Characterization of the Lipopolysaccharides (LPS) from Four Transposon Generated Symbiotic Mutants of Rhizobium trifolii

The LPSs from four symbiotic mutants of R. trifolii were isolated and characterized. The mutants were obtained from Dr. Barry Rolfe of The Australian National University. Three mutants have Tn5 insertions in the nod regions of the symbiotic plasmid. The fourth has a 30 kb deletion. This deletion includes the majority of the nod regions. All mutants fail to nodulate clover and fail to induce markedly curled root hairs, i.e. they are hac-nod-. The LPSs were purified as described by Carlson et al. 1978.7 The polysaccharide portion of the LPSs was purified by treating the LPSs in 1% HAc at 100° for 1 hour followed by Sephadex G-50 column chromatography. A larger and a smaller molecular weight polysaccharide (LPS1 and LPS2) are obtained. The LPS from one batch, but not from a second batch, of the insertion mutant ANU274 contains an additional polysaccharide which elutes in the void volume of the G-50 column (LPS3). Sugar compositions were determined by GC analysis of their alditol acetate derivatives. Uronic acid, 2-keto-3-deoxyoctonic acid (KDO), pyruvate and acetate were determined by colorimetric assays. Unusual sugars were identified by combined GC/MS analysis. The LPSs were also analyzed by PAGE.21 The LPS compositions from the mutants and the parent are similar, the major sugars being heptose galacturonic acid and glucuronic acid. The LPS from one batch of ANU274 was different in that it also contains rhamnose (not found in the other LPSs), increased levels of galactose and an unidentified molecule. The LPS from a second batch of ANU274 is the same as the parent and other mutant LPSs. The LPS1s from all strains, including both batches of ANU274, are very similar in composition. The LPS2s contain man, gal, glc and uronic acid, the major sugar being uronic acid. The LPS2s from all the mutants contain increased levels of glucose compared to the parent. We are uncertain if this glucose is an LPS2 component or due to contamination by small molecular weight glucans. The LPS3, present in the one batch of ANU274, contains largely rhamnose and galactose. PAGE analysis of the various LPSs show that they are very similar, except for the LPS from the one batch of ANU274. The banding patterns, except that of the one ANU274 LPS, suggest that the O-antigen may not consist of a repeating oligosaccharide but may be a single complex oligosaccharide. However, the banding pattern for the LPS from the one batch of ANU274 suggests an O-antigen which does consist of a repeating oligosaccharide. At the present time we do not know if the appearance of this different LPS in the one batch of ANU274 is due to the mutation and/or is due to slightly different growth conditions (although all batches were grown and harvested in a similar manner)