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 melilotibacteroids 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 dmemutants of the broad-host-range Sinorhizobiumsp. 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 pckAand single dmemutants fixed N 2 at reduced rates, a pckA dmedouble 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 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 N 2 fixation in A. caulinodansand S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).