Chain-length regulation by WzzE is necessary for, but genetically separable from, cyclic enterobacterial common antigen synthesis

Bacteria of the orderEnterobacterales(e.g.,Escherichia, Klebsiella, Yersinia) produce an invariant glycan, enterobacterial common antigen (ECA), that plays a role in maintaining outer membrane (OM) impermeability. ECA is found as the headgroup of a phospholipid and attached to LPS in the OM, and as a periplasmic cyclic form (ECA_CYC). WzyE polymerizes ECA repeat units to form the ECA chain with final chain length regulated by WzzE, a class 1 polysaccharide co-polymerase (PCP1). PCP1 function studies have shown interaction of the PCP1 transmembrane helices with the polymerase and a secondary activity found in the periplasmic domain of the PCP1 are required for chain-length regulation. However, WzzE is also necessary for ECA_CYCbiosynthesis, and it remains unclear why loss of WzzE prevents ECA_CYCproduction but not linear ECA production. Here, we constructed plasmid-based and chromosomalwzzEmutants inE. coliK-12 with alterations that affect chain-length regulation of other PCP1 and assessed their effects on ECA_CYCbiogenesis. Our data show loss of chain-length regulation and ECA_CYCsynthesis from mutations altering transmembrane helix 2 and mutations altering the periplasmic domain of WzzE. We also identified two WzzE variants to the same residue with identical, near wild type linear ECA chain-length regulation but differing effects on ECA_CYCproduction. Specifically, WzzE^F104Yproduces wild-type levels of ECA_CYC, while WzzE^F104Hproduces twofold less ECA_CYCthan wild type, demonstrating ECA_CYCsynthesis is genetically separable from chain length regulation. Overall, our results show that chain-length regulation by WzzE is necessary but not sufficient for normal production of ECA_CYC.