Peptidoglycan synthesis in Escherichia coli from a PBP1b perspective

Abstract

An essential and unique component of the bacterial cell envelope is the peptidoglycan layer. This layer, formed by glycan chains that are interlinked by peptide bridges, is essential for the maintenance of the specific cell shape of the bacterium, and protects it from rupture by osmotic pressure. Because this layer is so essential, easy accessible, on the outside of the bacterium, and we humans do not possess this structure, and hence its synthesis machinery, it has always been and still is an attractive target for antibacterial compounds. Since now a days more and more bacteria become resistant against the available antibiotics, it is of great importance to find new targets for the development of new classes of antibiotics. In this perspective, we focused on the cell division-specific bifunctional peptidoglycan synthase PBP1b of E. coli. By getting more insight in its interactions with and its regulation by (new) interacting proteins, the complex mechanism of peptidoglycan synthesis by PBP1b, and the regulation of this in space and time can be further clarified. This information can then be used for the rational development of new antibiotics. Site specific incorporation of an unnatural amino acid with cross-linking properties in PBP1b in vivo, is one strategy used to obtain information on the interaction profile of PBP1b. Using this technique, we found one new interacting protein, having a decreasing effect on the transpeptidase activity of PBP1b (CpoB), and we specified the interaction site of this, and an already known PBP1b regulator, FtsN, to be in a cleft between the UB2H and transpeptidase domain. Moreover, we also showed that a loop structure of the transpeptidase domain is an interaction hot spot which is also essential for proper functioning of this domain. We furthermore demonstrated the usefulness of a new immobilization strategy for SPR experiments, to allow measurements with fully active and correctly orientated proteins. This strategy should more reflect the in vivo situation, and prevent measurements of non-relevant interactions. Different fluorescently labeled substrate variants were also created, which can be used to analyze the activity of PBP1b, and other peptidoglycan synthases. These new variants allowed us to obtain results with a higher resolution and sensitivity. Finally, we describe an attempt to identify the part of PBP1b important for dimer formation. In vitro experiments have shown that dimerization increases the activity of PBP1b, however, the in vivo role of dimerization remains unclear. These results present new players in the vast amount of interactions in which PBP1b is involved, which reflects the complexity and importance of correct spatio-temporal regulation of peptidoglycan synthesis. Furthermore, the improvement of the assays to measure interaction kinetics and the activity of PBPs can be used in the ongoing research on bacterial cell wall synthesis, to find new targets for antibiotic development.