C4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. In
Sinorhizobium melilotibacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2fixation, 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 dmemutants of the broad-host-range Sinorhizobiumsp. strain NGR234 formed nodules whose level of N2fixation 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 pckAand single dmemutants fixed N2at reduced rates, a pckA dmedouble mutant had no N2-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 dmemutants may be specific to the alfalfa- S. melilotisymbiosis. We therefore examined the ME-like genes azc3656and azc0119from Azorhizobium caulinodans, as azc3656mutants 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 N2fixation in A. caulinodansand S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).