Dual binding sites for peanut lectin on Rhizobia

Abstract

Purified peanut lectin (peanut agglutinin, PNA) labelled with fluorescein isothiocyanate (FITC-PNA) or iodine-125 (¹²⁵I-PNA) bound to Rhizobium B. TG-3 and Rhizobium 5a, which nodulate peanut, but did not bind to R. japonicum or R. meliloti, which do not nodulate peanut, or to the non-nitrogen-fixing bacteria Escherichia coli and Bacillus subtilis. Washing the rhizobia in phosphate-buffered saline markedly decreased PNA binding. The decrease in binding could be correlated with removal of exopolysaccharides during washing. Dialysed culture filtrate could be labelled with ¹²⁵I-PNA and the radioactivity was recovered in an acetone-precipitable exopolysaccharide fraction, showing that the lectin complexed with exopolysaccharides. Residual binding which remained after washing could not be removed by further washing. Moreover, rhizobia could be labelled with ¹²⁵I-PNA after removal of exopolysaccharides and the radioactivity was recovered by extraction with hot phenol, indicating that the lectin complexed also with lipopolysaccharide on the bacterial cell wall. Rhizobial lipopolysaccharide not only inhibited haemagglutination by PNA but also dispersed preagglutinated human erythrocytes into free cell suspensions. The results demonstrate the presence of dual binding sites for PNA on rhizobia. Exopolysaccharides are the major sites and the remainder are lipopolysaccharides.