The impact of Elaeagnus angustifolia root exudates on Parafrankia soli NRRL B-16219 exoproteome.

Root exudates from host plant species are known to play a critical role in the establishment and maintenance of symbiotic relationships with soil bacteria. In this study, we investigated the impact of root exudates from compatible host plant species; Elaeagnus angustifolia on the exoproteome of Parafrankia soli strain NRRL B-16219. A total of 565 proteins were evidenced as differentially abundant, with 32 upregulated and 533 downregulated in presence of the plant exudates. Analysis of the function of these proteins suggests that the bacterial strain is undergoing a complex metabolic reprogramming towards a new developmental phase elicited in presence of host plant root exudates. The upregulation of Type II/IV secretion system proteins among the differentially expressed proteins indicates their possible role in infecting the host plant, as shown for some rhizobia. Additionally, EF-Tu, proteins upregulated in this study, may function as an effector for the T4SSs and trigger plant defense responses. These findings suggest that Parafrankia soli may use EF-Tu to infect the actinorhizal host plant and pave the way for further investigations of the molecular mechanisms underlying the establishment of symbiotic relationships.

Blastococcus brunescens sp. nov., a member of the Geodermatophilaceae isolated from sandstone collected from the Sahara Desert in Tunisia.

The taxonomic position of strain BMG 8361T, isolated from sandstone collected in the Sahara Desert of Southern Tunisia, was refined through a polyphasic taxonomic investigation. Colonies of BMG 8361T were pale-orange coloured, irregular with a dry surface and produced a diffusible pink or brown pigment depending on media. The Gram-positive cells were catalase-positive and oxidase-negative. The strain exhibited growth at 10-40 °C and pH values ranging from 5.5 to 9.0, with optima at 28-35 °C and pH 6.5-8.0. Additionally, BMG 8361T demonstrated the ability to grow in the presence of up to 1 % NaCl (w/v) concentration. The peptidoglycan of the cell wall contained meso-diaminopimelic acid, glucose, galactose, xylose, ribose, and rhamnose. The predominant menaquinones consisted of MK-9(H4) and MK-9. The main polar lipids were phosphatidylcholine, phosphatidylinositol, glycophosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, and two unidentified lipids. Major cellular fatty acids were iso-C16 : 0, iso-C16 : 1 h, and C17 : 1 ω8c. Phylogenetic analyses based on both the 16S rRNA gene and whole-genome sequences assigned strain BMG 8361T within the genus Blastococcus. The highest pairwise sequence similarity observed in the 16S rRNA gene was 99.5 % with Blastococcus haudaquaticus AT 7-14T. However, when considering digital DNA-DNA hybridization and average nucleotide identity, the highest values, 48.4 and 86.58 %, respectively, were obtained with Blastococcus colisei BMG 822T. These values significantly undershoot the recommended thresholds for establishing new species, corroborating the robust support for the distinctive taxonomic status of strain BMG 8361T within the genus Blastococcus. In conjunction with the phenotyping results, this compelling evidence leads to the proposal of a novel species we named Blastococcus brunescens sp. nov. with BMG 8361T (=DSM 46845T=CECT 8880T) as the type strain.

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.

Blastococcus carthaginiensis sp. nov., isolated from a monument sampled in Carthage, Tunisia.

A comprehensive polyphasic investigation was conducted to elucidate the taxonomic position of an actinobacterium, designated BMG 814T, which was isolated from the historic ruins of Carthage city in Tunisia. It grew as pink-orange pigmented colonies and displayed versatile growth capabilities, thriving within a temperature range of 20-40 °C, across a pH spectrum ranging from pH 5.5 to 10 and in the presence of up to 4 % NaCl. Chemotaxonomic investigations unveiled specific cell components, including diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, glycophosphatidylinositol, an unidentified aminoglycophospholipid, six unidentified aminolipids, two unidentified phospholipids and one unidentified lipid in its polar lipid profile. Furthermore, galactose, glucose and ribose were identified as the primary cell-wall sugars. Major menaquinones identified were MK-9(H4), MK-9(H2) and MK-9, while major fatty acids comprised iso-C15 : 0, iso-C16 : 0, C17 : 1 ω8c and C18 : 1 ω9c. Through phylogenetic analysis based on the 16S rRNA gene sequence, the strain was positioned within the genus Blastococcus, with Blastococcus capsiensis BMG 804T showing the closest relationship (99.1 %). In light of this, draft genomes for both strains, BMG 814T and BMG 804T, were sequenced in this study, and comparative analysis revealed that strain BMG 814T exhibited digital DNA-DNA hybridization and average nucleotide identity values below the recommended thresholds for demarcating new species with all available genomes of type strains of validly names species. Based on the polyphasic taxonomy assessment, strain BMG 814T (=DSM 46848T=CECT 8878T) was proposed as the type strain of a novel species named Blastococcus carthaginiensis sp. nov.

Exploring Bacillus amyloliquefaciens strain OM81 for the production of polyhydroxyalkanoate (PHA) bioplastic using olive mill wastewater.

In this study, bacterial strains isolated from olive oil mill wastewater assigned to Bacillus (n = 4) and Klebsiella (n = 1) genera, were evaluated for their ability to accumulate intracellular PHA granules using Sudan Black staining. A maximum PHA production of 0.14 g/L (i.e., 30.2% wt./wt. in dry biomass) was observed in Bacillus amyloliquefaciens strain OM81 after 72 h of incubation in the presence of 2% glucose (synthetic medium). To reduce bioplastic production costs and recover a polluting product, olive mill wastewater was tested as a carbon source. In this context, the maximum growth (1.45 g/L) was observed in the presence of 50% olive mill wastewater. After extracting the biopolymers with chloroform, quantitative and qualitative analyses were conducted using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). FTIR showed an absorption band at 1730 cm-1 assigned to the elongation of the PHB carbonyl groups. This approach offers a dual benefit of reducing pollution and bioplastic production costs. The Bacillus amyloliquefaciens strain OM81 showed promising results for PHAs production, making it a potential candidate for further investigation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03808-4.

Frankia colletiae sp. nov., a nitrogen-fixing actinobacterium isolated from Colletia cruciata.

A nitrogen-fixing actinobacterium strain (Cc1.17T) isolated from a root nodule of Colletia cruciata was subjected to polyphasic taxonomic studies. The strain was characterized by the presence of meso-diaminopimelic acid in its peptidoglycan, galactose, glucose, mannose, rhamnose, ribose and xylose as cell-wall sugars, phosphatidylinositol, diphosphatidylglycerol, glycophospholipids, phosphatidylglycerol, glycophospholipid and uncharacterized lipids as its polar lipids, and C16 : 0, iso-C16 : 0, C17 : 1 ω9 and C18 : 1 ω9 as major fatty acids (>10 %). Strain Cc1.17T showed 16S rRNA gene sequence similarities of 97.4-99.8 % to validly named Frankia species. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain Cc1.17T in a new lineage within the genus Frankia. Digital DNA-DNA hybridization and average nucleotide identity values between strain Cc1.17T and its closest phylogenomic neighbours were well below the thresholds recommended for prokaryotic species delineation. Therefore, strain Cc1.17T (=DSM 43829T=CECT 9313T) merits recognition as the type strain of a new species for which the name Frankia colletiae sp. nov. is proposed.

Root Nodule Microsymbionts of Native Coriaria myrtifolia in Algeria.

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.

Taxogenomic status of phylogenetically distant Frankia clusters warrants their elevation to the rank of genus: A description of Protofrankia gen. nov., Parafrankia gen. nov., and Pseudofrankia gen. nov. as three novel genera within the family Frankiaceae.

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.

Decrypting phytomicrobiome of the neurotoxic actinorhizal species, Coriaria myrtifolia, and dispersal boundary of Frankia cluster 2 in soil outward compatible host rhizosphere.

The actinorhizal plant, Coriaria myrtifolia, is a neurotoxic plant species endemic to the western Mediterranean area, which forms a nitrogen-fixing symbiosis with members of Frankia cluster 2. Contrarily to other Frankia clusters, the occurrence and mode of dispersal for infective cluster 2 units outside of the host plant rhizosphere remains controversial. The present study was designed to investigate the structure of the microbiomes of C. myrtifolia phytosphere, rhizosphere, and soil samples extending outward linearly up to 1 km. Results showed that the epiphyte and endophyte communities were not significantly different from each other for most of the plant tissues. The communities associated with the below-ground tissues (nodule and root) were significantly different from those found on the above-ground tissues (fruit, leaves, and stems) and had a higher community richness. Coriaria myrtifolia phytomicrobiomes were dominated by Cyanobacteria for leaf, stem, and fruit while Actinobacteria and Proteobacteria were dominant in the root and nodule organelles. The nodule, a special niche for nitrogen fixation, was mainly inhabited by Frankia but contained several non-Frankia bacteria. Beside Frankia cluster 2, the presence of clusters 1, 4, and large numbers of cluster 3 strains have been detected in nodules, roots, and rhizospheres of C. myrtifolia. Despite Frankia being found in all plots using plant trapping bioassays with C. myrtifolia seedlings, Frankia cluster 2 was not detected in soil metagenomes showing the limits of detection by this approach. This result also suggests that in the absence of appropriate host plant species, Frankia cluster 2 has a reduced number of infective units present in the soil outward from the rhizosphere.

Blastococcus tunisiensis sp. nov., isolated from limestone collected in Tunisia.

A new actinobacterium strain, designated BMG 823T, was isolated from a limestone sample collected in Tunisia. Its taxonomic position was scrutinized using a polyphasic approach. Colonies of strain BMG 823T were pink orange-coloured, regular and had a moist surface. Cells are Gram-stain-positive, catalase-negative and oxidase-negative. The strain grew at pH 5.5-9, 10-40 °C and in presence of up to 4 % NaCl (w/v). Chemotaxonomically, strain BMG 823T was characterized by cell-wall type III containing meso-diaminopimelic acid as diamino acid, glucose, ribose and rhamnose as whole-cell sugars, MK-9(H4) as predominant menaquinone, and phosphatidylcholine, diphosphadidylglycerol, phosphatidethanolamine, phosphatidylcholine, phosphatidylinositol, unidentified glycolipid, unidentified aminophospholipids and unidentified glycophospholipid as major polar lipids. The fatty acid profile consisted of iso-C16 : 0 and iso-C17 : 1 ω9. Phylogenetic trees based on 16S rRNA gene and genome sequences placed strain BMG 823T within the genus Blastococcus and separated it from all type strains of validly published species. Comparison of 16S rRNA gene sequence similarity, digital DNA-DNA hybridization and average nucleotide identity indicated that strain BMG 823T was most closely related to Blastococcus litoris DSM 106127T and Blastococcus colisei BMG 822T with pairwise values well below the species differentiation thresholds. The distinct phenotypic and genotypic features of strain BMG 823T (=DSM 46838T=CECT 8881T) within the genus Blastococcus warrant its recognition as the type strain for the new species for which we propose the name Blastococcus tunisiensis sp. nov.

Effect of actinorhizal root exudates on the proteomes of Frankia soli NRRL B-16219, a strain colonizing the root tissues of its actinorhizal host via intercellular pathway.

Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized.

Alone Yet Not Alone: Frankia Lives Under the Same Roof With Other Bacteria in Actinorhizal Nodules.

Actinorhizal plants host mutualistic symbionts of the nitrogen-fixing actinobacterial genus Frankia within nodule structures formed on their roots. Several plant-growth-promoting bacteria have also been isolated from actinorhizal root nodules, but little is known about them. We were interested investigating the in planta microbial community composition of actinorhizal root nodules using culture-independent techniques. To address this knowledge gap, 16S rRNA gene amplicon and shotgun metagenomic sequencing was performed on DNA from the nodules of Casuarina glauca. DNA was extracted from C. glauca nodules collected in three different sampling sites in Tunisia, along a gradient of aridity ranging from humid to arid. Sequencing libraries were prepared using Illumina NextEra technology and the Illumina HiSeq 2500 platform. Genome bins extracted from the metagenome were taxonomically and functionally profiled. Community structure based off preliminary 16S rRNA gene amplicon data was analyzed via the QIIME pipeline. Reconstructed genomes were comprised of members of Frankia, Micromonospora, Bacillus, Paenibacillus, Phyllobacterium, and Afipia. Frankia dominated the nodule community at the humid sampling site, while the absolute and relative prevalence of Frankia decreased at the semi-arid and arid sampling locations. Actinorhizal plants harbor similar non-Frankia plant-growth-promoting-bacteria as legumes and other plants. The data suggests that the prevalence of Frankia in the nodule community is influenced by environmental factors, with being less abundant under more arid environments.

Draft Genome Sequence for Frankia sp. Strain BMG5.11, a Nitrogen-Fixing Bacterium Isolated from Elaeagnus angustifolia.

Frankia sp. strain BMG5.11, which was isolated from Elaeagnus angustifolia nodules, is able to infect other actinorhizal plants, including Elaeagnaceae, Rhamnaceae, Colletieae, Gymnostoma, and Myricaceae Here, we report the 11.3-Mbp draft genome sequence of Frankia sp. strain BMG5.11, with a G+C content of 69.9% and 9,926 candidate protein-encoding genes.

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.

Elucidating the ecological networks in stone-dwelling microbiomes.

Stone surfaces are extreme environments that support microbial life. This microbial growth occurs despite unfavourable conditions associated with stone including limited sources of nutrients and water, high pH and exposure to extreme variations in temperature, humidity and irradiation. These stone-dwelling microbes are often resistant to extreme environments including exposure to desiccation, heavy metals, UV and Gamma irradiation. Here, we report on the effects of climate and stone geochemistry on microbiomes of Roman stone ruins in North Africa. Stone microbiomes were dominated by Actinobacteria, Cyanobacteria and Proteobacteria but were heavily impacted by climate variables that influenced water availability. Stone geochemistry also influenced community diversity, particularly through biologically available P, Mn and Zn. Functions associated with photosynthesis and UV protection were enriched in the metagenomes, indicating the significance of these functions for community survival on stones. Core members of the stone microbial communities were also identified and included Geodermatophilaceae, Rubrobacter, Sphingomonas and others. Our research has helped to expand the understanding of stone microbial community structure and functional capacity within the context of varying climates, geochemical properties and stone conditions.

Frankia soli sp. nov., an actinobacterium isolated from soil beneath Ceanothus jepsonii.

Actinobacterial strain CjT was directly isolated from soil beneath Ceanothus jepsonii growing in the USA. The strain formed cell structures typical of the genus Frankia including extensive hyphae, vesicles and sporangia, and it effectively nodulated members of the actinorhizal Colletieae, Elaeagnaceae and Myricaceae. The whole-cell hydrolysate of strain CjT was rich in meso-diaminopimelic acid and galactose, glucose, mannose, xylose, ribose and a trace of rhamnose. Tbe polar lipid profile contained phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. The menaquinone was predominantly MK-9(H4). The fatty acid profile predominantly consisted of C17 : 1ω8c, iso-C16 : 0, C15:0, C16 : 0 and C17 : 0. A multilocus sequence analysis phylogeny based on atp1, ftsZ, dnaK, gyrA and secA gene sequences positioned the strain within Elaeagnaceae- and Colletieae-nodulating species together with Frankia elaeagni DSM 46783T, Frankia discariae DSM 46785T and Frankia irregularis DSM 45899T. Pairwise 16S rRNA gene sequence similarities showed that strain CjT was most closely related to F. discariae DSM 46785T (99.78 %) while their digital DNA-DNA hybridization value was 41.1 %. Based on the overall analyses, strain CjT (=DSM 100623T=CECT 9041T) warrants classification as the type strain of a novel species, for which the name Frankia soli sp. nov. is proposed.

Genomic Insights Into Plant-Growth-Promoting Potentialities of the Genus Frankia.

This study was designed to determine the plant growth promoting (PGP) potential of members of the genus Frankia. To this end, the genomes of 21 representative strains were examined for genes associated directly or indirectly with plant growth. All of the Frankia genomes contained genes that encoded for products associated with the biosynthesis of auxins [indole-3-glycerol phosphate synthases, anthranilate phosphoribosyltransferases (trpD), anthranilate synthases, and aminases (trpA and B)], cytokinins (11 well-conserved genes within the predicted biosynthetic gene cluster), siderophores, and nitrogenases (nif operon except for atypical Frankia) as well as genes that modulate the effects of biotic and abiotic environmental stress (e.g., alkyl hydroperoxide reductases, aquaporin Z, heat shock proteins). In contrast, other genes were associated with strains assigned to one or more of four host-specific clusters. The genes encoding for phosphate solubilization (e.g., low-affinity inorganic phosphate transporters) and lytic enzymes (e.g., cellulases) were found in Frankia cluster 1 genomes, while other genes were found only in cluster 3 genomes (e.g., alkaline phosphatases, extracellular endoglucanases, pectate lyases) or cluster 4 and subcluster 1c genomes (e.g., NAD(P) transhydrogenase genes). Genes encoding for chitinases were found only in the genomes of the type strains of Frankia casuarinae, F. inefficax, F. irregularis, and F. saprophytica. In short, these in silico genome analyses provide an insight into the PGP abilities of Frankia strains of known taxonomic provenance. This is the first study designed to establish the underlying genetic basis of cytokinin production in Frankia strains. Also, the discovery of additional genes in the biosynthetic gene cluster involved in cytokinin production opens up the prospect that Frankia may have novel molecular mechanisms for cytokinin biosynthesis.

Contrasted evolutionary constraints on carbohydrate active enzymes (CAZymes) in selected Frankia strains.

Carbohydrate active enzymes (CAZymes) are capable of breaking complex polysaccharides into simpler form. In plant-host-associated microorganisms CAZymes are known to be involved in plant cell wall degradation. However, the biology and evolution of Frankia CAZymes are largely unknown. In the present study, we took a genomic approach to evaluate the presence and putative roles of CAZymes in Frankia. The CAZymes were found to be potentially highly expressed (PHX) proteins and contained more aromatic amino acids, which increased their biosynthetic energy cost. These energy rich amino acids were present in the active sites of CAZymes aiding in their carbohydrate binding capacity. Phylogenetic and evolutionary analyses showed that, in Frankia strains with the capacity to nodulate host plants, CAZymes were evolving slower than the other PHX genes, whereas similar genes from non-nodulating (or ineffectively nodulating) Frankia strains showed little variation in their evolutionary constraints compared to other PHX genes. Thus, the present study revealed the persistence of a strong purifying selection on CAZymes of Frankia indicating their crucial role.

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.

A novel phylogenetic tree based on the presence of protein domains in selected actinobacteria.

Protein functional domains are semi-autonomous parts of proteins capable of functioning independently. One protein may contain several domains and one domain may be present in different protein sequences. Thus, protein domains represent the niche specific adaptive nature of an organism. We hypothesized that the presence and absence of protein domains in an organism could be used to make a phylogenetic tree, which may better depict the biotope (niche). Here, we selected 100 actinobacteria and built a phylogenetic tree depending upon the presence and absence of protein domains. Strains of different genera from the same niche were found to cluster together suggesting niche specific domain acquisition among selected strains. Thus, the domain based phylogeny clustered the selected actinobacteria mainly according to their niche rather than their taxonomic classification.