SPORE PEPTIDOGLYCAN SYNTHESIS IN BACILLUS SUBTILIS

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The long-term objectives of the proposed studies are: 1) characterization of the enzymatic activities and gene products involved in synthesis of the peptidoglycan cell wall of bacterial endospores and 2) examination of the role of each type of peptidoglycan structural modification in determining endospore resistance properties. The specific aims are: 1) to determine the structure of the endospore peptidoglycan in the first stages of its synthesis and to track the structure to its final form in the dormant spore; 2) to identify the changes in synthesis of this structure produced by loss of penicillin- binding proteins, autolysins, and sporulation-associated gene products; and 3) to purify and characterize in vitro an enzyme involved in peptidoglycan side chain cleavage and muramic-lactam production. Knowledge of principles of endospore resistance properties, dormancy,and longevity may contribute to better decontamination methods and methods for storage and transport of drugs and vaccines. Peptidoglycan synthesis in general is an attractive target for antibiotic action, further studies of this process will contribute to methods for identification of new antibiotics. Peptidoglycan will be purified from sporulating cultures of Bacillus subtilis by chemical and enzymatic treatments, digested with muramidase, and analyzed by high-pressure liquid chromatography (HPLC) using methods previously developed for analysis of dormant sport peptidoglycan. Novel muropeptides will be identified using amino acid analysis and mass spectrometry. Appearance and loss of peptide side chain alanine and glycine residues, muramic- lactam production, and changes in peptide cross-linking will be quantified throughout the sporulation process. The analysis will be repeated using strains lacking individual penicillin-binding proteins, autolysins, and sporulation-associated gene products. The cwlD gene product will be purified and assayed in vitro for muramoyl-L-alanine activity and for muramic-lactam synthesis. Immature peptidoglycan samples from mutant strains and purified muropeptides will serve as substrates in these assays. Reaction progress will be monitored using the HPLC method and by adaptation of the method to capillary electrophoresis.