NAD⁺-malic enzyme affects nitrogenase activity of Mesorhizobium loti bacteroids in Lotus japonicus nodules

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A gram-negative bacterium, Mesorhizobium loti, contains a NADP⁺-malic enzyme (mlr5329) and a malate oxidoreductase (mlr0809) in the genome. We have screened transposon-induced mutants from the signature-tagged mutant library to survey their roles in nodule nitrogenase activity. The nodules induced by malate oxidoreductase mutants failed to fix N₂, although NADP⁺-malic enzyme (NADP⁺-ME) mutants induced nodules exhibiting no change in nodule nitrogenase activity. When malate-degrading enzyme activities were compared between malate oxidoreductase mutants and wild-type M. loti, NAD⁺-malic enzyme (NAD⁺-ME) activity was decreased significantly in malate oxidoreductase mutants, suggesting it is an NAD⁺-ME mutant. We found that NADP⁺-ME was not required for N₂ fixation. The fact that significant accumulations of sucrose, starch granules and malate were observed in the nodules induced by malate oxidoreductase mutant suggests that low nitrogenase activity of the nodules resulted in photosynthate accumulation. These data suggest that this malate oxidoreductase has a similar function to NAD⁺-ME in both Bradyrhizobium japonicum and Sinorhizobium meliloti.