Polysaccharide release factors drive lipid-carrier recycling in bacteria
Gram-negative bacteria build their cell envelope by transporting various sugar-based precursors across the inner membrane on the shared lipid carrier undecaprenyl (also known as C55). Because the cellular pool of undecaprenyl is finite and used by multiple competing pathways, it must be efficiently regenerated to sustain envelope assembly. Yet the enzymes that release polysaccharide cargo from the lipid carrier have remained elusive, leaving a major gap in our understanding of bacterial envelope biogenesis. Here we identify three periplasmic enzymes in Escherichia coli - UprA, UprB, and UprC - that hydrolyze the bond linking polysaccharide precursors to undecaprenyl. UprA releases elongating peptidoglycan glycan strands from the inner membrane, catalyzing the long-sought terminal step of peptidoglycan synthesis. UprB cleaves the undecaprenyl-linked initiating unit common to the enterobacterial common antigen and O-antigen pathways, while UprC is the likely release factor for the colanic acid pathway. These enzymes define a widespread Upr-like protein family (formerly DUF218) as polysaccharide release factors, revealing a unifying mechanism by which bacteria coordinate envelope assembly. Because all three Upr-like proteins share a conserved catalytic core, this family represents a potential target for antibiotics that simultaneously disrupt multiple envelope pathways.