An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

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.

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.

Rhizobioaugmentation of Casuarina glauca with N-fixing actinobacteria Frankia decreases enzymatic activities in wastewater irrigated soil: effects of Frankia on C. glauca growth.

The use of wastewater for irrigation in agroforestry is cost-effective for water management. It is well established that rhizospheric microorganisms such as N2-fixing bacteria are able to modulate rhizobioaugmention and to boost phyoremediation process. To date, no study has been conducted to evaluate biological effects of rhizobioaugmentation in Casuarina glauca trees induced by their symbiont N-fixing actinobacteria of the genus Frankia. The objective of the present study was to evaluate the main effects of rhizobioaugmentation on the biological activity in the C. glauca's rhizosphere and on C. glauca growth in soils irrigated with industrial wastewater. Two Frankia strains (BMG5.22 and BMG5.23) were used in a single or dual inoculations of C. glauca seedlings irrigated with industrial wastewater. Soil enzymes activity related to carbon, phosphorus, sulfur and nitrogen cycling were measured. Results revealed that the BMG5.22 Frankia strain increases significantly the size (dry weight) of C. glauca shoots and roots while dual inoculation increased significantly the root length. Surprisingly, ß-glucosidase (BG), cellobiohydrolase (CBH), ß-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER) activity in the rhizosphere decreased significantly in soils treated with the two strains of symbionts. This suggests no positive correlations between enzymatic activity and C. glauca growth.

Establishment of Actinorhizal Symbiosis in Response to Ethylene, Salicylic Acid, and Jasmonate.

Phytohormones play a crucial role in regulating plant developmental processes. Among them, ethylene and jasmonate are known to be involved in plant defense responses to a wide range of biotic stresses as their levels increase with pathogen infection. In addition, these two phytohormones have been shown to inhibit plant nodulation in legumes. Here, exogenous salicylic acid (SA), jasmonate acid (JA), and ethephon (ET) were applied to the root system of Casuarina glauca plants before Frankia inoculation, in order to analyze their effects on the establishment of actinorhizal symbiosis. This protocol further describes how to identify putative ortholog genes involved in ethylene and jasmonate biosynthesis and/or signaling pathways in plant, using the Arabidopsis Information Resource (TAIR), Legume Information System (LIS), and Genevestigator databases. The expression of these genes in response to the bacterium Frankia was analyzed using the gene atlas for Casuarina-Frankia symbiosis (SESAM web site).

Comparative Proteomic Analysis of Nodulated and Non-Nodulated Casuarina glauca Sieb. ex Spreng. Grown under Salinity Conditions Using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS).

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein-protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis.

Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster: They Come in Teams.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Stable Transformation of the Actinobacteria Frankia spp.

A stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genus Frankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into different Frankia strains by a filter mating with Escherichia coli strain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced into Frankia casuarinae strain CcI3 at a frequency of 4.0 × 10-3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced into F. casuarinae strain CcI3 and other Frankia strains at frequencies ranging from 10-2 to 10-4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerant Frankia sp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitive F. casuarinae strain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed in F. casuarinae strain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression in Frankia spp., overcoming a major hurdle in the field. This step in the development of genetic tools in Frankia spp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCE The absence of genetic tools for Frankia research has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids into Frankia spp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.

Comparative genomics and proteogenomics highlight key molecular players involved in Frankia sporulation.

Sporulation is a microbial adaptive strategy to resist inhospitable conditions for vegetative growth and to disperse to colonise more favourable environments. This microbial trait is widespread in Actinobacteria. Among them, Frankia strains are able to differentiate sporangia in pure culture, while others can sporulate even when in symbiosis with sporulation occurring within host cells. The molecular determinants controlling Frankia sporulation have not been yet described. In order to highlight, for the first time, the molecular players potentially involved in Frankia sporulation, we conducted (i) a comparison of protein contents between Frankia spores and hyphae and (ii) a comparative genomic analysis of Frankia proteomes with sporulating and non-sporulating Actinobacteria. Among the main results, glycogen-metabolism related proteins, as well as oxidative stress response and protease-like proteins were overdetected in hyphae, recalling lytic processes that allow Streptomyces cells to erect sporogenic hyphae. Several genes encoding transcriptional regulators, including GntR-like, appeared up-regulated in spores, as well as tyrosinase, suggesting their potential role in mature spore metabolism. Finally, our results highlighted new proteins potentially involved in Frankia sporulation, including a pyrophosphate-energized proton pump and YaaT, described as involved in the phosphorelay allowing sporulation in Bacillus subtilis, leading us to discuss the role of a phosphorelay in Frankia sporulation.

Metallophore profiling of nitrogen-fixing Frankia spp. to understand metal management in the rhizosphere of actinorhizal plants.

Frankia spp. are widespread nitrogen-fixing soil bacteria, which often live in symbiosis with a broad range of hosts. Metal homeostasis plays a crucial role in the success of the symbiosis regarding the acquisition of essential trace metals and detoxification of potentially toxic elements. We have hypothesised that Frankia releases many organic ligands with a broad spectrum of affinity for essential and toxic metals. We coined the term 'ligandosphere' to describe the entirety of excreted metal complexing agents and ligands derived from the dissolved organic matter. Using metal isotope-coded profiling (MICP); metallophores of physiological important and toxic trace metals were identified by the addition of stable metal isotope pairs such as 54Fe/58Fe, 63Cu/65Cu, 66Zn/68Zn or 95Mo/98Mo. Liquid chromatography coupled to a mass spectrometer revealed strong variations of the metallophore profile in between the 14 test-strains. In total, about 83 organic ligands were identified as binding to one of the tested metals. The predicted sum formula of the major Fe binding ligands and MS/MS experiments suggested that several metallophore candidates have a similar molecular backbone. Growth experiments with a hyper-producer of metallophores revealed a positive relationship between metallophore production and the concentration of Cu in the growth medium. The present study provides the first comprehensive overview of the complexity of Frankia's ligandosphere. It opens a path to a deeper understanding of mechanisms that regulate metal homeostasis in frankiae. Deciphering these mechanisms is important since the fitness of actinorhizal plants and their potential in ecological restoration relies heavily on their symbiosis with frankiae.

Actinorhizal trees and shrubs from Africa: distribution, conservation and uses.

Actinorhizal plants are a group of perennial dicotyledonous angiosperms, comprised of more than 200 species, most of which can establish root-nodule symbiosis with the nitrogen fixing actinobacteria of the genus Frankia. They are key providers of fundamental goods and services and can give a major contribution to mitigate the combined effects of climate changes, human population growth and loss of biodiversity. This aspect is particularly relevant for the developing economies of many African countries, which are highly exposed to climate and anthropogenic disturbances. In this work we have analyzed the distribution, conservation and uses of actinorhizal species native to or introduced in Africa. A total of 42 taxa distributed over six botanical families (Betulaceae, Casuarinaceae, Myricaceae, Elaeagnaceae, Rhamnaceae and Coriariaceae) were identified. The vast majority is able to thrive under a range of diverse environments and has multiple ecological and economic potential. More than half of the identified species belong to the genus Morella (Myricaceae), most of them native to Middle, Eastern and Southern Africa. Although the information about the conservation status and uses of Morella spp. is largely incomplete, the available data is indicative of their potential in e.g. forestry and agroforestry, food and medicine. Therefore, efforts should be made to upgrade actinorhizal research in Africa towards the sustainable use of biodiversity at the service of local (bio)economies.

An update on the taxonomy of the genus Frankia Brunchorst, 1886, 174AL.

Since the recognition of the name Frankia in the Approved Lists of bacterial names (1980), few amendments have been given to the genus description. Successive editions of Bergey's Manual of Systematics of Archaea and Bacteria have broadly conflicting suprageneric treatments of the genus without any advances for subgeneric classification. This review focuses on recent results from taxongenomics and phenoarray approaches to the positioning and the structuring of the genus Frankia. Based on phylogenomic analyses, Frankia should be considered the single member of the family Frankiaceae within the monophyletic order, Frankiales. A polyphasic strategy incorporating genome to genome data and omniLog® phenoarrays, together with classical approaches, has allowed the designation and an amended description of a type strain of the type species Frankia alni, and the recognition of at least 10 novel species covering symbiotic and non symbiotic taxa within the genus. Genome to phenome data will be shortly incorporated in the scheme for proposing novel species including those recalcitrant to isolation in axenic culture.

19th International Meeting on Frankia and Actinorhizal Plants.

It has been 40 years since the first meeting dedicated to Frankia and actinorhizal plants, which was held at Petersham, Massachusetts (reported in Torrey and Tjepkema, 1979). Since then biennial meetings have been organised and held in different venues around the globe (Table 1). The most recent meeting, the "19th International Meeting on Frankia and Actinorhizal Plants", organised in Hammamet, Tunisia from 17th to 19th of March, 2018, gathered scientists from Algeria, Argentina, Belgium, China, Egypt, France, India, Portugal, Senegal, Sweden, UK, USA and Tunisia. The event was a stimulating opportunity for active researchers to share many advances since the previous meeting held in Montpellier, France (Franche et al. 2016) and to discuss new perspectives in this research field.

The role of Frankia inoculation in casuarina plantations in China.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

Draft genome sequence of the symbiotic Frankia sp. strain BMG5.30 isolated from root nodules of Coriaria myrtifolia in Tunisia.

Frankia sp. strain BMG5.30 was isolated from root nodules of a Coriaria myrtifolia seedling on soil collected in Tunisia and represents the second cluster 2 isolate. Frankia sp. strain BMG5.30 was able to re-infect C. myrtifolia generating root nodules. Here, we report its 5.8-Mbp draft genome sequence with a G + C content of 70.03% and 4509 candidate protein-encoding genes.

In Planta Sporulation of Frankia spp. as a Determinant of Alder-Symbiont Interactions.

The Alnus genus forms symbiosis with the actinobacteria Frankia spp. and ectomycorrhizal fungi. Two types of Frankia lineages can be distinguished based on their ability to sporulate in planta Spore-positive (Sp+) strains are predominant on Alnus incana and Alnus viridis in highlands, while spore-negative (Sp-) strains are mainly associated with Alnus glutinosa in lowlands. Here, we investigated whether the Sp+ predominance in nodules is due to host selection of certain Frankia genotypes from soil communities or the result of the ecological history of the alder stand soil, as well as the effect of the sporulation genotype on the ectomycorrhizal (ECM) communities. Trapping experiments were conducted using A. glutinosa, A. incana, and A. viridis plantlets on 6 soils, differing in the alder species and the frequency of Sp+ nodules in the field. Higher diversity of Frankia spp. and variation in Sp+ frequencies were observed in the trapping than in the fields. Both indigenous and trapping species shape Frankia community structure in trapped nodules. Nodulation impediments were observed under several trapping conditions in Sp+ soils, supporting a narrower host range of Sp+ Frankia species. A. incana and A. viridis were able to associate equally with compatible Sp+ and Sp- strains in the greenhouse. Additionally, no host shift was observed for Alnus-specific ECM, and the sporulation genotype of Frankia spp. defined the ECM communities on the host roots. The symbiotic association is likely determined by the host range, the soil history, and the type of in plantaFrankia species. These results provide an insight into the biogeographical drivers of alder symbionts in the Holarctic region.IMPORTANCE Most Frankia-actinorhiza plant symbioses are capable of high rates of nitrogen fixation comparable to those found on legumes. Yet, our understanding of the ecology and distribution of Frankia spp. is still very limited. Several studies have focused on the distribution patterns of Frankia spp., demonstrating a combination of host and pedoclimatic parameters in their biogeography. However, very few have considered the in planta sporulation form of the strain, although it is a unique feature among all symbiotic plant-associated microbes. Compared with Sp- Frankia strains, Sp+ strains would be obligate symbionts that are highly dependent on the presence of a compatible host species and with lower efficiency in nitrogen fixation. Understanding the biogeographical drivers of Sp+ Frankia strains might help elucidate the ecological role of in planta sporulation and the extent to which this trait mediates host-partner interactions in the alder-Frankia-ECM fungal symbiosis.

Patterns of diversity, endemism and specialization in the root symbiont communities of alder species on the island of Corsica.

We investigated whether the diversity, endemicity and specificity of alder symbionts could be changed by isolation in a Mediterranean glacial refugium. We studied both ectomycorrhizal (EM) fungi and nitrogen-fixing actinobacteria associated with alders, and compared their communities in Corsica and on the European continent. Nodules and root tips were sampled on the three alder species present in Corsica and continental France and Italy. Phylogenies based on internal transcribed spacer (ITS) and a multilocus sequence analysis approach were used to characterize fungal and Frankia species, respectively. Patterns of diversity, endemism and specialization were compared between hosts and regions for each symbiont community. In Corsica, communities were not generally richer than on the mainland. The species richness per site depended mainly on host identity: Alnus glutinosa and Alnus cordata hosted richer Frankia and EM communities, respectively. Half of the Frankia species were endemic to Corsica against only 4% of EM species. Corsica is not a hotspot of diversity for all alder symbionts but sustains an increased frequency of poor-dispersers such as hypogeous fungi. Generalist EM fungi and host-dependent profusely sporulating (Sp+) Frankia were abundantly associated with Corsican A. cordata, a pattern related to a more thermophilic and xerophylic climate and to the co-occurrence with other host trees.

Molecular response to nitrogen starvation by Frankia alni ACN14a revealed by transcriptomics and functional analysis with a fosmid library in Escherichia coli.

The transcriptome of Frankia alni strain ACN14a was compared between in vitro ammonium-replete (N-replete) and ammonium-free dinitrogen-fixing (N-fixing) conditions using DNA arrays. A Welch-test (p < 0.05) revealed significant upregulation of 252 genes under N-fixing vs. N-replete (fold-change (FC) ≥ 2), as well as significant downregulation of 48 other genes (FC ≤ 0.5). Interestingly, there were 104 Frankia genes upregulated in vitro that were also significantly upregulated in symbiosis with Alnus glutinosa, while the other 148 genes were not, showing that the physiology of in vitro fixation is markedly different from that under symbiotic conditions. In particular,in vitro fixing cells were seen to upregulate genes identified as coding for a nitrite reductase, and amidases that were not upregulated in symbiosis. Confirmatory assays for nitrite reductase showed that Frankia indeed reduced nitrite and used it as a nitrogen source. An Escherichia coli fosmid clone carrying the nirB region was able to grow better in the presence of 5 mM nitrite than without it, confirming the function of the genome region. The physiological pattern that emerges shows that Frankia undergoes nitrogen starvation that induces a molecular response different from that seen in symbiosis.

The PEG-responding desiccome of the alder microsymbiont Frankia alni.

Actinorhizal plants are ecologically and economically important. Symbiosis with nitrogen-fixing bacteria allows these woody dicotyledonous plants to colonise soils under nitrogen deficiency, water-stress or other extreme conditions. However, proteins involved in xerotolerance of symbiotic microorganisms have yet to be identified. Here we characterise the polyethylene glycol (PEG)-responding desiccome from the most geographically widespread Gram-positive nitrogen-fixing plant symbiont, Frankia alni, by next-generation proteomics, taking advantage of a Q-Exactive HF tandem mass spectrometer equipped with an ultra-high-field Orbitrap analyser. A total of 2,052 proteins were detected and quantified. Under osmotic stress, PEG-grown F. alni cells increased the abundance of envelope-associated proteins like ABC transporters, mechano-sensitive ion channels and Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated (cas) components. Conjointly, dispensable pathways, like nitrogen fixation, aerobic respiration and homologous recombination, were markedly down-regulated. Molecular modelling and docking simulations suggested that the PEG is acting on Frankia partly by filling the inner part of an up-regulated osmotic-stress large conductance mechanosensitive channel.

Frankia Diversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity of Frankia Populations in Corresponding Rhizosphere Soils.

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia, and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa, Alnus cordata, Shepherdia argentea, Casuarina equisetifolia) while others were not (Alnus viridis, Hippophaë rhamnoides). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia, while another was detected in nodules from A. glutinosa Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia-specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils.IMPORTANCE The nitrogen-fixing actinobacterium Frankia forms root nodules on actinorhizal plants, with members of specific Frankia taxonomic clusters nodulating plants in corresponding host infection groups. We assessed Frankia diversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.