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ORIGINAL RESEARCH article

Front. Microbiol.

Sec. Microbial Physiology and Metabolism

Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1656964

This article is part of the Research TopicStructure and Mechanism of Microbial Membrane Active TransportersView all 5 articles

GerAB residues predicted to interfere with water passage based on Steered Molecular Dynamics are key to germinosome functionality

Provisionally accepted
Longjiao ChenLongjiao Chen1,2Houdijn BeekhuisHoudijn Beekhuis2Christina van den BoschChristina van den Bosch1Gianni VinayGianni Vinay1George KorzaGeorge Korza3Jocelyne VreedeJocelyne Vreede2Peter SetlowPeter Setlow3Stanley BrulStanley Brul1*
  • 1Universiteit van Amsterdam Swammerdam Institute for Life Sciences, Amsterdam, Netherlands
  • 2Universiteit van Amsterdam Van 't Hoff Institute for Molecular Sciences, Amsterdam, Netherlands
  • 3University of Connecticut Department of Molecular Biology and Biophysics, Farmington, United States

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Some Bacillales and Clostridiales bacteria form spores in unfavorable environments. These spores are dormant but can rapidly resume metabolism in germination. This process can be initiated by a variety of low molecular weight nutrients termed germinants. Structural modeling and mutagenesis studies showed that GerAB, an inner membrane (IM) protein of the Bacillus subtilis spore germinant receptor (GR) GerA, is involved in L-alanine-initiated spore germination. A previous molecular simulation study also suggested there is a water channel in GerAB. In the current work, Steered Molecular Dynamics (SMD) simulations were employed to force a single water molecule through GerAB, identifying three key amino acid residues, Y97, L199 and F342, that interfere with water passage. When these residues were altered to alanine, L-alanine germination no longer occurred in spores with L199A, F342A and triA (Y97A, L199A and F342A triple mutant), while Y97A mutant spores germinated ~61%. Additionally, except for Y97A, all other mutants showed compromised germination triggered by the AGFK mixture (L-asparagine, D-glucose, D-fructose and K+ ion). Western blotting found reduced levels of the GerA GR in the Y97A mutant, and an absence of the GerA GR in all other mutants. This proves that all three identified residues are crucial to the structural integrity of the GerA germinant receptor and also suggests they are essential for the formation of a fully functional GR complex, the germinosome.

Keywords: Bacterial spore, spore germination, Molecular Dynamics Simulation, APC transporter, water channel

Received: 30 Jun 2025; Accepted: 09 Sep 2025.

Copyright: © 2025 Chen, Beekhuis, van den Bosch, Vinay, Korza, Vreede, Setlow and Brul. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Stanley Brul, Universiteit van Amsterdam Swammerdam Institute for Life Sciences, Amsterdam, Netherlands

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Supplementary Material