Reconstitution and functional analysis of multi-enzyme complexes involved in Escherichia coli peptidoglycan synthesis

The shape and size of a bacterial cell is determined by the shape and size of the peptidoglycan sacculus, which forms an exoskeleton that is essential for the osmotic stability of the cell. Peptidoglycan is present in most bacterial species and its biosynthesis is the target of widely used, safe and effective antibiotics. Although many of the enzymes involved in peptidoglycan metabolism have been characterised, surprisingly little is known about how they co-operate to enlarge the peptidoglycan during growth. We will attempt to reconstitute and characterise the multi-enzyme complexes responsible for peptidoglycan synthesis in E. coli. The enlargement of the stress-bearing peptidoglycan sacculus requires the oligomerization of the lipid II precursor and its insertion into existing peptidoglycan by transglycosylation and transpeptidation. Various synthetic and hydrolytic enzymes are involved, and in previous studies we obtained evidence that the synthases and other proteins involved function as components of multi-enzyme complexes. We aim to reconstitute such complexes in vitro in their native forms from the purified components. With the help of a functional peptidoglycan synthesis assay involving the structural analysis of the resulting peptidoglycan, we will study the conditions required for the formation of complexes and the role of each protein. In particular, we will determine the substrate specificity of the monofunctional transpeptidases, and aim to identify and characterise as yet unknown protein-protein interactions involving the peptidoglycan synthases. Ultimately these studies will lead to an understanding of the molecular mechanisms involved in the enlargement of the peptidoglycan layer during cell growth. Because these reactions are specific to bacteria, the inhibition of the peptidoglycan synthesis represents an excellent target for antimicrobial chemotherapy. Knowledge of the molecular details of peptidoglycan synthesis will be crucial for the development of novel antibiotics required to treat drug-resistant pathogens.

The enlargement of the predominantly single-layered peptidoglycan (murein) sacculus during growth of Escherichia coli is a complex process involving peptidoglycan synthases, hydrolases and structural proteins. A fail-safe cell wall growth mechanism is essential because even a temporal weakening of the stress-bearing sacculus is likely to result in cell lysis and death. In addition, the incorporation of new wall material has to occur in such a way that the rod-shape of E. coli is maintained throughout the growth cycle. E. coli is known to encode six peptidoglycan synthases (penicillin-binding proteins or PBPs) and 12 known autolysins. The latter are periplasmic peptidoglycan hydrolases participating in cell growth and division. It is not known how these enzymes co-operate to insert the lipid II precursor at the cell wall growth sites. There is, however, evidence that the enlargement of the peptidoglycan layer is performed by multi-enzyme complexes containing a combination of synthases and other proteins. In the current proposal, we aim to unravel the mechanism(s) of peptidoglycan growth by studying the interactions and the reactions of the peptidoglycan synthases. Interactions involving peptidoglycan synthases will be studied in vivo and in vitro, and the reactions of the synthases as well as the activity of reconstituted synthases complexes and other cell division/elongation proteins will be studied in vitro.