Localization of Penicillin Binding Protein 1a Homologs in Caulobacter crescentus

How bacteria maintain their shape is not yet well understood. The bacterial cell wall is composed of peptidoglycan, which is a meshwork of glycan chains bonded covalently by short peptides. Since peptidoglycan is unique to bacteria, peptidoglycan synthesis proteins are well-known targets for antibiotic therapy against pathogenic bacteria.  We want to understand how peptidoglycan synthesis is regulated to produce Caulobacter crescentus’ shape. Unlike most bacteria, Caulobacter's genome encodes five homologs of penicillin binding protein 1a (PBP1a), an enzyme necessary for cell wall construction. The PBP1a's have a glycosyltransferase domain to synthesize the glycan strands and a transpeptidase domain that cross-links the peptides to form one large molecule, the sacculus. To understand how each gene contributes to the cell wall, we will localize each PBP1a within the cell. We hypothesize that their locations will correspond to their function, which can vary between stalk, division sites, lateral walls, or poles. We created a fusion protein between one of the PBP1a’s, CC0252 and the green fluorescent protein (GFP) and expressed it in Caulobacter. We then visualized the localization of the GFP-CC0252 fusion protein using fluorescence microscopy. This project will help us gain a better understanding of PBP1a proteins, which are involved in all bacterial cell wall synthesis. Our understanding of cell wall is based on a few species, and by understanding Caulobacter's cell wall construction, we can further understand more complex cell wall characteristics in bacteria.