cAs previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GRindependent germination by DPA with its associated Ca 2؉ divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP ؉ spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 M and 2 mM for L-alanine and <10 mM for L-valine, rates of gerP spore germination increased up to between 200 mM and 1 M L-alanine and 100 mM L-valine, and at 1 M L-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane.

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the "germinosome," and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with >75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ϳ50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ϳ2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds. Spores of Bacillus subtilis are metabolically dormant and resistant to harsh environmental conditions, allowing them to survive for many years (1). However, the presence of specific nutrients triggers the process of germination, in which spores lose their dormancy and resistance and then outgrow into vegetative cells. Normally, for germination to occur, nutrients must cross the coat, outer membrane, cortex, and germ cell wall of the dormant spore to reach nutrient germinant receptors (GRs) located in the spore's inner membrane (IM) (1-3). Lipid molecules in the B. subtilis spore IM are largely (ϳ70%) immobile, but upon completion of spore germination, the volume encompassed by the IM expands ϳ1.5-fold, and the amount of the immobile lipid fraction decreases to ϳ25%, similar to the value for vegetative cells, all in the absence of detectable ATP synthesis (4). Binding of specific germinants to their cognate GRs leads to the release of the spore core's large store (ϳ25% of core dry weight) of dipicolinic acid (DPA) chelated to divalent cations, mostly Ca 2ϩ (CaDPA); CaDPA release is accompanied by some water uptake into the core and some loss of spore resistance. CaDPA release also triggers the degradation of the spore's peptidoglycan (PG) cortex by cortexlytic enzymes (CLEs), allowing for core expansion, further core water uptake, completion of germination, and, ultimately, outgrowth to gen...