\u3cem\u3eRhizobium leguminosarum\u3c/em\u3e CFN42 Lipopolysaccharide Antigenic Changes Induced by Environmental Conditions

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

Cysteine proteases in nodulation and nitrogen fixation

1124-1132<span style="font-size: 14.0pt;mso-bidi-font-size:9.0pt;font-family:" times="" new="" roman","serif""="">The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells. They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death. They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature. wounding. Ethylene, and oxidative conditions) and plant-microbe interactions (including <span style="font-size: 14.0pt;mso-bidi-font-size:9.0pt;font-family:" times="" new="" roman","serif""="">nodulation). In the development and function of legume root nodules,  cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" times="" new="" roman","serif""="">(ii) <span style="font-size:14.0pt; mso-bidi-font-size:9.0pt;font-family:" times="" new="" roman","serif""="">protein turnover required during the <span style="font-size:14.0pt;mso-bidi-font-size:9.0pt;line-height:115%; font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">formation of new tissue; <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; line-height:115%;font-family:" times="" new="" roman","serif";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">(iii) <span style="font-size:14.0pt;mso-bidi-font-size: 9.0pt;line-height:115%;font-family:" times="" new="" roman","serif";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="">cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence. Because of their central importance to plant physiology,  cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level. Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.</span

Antigenic Change in the Lipopolysaccharide of \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42 Induced by Exudates of \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e

Growth of Rhizobium etli CE3 in the presence of exudates from Phaseolus vulgaris resulted in a modified lipopolysaccharide (LPS) that no longer reacted with monoclonal antibody JIM28. However, the overall LPS structure appeared not to be greatly altered, as revealed by unchanged mobility in gel electrophoresis and partial or unaltered reactivity with other antibodies. Activity that triggered LPS antigenic conversion was exuded from both seeds and roots, but reactivity with one of the antibodies indicated that the resulting alterations were not identical. Antibody binding to the LPS decreased as a function of the concentration of exudate present during growth of the bacteria. The antigenic change did not occur if purified LPS or nongrowing bacteria were incubated with the exudates. Exudate-induced LPS modification did not require the Sym plasmid

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

Six mutants of Rhizobium leguminosarum 3855 lacking uptake hydrogenase activity (Hup(−) phenotype) as a result of Tn5-mob mutagenesis of the hup-containing plasmid pRL6JI were tested for symbiotic performance on Pisum sativum L. and Vicia benghalensis L. Three pea cultivars and one vetch line, which induce four different levels of Hup activity in strain 3855, were grown to flowering under microbiologically controlled conditions in the absence of combined N. Direct Kjeldahl N measurements showed that in every case at least one Hup(−) mutant fixed as much N(2) as the isogenic Hup(+) strain. Measures of C(2)H(2) reduction, H(2) evolution, (3)H(2) incorporation, and plant dry weight were consistent with the interpretation that the oxidation of H(2) produced by the nitrogenase enzyme complex was not necessarily associated with increased N(2) fixation in these symbiotic associations. Tests with a smaller subset of the Hup(−) strains under four different root environments ranging from pH 5.0 to 8.2 likewise showed no significant advantage for the isogenic Hup(+) strain. It was concluded that the improvements in symbiotic N(2) fixation produced by pRL6JI are associated with some trait other than the Hup(+) phenotype

Glutathione and Homoglutathione Synthetases of Legume Nodules. Cloning, Expression, and Subcellular Localization

The thiol tripeptides glutathione (GSH) and homoglutathione (hGSH) are very abundant in legume root nodules and their synthesis is catalyzed by the enzymes γ-glutamylcysteine synthetase (γECS), GSH synthetase (GSHS), and hGSH synthetase (hGSHS). As an essential step to elucidate the role of thiols in N(2) fixation we have isolated cDNAs encoding the three enzymes and have quantified the transcripts in nodules. Assay of enzyme activities in highly purified nodule organelles revealed that γECS is localized in the plastids, hGSHS in the cytosol, and GSHS in the cytosol and mitochondria. These results are consistent with sequence analyses. Subcellular fractionation of nodules also showed that bacteroids contain high thiol concentrations and high specific γECS and GSHS activities. Results emphasize the role of nodule plastids in antioxidant protection and in control of thiol synthesis, and suggest that plastids may be important in the stress response of nodules. Overall, our results provide further evidence that thiol synthesis is critical for nodule functioning