Effect of gamma irradiation on the proteogenome of cold-acclimated Kocuria rhizophila PT10.

The effects of ionizing radiation (IR) on the protein dynamics of cold-stressed cells of a radioresistant actinobacterium, Kocuria rhizophila PT10, isolated from the rhizosphere of the desert plant Panicum turgidum were investigated using a shotgun methodology based on nanoflow liquid chromatography coupled to tandem mass spectrometry. Overall, 1487 proteins were certified, and their abundances were compared between the irradiated condition and control. IR of cold-acclimated PT10 triggered the over-abundance of proteins involved in (1) a strong transcriptional regulation, (2) amidation of peptidoglycan and preservation of cell envelope integrity, (3) detoxification of reactive electrophiles and regulation of the redox status of proteins, (4) base excision repair and prevention of mutagenesis and (5) the tricarboxylic acid (TCA) cycle and production of fatty acids. Also, one of the more significant findings to emerge from this study is the SOS response of stressed PT10. Moreover, a comparison of top hits radio-modulated proteins of cold-acclimated PT10 with proteomics data from gamma-irradiated Deinococcus deserti showed that stressed PT10 has a specific response characterised by a high over-abundance of NemA, GatD, and UdgB.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall.

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24.

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF.

Frankia umida sp. nov., isolated from root nodules of Alnus glutinosa L.

Frankia strain Ag45/Mut15T was isolated from a root nodule of Alnus glutinosa growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on A. glutinosa, in which it produces hyphae and clusters of N2-fixing vesicles. N2-fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15T contained meso-diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H6) and MK-9(H4). The major fatty acid profile (>10 %) consisted of iso-C16:0, C17 : 1 ω8c and C17 : 0. Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15T was most closely related to Frankia torreyi CpI1T and Candidatus Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on atpD, dnaA, ftsZ, pgk and rpoB amino acid sequences positioned the strain within cluster 1 of Alnus- and Myrica-nodulating species, close to Candidatus F. nodulisporulans AgTrST and F. canadensis ARgP5T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15T and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation. Candidatus F. nodulisporulans AgTrST, which cannot be cultivated in vitro, was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15T with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15T was not able to sporulate in nodule tissues like strain AgTrST.Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15T (=DSM 114737T=LMG 326O1T) to a novel species, with Ag45/Mut15T as type strain, for which the name Frankia umida sp. nov. is proposed.

Frankia alni Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Draft Genomes of Frankia strains AiPa1 and AiPs1 Retrieved from Soil with Monocultures of Picea abies or Pinus sylvestris using Alnus incana as Capture Plant.

The genomes of two nitrogen-fixing Frankia strains, AiPa1 and AiPs1, are described as representatives of two novel candidate species. Both strains were isolated from root nodules of Alnus incana, used as capture plants in bioassays on soils from a reforested site at Karttula, Finland, that was devoid of actinorhizal plants but contained 25 year-old monocultures of spruce (Picea abies (L.) Karsten) or pine (Pinus sylvestris L.), respectively. ANI analyses indicate that each strain represents a novel Frankia species, with genome sizes of 6.98 and 7.35 Mb for AiPa1 and AiPs1, respectively. Both genomes harbored genes typical for many other symbiotic frankiae, including genes essential for nitrogen-fixation, for synthesis of hopanoid lipids and iron-sulfur clusters, as well as clusters of orthologous genes, secondary metabolite determinants and transcriptional regulators. Genomes of AiPa1 and AiPs1 had lost 475 and 112 genes, respectively, compared to those of other cultivated Alnus-infective strains with large genomes. Lost genes included one hup cluster in AiPa1 and the gvp cluster in AiPs1, suggesting that some genome erosion has started to occur in a different manner in the two strains.

Correction: Pujic et al. The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules. Microorganisms 2022, 10, 651.

The authors wish to make the following corrections to this paper [...].

Draft Genome of Frankia sp. Strain R82, an Isolate from Root Nodules of Myrica gale.

The Frankia sp. strain R82 genome is described as representative of a novel candidate species within Frankia cluster 1, as indicated by average nucleotide identity (ANI) analyses, with its closest relatives being Frankia nodulisporulans AgTrs and strains Ag45/Mut15 and AgPM24 (86% identity).

A Nonspecific Lipid Transfer Protein with Potential Functions in Infection and Nodulation.

The response of Alnus glutinosa to Frankia alni ACN14a is driven by several sequential physiological events from calcium spiking and root-hair deformation to the development of the nodule. Early stages of actinorhizal symbiosis were monitored at the transcriptional level to observe plant host responses to Frankia alni. Forty-two genes were significantly upregulated in inoculated compared with noninoculated roots. Most of these genes encode proteins involved in biological processes induced during microbial infection, such as oxidative stress or response to stimuli, but a large number of them are not differentially modulated or downregulated later in the process of nodulation. In contrast, several of them remained upregulated in mature nodules, and this included the gene most upregulated, which encodes a nonspecific lipid transfer protein (nsLTP). Classified as an antimicrobial peptide, this nsLTP was immunolocalized on the deformed root-hair surfaces that are points of contact for Frankia spp. during infection. Later in nodules, it binds to the surface of F. alni ACN14a vesicles, which are the specialized cells for nitrogen fixation. This nsLTP, named AgLTP24, was biologically produced in a heterologous host and purified for assay on F. alni ACN14a to identify physiological effects. Thus, the activation of the plant immunity response occurs upon first contact, while the recognition of F. alni ACN14a genes switches off part of the defense system during nodulation. AgLTP24 constitutes a part of the defense system that is maintained all along the symbiosis, with potential functions such as the formation of infection threads or nodule primordia to the control of F. alni proliferation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Draft Genomes of Symbiotic Frankia Strains AgB32 and AgKG'84/4 from Root Nodules of Alnus Glutinosa growing under Contrasted Environmental Conditions.

The genomes of two nitrogen-fixing Frankia strains, AgB32 and AgKG'84/4, were isolated from spore-containing (spore+) and spore-free (spore-) root nodules of Alnus glutinosa, but they did not sporulate upon reinfection. The two strains are described as representatives of two novel candidate species. Phylogenomic and ANI analyses indicate that each strain represents a novel species within cluster 1, with genome sizes of 6.3 and 6.7 Mb smaller than or similar to those of other cultivated Alnus-infective cluster 1 strains. Genes essential for nitrogen-fixation, clusters of orthologous genes, secondary metabolite clusters and transcriptional regulators analyzed by comparative genomic analyses were typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. Compared to other cultivated Alnus-infective strains with large genomes, those of AgB32 and AgKG'84/4 had lost 380 or 409 genes, among which one hup cluster, one shc gene and the gvp cluster, which indicates genome erosion is taking place in these two strains.

Pseudomonas ST1 and Pantoea Paga Strains Cohabit in Olive Knots.

Two bacteria belonging to the Pseudomonas and Pantoea genera were isolated from olive knots. Both bacterial strains were omnipresent in this study's olive orchard with high susceptibility of the autochthonous olive genotypes indicating coevolution of bacteria with host plants. Genomes of two endemic bacteria show conserved core genomes and genome plasticity. The Pseudomonas ST1 genome has conserved virulence-related genes including genes for quorum sensing, pilus, and flagella biosynthesis, two copies of indole acetic acid biosynthesis (IAA) operons, type I-VI secretions systems, and genes for alginate and levan biosynthesis. Development of knots depends only on the presence of the Pseudomonas ST1 strain which then allows Pantoea paga strain co-infection and cohabitation in developed knots. The two bacteria are sensitive to a large number of antimicrobials, antibiotics, H2O2, and Cu (II) salts that can be efficiently used in propagation of bacterial free olive cultivars.

Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa.

The genomes of two nitrogen-fixing Frankia strains, Ag45/Mut15 and AgPM24, isolated from root nodules of Alnus glutinosa are described as representatives of a novel candidate species. Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated Alnus-infective cluster 1 strains but larger than that of the non-cultivated Alnus-infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated Alnus-infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe-Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Roots of the xerophyte Panicum turgidum host a cohort of ionizing-radiation-resistant biotechnologically-valuable bacteria.

Bacterial communities associated with roots of Panicum turgidum, exposed to arid conditions, were investigated with a combination of cultural and metataxonomic approaches. Traditional culture-based techniques were used and 32 isolates from the irradiated roots were identified as belonging to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria phyla. Four actinobacterial strains were shown to be ionizing-radiation (IR)-resistant: Microbacterium sp. PT8 (4.8 kGy (kGy)), Micrococcus sp. PT11 (4.4 kGy), Kocuria rhizophila PT10 (2.9 kGy) and Promicromonospora panici PT9T (2.6 kGy), based on the D10 dose necessary for a 90% reduction in colony forming units (CFU). Concerning the investigation of microbial communities in situ, metataxonomic analyses of the diversity of IR-resistant microorganisms associated with irradiated roots revealed a marked dominance of Actinobacteria (46.6%) and Proteobacteria (31.5%) compared to Bacteroidetes (4.6%) and Firmicutes (3.2%). Gamma irradiation not only changed the structure of bacterial communities, but also affected their functional properties. Comparative analyses of metabolic profiles indicated the induction of several pathways related to adaptation to oxidative stress in irradiated roots, such as DNA repair, secondary metabolites synthesis, reactive oxygen species (ROS)-mitigating enzymes, etc. P. turgidum is emblematic of desert-adapted plants. Until now, there is no other work that has focused on the microbial profile of irradiated roots of this xerophyte.

Draft genome sequence of Promicromonospora panici sp. nov., a novel ionizing-radiation-resistant actinobacterium isolated from roots of the desert plant Panicum turgidum.

A novel strain of the genus Promicromonospora, designated PT9T, was recovered from irradiated roots of the xerophyte Panicum turgidum collected from the Ksar Ghilane oasis in southern Tunisia. Strain PT9T is aerobic, non-spore-forming, Gram- positive actinomycete that produces branched hyphae and forms white to yellowish-white colonies. Chemotaxonomic features, including fatty acids, whole cell sugars and polar lipid profiles, support the assignment of PT9T to the genus Promicromonospora. The genomic relatedness indexes based on DNA-DNA hybridization and average nucleotide identity values revealed a significant genomic divergence between strain PT9T and all sequenced type strains of the taxon. Phylogenomic analysis showed that isolate PT9T was most closely related to Promicromonospora soli CGMCC 4.7398T. Phenotypic and phylogenomic analyses suggest that isolate PT9T represents a novel species of the genus Promicromonospora, for which the name Promicromonospora panici sp. nov. is proposed. The type strain is PT9T (LMG 31103T = DSM 108613T).The isolate PT9T is an ionizing-radiation-resistant actinobacterium (D10 value = 2.6 kGy), with resistance to desiccation and hydrogen peroxide. The complete genome sequence of PT9T consists of 6,582,650 bps with 71.2% G+C content and 6291 protein-coding sequences. This genome will help to decipher the microbial genetic bases for ionizing-radiation resistance mechanisms including the response to oxidative stress.

Ionizing-radiation-resistant Kocuria rhizophila PT10 isolated from the Tunisian Sahara xerophyte Panicum turgidum: Polyphasic characterization and proteogenomic arsenal.

A new strain belonging to the genus Kocuria, designed PT10, was isolated from irradiated roots of the xerophyte Panicum turgidum. Isolate PT10 is a Gram-positive, coccoid, aerobic and ionizing-radiation (IR)-resistant actinobacterium. PT10 has shown an ability to survive under extreme conditions, such as gamma irradiation, desiccation and high concentration of hydrogen peroxide. Phenotypic, chemotaxonomic and comparative genome analyses support the assignment of strain PT10 (LMG 31102 = DSM 108617) as Kocuria rhizophila. The complete genome sequence of PT10 consists of one chromosome (2,656,287 bps), with a 70.7% G + C content and comprises 2481 protein-coding sequences. A total of 1487 proteins were identified by LC-MS/MS profiling. In silico analyses revealed that the proteome of the oxidation-tolerant PT10 possesses several features explaining its IR-resistant phenotype and many adaptive pathways implicated in response to environmental pressures - desiccation, cold, reactive oxygen species and other stressors.

Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta.

We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alnobetula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5Mb instead of 7.5Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences reported in this study for AgTrS, AgUmASt1 and AgUmASH1 are MT023539/LR778176/LR778180 and NZ_CADCWS000000000.1/CADDZU010000001/CADDZW010000001, respectively.

Proposal of 'Candidatus Frankia alpina', the uncultured symbiont of Alnus alnobetula and A. incana that forms spore-containing nitrogen-fixing root nodules.

The members of the genus Frankia are, with a few exceptions, a group of nitrogen-fixing symbiotic actinobacteria that nodulate mostly woody dicotyledonous plants belonging to three orders, eight families and 23 genera of pioneer dicots. These bacteria have been characterized phylogenetically and grouped into four molecular clusters. One of the clusters, cluster 1 contains strains that induce nodules on Alnus spp. (Betulaceae), Myrica spp., Morella spp. and Comptonia spp. (Myricaceae) that have global distributions. Some of these strains produce not only hyphae and vesicles, as other cluster 1 strains do, but also numerous sporangia in their host symbiotic tissues, hence their phenotype being described as spore-positive (Sp+). While Sp+ strains have resisted repeated attempts at cultivation, their genomes have recently been characterized and found to be different from those of all described species, being markedly smaller than their phylogenetic neighbours. We thus hereby propose to create a 'Candidatus Frankia alpina' species for some strains present in nodules of Alnus alnobetula and A. incana that grow in alpine environments at high altitudes or in subarctic environments at high latitudes.

Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules.

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing "ecometabolome" approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.