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

Abstract

Lipopolysaccharide (LPS) was isolated from free-living Rhizobium leguminosarum bv. phaseoli CE3 cells grown at pH 4.8 (antigenically similar to bacteroid LPS) and compared with that from cells grown at pH 7.2 (free-living bacteria). Composition analysis revealed that pH 7.2 LPS differs from pH 4.8 LPS in that 2,3,4-tri-O-methylfucose is replaced by 2,3-di-0-methylfucose. The amount of 2-O-methylrhamnose is greater in the pH 4.8 LPS than in the pH 7.2 LPS. Analysis of the structural components of LPS (0-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the 0-chain polysaccharide. These structural variations between pH 4.8 and pH 7.2 LPSs provide a chemical basis for the observed lack of cross-reactivity with pH 4.8 LPS of two monoclonal antibodies, JIM28 and JIM29, raised against free-living bacteria grown at pH 7.2. An LPS preparation isolated from bacteroids contained both 2,3,4-tri-O-and 2,3-di-O-methylfucose residues. This result is consistent with the finding that the two monoclonal antibodies react weakly with bacteroid LPS. It is concluded that methylation changes occur on the LPS 0-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.Rhizobia are gram-negative soil bacteria that form nitrogen-fixing symbiotic associations with leguminous plants. In terms of host selection, the bacteria exhibit a great deal of specificity. The surface carbohydrates of rhizobia, which include the lipopolysaccharide (LPS), extracellular polysaccharide, and capsular polysaccharide, have all been hypothesized to play a role in symbiosis (9,11,18,20,22,23). The LPSs of Rhizobium species resemble their enterobacterial counterparts in having structurally distinct regions; the 0-chain polysaccharide, the core oligosaccharide, and the hydrophobic lipid A (10, 13-17); however, there are many differences in the structural details of Rhizobium LPSs compared with enterobacterial LPSs (1-3, 12, 25-27).Earlier reports on the role of LPSs in symbiosis were directed toward the possibility that LPSs were involved in the specific attachment of the symbiont to the host plant (8,9,30). Recent studies indicate involvement of LPSs at a later stage in the nodule development. Mutants which do not have the 0-chain region either fail to form normal infection threads or are not released properly from the infection thread into the host root cells (7,13,19,33,34,37).Using monoclonal antibodies to Rhizobium leguminosarum bv. 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). Similar results have been obtained for another strain of R. leguminosarum bv. viciae and for R leguminosarum bv. phaseoli (21, 38). These epitope changes occur only ...

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…NGR234 are more dramatic than the subtle changes we observe in the bacteroids of R. etli CE3. As mentioned previously, it has been reported that there is an increase in 2-O-methylfucosyl residues on the R. etli CE3 LPS O-chain during growth at low pH, from bacteria isolated from bean nodules and from bacteria grown in the presence of anthocyanin (9,11). In this study, we found that the O-chain from isolated R. etli CE3 bacteroids contains exactly one additional methyl group located on one of the five possible O-chain oligosaccharide repeating unit fucosyl residues.…”

“…In addition, it was shown that R. etli CE3, grown in the presence of P. vulgaris root or seed exudates, produced modified LPS that was no longer recognized by a particular monoclonal antibody (mAb), JIM28, specific for the O-chain polysaccharide of LPS from laboratory-cultured R. etli CE3 (10). Major compositional differences between LPS produced by CE3 cultures grown at pH 7.2 and that of pH 4.8 cultures included replacement of 2,3,4-tri-O-methylfucose by 2,3-di-O-methylfucose and an increase of 2-O-methylfucose content (11). These results showed the importance of determining the molecular/genetic basis for these subtle structural changes to R. etli LPS.…”

“…Prior reports have noted changes in the LPS during symbiosis by examining rhizobia obtained from their respective host root nodules (which consist of a mixture of bacteria and bacteroids) or during growth of rhizobial cultures under conditions that mimic those within the host root nodule (9,11,42,43). These changes have included differences in the DOC-PAGE banding pattern of the LPS, the production of a secondary rhamnan O-chain in the case of Rhizobium sp.…”

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

“…The CE358 mutant has an LPS that is devoid of all O-chain glycosyl components. The composition of the O-chain repeating unit of the parent strain has been reported (33,34). The complete structure of the lipid A moiety of the parent strain, including the fatty acid composition, has been published previously (15), and composition analysis indicates that the mutant LPSs contain a similar lipid A and fatty acid profile; i.e.…”

The Pea Nodule Environment Restores the Ability of a Rhizobium leguminosarum Lipopolysaccharide acpXL Mutant To Add 27-Hydroxyoctacosanoic Acid to Its Lipid A

“…Interestingly, there are marked differences between LPSs from free-living cultures and LPSs from nitrogen-fixing bacteroids in terms of size, composition, and antigenic properties (10,32,33). It has been shown that the rhizobial LPS undergoes structural modifications during the formation of bacteroids and that there are composition differences between the bacterial and bacteroid LPSs (10, 32).Variation in LPS structure due to environmental changes has been studied in cultured rhizobia by altering the growth conditions, such as lowering the oxygen level, lowering the pH, altering the carbon source, or adding plant-derived compounds (2,14,21,23,27). Such studies have shown that cues from the environment play an important role in LPS composition.…”