Structure-Based Functional Studies of the Effects of Amino Acid Substitutions in GerBC, the C Subunit of the Bacillus subtilis GerB Spore Germinant Receptor

Abstract

Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.