interaction of GerRB with GerD and with a GR B subunit possibly bound by L-alanine [7,28].…”

“…The correct construction of recombinant plasmids pHT315-f14 and pHT315-f15 was confirmed by sequencing, followed by electroporation into competent B. cereus ATCC 14579 cells, and selection and confirmation with colony PCR of an erythromycin-positive single colony. PR-gerR(A-C-B)-SGFP2 [7] strain 007 +pHT315-f05 Ery r PD-gerD-mScarlet-I [7] strain 010 +pHT315-f10 Ery r PD-gerD-SGFP2 [7] strain F06 +pHT315-f06 Ery Abbreviations: PEa, promoter of spoVA operon; PR, promoter of gerR operon; PD, promoter of gerD; Ery r , resistant to erythromycin.…”

“…cereus GerR GR has also been shown to interact with GerD in the dormant spore IM [7]. Another important group of spore germination proteins are the SpoVA proteins encoded by the spoVA operon which constitute a CaDPA channel in the IM of bacterial spores.…”

“…The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.10.475594 doi: bioRxiv preprint formation of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I foci, with significant heterogeneity in formation of germinosomes foci [7]. However, little information is known about the overall changes or behavior of germination proteins during the germination process of B. cereus spores.…”

“…Previous work suggests that GerR(A-C-B) is localized in the spore IM using fluorescent reporter protein fusions and the membrane dye FM 4-64, and is primarily responsible for L-alanine germination of B. cereus spores [7,20,21]. Recent work also showed that in the IM germinosome, GerR(A-C-B) and GerD could be visualized using fluorescent reporter proteins and this work suggested that the formation of germinosome foci could be significantly slower than the formation of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I foci, with significant heterogeneity in formation of germinosomes foci [7]. However, little information is known about the overall changes or behavior of germination proteins during the germination process of B. cereus spores.…”