Termination of Poly- N -acetylglucosamine (PNAG) Polymerization with N -Acetylglucosamine Analogues.

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli , the glycosyltransferase complex PgaCD produces poly- N -acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N 3 , SH, NH 2 ) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro , the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo , expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ . The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.