Amino Acid Residues in the GerAB Protein Important in the Function and Assembly of the Alanine Spore Germination Receptor of Bacillus subtilis 168

Abstract

The paradigm gerA operon is required for endospore germination in response to L-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for L-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-aminoacid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.In the model endospore-former Bacillus subtilis, endospore germination can be induced by exposure of the spores to L-alanine. This is mediated via a germinant receptor, a likely complex of three proteins, encoded by the gerA operon (31). The GerAA and GerAC components of this complex have been found in the inner membranes of the spores by Western blot analysis of spore fractions (14). The coexpressed GerAB protein, a predicted integral membrane protein with 10 membrane spans, would locate with the other two proteins, forming a membrane-associated receptor that initiates germination in response to L-alanine concentrations of Ն10 M.Homologous gene clusters, often organized as tricistronic operons, can be identified in the genomes of sporeformers across the genus Bacillus and in most clostridial species. The germinant specificity of various receptors has been elucidated in several species, and in all such cases, these receptors seem to be involved in the triggering of spore germination, individually or in combination. In B. subtilis, four other receptor operons have been identified as well as gerA. The gerB and gerK operons encode receptors that together mediate the germination response to the cogerminant mixture AGFK (L-asparagine, Dglucose, D-fructose, and K ϩ ions) (8). It has been suggested that the GerK receptor has a prominent role in the interaction with sugars, while GerB binds L-amino acids, but not suffi...

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

“…Indeed, a number of amino acid substitutions in the B. subtilis GerAA and GerAB proteins results in loss of GerAC from total spore extracts (29,30). Wild-type GerAC in the IM also appears likely to be essential for GerAA assembly in the IM, as loss of GerAC from the M fraction in spores lacking GerF, which encodes prelipoprotein diacylglycerol transferase, also results in loss of GerAA from this fraction (7).…”

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…It seems certain that it is the GRs that bind nutrient germinants, since a number of specific mutations in GR subunits affect the apparent affinity of the mutant GR for specific germinants (4,22,23,57). Similarly, spores with elevated levels of specific GRs germinate more effectively with low germinant concentrations (4).…”

Membrane Topology of the Bacillus anthracis GerH Germinant Receptor Proteins

“…Using this system, the membrane topologies of the B. anthracis GerH A , GerH B , and GerH C proteins were mapped, providing the first structural data on the A and B subunits of the nGRs. Although only the GerH protein topology was analyzed, the results are likely applicable to other nGR family members, as these proteins are predicted to have similar structures (4,6,26,29).…”

“…Both A-and B-type nGR subunits have been associated with germinant recognition (5,6,29,35). Site-directed mutagenesis of B-type nGR proteins in Bacillus subtilis and Bacillus megaterium has identified residues in the membrane-spanning regions of these proteins that are potentially involved in ligand binding (5,6). Both GerH A and GerH B contain membrane-spanning regions that could be available for interactions with nutrient germinants.…”