Membrane Topology of the Bacillus anthracis GerH Germinant Receptor Proteins

Bacillus anthracis spores are the etiologic agent of anthrax. Nutrient germinant receptors (nGRs) packaged within the inner membrane of the spore sense the presence of specific stimuli in the environment and trigger the process of germination, quickly returning the bacterium to the metabolically active, vegetative bacillus. This ability to sense the host environment and initiate germination is a required step in the infectious cycle. The nGRs are comprised of three subunits: the A-, B-, and C-type proteins. To date there are limited structural data for the A- and B-type nGR subunits. Here the transmembrane topologies of the B. anthracis GerHA, GerHB, and GerHC proteins are presented. C-terminal green fluorescent protein (GFP) fusions to various lengths of the GerH proteins were overexpressed in vegetative bacteria, and the subcellular locations of these GFP fusion sites were analyzed by flow cytometry and protease sensitivity. GFP fusion to full-length GerHC confirmed that the C terminus of this protein is extracellular, as predicted. GerHA and GerHB were both predicted to be integral membrane proteins by topology modeling. Analysis of C-terminal GFP fusions to full-length GerHB and nine truncated GerHB proteins supports either an 8- or 10-transmembrane-domain topology. For GerHA, C-terminal GFP fusions to full-length GerHA and six truncated GerHA proteins were consistent with a four-transmembrane-domain topology. Understanding the membrane topology of these proteins is an important step in determining potential ligand binding and protein-protein interaction domains, as well as providing new information for interpreting previous genetic work.