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Description
Endospores of multiple bacterial species carry alanine racemase on their envelopes, an enzyme that interconverts the germinant L-alanine and the germination inhibitor D-alanine1, 2. However, the role of alanine racemization in sporulation and spore ecology remains unclear. Here, we confirm that Bacillus subtilis spores possess two distinct alanine racemases3: AlrB, a sporulation-specific enzyme4, 5, and AlrA, the essential vegetative racemase that provides D-alanine for cell wall synthesis during growth. Both are recruited to the spore coat in a CotE-dependent manner and contribute to alanine racemization in purified spore preparations. Using cell-specific inducible protein degradation, we depleted both racemases during sporulation, bypassing the requirement for D-alanine supplementation during growth. Spores lacking both racemases developed normally, without premature germination within the mother cell, indicating that alanine racemization is not essential for sporulation in B. subtilis. However, these spores were insensitive to germination inhibition at high spore densities6. In structured environments within a microfluidic chamber, alanine racemization reduced germination in regions of high spore density. Wild-type spores exposed to intermittent L-alanine pulses remained dormant, whereas mutant spores incapable of racemizing alanine germinated under identical conditions. These findings suggest that alanine racemization enables B. subtilis spore populations to regulate germination, preventing it when the presence of L-alanine does not reliably indicate favorable conditions for vegetative growth.
