The penicillin-binding proteins (PBPs) synthesize and modify peptidoglycan (PG), the main structural element of the bacterial cell wall. PBPs and PG synthesis are highly conserved in all bacteria and both have been important targets for antibiotic and antibacterial development. In the Gram positive bacterium Bacillus subtilis, PBP1 is composed of the four domains S, N, P, and C in order from the N- to C-terminus. It plays important roles in vegetative PG synthesis. Compared to the wild type B. subtilis, the PBP1 null mutant has decreased growth rate, cell diameter, and PG crosslinking; the cell population has more long cells; and the colonies have raised and smooth edges. In this work, we constructed six mutant forms of PBP1 that were tagged with a C-terminal FLAG epitope, to complement the wild type gene. We examined the colony and cell morphologies, and PBP1 localization in the mutant strains. The removal of the cytoplasmic region of the PBP1 S domain and the replacement of PBP1 S domain by PBP4 S domain did not change the colony morphologies, and each of these two mutations had minor effects on growth rate, cell diameter, PG crosslinking and generation of long cells in the cell population. The single point mutation in the active site of the N or P domain presumably removed the enzymatic activity, and each mutation caused slower growth rate, decreased cell diameter and PG crosslinking. The point mutation in the P domain had a minor effect on the colony morphology and formation of long cells; while the mutation in the N domain altered the colony morphology, and resulted in high percentage of long cells that is comparable to the PBP1 null mutant. The C domain of PBP1 has no apparent enzymatic activity, but the loss of it altered the colony morphology, and caused slower growth rate, decreased cell diameter, and PG crosslinking. In the wild type B. subtilis, PBP1 localizes to the septum. This septum localization specificity was lost in strains expressing PBP1 without the C domain, with PBP4 S domain, or with a point mutation in the active site of the N domain. PBP1 with a point mutation in the active site of the P domain, or without the cytoplasmic region of the S domain, had decreased septum localization specificity. These findings were used to develop a model of how PBP1 domain functioning in B. subtilis. / Master of Science

Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didn't influence the normal membrane localizations of PBP2a or PBP2b. PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations. / Master of Science