Chain‐Length Regulation by <scp>WzzE</scp> Is Necessary for, but Genetically Separable From, Cyclic Enterobacterial Common Antigen Synthesis

ABSTRACT 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.