The cell wall glycolipid (somatic antigen) of the Re mutant of Salmonella minnesota is comparable in its endotoxic activity to the complete wild type lipopolysaccharide. The glycolipid is composed mainly of lipid A and 2-keto-3-deoxyoctonate (KDO) ; smaller amounts of ethanolamine and 4-aminoarabinose, and traces of amino acids are also present. The serological specificity is determined by KDO residues. In order to determine if non-lipid A constituents contribute to endotoxicity, amino groups and KDO units in the glycolipid were chemically modified by the introduction of substituents. When free amino groups were dinitrophenylated or succinylated the serological specificity was unaffected. By contrast, the original serological specificity was completely lost when the free hydroxyl groups of the KDO region were substituted with succinyl residues or the carboxyl groups converted to the methyl esters. In biological tests, however, the modified preparations exhibited full endotoxic activity. It is concluded that the polar groups, which determine the serological specificity of the KDO region of the glycolipid, do not play a specific role in endotoxicity.The cell wall lipopolysaccharides (endotoxins, 0 antigens) of gram-negative bacteria are biologically highly active substances. Their injection into animals causes a variety of physio-pathological effects and evokes the production of specific antibodies [1-31. Chemical studies over the past decade have provided sufficient information to establish a fairly detailed structure of lipopolysaccharides derived from Salmonella, Escherichia coli, Shigella and other genera [4,5]. They contain a polysaccharide portion, i.e. the 0-specific chains and the core, and a lipid portion, called lipid A. Many attempts have been made to correlate biological activity to specific chemical structures of the macromolecule. It has been shown clearly that discrete structures in the polysaccharide portion of lipopolysaccharides carry the serological determinants responsible for antibody specificity [1,2,4]. The question of the existence of a defined region in lipopolysaccharides responsible for endotoxic activity is still under discussion, however. The recent finding that the incomplete lipopolysaccharides and glycolipids of Salmonella R mutants, which lack 0-specific chains and large parts of the core, are also potent endotoxins [6--81, has demonstrated that the long polysaccharide chain is not essential to endotoxicity. The most deficient Unusual Abbreviation. KDO, 2-keto-3-deoxyoct~nate. The term glycolipid was introduced as a designation of defective R mutant lipopolysaccharides which contain oligosaccharide units instead of polysaccharide, linked to lipid A CSl.lipopolysaccharides, which have been obtained from R forms, are the glycolipids of Salmonella Re mutants. They contain lipid A, to which a trisaccharide of 2-keto-3-deoxyoctonate (KDO) is ketosidically bound [9,10]. Additional phosphate, ethanolamine, 4aminoarabinose and trace amounts (about O.lo/,) of amino acids are also present [l...
Salmonella typhimurium rfa-657 strains, including both mutants and transductants, were shown to contain in their lipopolysaccharides both the (usual) L and the D-iSOmer of glycero-Dnzanno-heptose. By the application, in sequence, of' two different extraction procedures S and R form lipopolysaccharides could be isolated scparately from a representative rfa-657 strain. The R lipopolysaccharide was predominantly of the Re chemotype, only a few 2-keto-3-deoxyoctonate (KDO) residues being substituted by heptose (Rd, chemotype) or by longer core chains. The D-heptose was enriched in the R lipopolysaccharide, while the S lipopolysaccharide contained mainly the natural L-isomer. It was found that D-hcptose can replace L-heptoses I to 111 of the wild-type core. However, an elongation to completeness of the core stubs containing D-heptose seldom occurs.The heterogeneity in structure of the lipopolysaccharide produced by this class of (leaky) mutants is assumed to be due to an altered 6-epimerase which, in the wild form, catalyses the conversion of NDP-D-glycero-D-manno-heptose into NDP-L-glyyeero-D-manno-heptose and which is determincd by the gene rfa-657 situated between cysE and pyrE. The symbol rfaD is proposed for this gene. Abbreviations. KDO, 2-keto-3-deoxyoctonate. Note. All values of the retention time, t~, refer to 2,3,4,6-tetra-0-methyl-glucose. MATERIALS AND METHODS Bacterial &rains, Phages and LipopolysaccharidesStrain SL 1027 is a smooth, genetically marked derivative of S. typhimurium strain LT2, and SL 3600 is a "part-rough'' (i.e. phenotypically intermediate between S and R ) mutant, rfa-657, of SL 1027, isolated from a mutagen-treated culture by selection for resistance to Felix 0 phage [3] a.nd SL 3149, the two SL 3600 reisolates, respectively.The first lot of lipopolysaccharide examined was extracted a t Preiburg from a batch of SL 3600 cells grown on nutrient agar, washed and acetone-dricd a t Stanford. Later one crop each of strains SL 3147,
Several species of Salmonella, Citrobacter, and Arizona were examined for the presence of 3-amino sugars, which were isolated from lipopolysaccharide hydrolysates by cation exchange chromatography and identified by paper and cation exchange chromatography in several systems and by specific colorimetric procedures. 3-Amino-3 ,6-dideoxyglucose was identified in C. freundii 8090, C. freundii 869, S. halle, S. telaviv, S. dakar, S. wandsworth, and S. champaign; 3-amino-3 ,6-dideoxygalactose was found in S. tranoroa and Arizona 24. Evidence suggests that these 3amino sugars occur in lipopolysaccharides as the N-acetyl derivatives. The composition of the lipopolysaccharides containing the 3-amino sugars was determined, and the lipopolysaccharides were compared serologically in order to correlate chemotypes with serotypes. The lipopolysaccharides containing 3-amino-3, 6dideoxyglucose reacted with serogroups 28 or 39; those containing 3-amino-3,6dideoxygalactose were found to react with group 55 antisera. The preparation of a lipopolysaccharide from the phenol phase of the 44% aqueous phenol extraction procedure is also reported. Ashwell and Volk (2) first reported the isolation and characterization of crystalline 3-acetamido-3,6-dideoxy-D-galactose, obtained from a phenol-soluble lipopolysaccharide prepared from Xanthomonas campestris, a member of the Pseudomonadaceae. Raff and Wheat (18) found a different 3-amino-3 ,6-dideoxy sugar in phenol-soluble cell wall lipopolysaccharide of the enterobacterium, Citrobacter freundii ATCC 8090. Jann et al. (Nature, in press) and Muller Seitz (Thesis, University of Freiburg,Germany) independently identified 3-amino-3, 6-dideoxy-D-galactose and 3-amino-3, 6-dideoxyglucose in Escherichia coli serotypes. The present work describes the tentative identification of 3-amino sugars found in the serologically cross-reactive C. freundii 8090, Citrobacter 869, Salmonella dakar, S. telaviv, S. champaign, S. wandsworth, S. tranoroa, and Arizona 24. MATERIALS AND METHODS Growth of cells. C. 1reundii ATCC 8090 was grown in 10-liter lots in 5-gal (18.9-liter) carboys at 25 C for 24 hr with constant aeration. The medium used was as follows, in grams per liter: yeast extract, 3; K2PHO4, 6.6; KH2PO4, 1.7; (NH4)2SO4, 2.5; MgSO4, 0.05; and glucose (autoclaved separately), 10. Cells were harvested with a DeLaval Gyrotest centrifuge,
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