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 O polymer, previously accumulated at 42 degrees C in the absence of core synthesis, into lipopolysaccharide following resumption of core formation at 30 degrees C. In addition, chase of the monomeric O-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.
McGrath
B C
BC
Department of Microbiology, University of Connecticut Health Center, Farmington 06030.
Osborn
M J
MJ
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D019081:O Antigens; D011105:Polyisoprenyl Phosphate Sugars / Q000302:isolation & purification*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000276:immunology* / Q000378:metabolism; D014316:Tritium
D019081:O Antigens; C062656:O-specific polysaccharide, Salmonella; D011105:Polyisoprenyl Phosphate Sugars; D011135:Polysaccharides, Bacterial; D014316:Tritium; D008358:Mannose; D005690:Galactose