title: Molecular dissection of structure and function in the lipopolysaccharide of Rhizobium leguminosarum strain 3841 using monoclonal antibodies and genetic analysis.
abstract: Following treatment with nitrosoguanidine, mutant derivatives of Rhizobium leguminosarum strain 3841 were isolated which failed to react with AFRC MAC 203. This monoclonal antibody normally recognizes a strain-specific lipopolysaccharide epitope which is developmentally regulated during legume nodule differentiation. Structural modification of lipopolysaccharide (LPS) was analysed by examining reactivity with a range of monoclonal antibodies with different epitope specificities, and also by analysis of LPS mobility changes after electrophoresis on polyacrylamide gels. One class of these LPS-defective mutants induced normal nitrogen-fixing (Fix+) nodules on peas (Pisum sativum), while another two classes of Fix- mutants were also identified, suggesting that a component of the LPS antigen that is part of the MAC 203 epitope is essential for normal nodule development leading to symbiotic nitrogen fixation. When grown under low-oxygen or low-pH culture conditions, one class of Fix- mutants completely lacked LPS-1 (the species that carries O antigen) and a second class showed a modified and truncated form of LPS-1. Mutants with defective LPS structure were also obtained after Tn5 mutagenesis of R. leguminosarum 3841 and all nine Fix- mutants were also found to lack the MAC 203 epitope. Three of these transposon-induced mutants synthesized a truncated form of LPS-1 that was structurally similar to that of the class of the NTG-induced mutants described above. These transposon-induced mutations, and the nitrosoguanidine-induced Fix- mutations, were closely linked and could be suppressed by the same cloned fragment of chromosomal DNA. The data presented here suggest that a precondition for normal nodule development of R. leguminosarum 3841 within pea nodules is the ability to synthesize relatively long-chain LPS-1 macromolecules under the physiological conditions encountered within the nodule. All mutants that lacked the ability to elongate LPS-1 macromolecules also failed to express the MAC 203 epitope.
authors.affiliation: John Innes Institute, John Innes Centre, Norwich, UK.
authors.forename: E L
authors.initials: EL
authors.lastname: Kannenberg
authors.forename: E A
authors.initials: EA
authors.lastname: Rathbun
authors.forename: N J
authors.initials: NJ
authors.lastname: Brewin
mesh_terms: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004251:DNA Transposable Elements / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000502:physiology*; D008040:Genetic Linkage; D007118:Immunoassay; D008070:Lipopolysaccharides / Q000737:chemistry*; D009154:Mutation / Q000235:genetics / Q000502:physiology*; D010100:Oxygen; D010641:Phenotype; D016961:Rhizobium leguminosarum / Q000737:chemistry* / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004251:DNA Transposable Elements; D000939:Epitopes; D008070:Lipopolysaccharides; D010100:Oxygen