Identification of compounds that inhibit late steps of peptidoglycan synthesis in bacteria

A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.