Chen, L.; Beekhuis, H.; Bosch, C. v. d.; Vinay, G.; Vreede, J.; Setlow, P.; Brul, S.

Some species in the Bacillales and Clostridiales bacterial orders form spores in unfavorable environments. These spores are metabolically dormant, and resistant to harsh conditions. However, upon triggering by a variety of small molecule nutrients, termed germinants, spores may germinate, increasing their core water content from 25%-45% in spores to ~80% in growing cells, thus restoring rapid metabolism and leading to outgrowth and then vegetative growth. Recent structural modeling and mutagenesis studies showed that a subunit of the prototypical Bacillus subtilis spore germinant receptor (GR) GerA, the integral inner membrane (IM) protein GerAB, is involved in spore germination initiated by L-alanine. Notably, a previous molecular simulation study showed the formation of a water channel in GerAB. In the current study we employed Steered Molecular Dynamics (SMD) simulations to force a single water molecule through GerAB. Examining the simulations revealed three key amino acid residues, Y97, L199 and F342, that interfere with water passage. These three residues were altered to alanine, followed by assays of germination. In the Y97A, L199A, F342A single mutants and the Y97A, L199A and F342A triple mutant (triA), all four types of mutant spores barely responded to L-alanine, while wild type spores germinated almost 100% under the same conditions. Y97A spores expressed the GerAA protein as determined by western blot analysis, corroborating the presence of the GerA GR. However, for the other three mutants, GerAA\'s presence in the spore could not be confirmed. In conclusion, Y97 plays a key role in GerAB mediated germination and suggests that GerAB indeed facilitates water passage and further indicating that water passage through GerAB is a crucial event in B. subtilis spore germination.