NAD-Malic Enzyme Affects Nitrogen Fixing Activity of Bradyrhizobium japonicum USDA 110 Bacteroids in Soybean Nodules

Abstract

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.

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

“…Although the physiological roles and biochemical mechanisms of malic enzymes have been reported to support nitrogenase activity in various microorganisms, such as E. coli, S. meliloti and B. japonicum (Dao et al 2008;Voegele et al 1999;Mitsch et al 2007), in this paper, we reported whether M. loti STM mutants, named NADP ϩ -dependent malic enzyme mutant (STM14, STM4) and malate oxidoreductase mutant (STM17, STM38), respectively, could infect L. japonicus. Both STM 4 and STM 14 mutants induced nodules like the wild type, indicating that NADP ϩ -ME was not required for symbiotic N 2 fixation (Figure 3).…”

“…A significant change in both sucrose and organic acid contents of the nodules was observed in Ljstm17 nodules (Figure 4). In our previous reports on the omega-cassette insertion mutant of NAD ϩ -ME in B. japonicum, the nitrogenase activities of nodules were reduced and the amount of sucrose and malate in nodules changed (Dao et al 2008). NAD ϩ -ME protein in S. meliloti is subjected to substrate inhibition, and shows allosteric regulation by acetyl-CoA, and its activity is positively regulated by succinate and fumarate (Voegele et al 1999).…”

“…For continuous supply of pyruvate to the TCA cycle, malic enzymes catalyze oxidative decarboxylation of malate to pyruvate in conjunction with the reduction of either NAD ϩ (EC 1.1.1.38 and EC 1.1.1.39) or NADP ϩ (EC 1.1.1.39 and EC 1.1.1.40) to NADH or NADPH, respectively (Driscoll and Finan 1996;Mitsch et al 1998;Provorov and Tikhonovich 2003). Genetic evidence of such physiological roles of a malic enzyme in bacteroids has been reported (Chen et al 1998;Dao et al 2008;Day et al 1994;Tajima et al 1990). Finan (1993, 1996) showed that Sinorhizobium meliloti, which forms indeterminate nodules, contains both NAD ϩ -dependent malic enzyme (NAD ϩ -ME) and a NADP ϩ -dependent malic enzyme (NADP ϩ -ME) in alfalfa root nodules, and found that the NAD Mitsch et al (2007) investigated the biochemical background of S. meliloti bacteroids using genetic techniques to determine various types of dme mutants.…”

“…The collected supernatant was centrifuged at 5,000ϫg. The pellet was used as crude bacteroids.The activities of nodule enzymes were measured as previously described for malic enzyme, MDH (Chen et al 1998;Dao et al 2008) and sucrose synthase (Morell and Copeland 1985). The organic acid content of nodules was analyzed using a L7100 HPLC system.…”

“…Bacteroids developed in infected plant cells are surrounded by a peribacteroid membrane and form a stable metabolic unit. This structure is believed to function like an intracellular organelle, and is designated the symbiosome (Dao et al 2008;Day and Copeland 1991;White et al 2007). Under this symbiotic condition, bacteroids have been reported to use C 4 dicarboxylic acids as their sole respiratory substrate.…”