Quantifying Bacillus subtilis Sporulation

To test for the effects of induced sigma factors on host sporulation, we diluted overnight B. subtilis cultures in fresh DSM (OD₆₀₀ = 0.05) and dispensed each culture into multiple wells of a 96-well plate that was then incubated in a BioTek Synergy H1 plate reader (37°C, fast and continuous shake setting). Under these conditions, we determined that cells enter stationary phase after approximately 4.5 h, marking the onset of sporulation. At this time, we induced expression of the cloned gene by adding IPTG (final concentration 1 mM) to half the cultures in the plate. We added water to the rest of the wells, which served as noninduced controls. At 24 h, we quantified the number of spores and vegetative cells in each well using a flow cytometry assay that distinguished spores from vegetative cells (nonspores) based on differential uptake of the nucleic acid stain SYBR green (97 (link)). We diluted each sample in Tris-EDTA buffer (pH 8) and then fixed the cells in 0.5% glutaraldehyde for 15 min at 4°C. We stained the fixed samples with SYBR green (20,000× dilution of commercial stock, Lonza) for 10 min at room temperature in the dark. We then enumerated cells using a volumetric NovoCyte 2000R flow cytometer (Acea; excitation, 488 nm; emission, 530/30 nm) and an automatic gating pipeline (see Fig. S6).