Co‐catabolism of arginine and succinate drives symbiotic nitrogen fixation
2020; Springer Nature; Volume: 16; Issue: 6 Linguagem: Inglês
10.15252/msb.20199419
ISSN1744-4292
AutoresCarlos Eduardo Flores-Tinoco, Flavia Tschan, Tobias Fuhrer, Céline Margot, Uwe Sauer, Matthias Christen, Beat Christen,
Tópico(s)Wastewater Treatment and Nitrogen Removal
ResumoArticle3 June 2020Open Access Source DataTransparent process Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation Carlos Eduardo Flores-Tinoco Carlos Eduardo Flores-Tinoco Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Flavia Tschan Flavia Tschan Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Tobias Fuhrer Tobias Fuhrer orcid.org/0000-0001-5006-6874 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Céline Margot Céline Margot Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Uwe Sauer Uwe Sauer orcid.org/0000-0002-5923-0770 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Matthias Christen Corresponding Author Matthias Christen [email protected] orcid.org/0000-0001-7724-4562 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Beat Christen Corresponding Author Beat Christen [email protected] orcid.org/0000-0002-9528-3685 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Carlos Eduardo Flores-Tinoco Carlos Eduardo Flores-Tinoco Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Flavia Tschan Flavia Tschan Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Tobias Fuhrer Tobias Fuhrer orcid.org/0000-0001-5006-6874 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Céline Margot Céline Margot Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Uwe Sauer Uwe Sauer orcid.org/0000-0002-5923-0770 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Matthias Christen Corresponding Author Matthias Christen [email protected] orcid.org/0000-0001-7724-4562 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Beat Christen Corresponding Author Beat Christen [email protected] orcid.org/0000-0002-9528-3685 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Search for more papers by this author Author Information Carlos Eduardo Flores-Tinoco1, Flavia Tschan1, Tobias Fuhrer1, Céline Margot1, Uwe Sauer1, Matthias Christen *,1 and Beat Christen *,1 1Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland *Corresponding author. Tel: +41 44 633 76 58; Email: [email protected] *Corresponding author. Tel: +41 44 633 64 44; Email: [email protected] Molecular Systems Biology (2020)16:e9419https://doi.org/10.15252/msb.20199419 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism—two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation. Synopsis This study challenges the current model of nitrogen exchange in rhizobia-legumes symbiosis and describes the CATCH-N cycle, which operates on the provision of arginine and succinate by the plant as part of a metabolic network driving symbiotic nitrogen fixation in rhizobia. The CATCH-N cycle co-catabolises plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. The CATCH-N cycle functions as an effective mechanism to promote the survival of bacteroids within infected plant cells and results in a net gain of assimilated nitrogen that subsequently amplifies the plant's arginine biosynthesis capacity. The study represents an important step towards the rational engineering of artificial nitrogen-fixing microbes. Introduction Nitrogen is a fundamental element of all living organisms and the primary nutrient that impacts crop yield (Socolow, 1999). Despite being highly abundant in the atmosphere, plants can only assimilate nitrogen in reduced forms such as ammonium. More than 125 megatons of nitrogen are fixed annually by the industrial Haber–Bosch process into ammonium and applied to increase agricultural crop production (Graham & Vance, 2000). Endosymbiosis between legumes and soil bacteria termed rhizobia is capable to fix nitrogen biologically. On a global scale, anthropogenic nitrogen delivered to the environment surpasses annual supplies by natural biological nitrogen fixation on land (Gruber & Galloway, 2008) leading to serious environmental impacts from climate change to the disruption of eco-systems and pollution of coastal waters. Improving the ability of plants and plant-associated organisms to fix atmospheric nitrogen has inspired biotechnology for decades (Beatty & Good, 2011; Bhardwaj et al, 2014; Gupta et al, 2015), not only for the apparent economic and ecological benefit that comes with the replacement of chemical fertilizers but also more recently for opportunities toward more sustainable agriculture and the potential to reduce greenhouse gas emissions. To catalyze atmospheric nitrogen fixation, rhizobia use a specific enzyme termed nitrogenase. Attempts to transfer and improve nitrogenase genes clusters have to date focused largely on organisms such as Escherichia coli (Dixon & Postgate, 1972; Wang et al, 2013). More recently, the emerging field of synthetic biology provides an alternative approach to engineer designer nitrogenase gene clusters in bacteria (Temme et al, 2012; Li et al, 2016; Burén et al, 2018; Yang et al, 2018). Despite these promising results, engineered organisms based on heterologous expression of nitrogenase genes have not yet come close to the efficiency of natural rhizobia–legume symbiosis systems (Beatty & Good, 2011; Good, 2018). While the molecular mechanism of the nitrogenase reaction has been resolved with atomistic detail (Hoffman et al, 2009, 2014; Seefeldt et al, 2009; Sippel & Einsle, 2017), the precise nature of metabolic interactions between plants and bacteria to sustain the energy-intensive process of nitrogen fixation has remained an open question. The current model of nutrient exchange in rhizobia–legume symbiosis postulates that, in exchange for fixed nitrogen, the plant provides C4-dicarboxylic acids such as succinate, which is metabolized through the tri-carboxylic acid (TCA) cycle to generate ATP and reduction equivalents needed for the nitrogenase reaction (Watson et al, 1988; Yurgel & Kahn, 2004; Clarke et al, 2014). However, multiple lines of evidence argue against a simple exchange of succinate for ammonium during symbiosis (Udvardi & Kahn, 1993; Kahn et al, 1985). The nitrogenase is highly sensitive to oxygen, which irreversibly inactivates the enzyme. While the microoxic environment encountered by rhizobia inside root nodules promotes nitrogenase activity, it also inhibits the catabolism of succinate through the TCA cycle. This is because increases in NADH and NADPH levels inhibit key enzymes of the TCA cycle including citrate synthase, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase, a process termed redox inhibition (Dunn, 1998; Prell & Poole, 2006). Thus, the TCA cycle probably operates below its full aerobic potential. Furthermore, if the metabolism of symbiotic nitrogen-fixing bacteria is based exclusively on the provision of succinate, then the bacterial nitrogen requirement must be covered solely by the nitrogenase reaction. However, nitrogen-fixing root-nodule bacteria (termed bacteroids) do not self-assimilate but rather secrete large quantities of ammonium (Bergersen & Turner, 1967; Brown & Dilworth, 1975; Udvardi & Poole, 2013) suggesting that the plant provides the bacteroids with a nitrogen-containing nutrient to cover their nitrogen needs. Finally, the degradation product of succinate through a fully operational TCA cycle is carbon dioxide. However, it has been reported that nitrogen-fixing bacteroids also secrete the amino acids alanine and aspartate (Kretovich et al, 1986; Waters et al, 1998; Allaway et al, 2000) suggesting a partially operating TCA cycle to yield alanine or aspartate. Based on metabolic considerations of inefficient TCA cycle operation under microaerobic conditions, the inability of bacteroids to self-assimilate nitrogen, and evidence for secretion of alanine or aspartate by nitrogen-fixing bacteroids, we postulated a nitrogen-containing nutrient that is plant-provided in addition to dicarboxylic acids. Since the plant must provide the N-containing compound in sufficient quantities, we reasoned that an amino acid might be a likely candidate. Based on the finding that nitrogen-fixing bacteroids utilize succinate and secrete the amino acids alanine and aspartate (Kretovich et al, 1986; Waters et al, 1998; Allaway et al, 2000; Day et al, 2001), we concluded that the plant-provided compound must comprise at least two nitrogen atoms to enable two consecutive transamination reactions. The first nitrogen is used for transamination of the ketoacid derived from succinate while the second nitrogen atom is utilized for transamination of the ketoacid derived from the plant-provided compound. Six out of the twenty natural amino acids (Arg, His, Lys, Gln, Asn, and Trp) contain two or more nitrogen atoms and thus are likely candidates. Thereof, His, Lys, and Gln can be excluded because their degradation involves a compulsory 2-oxoglutarate dehydrogenase step, which is subjected to redox inhibition and disfavored under microoxic conditions (Salminen & Streeter, 1990, 1992). Furthermore, we also excluded Trp and Asn because their catabolism enters the TCA cycle at the level of pyruvate and oxaloacetate, respectively, which limits energy metabolism within a partially operating TCA cycle. Based on these theoretical considerations, we postulated that the remaining amino acid arginine is a likely candidate for the nitrogen-containing compound provided upon symbiosis. Here, we report on the CATCH-N cycle based on the co-catabolism of plant-provided arginine and succinate as part of a specific metabolic network to sustain symbiotic nitrogen fixation as a synergistic interaction. Using 13C and 15N isotope tracing experiment in Bradyrhizobium diazoefficiens in conjunction with in planta transposon-sequencing analyses and enzymatic reaction network characterization in Sinorhizobium meliloti, we uncovered the principle of the metabolic inter-species interaction leading to the nitrogen-fixing symbiosis between plants and bacteria. Collectively, we demonstrate that the CATCH-N metabolism is governed by highly redundant functions comprised of at least 10 transporter systems and 23 enzymatic functions. In sum, our systems-level findings provide the theoretical framework and enzymatic blueprint for the optimization and redesign of improved symbiotic nitrogen-fixing organisms. Results The co-feeding of arginine and succinate stimulates nitrogenase activity To probe whether arginine functions as co-substrate to drive symbiotic nitrogen fixation, we assayed nitrogenase activity of mature bacteroids from B. diazoefficiens (strain 110 spc4) and S. meliloti (strain CL 150) in the presence of nodule crude extracts and upon supplementation of succinate and arginine (Materials and Methods). The addition of nodule crude extracts to isolated bacteroids resulted in strong stimulation of nitrogenase activity (Fig 1A), supporting the idea that plant-provided nutrients are necessary for symbiotic nitrogen fixation. While the stimulation of nitrogenase only poorly occurred in the presence of succinate as the sole nutrient, we found that the addition of arginine stimulated nitrogenase activity in B. diazoefficiens and S. meliloti by 46% ± 4% and 116% ± 2%, respectively, as compared to nodule extracts (Appendix Table S1). The co-feeding of arginine in combination with succinate restored nitrogenase activity to the same extent as nodule extracts (91% ± 6% and 92% ± 6%) for B. diazoefficiens (Fig 1A) and S. meliloti bacteroids, respectively. Furthermore, adding solely malate or co-supplementing malate and arginine inhibited or only poorly stimulated the nitrogenase activity in isolated B. diazoefficiens bacteroids (−20% ± 5%, and 9% ± 4%, Fig 1A, Appendix Table S1). Therefore, we concluded that the co-feeding of arginine and succinate is sufficient to stimulate nitrogenase activity in bacteroids. Figure 1. The co-feeding of arginine and succinate promotes nitrogen fixation and ATP production in isolated bacteroids Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represent the mean and standard error of the mean of at least eight independent replicates. Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the four individual measurements (points) together with the regression trajectory of each experiment are shown. Source data are available online for this figure. Source Data for Figure 1 [msb199419-sup-0008-SDataFig1.xlsx] Download figure Download PowerPoint The nitrogenase enzyme complex catalyzes one of the most energy-consuming enzymatic reactions found in nature with 16 ATP molecules and 8 low-potential ("high-energy") electrons required for the reduction of a single nitrogen molecule. Nitrogenase is irreversibly inactivated in the presence of oxygen, which restricts the reduction of atmospheric nitrogen to low-oxygen conditions. Thus, to support nitrogen fixation, bacteroids must produce substantial amounts of ATP under microoxic conditions. The finding that succinate as the sole nutrient did not result in nitrogenase stimulation suggested that succinate catabolism via the TCA cycle does likely not generate sufficient ATP to support efficient nitrogenase reaction. To measure the ATP level produced in isolated B. diazoefficiens bacteroids, we quantified the increase in intracellular ATP through ATP-dependent luciferase assays (Materials and Methods). In agreement with the absence of an operational TCA cycle, we observed that the addition of succinate alone failed to stimulate ATP production. In contrast, we found that co-feeding of succinate together with arginine caused an increase from 1.53 ± 0.04 to 4.03 ± 0.09 attomole ATP per cell corresponding to a 2.61 ± 0.03-fold increase in intracellular ATP levels (Fig 1B). In sum, these findings demonstrate that co-catabolism of arginine and succinate supports biological nitrogen fixation in vitro in B. diazoefficiens and S. meliloti. Isotope tracing experiments reveal the presence of three parallel arginine degradation pathways To gain further insights into the bacteroid metabolism and possible routes of arginine degradation operating during nitrogen fixation, we performed stable isotope labeling studies with B. diazoefficiens. We incubated isolated bacteroids under stringent microoxic conditions with 13C arginine in the presence of unlabeled succinate and quantified isotope labeling pattern of arginine degradation intermediates by LC-MS/MS (Table 1). Upon the addition of 13C arginine, we observed a rapid increase in the labeled intracellular arginine pool (99.43% 13C), demonstrating active arginine transport into nitrogen-fixing bacteroids. Table 1. Arginine catabolism in Bradyrhizobium diazoefficiens bacteroids fed with 13C arginine and unlabeled succinate Metabolite Fractional labeling (%)a Arginine (ARG) 99.43 ± 0.10 Citrulline (CIT) 60.22 ± 5.26 Ornithine (ORN) 81.25 ± 5.01 Proline (PRO) 23.19 ± 3.58 Glutamate (GLU) 7.67 ± 0.82 4-guanidinobutanoate (GBA) 90.40 ± 3.03 4-aminobutanoate (GABA) 6.26 ± 1.06 a 13C Fractional labeling after 150 min incubation with 13C L- arginine. Shown are the average and the standard error of the mean (SEM). Upon further incubation, we found 13C isotope labels in key intermediates of multiple arginine degradation pathways steadily increasing. After 150 min, ornithine, proline, and glutamate, the intermediates of the classical arginase-mediated degradation pathway, were labeled to 81.25%, 23.19%, and 7.67% 13C (Table 1). In addition, we found that citrulline, which represents the first step of the arginine deiminase pathway, was labeled to 60.22% 13C. The presence of an arginine deiminase pathway in isolated bacteroids is in agreement with the previously proposed enzymatic production of ATP by the enzyme carbamate kinase (Dunn, 2015). Furthermore, we also observed fractional labeling of 90.40% for 4-guanidinobutanoate and 6.26% for 4-aminobutanoate (GABA), suggesting the presence of a functional arginine transaminase pathway operating in bacteroids (Table 1) that yields alanine or aspartate. The observation that bacteroids possess an arginine transamination pathway was intriguing because it provides a functional link between arginine degradation and alanine or aspartate secretion, which was previously reported as part of the metabolite exchange occurring during symbiotic nitrogen fixation (Day et al, 2001). In agreement with this hypothesis, upon incubation of isolated bacteroids under stringent microoxic conditions with 15N arginine, we observed fractional labeling of aspartate of 90.34% (Appendix Fig S1). These findings suggest that at least three independent arginine degradation pathways operate simultaneously in nitrogen-fixing B. diazoefficiens bacteroids causing release of ammonium independent from the nitrogenase reaction. Transposon sequencing reveals symbiosis genes involved in the uptake and catabolism of arginine To gain further insights into the gene sets and enzymatic functions responsible for uptake and degradation of arginine, we conducted a functional genetic screen in planta using transposon sequencing (TnSeq; van Opijnen et al, 2009; Christen et al, 2011). TnSeq measures genome-wide changes in transposon insertion abundance prior and after subjecting large mutant populations to selection regimes (Christen et al, 2016) and allows systems-level definition of conditional essential gene sets for a given environment (Ochsner et al, 2017; Québatte et al, 2017). We reasoned that TnSeq provides a unique opportunity to identify specific metabolic pathways including arginine transport and degradation genes that become essential upon engagement in symbiosis. We choose S. meliloti-Medicago truncatula as the rhizobia–legume symbiosis system, because supernodulating M. truncatula lss plants (Schnabel et al, 2010) provided a high frequency of nodules increasing the resolution of the TnSeq analysis. In total, we infected 4,500 M. truncatula lss plants with a high-density S. meliloti transposon mutant library of 750,128 unique Tn5 insertions (Fig 2A, Dataset EV1, Materials and Methods). Six weeks post-inoculation, we recovered 99,623 unique Tn5 mutants from 375,000 root nodules (Dataset EV2). By comparing the TnSeq dataset obtained from in planta infection assays and input transposon mutant libraries, we mapped a set of 977 symbiosis genes corresponding to 15.71% of the tripartite 6.7-megabase (Mb) genome (Dataset EV3, Materials and Methods). A gene is classed as a symbiosis gene when the fractional representation in the library recovered from nodules is significantly less that the fractional representation in the original library (Dataset EV3, Materials and Methods), implying that strains carrying the mutation do not prosper in nodules. Among the identified 977 symbiosis genes, 435 genes were located on the chromosome, 295 on pSymA, and 247 on pSymB indicating that all three replicons of S. meliloti contribute to symbiotic nitrogen fixation (Fig 2B). Figure 2. The symbiosis genome of S. meliloti revealed by transposon sequencing (TnSeq) Schematic representation of the plant infection screen that was used to map the S. meliloti symbiosis genome. Tn5 transposon mutant pools were selected for their ability to establish symbiosis with M. truncatula. After selection, Tn5 mutants recovered from root nodules were identified by TnSeq. Genome map visualizing the distribution of essential symbiosis genes among the three S. meliloti replicons. Symbiosis genes are plotted as lines on the chromosome (gray) and the mega-plasmids pSymA (blue) and pSymB (green). Functional classification of essential symbiosis genes located on the chromosome (gray), pSymA (blue), and pSymB (green). Source data are available online for this figure. Source Data for Figure 2 [msb199419-sup-0009-SDataFig2.xlsx] Download figure Download PowerPoint Functional classification revealed that the large majority of symbiosis genes comprise cellular functions such as metabolism (507 genes, 51.89%), gene regulation (196 genes, 20.06%), and other cellular processes (228 genes, 23.34%; Fig 2C, Dataset EV4, Materials and Methods). The identified gene set included well-characterized symbiosis factors involved in nodulation (34 genes, 3.48%) as well as functions associated with the nitrogenase enzyme complex (12 genes, 1.23%). Collectively, a set of 177 symbiosis genes, corresponding to over one-third of the 507 metabolic symbiosis genes (34.91%), was associated with nitrogen metabolism including genes for the transport (59 genes), biosynthesis (78 genes), and degradation (40 genes) of amino acids and other nitrogen-containing compounds. While only 3 out of 78 essential biosynthesis genes (3.85%) were involved in the synthesis of arginine, we found a large fraction of 18 out of 59 essential transport genes (30.51%) and 22 out of 40 essential catabolic genes (55.00%) annotated as being involved in the uptake and catabolism of arginine and its derivatives. In sum, these findings from in planta TnSeq analysis highlight that the provision of arginine and its consecutive degradation is of fundamental importance to drive symbiotic nitrogen fixation in planta. TnSeq identifies multiple arginine transport systems mediating acid tolerance Among the 18 transport genes essential for symbiosis, we found two putative arginine and four putrescine ABC transport systems that we named artABCDE (SMc03124-28) for arginine transporter; satABC (SMa2195-97) for symbiotic arginine transporter; and potFGHI (SMc00770-3), potABCD2 (SMa0799-803), potABCD3 (SMa0951-3), and potABCD4 (SMa2203-9) for the putrescine uptake systems (Dataset EV3). In addition, two arginine/agmatine antiporter genes adiC (SMa0684) and adiC2 (SMa1668) encoded on pSymA were also essential during symbiosis (Dataset EV3). Interestingly, the identified transport systems participate in urease reactions and arginine deiminase pathways that mediate acid tolerance. Cross-comparing expression profiles using previously published RNA-seq datasets (Roux et al, 2014), we found that artABCDE was the only transport system to be constitutively expressed during all stages of symbiosis, while the expression of all other transporters was specifically induced during development into nitrogen-fixing compartments (symbiosomes). To gain further insights, we searched for additional symbiosis genes related to acid tolerance and indeed found multiple essential components in the TnSeq dataset (Dataset EV3). From the urease pathway, we identified two arginase genes argI1 (SMc03091) and argI2 (SMa1711) and the urease gene ureA (SMc01941) and ureE (SMc01832). From the arginine deiminase system, we found the arcABC operon (SMa0693, SMa0695, SMa0697) to be essential for symbiosis. Both systems catalyze the conversion of arginine into ornithine leading to the production of ammonia as part of the acid tolerance mechanism. Furthermore, the arginine deiminase system also provides ATP via the enzymatic step of ornithine carbamoyltransferase arcB (Cunin et al, 1986). Interestingly, two additional copies of ornithine carbamoyltransferase were also essential (argF1, encoded by SMc02137, and arcB2, encoded by SM_b20472), emphasizing the importance of genetic redundancy in arginine deiminase-dependent ATP synthesis during symbiosis. The urease and arginine deiminase acid tolerance mechanisms rely on the efflux of ammonium (Marquis et al, 1987). Indeed, the ammonium efflux pump encoded by amtB (SMc03807) was among the top-ranked symbiosis genes. These findings underscore the importance of ammonium secretion as a compulsive property of bacteroids independent of the nitrogenase reaction. Arginase gene deletions show nitrogen starvation phenotypes during plant infection assays To validate the importance of the identified arginine-dependent acid tolerance systems for symbiosis, we constructed a panel of deletion mutants of the urease and arginine deiminase pathways and assessed nitrogen starvation phenotypes during plant infection assays (Materials and Methods, Fig 3D, Appendix Table S2). Out of the 8 mutants evaluated, all displayed symbiotic defects. On the level of the arginine transport systems, we found that artABCDE and satABC showed a reduction in nitrogenase activity of 47.06% ± 7.27% and 55.45% ± 10.99%. Similarly, gene deletions in the urease pathway such as the arginase mutants argI1 and argI2 exhibited a reduction of 71.18% ± 5.21% and 70.97 ± 5.16% for single deletions and 80.89% ± 3.15% for the double deletion mutant. Deletion of the urease ureGFE and the ammonium efflux system amtB resulted in a 64.68% ± 5.50% and 80.90% ± 4.81% reduction in nitrogenase activity. Figure 3. Assessment of nitrogen starvation phenotypes of M. truncatula upon infection with S. meliloti mutants impaired in arginine transport and catabolism The aerial part of M. truncatula upon infection with S. meliloti ∆argI1, ∆argI2, ∆argI1,2 and the dicarboxylate transport mutant ∆dctAB were smaller than those inoculated with the wild-type strain, highlighting the importance of arginine catabolism for nitrogen fixation. Cross sections of nodules bearing S. meliloti WT or arginine catabolism mutant ∆argI1, ∆argI2 reveals the yellowish color of non-functional nodules induced by the ∆argI1 ∆argI2 double deletion mutant. Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆argI1 ∆argI2 double deletion mutant defective in arginine catabolism. Nitrogenase activity in M. truncatula nodules inoculated with S. meliloti strains defective in arginine catabolism. Data points are the mean of at least 30 plants measured after 8 weeks post-inoculation; error bars indicate standard error of the mean. Source data are available online for this figure. Source Data for Figure 3 [msb199419-sup-0010-SDataFig3.xlsx] Download figure Download PowerPoint Plants inoculated with the argI1, argI2 single and double deletion mutant harbored a typical phenotype of nitrogen starvation. The aerial part of infected plants was smaller than those inoculated with WT strain (Figs 3A and EV1). Nodules induced by the argI1, argI2 double deletion mutant displayed the yellowish color of non-functional M. truncatula nodules (Fig 3B). Furthermore, observations of argI1, argI2 nodule sections by scanning electron
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