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 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. 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 define WzzE functions necessary for ECACYC biosynthesis, we generated plasmid-borne wzzE mutants in Escherichia coli K-12 and quantified their effects on linear ECA regulation and ECACYC synthesis, determining that mutations disrupting linear ECA regulation in either transmembrane helix 2 or the periplasmic domain abolished ECACYC synthesis. Moreover, we identified two mutations residue F104 that differed in ECACYC abundance despite indistinguishable linear ECA regulation: wzzEF104H caused an approximately 2-fold decrease in ECACYC abundance, whereas wzzEF104Y retained wild-type abundance. Chromosomal wzzE mutants recapitulated this phenotype, demonstrating that these substitutions genetically uncouple levels of ECACYC synthesis from linear ECA regulation. Thus, although WzzE-mediated chain-length regulation is necessary for ECACYC biogenesis, it is not sufficient.