Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin

Abstract

The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase. These new antigens were not detected on cells in midexponential or late stationary phase. Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase. Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase. In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS. White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase. The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis. In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.