The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli , the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance.

Highlights d Crystal structure of GIL01 gp7 has been solved d A hybrid approach provides a model for gp7 scaffolding of LexA d gp7 is seen to interact with phylogenetically distinct Staphylococcus aureus LexA d Structural evidence of a phage factor associating with LexA to modulate the SOS response

The bacterial cell wall plays a key role in viability and is an important drug target. The cell wall is made of elongated polymers that are crosslinked to one another to form a load bearing mesh. An alternate cell wall crosslinking mechanism performed by the L,D-transpeptidase YcbB has been implicated in the stress regulated roles of β-lactam resistance, outer membrane defect rescue, and typhoid toxin release. The role for this stress linked crosslinking in the context of a host infection was unclear. Here we resolve the crystallographic structures of both S. Typhi YcbB and C. rodentium YcbB acylated with ertapenem that delineate the conserved structural characteristics of YcbB. In parallel, we show that the general role of YcbB in peptidoglycan reinforcement under bacterial outer envelope stress does not play a significant role in acute infections of mice by C. rodentium and S. Typhimurium. Cumulatively, in this work we provide a foundation for the development of novel YcbB specific antibacterial therapeutics to assist in treatment of increasingly drug resistant S. Typhi infections.