Gram-negative bacteria are to a large extent covered by lipopolysaccharide (LPS) anchored in the outer leaflet of their outer membrane. There are presently four described pathways for the O-antigen assembly of LPS, viz., synthase-, Wzk-, ABC-transporterand Wzx/Wzy-dependent pathways, where the latter two are used in Escherichia coli, subject to the O-antigen polysaccharide to be made. NDP-sugar monosaccharides are used by glycosyltransferases in the process of linking sugar residues together in

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... the cytoplasm and depending on the biosynthetic pathway polymerization of the O-antigen takes place either in the cytoplasm (ABCtransporter pathway) or in the periplasm, where an oligosaccharide anchored in the inner membrane is flipped to the periplasmic side to this end (Wzx/Wzy-dependent pathway). Additions of sugars to form side-chains on the O-antigen may also occur in the periplasmic space. The degree of polymerization of the O-antigen is regulated to give a modal distribution, i.e., a narrow distribution around the most probable chain-length. The O-antigen is subsequently conjugated to the lipid A-core to form the LPS, which is transported across the periplasmic region by an ATP-driven mechanism as part of an LPS transport (Lpt) system. By using predictions of NDPmonosaccharide pathways and glycosyltransferase function it is shown how O-antigen structure can be elucidated rapidly by the computer program CASPER using bioinformatics data in conjunction with unassigned NMR data of the polysaccharide.