Spore germination and outgrowth experiments

Spores were heat activated at 70°C for 30 min in order to ensure synchronized germination. Germination and outgrowth experiments were performed in germination media composed of Spizizen Minimal Medium (SMM; as described in Nicholson and Setlow, 1990) with or without 1.2 M NaCl, which additionally contained 50.5 mM D-glucose, 0.5 mM L-tryptophan, and 10 mM of the germination trigger L-alanine.

The transcriptomics outgrowth experiments were performed in 45 ml germination medium (500 ml flasks). The medium was inoculated with 1.2 × 10¹⁰ heat-activated spores (in total) and 15 ml samples were withdrawn at 30, 60, and 90 min after inoculation. The samples were immediately mixed with ice-cold killing buffer (Nicolas et al., 2012) and washed with ice cold water by centrifugation (1 min at 10,000 x g at 4°C). The pellet was resuspended in 400 μl ice-cold LETS buffer (0.1 M LiCl, 0.01 M Na₂EDTA, 0.1 M Tris-HCl pH 7.4, 0.2% SDS), transferred to a pre-cooled Lysing Matrix B tube (MP Biomedicals, Santa Ana, CA, USA) containing 500 μl phenol:chloroform (1:1) and 25 μl 10% SDS, and used for RNA isolation. For the dormant spore RNA samples, spore suspensions were also heat-treated for consistency. Subsequently they were centrifuged, the pellets were resuspended in LETS buffer, and transferred to Lysing Matrix B tube for RNA isolation. The transcriptomics outgrowth experiments were performed in duplicate using two independent spore batches.

For spectrophotometric measurements, germination was carried out in triplicate in 96-well plates, each containing 200 μl of germination media. Each well was inoculated with 40 μl heat-activated spores to a starting optical density of ca. 0.5 at 600 nm (OD_600nm) corresponding to a total of ca. 4 × 10⁷ spores per well. The plate was incubated at 37°C in a multi-plate reader (ELx808IU, BioTek, Bad Friedrichshall, Germany) that read the OD_600nm of the culture, with 5 s of shaking before all readings. The OD_600nm data was normalized by division of each reading by the first measured value (t_0min), yielding the relative OD_600nm given in %. A 60% decrease in relative OD_600nm corresponds to germination of the whole spore population (Atluri et al., 2006; Nagler et al., 2014).