Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium

Previous immunoelectron microscopic studies have shown that both the final intermediate in O-antigen synthesis, undecaprenol-linked O polymer, and newly synthesized O-antigenic lipopolysaccharide are localized to the periplasmic face of the inner membrane (C. A. Mulford and M. J. Osborn, Proc. Natl. Acad. Sci. USA 80: 1159-1163, 1983). In vivo pulse-chase experiments now provide further evidence that attachment of O antigen to core lipopolysaccharide, as well as polymerization of O-specific polysaccharide chains, takes place at the periplasmic face of the membrane. Mutants doubly conditional in lipopolysaccharide synthesis [kdsA(Ts) pmi] were constructed in which synthesis of core lipopolysaccharide and O antigen are temperature sensitive and mannose dependent, respectively. Periplasmic orientation of O antigen:core lipopolysaccharide ligase was established by experiments showing rapid chase of undecaprenol-linked D polymer, previously accumulated at 42°C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30°C. In addition, chase of the monomeric G-specific tetrasaccharide unit into lipopolysaccharide was found in similar experiments in an O-polymerase-negative [rfc kdsA(Ts) pmi) mutant, suggesting that polymerization of O chains also occurs at the external face of the inner membrane.