Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum

Abstract

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.Lipopolysaccharides (LPS) are major immunogenic components of the outer membrane of rhizobia and other gramnegative bacteria (24,26,47,56). The composition of Rhizobium LPS varies greatly among different species as well as among strains of a single species (18,22,61). The LPS molecules from Rhizobium leguminosarium are divided into two broad groups: complex macromolecules (LPS-1), which vary in composition from strain to strain and carry variable numbers of repeating oligosaccharides (O antigen); and smaller macromolecules, termed core LPS (LPS-2), which do not vary among strains of R. leguminosarum (18)(19)(20)(21). LPS-1, including 0 ant...

Chemical characterization of pH-dependent structural epitopes of lipopolysaccharides from Rhizobium leguminosarum biovar phaseoli

“…viciae bacteroids, it has been shown that there are changes in LPS epitopes which occur during differentiation of bacteria into bacteroids (6,35,39,42). Some of these changes can be produced ex planta, by growing the bacteria at low pH or low 02 tension, conditions which mimic, in part, the microenvironment of the root nodule (28).…”

Concomitant inhibition of nitrogen fixation and N-Phenyl-1-Naphthylamine uptake activity of bacteroids of aBradyrhizohium japonicumstrain by nitrate

“…Several reports have indicated that the amount of LPS in the outer membrane decreases in a cell (Van Brussel et al 1977) and that the composition of the membrane changes during the differentiation from free-living bacterium to bacteroid (Brewin et al 1986;Sindhu et al 1990;VandenBosch et al 1989). Goosen-de Roo et al (1991) demonstrated by electron microscopy with monoclonal antibody of O-antigens of LPS of Rhizobium leguminosarum bv.…”

An exoB mutant of Rhizobium sp. is effective in indeterminate nodules of Hedysarum coronarium

“…2a). According to other authors (Sindhu et al, 1990), LPSI is presumably the entire LPS (lipid A, core and O-antigen) and LPSII, that migrated just behind the solvent front, consists of only lipid A and core. Mutant CallO produced a LPS, which also consists of two parts, but both migrated faster than LPSI and LPSII of parental RB19 strain (Fig.…”

“…Bacterial cells were harvested after growth to stationary phase in T Y broth by centrifugation of 1 ml samples. Pelleted cells were resuspended in 300 pl Laemmli solubilization buffer (Laemmli, 1970), treated with proteinase K (50 pg ml-l) according to Sindhu et al (1990) An effective exoB mutant of Rhixobztrm sp.…”