The germination operon, gerS of Bacillus anthracis and its role in aromatic-triggered and cell-associated germination

This thesis focuses on the germinants of B. anthracis endospores and their relevance to cell-associated germination. The infective entity of anthrax is the dormant endospore of the bacterium Bacillus anthracis. To initiate an infection the endospore must first break dormancy through a process termed germination, which occurs in the host organism. The germination process is a rapid response to certain, specific chemical entities, termed germinants, that bind to protein receptors found on the inner membrane of the dormant endospore. The binding of germinant to receptor causes an irreversible cascade of events that leads to the breakdown of endospore dormancy and loss of endospore resistances.; The germinants of B. anthracis have been little studied, and we attempted to define distinct chemical moieties that triggered germination in the endospore. Through germinant surveys we found that a variety of amino acids and ribonucleosides serve as strong germinants in B. anthracis . In particular, there are five distinct germination pathways: L-alanine, L-alanine with an aromatic amino acid ( AEA), L-alanine with proline, aromatic amino acids and inosine dependent (AAID1), and certain nonaromatic amino acids with inosine (AAID2).; Through genetic manipulation and bioinformatics we discovered a chromosomally located gerA-type locus. The gerA-type loci are the putative germinant receptors in Bacillus species. This is the first reported chromosomal-encoded germinant receptor in B. anthracis. We discovered that the locus, named gerS, encodes a putative receptor that is essential to both the AAID and AEA germination pathways. Further work revealed that the gerS locus was responsible for binding to aromatic chemical structures.; Investigations with cell cultures showed that B. anthracis endospores responded rapidly and vigorously to the presence of macrophage. This rapid cell-associated germination was eliminated by a mutation that inactivates gerS. Several other mutants were produced that were also deficient in cell-associated germination. All cell-associated germination deficient mutants shared the common characteristic of being defective in AAID germination pathways.; The germination pathways of B. anthracis were investigated through a variety of genetic and biochemical methods. This work has shown that there are several distinct germination pathways. One of the germination receptors for B. anthracis was isolated from the chromosome and found to be an aromatic specific receptor, which is also necessary for both AAID and AEA germination responses. The AAID response was essential for rapid cell-associated germination. We hypothesize that the pathogenicity of B. anthracis is dependent on the AAID germination response and that one way to terminate this response is to eliminate the gerS locus on the B. anthracis chromosome.