The dynamic interplay between peptidoglycan synthases by in vitro reconstitution

• Applicant: Dr. Sofia Ainatzi
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 570424261

The cell wall is highly conserved among bacteria and essential to their survival, that is why enzymes synthesizing it are a favoured target of antibiotics. Recent studies indicate that the bacterial cell wall, also called peptidoglycan (PG), is synthesized by two distinct classes of PG synthases: the bifunctional class A penicillin binding proteins (PBPs) that possess both the glycosyltransferase (GTase) and transpeptidase (TPase) activities and the SEDS (shape, elongation, division, sporulation) GTases with their cognate class B PBP TPases. During division, the PG layer must grow inward to form a septum and both classes of PG synthases, i.e. the SEDS protein FtsW and the class A PBP1b, have been associated with the septal PG synthesis. It was recently demonstrated that FtsW, with its associated TPase, FtsI, purified as FtsWIQLB pentameric protein complex is active to produce PG in vitro, however, the exact kinetics of the PG synthesis by this complex remains uncharacterized. Additionally, the interactions and crosstalk between these two classes of PG synthases remain poorly studied. In this project, we aim to determine the dynamics of PG synthesis by FtsWIQLB complex reconstituted in polymer-supported lipid membranes. Additionally, we will characterize the composition of the PG product using a mass-spectrometry based pipeline. Lastly, by using this platform we will investigate the crosstalk between the two classes of PG synthases. Overall, this project aims to enhance our mechanistic understanding of the PG synthesis by the two classes of PG synthases and their synergistic effects on PG assembly. This new platform can further be applied for the identification of novel inhibitors of these enzymes and thus facilitate the identification of new antibiotics.

• DFG Programme: WBP Fellowship
• International Connection: Spain
• Host: Dr. Natalia Baranova