Malic Enzyme Cofactor and Domain Requirements for Symbiotic N ₂ Fixation by Sinorhizobium meliloti

Abstract

The NAD؉ -dependent malic enzyme (DME) and the NADP ؉ -dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N 2 (Fix ؊ ) in alfalfa root nodules, whereas tme mutants are unimpaired in their N 2 -fixing ability (Fix ؉ ). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N 2 fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N 2 fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD ؉ -dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N 2 fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH ؉ H ؉ to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N 2 -fixing bacteroids.Members of the Rhizobiaceae enter into a symbiotic association with a plant host, during which time the free-living bacteria differentiate into nitrogen-fixing cells known as bacteroids. This transition is accompanied by a number of important physiological adaptations, among which are alterations to the metabolic pathways of the microbe (14). There is much evidence to suggest that bacteroids within nodules utilize C 4 -dicarboxylic acids as their primary carbon and energy sources and that these acids are metabolized via the tricarboxylic acid (TCA) cycle (13,30,31,43). A number of reports have suggested that cycling of amino acids between the plant and bacteroid also plays a key role in carbon and nitrogen flow in N 2 -fixing root nodules (1,24,40). These reports have demonstrated that much remains to be resolved concerning C and N flow between the bacteroid and plant during symbiotic N 2 fixation.In N 2 -fixing bacteroids metabolizing C 4 -dicarboxylic acids via the TCA cycle, a pathway for the generation of acetyl coenzyme A (acetyl-CoA) is essential. One route for the formation of acetyl-CoA is via malic enzyme and pyruvate dehydrogenase (7, 8). Malic enzymes are responsible for the conversion of malate to pyruvate with the concomitant reduction of a nicotinamide cofactor. Sinorhizobium meliloti contains two malic enzymes, DME (diphosphopyridine nucleotide-dependent malic enzyme), wh...

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

“…Preparation of a crude enzyme solution and the malic enzyme assay were performed as we have reported1 1,14) . Malate dehydrogenase activity was determined as reported by Smith 25) .…”

“…Although the conversion of pyruvate and malate was similar for DME and TME, their data showed that the high expression level of TME did not restore DME activity. In addition, Escherichia coli NAD + -dependent malic enzyme restored wild-type N 2 fixation by the dme mutant 14) . In Bradyrhizobium japonicum, however, there has been no genetic evidence for roles of DME and TME in the nitrogenase activity of bacteroids in nodules.…”

“…They found that dme mutants of Sinorhizobium meliloti were Nod + and Fix − , and conversely, that a NADP + -dependent malic enzyme (TME)-defective mutant was Nod + and Fix + . In a recent report 14) , they examined the biochemical background of S. meliloti bacteroids by preparing various types of dme mutants. Although the conversion of pyruvate and malate was similar for DME and TME, their data showed that the high expression level of TME did not restore DME activity.…”

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).…”

“…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. These results showed that NADP ϩ -ME activity in nodules did not replace NAD ϩ -ME activity.…”