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 (ECACYC). Intriguingly, ECACYC 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 ECACYC biogenesis and no ECACYC is synthesized in its absence. To uncover WzzE functions necessary for ECACYC 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 ECACYC synthesis. Mutations affecting linear ECA chain-length regulation in either transmembrane helix 2 or the periplasmic domain abolished ECACYC 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 ECACYC production: chromosomal WzzEF104Y produced near wild-type levels of ECACYC, whereas WzzEF104H produced approximately two-fold less ECACYC. This difference in ECACYC production vs. linear chain-length regulation demonstrates ECACYC synthesis is genetically separable from this regulation. Thus, WzzE-mediated chain-length regulation is necessary for ECACYC biogenesis, but not sufficient.