Fig. 3. Mutations in the putative l-alanine-binding pocket of GerAB enable spores to respond to l-leucine or l-serine.

a Spores harboring gerAB(V101A) or gerAB(V101C) germinate in response to 1 mM l-alanine with kinetics similar to wild-type (WT). b V101A and V101C mutant spores but not wild-type also germinate in response to 5 mM l-leucine. c Representative phase-contrast images of wild-type and gerAB(V101C) spores after 100 min incubation with buffer, 1 mM l-alanine, or 5 mM l-leucine. Larger fields can be found in Supplementary Fig. 9. Scale bar, 2 µm. d d-alanine inhibits germination of gerAB(V101C) spores in response to l-leucine. Spores were incubated with 5 mM l-leucine with or without d-alanine at 0.625, 1.25, 2.5 mM, and OD₆₀₀ was monitored. The decrease in optical density in the V101 mutants was reproducibly less than wild-type in the germination assays and is likely due to spontaneous germination of a subset of the spores during their purification. e Spores harboring gerAB(L199S) germinate in response to 5 mM l-serine. Representative data from one of three biological replicates with two technical replicates are shown. Error bars in a, b, d, e indicate ± SD of two technical replicates.