Breukink, E. | Search bioRxiv

bioRxiv2020

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as {beta}-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

biochemistry↗

bioRxiv2020

Biochemical and structural insights into the activation of PBP1b by the essential domain of FtsN

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division PG synthesis localizes at mid-cell under the control of a multiprotein complex, the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. The region L75 to Q93 of FtsN (EFtsN) was shown to be essential and sufficient for its functioning in vivo but the specific target and the molecular mechanism remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its GTase activity. We also report the crystal structure of PBP1b in complex with EFtsN which provides structural insights into the mode of binding of EFtsN at the junction between the GTase and UB2H domains of PBP1b. Interestingly, the mutations R141A/R397A of PBP1b, within the EFtsN binding pocket, reduce the activation of PBP1b by FtsN. This mutant was unable to rescue {Delta}ponB-ponAts strain at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that PBP1b is a target of EFtsN and suggest that binding of FtsN to PBP1b contributes to trigger septal PG synthesis and cell constriction.

microbiology↗