The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

Abstract

The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.