Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation

Scott, John D.; Ludwig, Robert A.

doi:10.1099/mic.0.26603-0

Cited by 21 publications

(22 citation statements)

References 34 publications

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“…Since this complex is not present in rhizobia, an alternative pathway is required. One possibility is that the electron-transferring flavoprotein (ETF) complex, FixABCX, interacts with pyruvate dehydrogenase, as shown by genetic suppressor analysis in A. caulinodans (61). ETF complexes use electron bifurcation in anaerobic bacteria (62,63), which might enable FixABCX to generate low-potential electrons for reduction of ferredoxin and then N 2 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

et al. 2016

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Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N 2 . Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [ 13 C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N 2 -fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N 2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-␤-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N 2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. IMPORTANCEBiological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N 2 . However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-␤-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N 2 in all legume nodules.

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Section: Discussionmentioning

confidence: 99%

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

et al. 2016

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“…However, it is also possible that DME is required to generate pyruvate as a precursor for some other important metabolic pathways in bacteroids. Pyruvate could be a precursor in the yet-to-be-defined pathway leading to the generation of reductant for nitrogenase (35). Another possible pathway could be alanine synthesis in nodules.…”

Section: Why Nadmentioning

confidence: 99%

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

2007

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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...

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“…1). Group II ETF genes ( fixB and fixA) are found in nitrogen-fixing bacteria where they divert electrons from dehydrogenases to nitrogenases, bypassing the electron transport chain (Scott & Ludwig, 2004) (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

“…These soluble FAD-containing heterodimeric proteins are distributed across all domains of life, and are responsible for funnelling electrons from dehydrogenases to the membrane-bound respiratory chain of bacteria and mitochondria (Toogood et al, 2007) or to nitrogen fixation in some bacteria (Scott & Ludwig, 2004). ETFs consist of a large (ETF-a) and small (ETF-b) subunit, and based on comparative amino acid sequence analysis, they are traditionally divided into three groups, which have different biological functions (Tsai & Saier, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Group II ETFs divert electrons away from primary dehydrogenases towards nitrogenase reductase. Single knockouts of primary dehydrogenases do not impair symbiotic nitrogen fixation and therefore it is expected that group II ETFs have multiple electron donors, although the only confirmed partner is pyruvate dehydrogenase in Azorhizobium caulinodans (Scott & Ludwig, 2004). The only studied group III ETF is known to be required for the anaerobic reduction of carnitine, although the direction of electron flow is unknown (Walt & Kahn, 2002).…”

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An electron transfer flavoprotein is essential for viability and its depletion causes a rod-to-sphere change in Burkholderia cenocepacia

2015

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Essential gene studies often reveal novel essential functions for genes with dispensable homologues in other species. This is the case with the widespread family of electron transfer flavoproteins (ETFs), which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions highthroughput screens have identified ETFs as putatively essential in several species. In this study, we constructed a conditional expression mutant of one of the ETFs in Burkholderia cenocepacia, and demonstrated that its expression is essential for growth on both complex media and a variety of single-carbon sources. We further demonstrated that the two subunits EtfA and EtfB interact with each other, and that cells depleted of ETF are non-viable and lack redox potential. These cells also transition from the short rods characteristic of Burkholderia cenocepacia to small spheres independently of MreB. The putative membrane partner ETF dehydrogenase also induced the same rod-to-sphere change. We propose that the ETF of Burkholderia cenocepacia is a novel antibacterial target.

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Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation

Cited by 21 publications

References 34 publications

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

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

An electron transfer flavoprotein is essential for viability and its depletion causes a rod-to-sphere change in Burkholderia cenocepacia

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Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation

Cited by 21 publications

References 34 publications

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

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

An electron transfer flavoprotein is essential for viability and its depletion causes a rod-to-sphere change in Burkholderia cenocepacia

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