Biomedical subjects
Publications and source records attributed to S G Wilkinson.
After acidic hydrolysis of lipid A preparations from pseudomonads, products containing both hydroxy and nonhydroxy fatty acids were obtained. The major products were alkanoate esters of the hydroxy acids. Similar compounds were formed when a 3-hydroxy acid was heated with a nonhydroxy acid under the same conditions.
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Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the beta-(1-->6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.
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1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35-40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as P(i) by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.
A careful examination of the low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa (N.C.T.C. 1999) revealed the presence of ethanolamine triphosphate. During storage, the compound decomposed to give ethanolamine pyrophosphate, identified in a previous study (Drewry et al., 1971); PP(i) may be a further decomposition product. Evidence for the attachment of ethanolamine triphosphate to a polysaccharide fraction was obtained, but the possibility that some was attached to the lipid A moiety was not excluded. Basic compounds released during the hydrolysis of lipopolysaccharide included amino acids, polyamines and oligopeptides.
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1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.
1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70-75% of the lipopolysaccharide and 86-90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material.
Cell walls of 12 pseudomonads considered to be sensitive to ethylenediaminetetraacetic acid (EDTA) were prepared and analyzed. The wall of each species contained protein, peptidoglycan, loosely bound lipid, and lipopolysaccharide. The walls of Pseudomonas stutzeri and P. syncyanea were unusually susceptible to mechanical disintegration. The wall of P. syncyanea had an unusually high content of lipid and low contents of protein and peptidoglycan. Except for P. syncyanea, all the walls contained less phosphorus than the walls of the highly EDTA-sensitive P. aeruginosa and P. alcaligenes, but more than the walls of EDTA-resistant pseudomonads. The amino acid compositions of wall proteins were similar for all species. Amino sugars detected were glucosamine, galactosamine, muramic acid, and at least five unidentified components (possibly including fucosamine and quinovosamine). Glucose and rhamnose were the major neutral sugars in most walls. Galactose, mannose, fucose, and ribose were also detected, the last two each in a single species. Except for P. stutzeri and P. syncyanea, the walls had rather low contents of phospholipids (mainly cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol in all species). An ornithine-containing nonphospholipid was present in all walls, and a hexuronosyldiglyceride was probably present in most walls. The fatty acid compositions of loosely bound lipids were qualitatively similar for all species: saturated C(16) and monoenoic C(16) and C(18) acids were the major components. Except for P. aureofaciens, the extraction of phosphorus on treatment of walls with EDTA at pH 9.2 was much less than for P. aeruginosa and P. alcaligenes.
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