NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities

Abstract

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).

Fig 1

C₄-dicarboxylic acid-related metabolism. Abbreviations: DME, NAD(P)⁺-dependent malic enzyme; TME, NADP⁺-dependent malic enzyme; PCK, phosphoenolpyruvate carboxykinase; PYK, pyruvate kinase; PDH, pyruvate dehydrogenase; CS, citrate synthase; MDH, malate dehydrogenase; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

Fig 2

Sinorhizobium sp. NGR234 dme gene region. The dme gene is shown together with relevant restriction sites and the locations of the dmeΔ14::Ω Sp^r and dme-9::Ω Sp^r insertion mutations. NGR_c16870 encodes a putative metallophosphoesterase protein, and NGR_c16890 encodes a transcriptional regulator (NgrR). The thick line indicates the 3,328-bp region sequenced in this work, and the lower line indicates the dme region cloned into the complementing plasmid pTH2584.

Fig 3

DME and TME proteins in bacteroid extracts from NGR234 strains. Western blots were probed with anti-DME antibody (A) and anti-TME antibody (B). In addition to the TME protein, anti-TME antiserum detects an additional protein band that migrates at 95 kDa and is present in extracts from S. meliloti and NGR234. Each lane was loaded with 25 μg of protein. Lane 1, free-living S. meliloti (wild type); lane 2, free-living S. meliloti dme-3::Tn5 tme-4::Ω Sp^r. Lanes 3 to 14 show bacteroid extracts from nodules, as follows: lane 3, C. cajan plus NGR234; lane 4, C. cajan plus NGR234 dme-9; lane 5, C. cajan plus NGR234 dmeΔ14; lane 6, L. purpureus plus NGR234; lane 7, L. purpureus plus NGR234 dme-9; lane 8, L. purpureus plus NGR234 dmeΔ14; lane 9, M. atropurpureum plus NGR234; lane 10, M. atropurpureum plus NGR234 dme-9; lane 11, M. atropurpureum plus NGR234 dmeΔ14; lane 12, V. unguiculata plus NGR234; lane 13, V. unguiculata plus NGR234 dme-9; and lane 14, V. unguiculata plus NGR234 dmeΔ14. Data for L. leucocephala are not shown. Prestained protein standards of 95 kDa and 72 kDa are shown in the outer left lane. wt, NGR234; mt1, NGR234 dme-9; mt2, NGR234 dmeΔ14.

Fig 4

AZC3656 protein ME activity in response to various concentrations of l-malate and the cofactors NAD⁺ and NADP⁺. (A) ME specific activity (S.A.) in response to l-malate was measured at an NAD⁺ concentration of 1.5 mM. (B) ME specific activity (S.A.) in response to NAD⁺ (squares) and NADP⁺ (triangles) was measured at a concentration of 30 mM l-malate. Insets show Lineweaver-Burk plots (1/V versus 1/S). Data were analyzed using the equation 1/V = 1/V_max + (K_m/V_max)(1/S) to determine the K_m and V_max with respect to malate and NAD(P)⁺. Enzyme assay mixtures contained 1.1 μg purified AZC3656, 100 mM Tris-HCl, pH 7.8, 3 mM MnCl₂, 5 mM NH₄Cl, and 1.5 mM NAD⁺ in a final volume of 1 ml. Error bars represent standard errors of the means for triplicate samples.

Fig 5

AZC3656 malic enzyme activity is stimulated by fumarate and succinate and inhibited by acetyl-CoA. (A) ME specific activity (S.A.) with 1.5 mM NAD⁺ and 0 mM (■), 0.1 mM (●), or 1 mM (▲) fumarate. The inset shows a Lineweaver-Burk plot (1/V versus 1/S). (B) ME specific activity (S.A.) with 1.5 mM NAD⁺ and 0 mM (■), 1 mM (●), or 10 mM (▲) succinate. The inset shows a Lineweaver-Burk plot (1/V versus 1/S). (C) ME specific activity (S.A.) with 1.5 mM NAD⁺ and 0 μM (■), 25 μM (●), 50 μM (▲), or 100 μM (★) acetyl-CoA. (D) Lineweaver-Burk plot (1/V versus 1/S) of the data in panel C.