Heterogeneity of Lipopolysaccharides. Analysis of Polysaccharide Chain Lengths by Sodium Dodecylsulfate‐Polyacrylamide Gel Electrophoresis

Abstract

Lipopolysaccharide preparations from R (rough) Escherichia coli 0 8 -, SR (semirough) Salmonella typhimurium and S (smooth) strains E. coli 0 8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1 % sodium dodecylsulfate. The results obtained were compared with those obtained from the same lipopolysaccharide preparations by degradation analysis. In dodecylsulfate gel electrophoresis the lipopolysaccharide preparation from the E. coli R mutant and the S. typhimurium SR mutant showed one band each (R-and SR-band, respectively) with different electrophoretic mobilities. The lipopolysaccharide preparation from the E. coli 0s-strain exhibited two bands, one of which had the same electrophoretic mobility as the R-band and the other was identified as S-band. The lipopolysaccharide preparation from the Citrobacter 396 S-strain exhibited four bands: one R-band, one SR-band and two S-bands. The results showed that wild-type S strains contain more than one type of lipopolysaccharide. They differ in the length of their 0-specific polysaccharide chains. The lipid A content of the different lipopolysaccharide was expressed in their electrophoretic mobilities.For the characterization of enterobacterial lipopolysaccharides we have been using mild acid hydrolysis (removal of lipid A) and fractionation of the polysaccharide moiety by gel permeation chromatography [l -31. The carbohydrate moiety of the lipopolysaccharides from most smooth (S) bacteria can be separated on Sephadex into two main fractions, one representing the core oligosaccharide substituted with the 0-specific polysaccharide and the other representing the unsubstituted core oligosaccharide (for definitions see . More recently, we found that in some cases the fractionation pattern was more complex. Besides unsubstituted core oligosaccharide (R) and core oligosaccharide substituted with the O-specific polysaccharide (S) we have also found core oligosaccharide substituted with only one 0-specific repeating unit. Other separations revealed the presence of core oligosaccharides substituted with 0-specific polysaccharides of different chain lengths. Thus, bacteria may exhibit on their surface several types of lipopolysaccharides. The heterogeneity was only observed after degradation of the lipopolysaccharide preparations and fractionation of the degraded polysaccharides. Because these procedures are time consuming, we have sought for a simpler method to analyze the molecular state (S-, SR-, R-forms) of lipopolysaccharides in bacterial wild types and mutants. We have found that electrophoresis of lipopolysaccharides in polyacrylamide gels in the presence of the dissociating surfactant sodium dodecylsulfate is a simple and useful tool for the molecular characterization of enterobacterial lipopolysaccharides. MATERIALS AND METHODS Bacteria and CultivationEscherichia coli 0 8 K27-: H-(F 492) and the R mutant derived from it (F 470), a Salmonella typhimurium SR strain (F 971) and Citrobacte...

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

“…These populations represent sets of molecules with 0 antigens of different lengths which are made in large amounts. Other investigators have also demonstrated, using gel permeation chromatography in combination with other methods, that the LPS from members of the family Enterobacteriaceae and other gram-negative bacteria could be resolved into at least two main populations of LPS, differing in the length of their O-polysaccharide chain (9,26,30,32,34). In the results presented here, we found that the LPS from strains 503 and 1715 ( Fig.…”

“…4). Although SDS-PAGE separates LPS molecules according to size (26,45,48), we propose that the anomalous migration in SDS-PAGE of the A bands is due to a'difference in the charge in the core-lipid A region of the molecules. The lack of phosphate substituents in the A-band sample would make the molecules much less negatively charged than the B-band fractions, which are high in phosphate groups.…”

“…Structural microheterogeneity in several regions of LPS molecules from members of the family Enterobacteriaceae (2,14,22,37,38,48,51) and Pseudomonas aeruginosa strains (33,57) has been demonstrated. Of the several methods used to separate the subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20,24,45,48) and gel filtration (9,26,30,32,34,48) are the best. Either of these two methods by themselves, however, may be insufficient to completely characterize the high-and low-molecular-weight fractions of LPS.…”

Characterisation and application of a murine monoclonal antibody specific for the serogroup C2 Salmonella

“…wt species (fig. 1, lane B) (Jann et al, 1975;Goldman and Leive, 1980). The 0 polysaccharide side chains of the LPS of serogroups Cl and C2 Salmonella are antigenically related in that they share a common antigenic determinant of 0 factor 6 which is believed to correspond structurally to the disaccharide Dglucose + D-mannose (Fuller and Staub, 1968).…”

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3

“…It is not clear, however, whether this coexistence is full or if it is only a fraction of OPS-substituted molecules that are also ECA LPS -substituted. The simultaneous stepwise biosynthesis of LPS and turnover on the bacterial surface result in a heterogeneous population of molecules that makes the chemical and structural analysis of LPS challenging and, on top, isolation and fractionation methods may cause further alterations in LPS (Hitchcock & Brown, 1983;Jann et al, 1975;Munford et al, 1980;Nowotny, 1984;Tavío et al, 2000).…”