NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2 Fixation Capabilities

C-4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N-2-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N-2 fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N-2 fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N-2 at reduced rates, a pckA dme double mutant had no N-2-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N-2 fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).