The morphogenetic events required to form a unique microbial structure, the B. subtilis spore coats, will be studied. Specific coat polypeptides are being purified, antibodies prepared and used as probes to detect precursors, processing steps and to look for alternations in coat mutants. The mechansim of cross linking of coat polypeptides via dityrosine residues to form an insoluble coat fraction will be further studied. The antibodies will be used to detect clones of spore coat structural protein genes. We shall also attempt to clone a gene affecting germination and spore coat structure by using as a probe a clone of the closely linked leucine genes. Mutants altered in one or more post exponential proteases will be isolated, mapped and isogenic strains constructed. The physiological effects of such mutations on protein turnover, shift down adaptation and spore coat polypeptide (or other protein) processing will be studied. We have evidence for one protease involved in the latter events and shall purify the enzyme to determine specificity, inhibition and to study in vitro processing. We shall also further examine the function of a protein protease inhibitor produced early during sporulation by trying to obtain mutants with an altered inhibitor. Glutamine synthetase in B. subtilis, in contrast to many other organisms, does not appear to be controlled by an elaborate cascade mechanism nor by a series of regulatory genes. All mutants isolated to date map in or close to the structural gene and yet there is some regulation of the amount of enzyme produced, especially by a trans acting factor detectable in partial diploids. By appropriate cloning we shall define this trans acting factor, and sequence it as well as potential regulatory regions close to the structural gene. We shall also construct more diploids to do complementation studies with various classes of mutants. Clones of the structural gene will also be used for localized nutagenesis, especially deletions to construct strains devoid of any activity or antigen. The pleiotropic effects of such mutants on sporulation and catabolism will be examined.
