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Malate metabolism in R. leguminosarum. Dme, NAD⁺ malic enzyme; GltA, citrate synthase; Mdh, malate dehydrogenase; PckA, phosphoenolpyruvate carboxykinase; Pdh, pyruvate dehydrogenase; PykA, pyruvate kinase; Tme, NADP⁺ malic enzyme.
Source publication
Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation
to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD+ malic enzyme (Dme) but not NADP+ malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate fo...
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Citations
... The downstream metabolic pathways, in which the products of the degradation of these aromatic compounds may be used, included the citrate cycle, propanoate, and pyruvate metabolism. The latter pathway is closely linked to symbiotic nitrogen fixation, which is energized via the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate [62]. There is a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth, generally associated with the promotion of ammonia secretion (derived from fixed nitrogen) into the plants [63,64]. ...
The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.
... Through the catalysis by malic enzyme, malate is decarboxylated into pyruvate, and the latter is further decarboxylated and then coenzyme A is added to generate acetyl CoA . In R. leguminosarum, there are two forms of malic enzyme (Table 2): Dme (NAD + dependent) and Tme (NADP + dependent) (Mulley et al. 2010). Mutations of dme or dme-tme of R. leguminosarum induced nitrogen-fixing phenotypes in peas (Driscoll and Finan 1996;Mulley et al. 2010). ...
... In R. leguminosarum, there are two forms of malic enzyme (Table 2): Dme (NAD + dependent) and Tme (NADP + dependent) (Mulley et al. 2010). Mutations of dme or dme-tme of R. leguminosarum induced nitrogen-fixing phenotypes in peas (Driscoll and Finan 1996;Mulley et al. 2010). However, when the dme mutation was accompanied by mutations in pyruvate kinase or phosphoenolpyruvate carboxykinase, the nitrogen-fixing ability of R. leguminosarum was abolished (Driscoll and Finan 1996;Mulley et al. 2010). ...
... Mutations of dme or dme-tme of R. leguminosarum induced nitrogen-fixing phenotypes in peas (Driscoll and Finan 1996;Mulley et al. 2010). However, when the dme mutation was accompanied by mutations in pyruvate kinase or phosphoenolpyruvate carboxykinase, the nitrogen-fixing ability of R. leguminosarum was abolished (Driscoll and Finan 1996;Mulley et al. 2010). This indicates that R. leguminosarum can produce acetyl CoA from pathways catalyzed by Dme, pyruvate kinase or phosphoenolpyruvate carboxykinase. ...
Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.
... The carbon source for rhizobia is predominately C4-dicarboxylic acids, primarily succinate and malate (129). Dicarboxylates are consumed by an NAD 1 -dependent malic enzyme paired with PEP-carboxykinase to produce acetyl-CoA, which can be used for carbon storage or consumption in the TCA cycle (130)(131)(132). Rhizobia express FixABCX encoded on the Sym plasmid and immediately upstream and regulated by nifA (133,134). ...
The availability of fixed nitrogen is a limiting factor in the net primary production of all ecosystems. Diazotrophs overcome this limit through the conversion of atmospheric dinitrogen to ammonia. Diazotrophs are phylogenetically diverse bacteria and archaea that exhibit a wide range of lifestyles and metabolisms, including obligate anaerobes and aerobes that generate energy through heterotrophic or autotrophic metabolisms. Despite the diversity of metabolisms, all diazotrophs use the same enzyme, nitrogenase, to reduce N2. Nitrogenase is an O2-sensitive enzyme that requires a high amount of energy in the form of ATP and low potential electrons carried by ferredoxin (Fd) or flavodoxin (Fld). This review summarizes how the diverse metabolisms of diazotrophs utilize different enzymes to generate low potential reducing equivalents for nitrogenase catalysis. These enzymes include substrate-level Fd oxidoreductases, hydrogenases, photosystem I or other light-driven reaction centers, electron bifurcating Fix complexes, proton motive force-driven Rnf complexes, and Fd:NAD(P)H oxidoreductases. Each of these enzymes is critical for generating low potential electrons while simultaneously integrating the native metabolism to balance nitrogenase's overall energy needs. Understanding the diversity of electron transport systems to nitrogenase in various diazotrophs will be essential to guide future engineering strategies aimed at expanding the contributions of biological nitrogen fixation in agriculture.
... Inside nodules, rhizobia differentiate into bacteroids that reduce atmospheric N 2 into ammonia for secretion to the plant host in exchange for dicarboxylates, primarily succinate and malate (3,4). Succinate and malate are typically metabolized via malic enzyme and pyruvate dehydrogenase, yielding acetyl-coenzyme A that can be oxidized in the tricarboxylic acid (TCA) cycle (5). Whether bacteroids need a complete TCA cycle remains unclear, as it is essential for Rhizobium and Sinorhizobium species, but mutants of Bradyrhizobium japonicum lacking 2-oxoglutarate dehydrogenase activity achieve wild-type levels of nitrogen fixation on a per-bacteroid basis (6,7). ...
... The obtained flux distribution captured key features of bacteroid metabolism, including use of the TCA cycle, pyruvate synthesis via malic enzyme, and minor activity of gluconeogenesis ( fig. S4) (5,8,37). ...
Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N 2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in mi-croaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD + ratio than sugars. Modeling and 13 C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N 2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.
... However, in alfalfa rhizobia, Sinorhizobium meliloti, deletion of the phosphotransacetylase domain from the malic enzyme leads to a significant (40-50 %) increase of nitrogenase activity and of the root nodule mass. Moreover, activation of phosphoenolpyruvate carboxykinase in bacteroids leads to a 70 % reduction of N 2 -fixing activity compared to wild-type bacteroids (Mulley et al., 2010), suggesting that operation of this enzyme is disadvantageous for the symbiosis operation. Earlier, we revealed in alfalfa rhizobia (Sinorhizobi um meliloti) a number of eff genes whose inactivation by Tn5 insertions leads to an increased symbiotic efficiency (SE) -the impact of bacteria inoculation on host plant productivity Onishchuk, Sharypova and Simarov, 1994). ...
Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.
... While all bacteroids are required to convert TCA cycle intermediates into acetyl-CoA, the synthesis routes differ. Bacteroids of S. meliloti and A. caulinodans exclusively use ME (190,191), whereas Sinorhizobium fredii and R. leguminosarum utilize both ME and PCK/PK (191,192). In addition, the activity of PCK is important for bacteroids to sustain gluconeogenesis, which is essential for the production of precursors for various metabolites (63,193). ...
Rhizobia are a phylogenetically diverse group of soil bacteria that engage in mutualistic interactions with legume plants. Although specifics of the symbioses differ between strains and plants, all symbioses ultimately result in the formation of specialized root nodule organs which host the nitrogen-fixing microsymbionts called bacteroids. Inside nodules, bacteroids encounter unique conditions that necessitate global reprogramming of physiological processes and rerouting of their metabolism. Decades of research have addressed these questions using genetics, omics approaches, and more recently computational modelling. Here we discuss the common adaptations of rhizobia to the nodule environment that define the core principles of bacteroid functioning. All bacteroids are growth-arrested and perform energy-intensive nitrogen fixation fueled by plant-provided C 4 -dicarboxylates at nanomolar oxygen levels. At the same time, bacteroids are subject to host control and sanctioning that ultimately determine their fitness and have fundamental importance for the evolution of a stable mutualistic relationship.
... Our previous INSeq screen in Rlv3841 similarly reported the termini of RL3424 (dctA) to tolerate a large number of insertions (62). RL0037 (pckA), encoding phosphoenolpyruvate carboxykinase, which is essential for growth on dicarboxylates (63), was also specifically required in nodule bacteroids (SI Appendix, Table S8). This fits with evidence that dicarboxylates drive bacteroid metabolism but not bacterial growth during nodule infection, where multiple carbon sources are probably available. ...
Significance
Rhizobia are soil-dwelling bacteria that form symbioses with legumes and provide biologically useable nitrogen as ammonium for the host plant. High-throughput DNA sequencing has led to a rapid expansion in publication of complete genomes for numerous rhizobia, but analysis of gene function increasingly lags gene discovery. Mariner-based transposon insertion sequencing has allowed us to characterize the fitness contribution of bacterial genes and determine those functionally important in a Rhizobium– legume symbiosis at multiple stages of development.
... Therefore, WSM1521 was used as a positive control for nitrogenase activity, with the prediction that GFP fluorescence from WSM1521 nodules should be higher than WSM1475, Rlv3841, and UPM791. GFP detection, ARA, and shoot DW biomass were normalized by the number of nodules per plant (SI Appendix, GFP fluorescence and nitrogenase activity correlated, with a peak at 28 dpi (Fig. 2C), in agreement with maximum ARA occurring at flowering in peas (34). Dry biomass measurements at 35 dpi and 42 dpi (SI Appendix, Table S6) showed that strain WSM1521 is highly effective and confirm that sfGFP fluorescence is an easy method for quantification of nitrogenase activity in individual nodules. ...
Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N 2 ) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N 2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N 2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium -legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.
... It appears to be the result of a duplication or insertion event, as it is located close to a transposable element. Rlv 3841 has two pathways for malate conversion to pyruvate: Dme and phosphoenolpyruvate (PEP) carboxykinase (PckA)/pyruvate kinase (PykA) [66]. These pathways are also present in Rlv UPM791: Dme, RLV_2776; and PckA, RLV_7044, with two copies of PykA: RLV_6265, similar to the protein of Rlv 3841, and RLV_3363, both in the chromosome. ...
Rhizobium leguminosarum
bv.viciaeis a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of theFabeaetribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome ofR. leguminosarumbv.viciaeUPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains ofR. leguminosarumbv.viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.
... Symbiotic N 2 fixation relies on precise and efficient integration of bacterial and plant metabolism. The plant provides dicarboxylic acids, predominately malate and succinate, and these are taken up by bacteroids via the dicarboxylic transport system (Dct), which is essential for N 2 fixation (7)(8)(9). Furthermore, either NAD ϩ malic enzyme (diphosphopyridine nucleotide-dependent malic enzyme [Dme]) or the combined activities of Dme, phosphoenolpyruvate (PEP) carboxykinase, and pyruvate kinase are essential for dicarboxylate metabolism and N 2 fixation in bacteroids (9,10). ...
... The plant provides dicarboxylic acids, predominately malate and succinate, and these are taken up by bacteroids via the dicarboxylic transport system (Dct), which is essential for N 2 fixation (7)(8)(9). Furthermore, either NAD ϩ malic enzyme (diphosphopyridine nucleotide-dependent malic enzyme [Dme]) or the combined activities of Dme, phosphoenolpyruvate (PEP) carboxykinase, and pyruvate kinase are essential for dicarboxylate metabolism and N 2 fixation in bacteroids (9,10). ...
... This suggests that for optimal growth on succinate, there is a requirement for increased nucleotide recovery or synthesis compared to growth on glucose. RL0037 (pckA) encodes phosphoenolpyruvate carboxykinase, which converts oxaloacetate into phosphoenolpyruvate, essential for gluconeogenesis ( Fig. 2) (9). RL2990 (ubiA) encodes a 4-hydroxybenzoate octaprenyltransferase required for ubiquinone biosynthesis and electron transport. ...
Importance:
Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium that adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.
