Revisiting bacterial spore germination in the presence of peptidoglycan fragments

Bacterial spores of Bacillus species are metabolically inert cell types formed in response to nutrient starvation. Spores must undergo the process of germination to resume vegetative growth. This process is stimulated by the interaction of various nutrient molecules with specialized clusters of membrane-localized germinant receptors (GRs) present within spores. A second route to spore germination involving the stimulation of the PrkC Ser/Thr kinase by soluble peptidoglycan fragments was proposed in 2008 and has been subject to much less scrutiny. The current study examined the germinative response of spores of Bacillus subtilis, Bacillus cereus, and Bacillus megaterium when incubated in the presence of complex mixtures of peptidoglycan fragments or purified peptidoglycan fragments previously identified as germinants. The spore suspensions did not show any appreciable germination, as determined by fluorometric dipicolinic acid release, flow cytometry, or microscopy. However, the purified peptidoglycan fragments displayed a stimulatory effect on germination triggered by amino acids and nucleosides with spore GRs. In contrast, GR-mediated germination was inhibited to varying degrees by unidentified components of the complex peptidoglycan fragment mixtures derived from enzymatic digests of B. subtilis vegetative sacculi. Collectively, our results indicate that soluble peptidoglycan fragments cannot initiate spore germination but may influence germination via mechanisms that have yet to be established.IMPORTANCEStimuli and mechanisms that underpin bacterial spore germination are fairly well characterized. The physiological route relies upon the interaction of various small nutrient molecules with receptor proteins buried within spores. An alternative route to germination, whereby fragments of bacterial cell wall material-peptidoglycan-were proposed to stimulate a different receptor system, was proposed more recently (I. M. Shah, M. H. Laaberki, D. L. Popham and J. Dworkin, Cell 135:486-496, 2008, https://doi.org/10.1016/j.cell.2008.08.039). Results from the current study, where spores of several species of Bacillus were exposed to various peptidoglycan fragment-containing solutions, do not support this model of germination. This is significant since knowledge of germination can be exploited in a practical sense, as germinated spores are much easier to eradicate-in food processing and healthcare settings, for example-than their dormant counterparts.