Synthesis of the peptidoglycan cell wall is an essential process in most bacteria and is an effective target for antibiotics. The activities of the major enzymes, the penicillin-binding proteins (PBPs), involved in peptidoglycan polymerization are known, but the specific mechanisms by which the multiple proteins in this family control cell shape and division are not clear. The long-term objectives of the proposed studies are characterization of the functional domains of the major PBPs of Bacillus subtilis and identification of interactions between these and other proteins. Studies of peptidoglycan synthesis during both vegetative growth and spore formation will be undertaken. An understanding of the network of proteins involved in polymerizing particular cell wall structures will reveal new potential targets for antibiotic development. Knowledge of the processes of spore peptidoglycan formation and of spore peptidoglycan degradation during germination will contribute to the development of methods for spore killing. Such methods will be generally applicable to defense against spore-based biological weapons. Variant class A PBPs will be produced, including active site mutants, truncated proteins, and chimeric proteins containing domains from related proteins, and their abilities to carry out the various functions of the major B. subtilis class A PBP, PBP1, will be assessed. Enzymatic activities and effects on phenotypic properties (cell length, cell diameter, growth rate, peptidoglycan structure, and localization of PBP1) will be measured. Similar studies will examine the role of PBP2c in spore wall synthesis. Immunoprecipitation, assays for PBP and autolysin activities, defined mutant strains, western blotting, and mass spectrometry will be used to identify proteins that interact with class A PBPs during cell growth, division, and sporulation. Correlations will be made between disruptions of protein-protein interactions and changes in phenotypic properties. Cellular localization and protein-protein interactions of class B PBPs required for cell shape determination, cell division, and spore formation will be determined using immunofluorescence microscopy and immunoprecipitation.
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