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 ∼ 50% 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 ∼ 2-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.