Reasoning that the growth of bacteria could serve as a marker of an environment that is conducive to growth, the authors set out to probe whether growing bacteria produced signals that could induce spore germination. Filtered bacterial culture supernatants from
Bacillus subtilis
and
Escherichia coli
, but not
Staphylococcus aureus
, induced spore germination. Intriguingly, only growing cells produced the spore-germination signal. During growth, enzymes lyse the mature peptidoglycan sac that maintains bacterial cell shape to allow insertion of new peptidoglycan monomers, releasing muropeptides in the process. Gram-negative bacteria can recycle muropeptides, whereas Gram-positive bacteria simply release them into the environment. Because B. subtilis supernatants were more efficient germinants than E. coli supernatants, the authors proposed that liberated peptidoglycan fragments might induce germination.
Tiny amounts of purified and digested B. subtilis peptidoglycan were sufficient to induce spore germination, and high-performance liquid chromatography was used to pinpoint disaccharide tripeptide as the smallest molecule that could induce germination. Purification of peptidoglycan from a selection of Gram-positive and Gram-negative bacteria confirmed that only peptidoglycan with a meso-diaminopimelic acid residue in the third position of the stem peptide induced germination, which explained why S. aureus supernatant (L-Lys in the third position of the stem peptide) failed to induce germination.
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