Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI3 produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI3 produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI3 had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI3 were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection.

Mongolia's fertilizer industry produces 2,000 tons of fertilizer in 10 years, while imports amount to 6-8 tons a year. One of the main biological features of the root system of sea buckthorn is the ability to absorb nitrogen from the air with the help of its root nodules. The main goal of the research is to identify the native nitrogen-absorbing bacteria of sea buckthorn and to increase their activity by preparing bacterial fertilizers and determining their effects on plants. In our study, we obtained 2 types of nitrogen-fixing bacteria using common microbiological methods. Sea buckthorn mongolica subspecies was used in this study. The study of root nodules of sea buckthorn (Hippophae rhamnoides L.) aims to identify symbiotic nitrogen-fixing bacteria. Sea buckthorn root nodules contain Frankia actinorrhizal microorganisms. Sea buckthorn seedlings, including those fertilized with nitrogen-fixing bacterial cultures, showed a positive correlation with plant growth. From June to September, when air temperatures are high and photosynthesis active in the field, the number of bacterial cells and nitrogenase activity were found to elevate in the root nodules of sea buckthorn plants. During this period, the roots extended 1.4 to 2.0 meters in length. The first- and second-order small roots, along with the main root, began forming small nodules filled with microorganisms. These microorganisms play a crucial role in capturing nitrogen in its molecular form from the air and converting it into a form usable by plants. However, studies on the diversity and distribution of Frankia strains have been hindered by challenges in isolating them from field-collected root nodules. In the field, nitrogenase activity in root nodules remained high from May to September, corresponding with the periods of high air temperatures and active photosynthesis.

There have been frequent reports of more than one strain of the nitrogen-fixing symbiont, Frankia, in the same root nodule of plants in the genus Alnus, but quantitative assessments of their relative contributions have not been made to date. Neither has the diversity of other microbes, having potential functional roles in symbiosis, been systematically evaluated. Alnus rubra root nodule microbiota were studied using Illumina short read sequencing and kmer-based read classification. Single end 76 bp sequencing was done to a median depth of 96 million reads per sample. Reads were assigned to taxa using KrakenUniq, with taxon abundances being estimated using its companion program Bracken. This was the first high resolution study of Alnus root nodules using next generation sequencing (NGS), quantifying multiple Cluster 1 A Frankia strains in single nodules, and in some cases, a Cluster 4 strain. Root nodules were found to contain diverse bacteria, including several genera containing species known to have growth-promoting effects. Evidence was found for partitioning of some bacterial strains in older versus younger lobes.

Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss.

Nearly 50 years after the ground-breaking isolation of the primary Comptonia peregrina microsymbiont under axenic conditions, efforts to isolate a substantial number of Protofrankia and Frankia strains continue with enduring challenges and complexities. This study aimed to streamline genomic insights through comparative and predictive tools to extract traits crucial for isolating specific Frankia in axenic conditions. Pangenome analysis unveiled significant genetic diversity, suggesting untapped potential for cultivation strategies. Shared metabolic strategies in cellular components, central metabolic pathways, and resource acquisition traits offered promising avenues for cultivation. Ecological trait extraction indicated that most uncultured strains exhibit no apparent barriers to axenic growth. Despite ongoing challenges, potential caveats, and errors that could bias predictive analyses, this study provides a nuanced perspective. It highlights potential breakthroughs and guides refined cultivation strategies for these yet-uncultured strains. We advocate for tailored media formulations enriched with simple carbon sources in aerobic environments, with atmospheric nitrogen optionally sufficient to minimize contamination risks. Temperature adjustments should align with strain preferences-28-29°C for Frankia and 32-35°C for Protofrankia-while maintaining an alkaline pH. Given potential extended incubation periods (predicted doubling times ranging from 3.26 to 9.60 days, possibly up to 21.98 days), patience and rigorous contamination monitoring are crucial for optimizing cultivation conditions.

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.

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for "Alnus-infective strains" (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "Alnus-infective strains", Sp+ Frankia genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).

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.

The genus Frankia is at present the sole genus in the family Frankiaceae and encompasses filamentous, sporangia-forming actinomycetes principally isolated from root nodules of taxonomically disparate dicotyledonous hosts named actinorhizal plants. Multiple independent phylogenetic analyses agree with the division of the genus Frankia into four well-supported clusters. Within these clusters, Frankia strains are well defined based on host infectivity range, mode of infection, morphology, and their behaviour in culture. In this study, phylogenomics, overall genome related indices (OGRI), together with available data sets for phenotypic and host-plant ranges available for the type strains of Frankia species, were considered. The robustness and the deep radiation observed in Frankia at the subgeneric level, fulfilling the primary principle of phylogenetic systematics, were strengthened by establishing genome criteria for new genus demarcation boundaries. Therefore, the taxonomic elevation of the Frankia clusters to the rank of the genus is proposed. The genus Frankia should be revised to encompass cluster 1 species only and three novel genera, Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov., are proposed to accommodate clusters 2, 3, and 4 species, respectively. New combinations for validly named species are also provided.

Novel species of Frankia, Frankia gtarii sp. nov. and Frankia tisai sp. nov., isolated from a root nodule of Alnus glutinosa

The cadCA and cadB/DX operons are possibly induced in cadmium resistance mechanism by Frankia alni ACN14a

Since their introduction at the beginning of the 19th century, Casuarina species have become important components of the Algerian landscape, yet the numbers of species present in Algeria and their identity, distribution, and symbiotic status are still not well understood. A survey conducted on 2,304 trees in 96 Casuarina stands across Algeria indicates that they are represented exclusively by five species: Casuarina cristata Miq., C. cunninghamiana Miq., C. equisetifolia L., C. glauca Sieber ex Spreng. and C. junghuhniana Miq. Two species, C. cunninghamiana and C. glauca, were the most common – the former occurring more often in the relatively humid and temperate areas of the country while the latter is most frequent in the drier and warmer areas. Production of seedlings of C. cunninghamiana and C. glauca in 12 nurseries resulted in low rates of spontaneous mycorrhizal infection frequency and nodulation, suggesting that inoculation of seedlings with selected mycorrhizal fungi and/or Frankia strains in the nursery would be necessary to improve their establishment in the field. This study is the first survey of the geographical distribution of Casuarina species in Algeria and should lead to more efficient utilization of this widely used tree genus in Algeria.

Competition of two inoculated Frankia strains in root nodulation of Alnus glutinosa seedlings and associated Frankia-strain growth in rhizospheric and non-rhizospheric soils

ABSTRACT Metabolic-profiling (metabolomics/metabonomics) is a measurement strategy among biological systems for low-molecular-weight metabolites and their corresponding intermediates acting as dynamic physiological, pathophysiological and developmental stimuli. Information regarding the metabolomics of Frankia is elusive. In this study, we analyzed 48 Frankia whole-genome sequences. Kyoto Encyclopedia of Genes and Genomes (KEGG) database and KEGG Automatic Annotation Server (KAAS) were used for the prediction and functional annotation of genes associated with diverse metabolic pathways. Comprehensive codon and amino-acid usage analyses along with tRNA adaptation index and dN/dS-based evolutionary inquest were performed. We have used the Database-of-Essential-Genes (DEG) server to predict the essentiality of the metabolic pathway-related-genes. We found a strong effect of compositional constraints on the metabolic pathway genes of Frankia. The evolutionary analysis revealed the conserved nature of the studied genes. Since the metabolic pathway-related genes were found to be housekeeping genes, they are conserved and less evolved. This was further validated with a dN/dS value of less than 1. Moreover, the number of synonymous changes was more than non-synonymous changes so that the amino acid sequence remains identical. The cluster of orthologs (COG) analysis revealed “metabolism” as the main functional category of the studied gene set. The core metabolic pathway-related genes among considered Frankia strains accumulated 115 genes. This study for the first time reveals the genomic characteristics of the metabolic pathway-related genes among Frankia. From all the analysis, we could hypothesize that in spite of their host specificity and differential taxonomical distribution, Frankia maintain a core metabolic proficiency among them.

Coriaria myrtifolia occurs as natural flora of warm temperate climates of northern Algeria which commonly found in hedges, forest and ravine edges. This actinorhizal species was known to establish a mutualistic symbiosis with members of phylogenetic cluster 2 (including strains associated to Coriaria spp., Ceanothus, Datiscaceae, and Dryadoideae) within the genus Frankia. Attempts to isolate C. myrtifolia microsymbionts from native plants growing in 4 locations in Algeria permitted to only recover asymbiotic Frankia strains (unable to reestablish nodulation and to fix nitrogen) from phylogenetic cluster 4 and several non-Frankia actinobacteria including members of Micrococcus, Micromonospora, Nocardia, Plantactinospora, and Streptomyces genera. The biodiversity of Frankia microsymbionts of C. myrtifolia root nodules was assessed using PCR-amplification followed by partial nucleotide sequencing of glnA1 (glutamine synthetase type 1) gene. On the 12 different glnA1 gene sequences obtained in this study, 9 were detected for the first time, and were mainly closelyrelated to Mediterranean genotypes previously described in the Grand Maghreb countries (Morocco and Tunisia) and in Europe (France) but without clear separations from other cluster 2 genotypes.

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 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.

Shepherdia × utahensis ‘Torrey’ (hybrid buffaloberry) is an actinorhizal plant that can form symbiotic nodules with the actinobacterial genus Frankia . However, little research has been conducted to investigate the presence of Frankia in their nodules and the effects on plant growth. In this study, plants were grown in a Metro-Mix ® 820 substrate and inoculated with soils collected from Mohave County, AZ, or in a low organic-matter substrate inoculated with soils from North Logan, UT. The presence of Frankia was quantified using PolF/PolR primers to amplify their nitrogenase ( nif H) gene sequences. In the Metro-Mix 820 substrate, plants irrigated with nitrogen (N)-free Hoagland’s solution at pH 6.5 formed nodules at week 12 after experiment initiation, whereas those receiving the same solution with 2 m m ammonium nitrate (NH 4 NO 3 ) appeared healthy, but no nodules formed. In the low organic-matter substrate, nodules formed in 5 weeks when plants were irrigated with N-free Hoagland’s solution at pH 7.5. Four 300-bp fragments of query sequences (SU1, SU2, SU3, and SU4) were obtained from nodules. When compared with nif H gene sequences reported in the literature using the Basic Local Alignment Search Tool (BLAST), more than 90% similarity to the nif H of Frankia spp. was obtained. The Frankia strains in the nodules shared nif H sequences similar to those of the same host-specific group of Shepherdia . Furthermore, Frankia strains with similar nif H genes have been reported in nodules of Shepherdia argentea (silver buffaloberry). Additionally, Frankia strains belonging to cluster 3 infective strains consisting of Elaeagnaceae and Rhamnaceae infective Frankia showed high similarity to the query sequences. This research demonstrates that nodulation of S. ×utahensis is inhibited at 2 m m NH 4 NO 3 . Apart from N, nodule formation may be associated with the substrate type and pH of the nutrient solution. Based on nif H gene sequence amplification, Frankia strains in the root nodules may have the potential to fix atmospheric nitrogen (N 2 ). These Frankia strains have signature gene sequence characteristics of Elaeagnaceae-infective Frankia , suggesting that S. × utahensis shares Frankia strains similar to its parents.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases,MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

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, β-glucosidase (BG), cellobiohydrolase (CBH), β-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.