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In lipopolysaccharide (LPS) biosynthesis of Gram‐negative bacteria the lipid A‐core oligosaccharide (LA‐core) and O‐polysaccharide (O‐PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL‐encoded ligase joins O‐PS onto LA‐core. Enterobacterial common antigen (ECA) biosynthesis follows that of O‐PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA‐core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA‐inner core (IC) onto which a homopolymeric O‐PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O‐PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O‐PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single …