The purpose of this project is to examine and evaluate the penicillin-binding proteins (PBPs) of Bacillus subtilis under a variety of physiological conditions. The long range goal is to correlate the activities of these proteins with such biological processes as growth of the cell wall, formation of the septum during cell division or sporulation, and synthesis of the cortex layer in the developing forespore. Since all these events are susceptible to inhibition by penicillin, it is assumed that the action of at least some of the PBPs must be essential for their normal completion. A major effort will be made to evaluate the PBP changes that occur during sporulation because there are 2 penicillin-sensitive steps in this developmental process and because the early stages of sporulation are very similar to events occuring in a vegetative cell division. The nature of the PBP fluctuations, their physical location, their timing, and their universality will be examined. As a complement to these studies, the topographical distribution of the vegetative PBPs within the cell membrane will be determined by a comparison of membranes from minicells (primarily cell ends) with those from rod-shaped cells (primarily cell sidewalls). Furthermore, a phenotypic characterization of the 2 existing PBP-deficient mutants will be done to look for some biological property or function that can be attributed to the specific PBP defect. These PBP mutants and others that may be found in our proposed survey of the available cell shape and cell division mutants of B. subtilis will be the subjects of genetic analysis to begin to map the PBP genes, to determine their linkage to one another, and to correlate them with specific phenotypic properties. The various approaches that are proposed here should significantly add to our sparse knowledge of the in vivo roles of these PBPs. This information will both complement and extend the understanding we have gained primarily from in vitro biochemical analyses. The results should provide some important and useful insights into the mechanisms of cell division and sporulation, their regulation, and their inhibition by penicillin. On another level, additional information about sporulation could ultimately enable us to manipulate the process better for the production of various sporulation-specific products such as proteolytic enzymes, exotoxins, and antibiotics for industrial and medical uses.
