Protein-lipid-lipopolysaccharide association in the superficial layer of Spirillum serpens cell walls.

The backing layer of the Spirillum serpens VHA cell wall, which supports and is bonded to the outer, structured protein layer, was isolated and shown to be similar in composition to the same elements of the outer membrane. It contained a lipopolysaccharide that was similar, but not identical, to that of the intact wall and the same phospholipids. The interaction of the isolated wall lipopolysaccharide with the loosely bound wall lipids provided lamellae, whose surfaces were an effective template for a lifelike reassembly of the isolated outer-layer hexagonal protein in the presence of Ca2+. Assembly did not take place on pure lipopolysaccharide, which dispersed in differing forms. A lipid-lipopolysaccharide-water interface appeared to be required as a template surface for the assembly. Lipopolysaccharide from Pseudomonas aeruginosa was able to replace that of S. serpens in the template. These observations suggest that lipid-lipopolysaccharide complexes are highly ordered, and this order is important to the nucleation and assembly of the protein array.

Analysis of the cell wall and lipopolysaccharide of Spirillum serpens.

Isolated walls of Spirillum serpens VHA contained lipid, lipopolysaccharide, and protein in amounts similar to those of other gram-negative organisms. The loosely bound lipids consisted mainly of phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Lipopolysaccharide was tightly bound to the wall and could only be removed in a substantial amount after digestion of the wall with Pronase. The lipopolysaccharide contained L-glycero-D-mannoheptose, rhamnose, glucosamine, ethanolamine, and phosphate in common with many of the lipopolysaccharides isolated from the Enterobacteriaceae. However, 2-keto-3-deoxyoctonic acid was not detected. Several unidentified sugars were present. The fatty acid composition resembled that found in lipopolysaccharides isolated from various pseudomonads. Two major regions were identified in the polysaccharide moiety, one apparently corresponding to the core polysaccharide and the other corresponding to the side-chain polysaccharide as in enterobacterial and pseudomonad lipopolysaccharides. The side chains were obtained as low-molecular-weight material and their structure was partially elucidated by the isolation and partial characterization of N-acetylglucosaminyl-(1 leads to 4)-rhamnose.

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.

Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa.

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.