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by claude@2026-07, 2026-07-04
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The study uses molecular dynamics simulations and follow-up mutational analysis to examine how water interacts with GerAB, the B subunit of the Bacillus subtilis GerA germinant receptor that functions as a L-alanine sensor during spore germination. Ten parallel MD simulations predicted a low but persistent water permeation pathway through GerAB (1–121 water molecules/µs), lined by eight hydrophilic residues, distinct from the L-alanine binding pocket. Substituting high water-contact residues with similarly sized non-polar residues impaired L-alanine germination and disrupted GerAB structural integrity, and these mutants showed altered slower kinetics in response to an AGFK germinant mixture. This paper is not about endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
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, while progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA GR (germinant receptor), 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 1-121 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 this 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 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.
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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, while progressing through outgrowth to vegetative growth. GerAB is the B subunit of the prototypical Bacillus subtilis GerA GR (germinant receptor), 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 1-121 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 this 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 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.
Competing Interest Statement
The authors have declared no competing interest.
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