Structural and functional analysis of the Bacillus cereus GerI inosine-responsive spore germinant receptor

Bacterial endospores are metabolically dormant yet can rapidly return to vegetative growth upon exposure to nutrients through the process of germination. Spore germination is triggered by specific chemical nutrients binding to cognate germinant receptors (GRs) in spores’ inner membrane. These GRs function as ligand-gated ion channels and are composed of clusters of at least three subunits. Given their central role in germinant recognition and discrimination, elucidating 3D structures of GR subunits is a key part of efforts to understand the mechanism(s) of spore germination. Here we present the crystal structure of the N-terminal domain of the Bacillus cereus GerIA protein (GerIANTD), a component of the inosine-responsive GerI GR. GerIANTD adopts a conformation homologous to substrate-binding proteins in bacterial ABC transporters. NMR chemical shift perturbation and site-directed mutagenesis identified GerIANTD residues potentially involved in inosine binding or critical for germinosome assembly in B. cereus spores, the absence of which abrogated inosine-induced germination. Molecular modelling and mutagenesis identified residues in the GerIB subunit forming germinant and cation-binding sites. GerQ, the second GR that contributes to inosine germination in B. cereus spores, was capable of complementing hypomorphic gerI alleles in several instances, demonstrating cooperative restoration of function despite being incapable of initiating germination to inosine in gerI null spores. Collectively, our results provide new insights into GR subunit function and the molecular basis of the B. cereus germinative response to inosine.