Camaione, S.; Thompson, C. M.; Briggs, N. S.; Roper, D.

Bacterial cell wall processing enzymes have a central role in the control of the construction, remodelling and breakdown of the peptidoglycan wall that protects bacteria from osmotic rupture. Many of these enzymes have biologically essential functions and exert mechanistically diverse activities on a complex polymer substrate. Despite this biological importance they are relatively understudied and there are significant technical challenges in measuring the activity of individual enzymes against the complex chemical structure of the bacterial cell wall peptidoglycan. To generate a suitable substrate, bacterial cells are grown with a fluorescent D-alanine analogue, allowing the isolation of natively fluorescently labelled PG. This material is then used to measure interactions with PG-targeting enzymes and regulatory proteins. The resulting assay enables analysis of multiple enzyme-substrate combinations within minutes while retaining molecular specificity. This approach provides a robust, cost-effective alternative to conventional PG activity assays and could accelerate the identification and characterisation of novel antimicrobial compounds targeting bacterial cell wall metabolism. Our results indicate this assay has the potential to be employed in high-throughput inhibitor discovery, making it effective for a broad-spectrum assay for bacterial peptidoglycan research applications.