Rhizobium leguminosarum CFN42 lipopolysaccharide antigenic changes induced by environmental conditions

Abstract

Four monoclonal antibodies were raised against the lipopolysaccharide of Rhizobium leguminosarum bv. phaseoli 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 planta of strain CFN42 at low pH, high temperature, low phosphate, or low oxygen concentration also eliminated binding of one or both of these antibodies. 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. The antigenic changes at low pH were dependent on growth of the bacteria but were independent of nitrogen and carbon sources and the rich or minimal quality of the medium. The Sym plasmid of this strain was not required for the changes induced ex planta. Analysis of bacterial mutants inferred to have truncated O-polysaccharides indicated that part, but not all, of the lipopolysaccharide O-polysaccharide portion was required for binding of these two antibodies. In addition, this analysis suggested that O-polysaccharide structures more distal to lipid A than the epitopes themselves were required for the modifications at low pH that prevented antibody binding. Two mutants were antigenically abnormal, even though they had abundant lipopolysaccharides of apparently normal size. One of these two mutants was constitutively unreactive toward three of the antibodies but indistinguishable from the wild type in symbiotic behavior. The other, whose bacteroids retained an epitope normally greatly diminished in bacteroids, was somewhat impaired in nodulation frequency and nodule development.

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

“…fucose, 3-O-methylrhamnose, glucuronic acid, N-acetylquinovosamine, and additional mannose). This result, together with the increased electrophoretic mobility compared with the higher molecular weight LPS I of the parent strain, shows that CE359 LPS V contains a truncated version of the O-chain and is consistent with previous reports showing that LPS V binds monoclonal antibodies that are specific for the O-chain (12).…”

“…DOC-PAGE analysis showed the presence of typical high molecular weight LPS (LPS I, containing O-chain polysaccharide) and low molecular weight LPS (LPS II, which lacks the O-chain, and various LPSs that carry a truncated O-chain) (12,17). As described in previous reports (12,17), mutant CE358 produced only one type of LPS, having an electrophoretic mobility identical to LPS II. Mutant CE359 also produced an LPS II species, as well as an LPS of slightly higher molecular weight, which carries a truncated O-chain polysaccharide (17).…”

“…Mutant CE359 also produced an LPS II species, as well as an LPS of slightly higher molecular weight, which carries a truncated O-chain polysaccharide (17). The various forms of LPS that carry a truncated O-chain have been designated LPS III, IV, and V and are distinguishable based on their differential reactivity with monoclonal antibodies (12). Mutant CE359 produces LPS V in addition to LPS II (12).…”

“…The various forms of LPS that carry a truncated O-chain have been designated LPS III, IV, and V and are distinguishable based on their differential reactivity with monoclonal antibodies (12). Mutant CE359 produces LPS V in addition to LPS II (12).…”

Identification of an ATP-binding Cassette Transporter for Export of the O-antigen across the Inner Membrane inRhizobium etli Based on the Genetic, Functional, and Structural Analysis of an lps Mutant Deficient in O-antigen

“…Fig. 7 (B-D) shows the results of immunoblots using mAbs JIM26, JIM27, and JIM28, which specifically recognize the O-chain polysaccharide of CE3 LPS (48). As would be expected, no reaction with any of these mAbs was observed for the FAJ1200 aqueous extract.…”