C4-dicarboxylic Acid Transport Research Articles

Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA.

Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that nodulate the agriculturally important Phaseolus vulgaris and several other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix−) and increased in number compared to P. vulgaris plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites’ changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix− nodules, the level of indole-acetamide (IAM) and brassinosteroids increased. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differentially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hypernodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress response. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix− rhizobia.

Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct)

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Effect of divalent cations on succinate transport in Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti

Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C4-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca(2+) or Mg(2+). However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca(2+) or Mg(2+) had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca(2+) to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca(2+) and Mg(2+). A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.

Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti

The relationship between chemotaxis and transport of C4 dicarboxylic acids was analyzed with Rhizobium meliloti dct mutants defective in one or all of the genes required for dicarboxylic acid transport. Succinate, malate, and fumarate were moderately potent chemoattractants for wild-type R. meliloti and appeared to share a common chemoreceptor. While dicarboxylate transport is inducible, taxis to succinate was shown to be constitutive. Mutations in the dctA and dctB genes both resulted in the reduction, but not elimination, of chemotactic responses to succinate, indicating that transport via DctA or chemosensing via DctB is not essential for C4 dicarboxylate taxis, although they appear to contribute to it. Mutations in dctD and rpoN genes did not affect taxis to succinate. Aspartate, which is also transported by the dicarboxylate transport system, elicited strong chemotactic responses via a chemoreceptor distinct from the succinate-malate-fumarate receptor. Taxis to aspartate was unaltered in dctA and dctB mutants but was considerably reduced in both dctD and rpoN mutants, indicating that aspartate taxis is strongly dependent on elements responsible for transcriptional activation of dctA. Methylation and methanol release experiments failed to show a significant increase in methyl esterification of R. meliloti proteins in response to any of the attractants tested.

Induction of C4-dicarboxylate transport genes by external stimuli in Rhizobium meliloti.

We have characterized two mutants of Rhizobium meliloti L5-30 obtained by random mutagenesis using Mu-lacZ that were defective in transport of C4-dicarboxylic acids. These mutants induced ineffective nodules on alfalfa. Mutations in the two strains appeared to be located in a dctA gene. Levels of dctA gene expression were determined under different environmental conditions using dctA-lacZ fusions, and beta-galactosidase activities increased in response to osmotic stress and also when the cells were incubated in medium low in calcium. The transcriptional induction of the dctA gene by environmental signals was decreased by DNA gyrase inhibitors such as novobiocin and coumermycin.

C4-Dicarboxylate Metabolism in Free-living and Bacteroid Forms of Rhizobium leguminosarum MNF3841

The transport and catabolism of C4-dicarboxylic acids have been further studied in Rhizobium leguminosarum MNF3841. Uptake of [14C]succinate was induced in free-living cells of strain MNF3841 within 12 min of exposure to succinate and reached a maximum rate within 60 min. Free-living cells of strain MNF3841 oxidizing fumarate accumulated pyruvate when treated with arsenite, an inhibitor of pyruvate dehydrogenase. Generation of pyruvate from C4-dicarboxylates was accomplished by malic enzyme. Although malic enzyme was present in free-living cells grown on sucrose, higher activities were observed when fumarate or l-arabinose was the growth substrate. In crude extracts, malic enzyme activity required either NAD+ or NADP+ as cofactor, together with Mn2+, and was stimulated by K+. Bacteroids from pea nodules transported [14C]succinate immediately after isolation, and contained twofold higher activities of both malic enzyme and pyruvate dehydrogenase than those found in fumarate-grown free-living cells. These data indicate that C4-dicarboxylates are available to nodule bacteroids and that they are catabolized through the tricarboxylic acid cycle using malic enzyme and pyruvate dehydrogenase to generate acetyl-CoA.

Properties of Double Mutants of Rhizobium leguminosarum Which Are Defective in the Utilization of Dicarboxylic Acids and Sugars

SUMMARY: Rhizobium leguminosarum MNF3201 is a mutant defective in C4 dicarboxylic acid transport (dct). which forms ineffective nodules on pea. The Tn5-induced mutants MNF3041 (ribokinase-negative), MNF3045 (arabinonate dehydratase-negative), MNF3064 (defective in the “Entner-Doudoroff enzymes”) and MNF3070 (pyruvate carboxylase-negative) nodulate peas and fix N2. The carbohydrate-utilization mutations of these strains were each transduced into MNF3201 using phage RL38. The double mutants were defective in the utilization of both C4 dicarboxylic acids and the respective carbohydrates. All the double mutants formed nodules on peas, although these were still ineffective. Thus it appears that R. leguminosarum can use carbon sources other than dicarboxylic acids or common classes of sugars to fuel the nodulation process.

Le vie fotosintetiche C3, C4e CAM nel contesto ecologico

Abstract Ecological aspects of C3, C4 and CAM photosynthetic pathways. - Three different photosynthetic CO2 fixation pathways are known to occur in higher plants. However all three pathways ultimately depend on the Calvin-Benson cycle for carbon reduction. The oxygenase activity of RuBP carboxilase is responsible for photorespiratory CO2 release. Both C4 and CAM pathways behave as a CO2 concentrating mechanism which prevent photorespiration. The CO2-concentrating mechanism in C4 plants is based on intracellular symplastic transport of C4 dicarboxylic acids from mesophyll-cells to the adjacent bundle-sheath cells. On the contrary in CAM plants the CO2-concentrating mechanism is based on the intracellular transport of malic acid into and out of the vacuole. The C4 photosynthetic pathway as compared to the C3 pathway permits higher rates of CO2 fixation in high light and high temperature environments at low costs in terms of water loss, given the stability of the photosynthetic apparatus under such conditions. CAM is interpreted as an adaptation to arid environments because it enables carbon assimilation to take place at very low water costs during the night when the evaporative demand is low. Nevertheless many aquatic species of Isoetes and some relatives are CAM, suggesting the adaptive role of CAM to environments which become depleted in CO2. The photosynthetic carbon fixation pathway certainly contributes to the ecological success of plants in different environments. However the distribution of plants may also reflect their biological history. On the other hand plants with different photosynthetic pathways coexist in many communities and tend to share resources in time. In any case some generalizations are possible: C4 plants enjoy an ecological advantage in hot, moist, high light regions while the majority of species in desert environments are C3; CAM plants are more frequent in semiarid regions with seasonal rainfall, coastal fog deserts, and in epiphytic habitats in tropical rain forests.

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.

Properties of mutants of Escherichia coli lacking malic dehydrogenase and their revertants.

Mutants of Escherichia coli lacking malic dehydrogenase activity (mdh) were incapable of growth on acetate", succinate- or malate/mineral medium. Revertants of mdh strains which had regained the ability to grow on C4-dicarboxylic acids could be divided into two distinct classes. One type of revertant had regained the ability to synthesize functional malic dehydrogenase. The other type of revertant still lacked malic dehydrogenase activity but possessed a suppressor mutation which altered the regulation of the synthesis or activity of the C4-dicarboxylic acid transport system, resulting in increased C4-dicarboxylic acid transport activity. This latter class of revertants apparently synthesized oxalacetate from malate via the sequential actions of the NAD-linked malic enzyme, phosphoenolpyruvate synthetase, and phosphoenolpyruvate carboxylase. Evidence has been presented that is consistent with the hypothesis that oxalacetate is the inducer of the C4-dicarboxylic acid transport system. The inability of mutants lacking malic dehydrogenase to grow with a C4-dicarboxylic acid as the carbon source can be attributed to the difficulty such mutants have in synthesizing oxalacetate.

Regulation of C4-dicarboxylic acid transport in Bacillus subtilis.

Various mutant strains of Bacillus subtilis were used to study the induction and regulation of the transport of tricarboxylic acid cycle C4-dicarboxylates. L-Malate was the only physiological inducer and bromosuccinate was a gratuitous inducer of dicarboxylic acid transport in a succinic dehydrogenase deficient mutant. Several mutants were isolated with alterations in the ability to transport dicarboxylic acids. One of these (WK6) was defective in the ability to take up succinate when grown on glutamate minimal medium, whereas another (WK1) was inducible to high levels by extremely low levels of L-malate. Alpha-Ketoglutarate dehydrogenase mutants were unable to take up dicarboxylates because of repression of transport by glutamate and (or) alpha-ketoglutarate. A mutation which resulted in increased levels of alpha-ketoglutarate dehydrogenase partially overcame this inhibition. Glutamate did not prevent the induction of dicarboxylic acid transport by L-malate in succinic dehydrogenase mutants but was markedly inhibitory in alpha-ketoglutarate dehydrogenase mutants.

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis.

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.