Chain-Length Regulation by WzzE Is Necessary for, but Genetically Separable From, Cyclic Enterobacterial Common...

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 (ECA_CYC). Intriguingly, ECA_CYC 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 ECA_CYC biogenesis and no ECA_CYC is synthesized in its absence. To define WzzE functions necessary for ECA_CYC biosynthesis, we generated plasmid-borne wzzE mutants in Escherichia coli K-12 and quantified their effects on linear ECA regulation and ECA_CYC synthesis, determining that mutations disrupting linear ECA regulation in either transmembrane helix 2 or the periplasmic domain abolished ECA_CYC synthesis. Moreover, we identified two mutations residue F104 that differed in ECA_CYC abundance despite indistinguishable linear ECA regulation: wzzE_F104H caused an approximately 2-fold decrease in ECA_CYC abundance, whereas wzzE_F104Y retained wild-type abundance. Chromosomal wzzE mutants recapitulated this phenotype, demonstrating that these substitutions genetically uncouple levels of ECA_CYC synthesis from linear ECA regulation. Thus, although WzzE-mediated chain-length regulation is necessary for ECA_CYC biogenesis, it is not sufficient.