Identification of key residues in Bacillus subtilis germinant receptor protein GerAB involved in spore water uptake

Some Bacillales and Clostridiales bacteria form spores in unfavorable environments. These spores are dormant but can rapidly resume metabolism in response to certain small molecule nutrients. This process is termed germination and can be initiated by a variety of low molecular wt 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 was minimal in spores with L199A, F342A and triA (Y97A, L199A and F342A triple mutant), while Y97A mutant spores germinated [~]61%. And besides Y97A, all other mutants showed compromised germination triggered by AGFK mixture. 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 proved that all three identified residues are crucial to the structural integrity of the GerAB protein and therefore probably the formation of the GR complex, the germinosome.