We report on the cloning and characterization of the rJb gene cluster of the 075 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the 075 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized 0 chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized 0 chain in E. coli S0874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short 0 chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rib region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate 0-chain distribution. We propose to call this regulator gene rol, for regulator of 0 length.The lipopolysaccharide (LPS) antigen of Escherichia coli and Salmonella spp. consists of three different molecular species (lipid A, core oligosaccharide, and 0 polysaccharide) that are synthesized independently of each other and are ligated together in or on the inner membrane (17). The O-polysaccharide subunit typically consists of one to five sugar molecules linked together to form a macromolecular polymer of repeated units that vary from 1 to more than 40 in number (11). After assembly, the entire LPS molecule is translocated to the outer membrane by an unknown mechanism (11, 15). The lipid A and core units are embedded in the outer membrane, while the 0 antigen extends outward from the cell. The 0 antigen has been shown to be important in mediating the effects of serum resistance, infection by certain bacteriophages, and interaction with the microenvironment (4,20,22,25). The presence of 0 antigen on the surface of these bacteria confers a smooth phenotype and has been correlated to increased virulence (18).On sodium dodecyl sulfate (SDS)-polyacrylamide gels, silver-stained or radiolabeled LPS has a ladderlike appearance, with each rung or step of the ladder corresponding to a complete lipid A-core unit plus an 0 chain of a specific length. Several groups of investigators have noted a bimodal distribution of LPS from wild-type strains in stained SDSpolyacrylamide gels (3, 10, 21). High-molecular-weight 0 antigens with chain lengths of 19 to 34 make up to 77% of the total antigen, with low-molecular-weight 0 antigen (1 to 5 chain lengths) forming the bulk of the remainder. Intermediate sizes of 0 antigen ...

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In previous studies it had been shown that lipopolysaccharide from 04-specific recombinant Escherichia coli, had serological reactivities and a chemical composition that differed from wildtype 0 4 LPS [Haraguchi, G. E., Zahringer, U., Jann, B., Jann, K., Hull, R. A. & Hull, s. I. (1991) Microb. Pathog. 10,. Here we present the structural elucidation of the 0-specific moieties from lipopolysaccharides of some of the recombinant strains obtained in previous studies. Compositional analysis, methylation, chemical reactions and NMR spectroscopy showed that, during genetic manipulations (recombination, cosmid cloning, plasmid subcloning), a gradual structural change in the 0-specific polysaccharides was observed in the recombinant strains. These changes comprised of an alteration in the position of glucose (side chain) substitution, a change in the anomeric configuration of the main-chain N-acetylglucosamine and an exchange of a-L-rhamnopyranose for p-Dgalactofuranose. The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.The 0-antigenic lipopolysaccharides (LPS) are amongst the most important virulence factors of Gram-negative bacteria, such as Escherichia coli. Their role in pathogenicity seems to be in bacterial resistance against the nonspecific host defense mechanism, as mediated by complement and phagocytes [1-41. In E. coli, certain 0 antigens are associated with certain diseases. Thus, out of more than 150 different 0 antigens, only a few, mainly 01, 02, 06, 018 and 075, prevail in urinary tract infections. E. coli strains expressing the 0 4 antigen are frequently isolated from pyelonephritis cases [5 -81. Serological cross reactivity between the 0 4 and 018 antigens has been reported [9].The 0-antigenic LPS consist of three structurally and functionally distinct regions, which are interlinked by glycosidic bonds. These are lipid A (the mediator of endotoxicity), the core oligosaccharide and the 0-specific polysaccharide chain (the latter consists of oligosaccharide repeating units which determine the serological 0-specificity of the respective bacteria). These structural regions are encoded from difCorrespondence to K. Jann, Max-Planck-Institut fur ImmunbioFax: + 49 0761 5108 221.