Partial Characterization of LPSs from Rhizobium fredii USDA 205 Before and After NOD Gene Induction

The lipopolysaccharides (LPS) from Rhizobium fredii USDA 205 and its nod- mutant, HC 205, which has been cured of the 192-MDa symbiotic plasmid, were isolated by the phenol-water extraction method. The LPS from the wild-type strain which was grown in association with host-plant root extract or a flavone analog to a constituent of the root extract, which is required for the induction of at least a portion of the nodulation genes, was also isolated. The conditions required for the induction of the nod genes were determined by work with a nod- mutant of R. fredii USDA 201 which contains a Mu-lac insertion under the control of a nodulation gene promoter. The isolated LPSs were purified by column chromatography and partially analyzed by proton-NMR, gas chromatography (GC), colormetric assays, and polyacrylamide gel electrophoresis (PAGE) employing either sodium dodecyl sulfate (SDS) or 7-deoxycholic acid (DOC) as a detergent. Significant differences were seen in the DOC-PAGE banding patterns of the induced LPS samples when compared to the non-induced LPSs. The most important distinction being a relative reduction in overall LPS molecular weight by discreet increments for the induced LPS from USDA 205. This was accompanied by small molecular weight differences in the predominant bands of similar molecular weight. GC and colormetric analysis indicate that the polysaccharide portion of the LPSs of all samples is composed predominantly of galactose and 2-keto-3-deoxyoctonic acid (KDO), a common constituent of gram-negative bacterial LPSs. There were some compositional differences between the induced and non-induced LPSs but the significance of these differences remains unclear at this time