A gram-negative bacterium, Mesorhizobium loti, contains a NADP ϩ -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 N 2 , although NADP ϩ -malic enzyme (NADP ϩ -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 ϩ -malic enzyme (NAD ϩ -ME) activity was decreased significantly in malate oxidoreductase mutants, suggesting it is an NAD ϩ -ME mutant. We found that NADP ϩ -ME was not required for N 2 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 ϩ -ME in both Bradyrhizobium japonicum and Sinorhizobium meliloti. Plant Biotechnology 27, 311-316 (2010) Original Paper Transposon insertion mutants, generated using signature-tagged mutagenesis (STM), are a powerful technique that can be used to identify the genes required for symbiotic nitrogen fixation function in root nodules (Shimoda et al. 2008). We have selected STM mutants that had a transposon inserted in the malic enzyme genes from the collection of transposon mutants and studied the function of these genes. Key words: Lotus japonicus, Mesorhizobium loti, NAD Materials and methods Bacterial strains and mediaTransposon insertion mutants (STM mutant) of M. loti MAFF303099 were obtained from Kazusa DNA Research Institute (Shimoda et al. 2008). M. loti strains were cultured in tryptone-yeast extract (TY) liquid medium (Beringer 1974) at 28ºC. Antibiotics were added to the media in the following concentrations: spectinomycin 100 µg mL Ϫ1, streptomycin 100 µg mL -1 and phosphomycin 100 µg mL Ϫ1. Plant materialsSeeds of L. japonicus B-129 Gifu (Handberg and Stougaard 1992) were sterilized with a sodium hypochlorite solution, rinsed with sterile water, and allowed to germinate in sterile vermiculite with liquid B&D medium (Broughton and Dilwoth 1971) in double Magenda jars. Then 1ϫ10 9 cells mL Ϫ1 was added in the jars. These plants were grown in an artificially growth cabinet controlled at 22ºC (16 hr/8hr, light/dark). Acetylene reduction activityFor acetylene reduction activity, plants at 35 days after infection were used. The nodules were placed in a 20 ml vial and incubated at 25ºC by adding 2.6 ml of acetylene. After 30 min, the amount of ethylene formed was measured by gas chromatography (Shimazu GC-8A) to determine ethylene production as described previously (Kouchi et al. 1989) Light microscopyNodules were fixed in 4% paraformaldehyde and 25% glutaraldehyde in 0.5 M Sodium phosphate buffer (pH 7.2...
Mesorhizobium loti is a Gram negative bacterium that induces N 2 -fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.
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