The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.
The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a β‐GlcN‐(1–6)‐GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O‐1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O‐3 and O‐4 of β‐GlcN, and no substituent at O‐4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure:
where Δ‐GalNA‐(1–3)‐β‐QuiNAc represents a modified fragment of the O‐chain repeating unit. Two shorter oligosaccharides lacking the O‐chain fragment were also identified. A minor amount of the disaccharide β‐GlcN‐(1–6)‐α‐GlcN‐1‐P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end.
The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2‐N‐(3‐O‐acyl4‐acyl2)‐β‐GlcN‐(1–6)‐2‐N‐acyl1−3‐O‐acyl3‐GlcN where acyl1, acyl2 and acyl3 are 3‐hydroxyhexadecanoic or 3‐hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH‐1 of the reducing end glucosamine, and OH‐3 and OH‐4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.
Liopolysaccharides were prepared from six organisms by the use of two cell-disruption procedures before conventional phenol-water extraction. Disruption of cells by grinding with glass beads or by digestion with hen egg white lysozyme before phenol extraction facilitated rapid purification and greater yields of lipopolysaccharide. Pretreatment of cells with lysozyme in the presence of ethylenediaminetetraacetic acid was the most efficient method in terms of lipopolysaccharide yield and ease of preparation. Increase in lipopolysaccharide yield achieved by use of the lysozyme method, compared with the conventional phenol extraction, varied from 1.7- to 12.4-fold. Preparations were designated as pure according to several criteria and were judged not to have undergone changes as a result of prephenol extraction procedures.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure 32)-
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