The NAD + -dependent malic enzyme (DME) has been reported to play a key role supporting nitrogenase activity in bacteroids of Sinorhizobium meliloti. Genetic evidence for a similar role in Bradyrhizobium japonicum USDA110 was obtained by constructing a dme mutant. Soybean plants inoculated with a dme mutant did not show delayed nodulation, but formed small root nodules and exhibited significant nitrogen-deficiency symptoms. Nodule numbers and the acetylene reducting activity per nodule as a dry weight value 14 and 28 days after inoculation with the dme mutant were comparable to those of plants inoculated with wild-type B. japonicum. However, shoot dry weight and acetylene reducting activity per nodule decreased to ca. 30% of the values in plants with wild-type B. japonicum. The sucrose and organic acid (malate, succinate, acetate, α-ketoglutarate and lactate) contents of the nodules were investigated. Amounts of sucrose, malate and α-ketoglutarate increased on inoculation with the dme mutant, suggesting that the decreased DME and nitrogenase activities in the bacteroids resulted in a reduction in the consumption of these respiratory metabolites by the nodules. The data suggest that the DME activity of B. japonicum bacteroids plays a role in nodule metabolism and supports nitrogen fixation.

During nodule development of legumes, together with the morphological alteration of plant cells, the rhizobia undergo marked biochemical and physiological changes and differentiate to bacteroids. Bacteroids invading a nodule are no longer free-growing cells, instead depending on the plant cell for all resources. To elucidate the molecular mechanism of the bacteroid differentiation of Bradyrhizobium japonicum, a time-course analysis of bacteroid protein profiles was studied by 2-D gel electrophoresis in soybean nodules. Using proteomic analysis, protein expressions in soybean nodule bacteroids 7, 10, 14, 28, and 49 days after inoculation (DAI) were monitored. The time points coincide with the early stage of nodule formation (7-10 DAI), the onset of nitrogen fixation (14-28 DAI), and nodule senescence (49 DAI). In this study, 275 annotated protein spots were successfully identified, and a cluster analysis of their expression was performed. A large portion of putative, upregulated proteins were observed in bacteroids at 7 or 10 DAI, and these upregulated proteins were mainly related to transcription, translation, protein folding, and degradation. In the later period (14-28 DAI) of bacteroid differentiation, a number of Nif and Fix proteins were upregulated. In addition, proteins related to the chaperonin and a synthetic enzyme of the poly-beta-hydroxybutyrate were expressed at high abundance. A number of proteins related to solute transporter were upregulated in the bacteroids and dominantly detected throughout nodule development. Changes of relative abundance of these proteins are discussed in relation to symbiosis.

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