Visualization of SpoVAEa Protein Dynamics in Dormant Spores of <i>Bacillus cereus</i> and Dynamic Changes in Their Germinosomes and SpoVAEa during Germination

Bacillus cereus spores, like most Bacillus spores, can survive for years and germinate when their surroundings become suitable, and germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of dormant spores, 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 B. subtilis spores and (ii) the formation of the foci termed germinosomes between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing B. cereus spores is slower than foci formation by GerR and GerD individually. However, the movement dynamics of SpoVAEa in B. cereus spores, and the behavior of the germinosome upon B. cereus spore germination, are not known. In this study, we found that SpoVAEa fluorescent foci in dormant B. cereus spores move on the IM, but slower than in B. subtilis spores, and they likely co-localize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that (i) the 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 can be lost soon after the spore-phase transition. However, this is not always the case, as some GerR-SGFP2 and GerD-mScarlet-I foci continued to exist, co-localize, and even show a weak FRET signal. These data highlight the heterogeneous behavior of spore germination protein complexes and indicate that some complexes may persist beyond the initiation of germination. IMPORTANCE Bacillus cereus is commonly present in soil and infects humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific inner membrane (IM) channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, and the dynamics of germinosomes with GerR and GerD in spore germination. Our results expand upon observations of interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B, and C subunits and GerD, as well as those 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

Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of <i>Bacillus</i> <i>subtilis</i> spores

The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca(2+)-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs’ spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores’ rapid phase darkening, which is considered as due to rapid Ca(2+)DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40–80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events

Asynchronous division by non-ring FtsZ in the gammaproteobacterial symbiont of <em>Robbea hypermnestra</em>

The reproduction mode of uncultivable microorganisms deserves investigation as it can largely diverge from conventional transverse binary fission. Here, we show that the rod-shaped gammaproteobacterium thriving on the surface of the Robbea hypermnestra nematode divides by FtsZ-based, non-synchronous invagination of its poles-that is, the host-attached and fimbriae-rich pole invaginates earlier than the distal one. We conclude that, in a naturally occurring animal symbiont, binary fission is host-oriented and does not require native FtsZ to polymerize into a ring at any septation stage