Abstract
Some species in the
Bacillales
and
Clostridiales
orders form spores under unfavourable environmental conditions. These spores are metabolically dormant and highly resistant to extreme stress. The spore core—analogous to the protoplast of vegetative cells—contains only 25–45% water by wet weight, compared to ~80% in vegetative cells. Upon activation by small-molecule nutrients, spores germinate, restoring their core water content, restoring metabolism and becoming easy to kill and then progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical
Bacillus subtilis
GerA germinant receptor (GR), a membrane protein belonging to the Amino Acid–Polyamine–Organocation (APC) superfamily of transporters. It functions as the L-alanine sensor that initiates germination and was previously predicted, based on molecular dynamics (MD) simulations, to contain a putative water channel. Using MD simulations, we identified low amount of water permeating through GerAB (ranging from 1 to 121 water molecule/μs in 10 parallel MD simulations), thus revealing a water pathway in GerAB that diverges from the L-alanine binding pocket, suggesting that water transport may play roles in germination beyond facilitating ligand binding. Analysis of water–residue contact frequencies identified eight hydrophilic residues lining the water’s path. Individual substitution of high-contact residues with similarly sized non-polar residues impaired L-alanine germination and disrupted GerAB structural integrity as assessed by Western Blotting. These mutants also respond to the AGFK germinant mixture (L-asparagine, D-glucose, D-fructose and potassium) in slower, yet individually distinct kinetics compared to that of wild-type (wt) spores. These findings prove that water contact residues in GerAB predicted by MD simulations are crucial for the stability of this protein and thus the germinosome complex with all GRs.