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

Enterobacterial common antigen (ECA) is a conserved glycan that supports outer membrane impermeability and intrinsic antibiotic resistance in Enterobacterales. ECA exists in outer membrane diacylglycerol-phosphate- and lipopolysaccharide-linked forms, and a cyclic periplasmic form (ECA _CYC ). Intriguingly, ECA _CYC both affects the outer-membrane permeability barrier and functions in regulation of diacylglycerol-phosphate-linked ECA levels. While the length of linear ECA polymers generated by WzyE is regulated by the co-polymerase WzzE, WzzE is also required for ECA _CYC biogenesis and no ECA _CYC is synthesized in its absence. To uncover WzzE functions necessary for ECA _CYC biosynthesis, we generated plasmid-borne and chromosomal wzzE mutants in Escherichia coli K-12 and quantified their effects on linear chain-length regulation and on ECA _CYC synthesis. Mutations affecting linear ECA chain-length regulation in either transmembrane helix 2 or the periplasmic domain abolished ECA _CYC synthesis. Notably, two variants at residue F104 exhibited identical linear ECA chain-length regulation which was similar to wild type, but resulted in sharply different ECA _CYC production: chromosomal WzzE _F104Y produced near wild-type levels of ECA _CYC , whereas WzzE _F104H produced approximately two-fold less ECA _CYC . This difference in ECA _CYC production vs. linear chain-length regulation demonstrates ECA _CYC synthesis is genetically separable from this regulation. Thus, WzzE-mediated chain-length regulation is necessary for ECA _CYC biogenesis, but not sufficient.