Date of Award

Fall 1991

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Four monoclonal antibodies, obtained from N. Brewin, had been raised against the lipopolysaccharide of Rhizobium 7eguminosarum bv. phaseo7i CFN42 grown in tryptone and yeast extract. Two of these antibodies reacted relatively weakly with the lipopolysaccharide of bacteroids of this strain isolated from bean nodules. Growth ex p7anta of strain CFN42 at low pH, high temperature, low phosphate, or low oxygen concentration also eliminated binding of one or both of these antibodies. The antigenic changes at low pH were dependent on growth of the bacteria, but I independent of nitrogen and carbon sources and the rich/minimal quality of the medium. Nor was the Sym plasmid of this strain required for the changes induced ex p7anta. Lipopolysaccharide mobility on gel electrophoresis and reaction with other monoclonal antibodies and polyclonal antiserum indicated that the antigenic changes detected by these two antibodies did not represent major changes in lipopolysaccharide structure. Analysis of bacterial mutants inferred to have truncated 0-antigens indicated that part, but not all, of the lipopolysaccharide 0-antigen portion was required for binding of these two antibodies. 0-antigen structures more d i sta 1 to 1 i pi d A than the ep i topes themse 1 ves were required for the modifications at low pH that prevented antibody binding. Two mutants had abundant lipopolysaccharides of apparently normal size. One of these two mutants was constitutively unreactive toward t!l-ree of the antibodies but indistinguishable from the wild type in sYmb.iotic behavior. The other, whose bacteroids retained an epitope normally greatly diminished in bacteroids, was slightly impaired in nodulation frequency and nodule development. The mutant gene(s) was in the lps region a. Bean exudate induced the loss of reactivity of LPS I with one of the two antibodies which reacted weakly with the bacteroid LPS. Naringenin (1 ~) and pea exudate did not induce this LPS modification, although in control experiments they were as potent as bean exudate in inducing nod-lac fusions (Cevallos et al, 1989) in a wild-type genetic background. pSym- strain CE144 did not alter its LPS in the presence of bean exudate. Response to exudate was restored when CE144 carried recombinant plasmids harboring a locus closely linked to nodBC.

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