Epitope identification for a panel of anti-Sinorhizobium meliloti monoclonal antibodies and application to the analysis of K antigens and lipopolysaccharides from bacteroids

Abstract

In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506-509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654-661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. meliloti and Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids.

FIG. 1

Immunoblot analysis of the polysaccharide extracts from cultured cells of S. meliloti. The immunoblots were probed with the MAbs shown in Table 2. The MAb designations are given above the lanes. The strains are identified in abbreviated form beneath the lane as follows: 23, S. meliloti NRG23; 34, S. meliloti NRG34; 43, S. meliloti NRG43; 53, S. meliloti NRG53; 133, S. meliloti NRG133; 185, S. meliloti NRG185; 247, S. meliloti NRG247; 282, S. meliloti NRG282; 286, S. meliloti NRG286; 289, S. meliloti NRG289. The R-LPS, S-LPS, and K-antigen labels identify the antigens bound by the MAbs (the S-LPS and K antigens comigrate on polyacrylamide gels, so those regions overlap). Only a few examples of negative results are shown.

FIG. 2

Immunoblot analysis of the polysaccharide extracts from alfalfa bacteroids of S. meliloti NRG185 (185b) and S. meliloti NRG247 (247b). The leftmost lane contains the extract from cultured cells of S. meliloti NRG247 (247c) for comparison. The immunoblots were probed with the MAbs shown in Table 2. The two arrows indicate the migration positions of two bacteroid-specific S-LPS banding regions not found in the analysis of cultured cells (the fact that these bands were recognized by MAb 10 showed that it was LPS).