Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

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Abstract
Bacillus cereus 569 spores germinate either with inosine as a sole germinant or with a combination of nucleosides and l-alanine. Whereas the inosine-only germination pathway requires the presence of two different germination receptors (GerI and GerQ) to be activated, the nucleoside/alanine germination pathway only needs one of the two receptors. To differentiate how nucleoside recognition varies between the inosine-only germination pathway and the nucleoside/alanine germination pathway, we tested 61 purine analogues as agonists and antagonists of the two pathways in wild-type, ΔgerI and ΔgerQ spores. The structure–activity relationships of germination agonists and antagonists suggest that the inosine-only germination pathway is restricted to recognize a single germinant (inosine), but can be inhibited in predictable patterns by structurally distinct purine nucleosides. B. cereus spores encoding GerI as the only nucleoside receptor (ΔgerQ mutant) showed a germination inhibition profile similar to wild-type spores treated with inosine only. Thus, GerI seems to have a well-organized binding site that recognizes inosine and inhibitors through specific substrate–protein interactions. Structure–activity analysis also showed that the nucleoside/alanine germination pathway is more promiscuous toward purine nucleoside agonists, and is only inhibited by hydrophobic analogues. B. cereus spores encoding GerQ as the only nucleoside receptor (ΔgerI mutant) behaved like wild-type spores treated with inosine and l-alanine. Thus, the GerQ receptor seems to recognize substrates in a more flexible binding site through non-specific interactions. We propose that the GerI receptor is responsible for germinant detection in the inosine-only germination pathway. On the other hand, supplementing inosine with l-alanine allows bypassing of the GerI receptor to activate the more flexible GerQ receptor.