This application proposes three major areas of work on aspects of spore formation, dormancy, resistance and germination with the bacterium Bacillus subtilis. These areas are: 1) determine the structure and function of the complex between alpha/beta-type small, acid-soluble spore proteins (SASP) and DNA; 2) determine the structure, mechanism and function of the protease (GPR) that degrades SASP during spore germination; and 3) investigate various aspects of spore germination.
Specific aims i nclude: a) determine the structure of SASP-DNA complexes by X-ray crystallography and use these data as well analysis of SASP with further site directed changes to determine the mechanism whereby SASP provide DNA protection; b) mutagenise the GPR coding gene to obtain variants that have lost catalytic activity but retain structure; c) determine the structure of both the active and zymogen forms of GPR; d) examine the role of another possible SASP-specific protease encoded by the yyaC gene; e) determine the mechanism of regulation of the gerK operon that encodes one of the spore's nutrient germinant (Ger) receptors essential for spore germination with nutrients; f) measure the relative level of expression of the three operons (gerA, gerB and gerK) that encode the spore's Ger receptors; g) elucidate the requirement for lipid addition to the proteins encoded by the C-cistrons of the operons that encode the Ger receptors; h) examine the function of the SpoVA proteins in movement of dipicolinic acid (DPA) and/or ions in spore germination; i) determine the location of the SpoVA proteins in spores; j) assess the interaction of various proteins that make up Ger receptors as well as different the Ger receptors themselves; k) determine the factors that affect the spontaneous spore germination that does not require nutrients; I) isolate and characterize mutants that are altered in spontaneous spore germination and mutants whose spores germinate well in the presence of ion channel blockers; and m) express Ger receptors and SpoVA proteins in E. coli to study their function. ? ? ?
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