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

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, DgerI and DgerQ 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 (DgerQ 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 (DgerI 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.

Kinetic Evidence for the Presence of Putative Germination Receptors in C lostridium difficile Spores

“…For taurocholate titrations, spores were exposed to various concentrations of taurocholate (8,10,14,20, and 30 mM) at specific constant glycine concentrations (8, 9, 10, 13, and 16 mM). For glycine titrations, spores were exposed to various concentrations of glycine (8,10,12,14, and 18 mM) at specific constant taurocholate concentrations (6, 7, 9, 12, and 15 mM). The concentration ranges for both cogerminants were selected to avoid data clusters in double reciprocal plots.…”

“…As an alternative to genetic approaches, we have used kinetic methods and molecular probes to study Bacillus and Clostridium spore germination (1,3,14,30). These techniques allow proposing possible mechanisms for spore germination even when the identity of the germination receptors is not known (30).…”

Requirements for In Vitro Germination of Paenibacillus larvae Spores

“…The nature of the spore germination signals has been studied widely in Bacilli and Clostridia (21,27,28,(44)(45)(46)(47)(48)(49)(50), but the triggers for P. larvae spore germination have not been identified. In order to cultivate P. larvae, specialized media have been produced (1,31,(51)(52)(53).…”

The Ger Receptor Family from Sporulating Bacteria

“…Structure activity relationship of nucleoside agonists and antagonists of B. cereus spore germination have shown that the GerI receptor is constrained to recognize a single germinant (inosine) and is inhibited in a predictable pattern by structurally related nucleosides. On the other hand, the GerQ receptor is more promiscuous toward nucleoside agonists and is inhibited by nucleosides containing hydrophobic groups at the 6-position (Dodatko et al, 2009a).…”

“…Germination agonists and antagonists have been used to characterize the binding sites of B. cereus nucleoside receptors (Dodatko et al, 2009a). This approach could be extended to map germinant/Ger receptor interactions in other complexes.…”