A polyclonal antiserum generated against the Bradyrhizobiumjaponicum lectin BJ38 was characterized to be specifically directed against the protein. Treatment of B. japonicum cells with this antiserum and subsequent visualization with transmission electron microscopy and both conventional and confocal fluorescence microscopy revealed BJ38 at only one pole of the bacterium. BJ38 appeared to be organized in a tuft-like mass, separated from the bacterial outer membrane. BJ38 localization was coincident with the attachment site for (i) homotypic agglutination to other B. japoncum cells, (it) adhesion to the cultured soybean cell line SB-1, and (iu) adsorption to Sepharose beads covalently derivatized with lactose. In contrast, the plant lectin soybean agglutinin labeled the bacteria at the pole distant from the bacterial attachment site. These results indicate that the topological distribution of BJ38 is consistent with a suggested role for this bacterial lectin in the polar binding ofB.japonium to other ceUs and surfaces.Bradyrhizobiumjaponicum normally infects roots of soybean plants leading to symbiotic nitrogen fixation (1-3). We have documented (4) that this bacterial strain exhibits four saccharide-specific binding activities: (i) adsorption to Sepharose beads derivatized with lactose (Lac-Sepharose), (ii) homotypic autoagglutination, (iii) heterotypic binding to cultured soybean (SB-1) cells, and (iv) heterotypic adhesion to soybean roots. In all four of these assays, galactose inhibited the binding but a C-2 derivative of the monosaccharide N-acetyl-D-galactosamine failed to yield the same effect. Mutants ofB. japonicum, designated as N4 and N6, that were isolated on the basis of a defect in one binding activity (SB-1 cell binding) showed a concomitant loss in the other three binding capacities (4). These observations suggested that all four of the carbohydrate-specific binding processes may be mediated by the same component(s) and mechanism(s). Consistent with this proposal, we have purified a carbohydrate binding protein, designated BJ38 (Mr 38,000), which has an affinity for galactose and lactose "13-and 240-fold higher, respectively, than its affinity for N-acetyl-D-galactosamine (5). This carbohydrate binding specificity correlates well with that of the B. japonicum binding activities. It was proposed, therefore, that BJ38 may mediate the carbohydrate binding activities of B. japonicum.In all four carbohydrate-specific binding activities tested, B. japonicum bound in a polar fashion (4). Thus, any hypothesis implicating a role of BJ38 in these binding assays would require that the lectin be exposed at the surface of the bacterium where attachment occurs. In the present communication, we report the characterization of a specific anti-BJ38 antibody that has allowed us to identify the topological distribution of BJ38 on the bacterial cell surface. MATERIALS AND METHODSCeUl Cultures. B. japonicum (RilOd) was originally obtained from Barry Chelm of Michigan State University. This bacterial strain was ...

Abstract. Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr ,030,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.