Figure 1. Spontaneous Germination of B. subtilis Spores.

(A) Spores germinate at high frequencies in response to a germinant such as alanine (“induced”) or at lower frequencies in the absence of any specific environmental signal (“spontaneous”).

(B) Spores of B. subtilis (JDB1772) were heat treated and then plated on LB (black), mISP4 (green), and mISP4 supplemented with 10 mM alanine (gray) to determine the fraction of germinating spores. CFU (mISP4) versus CFU (LB), p = 0.004; CFU (mISP4) versus CFU (mISP4ala), p = 0.008 (Mann-Whitney test). Shown is the mean, and error bars represent the SD.

(C) Measurement of the spontaneous germination rate. Wild-type spores (JDB1772, ~1.0 × 10⁹) maintained in distilled water at 37°C were plated at the indicated time points on mISP4, and the number of spontaneously germinated spores was determined. The regression line is: y = 3 × 10⁻⁵x; R² = 0.8963. Shown is the mean, and error bars represent the SD.

(D) Schema of experiment to demonstrate that colony formation on mISP4 is not due to mutation. B. subtilis spores are plated on mISP4 and a single colony from that plate is sporulated by exhaustion in DSM, and the resulting spores are plated on both mISP4 and LB to determine the frequency of spontaneous germination. This process is repeated several times.

(E) Single wild-type B. subtilis colonies (JDB1772) were cycled through three rounds of sporulation and successive spontaneous germination. The descendants after round 3 were plated on LB (black bar) and mISP4 (green bar) to determine the final spontaneous germination rate.

(F) Germination on mISP4 with heat-treated spores of wild-type strain (JDB1772) and strains lacking a subset or all of the five germination receptor mutants (“ΔgerA,” JDB2919; “ΔgerB,” JDB3118; “ΔgerK,” JDB3615; “ΔgerA ΔgerB,” JDB1366; “ΔGR3,” JDB3614; “ΔGR5,” JDB1914) were plated on LB (black), mISP4 (green), or mISP4 + 10 mM alanine (gray). Shown is the mean, and error bars represent the SD.