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.

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.

Permanent draft genome sequence of Frankia sp. NRRL B-16219 reveals the presence of canonical nod genes, which are highly homologous to those detected in Candidatus Frankia Dg1 genome.

Frankia sp. NRRL B-16219 was directly isolated from a soil sample obtained from the rhizosphere of Ceanothus jepsonii growing in the USA. Its host plant range includes members of Elaeagnaceae species. Phylogenetically, strain NRRL B-16219 is closely related to "Frankia discariae" with a 16S rRNA gene similarity of 99.78%. Because of the lack of genetic tools for Frankia, our understanding of the bacterial signals involved during the plant infection process and the development of actinorhizal root nodules is very limited. Since the first three Frankia genomes were sequenced, additional genome sequences covering more diverse strains have helped provide insight into the depth of the pangenome and attempts to identify bacterial signaling molecules like the rhizobial canonical nod genes. The genome sequence of Frankia sp. strain NRRL B-16219 was generated and assembled into 289 contigs containing 8,032,739 bp with 71.7% GC content. Annotation of the genome identified 6211 protein-coding genes, 561 pseudogenes, 1758 hypothetical proteins and 53 RNA genes including 4 rRNA genes. The NRRL B-16219 draft genome contained genes homologous to the rhizobial common nodulation genes clustered in two areas. The first cluster contains nodACIJH genes whereas the second has nodAB and nodH genes in the upstream region. Phylogenetic analysis shows that Frankia nod genes are more deeply rooted than their sister groups from rhizobia. PCR-sequencing suggested the widespread occurrence of highly homologous nodA and nodB genes in microsymbionts of field collected Ceanothus americanus.

Proteogenomics data for deciphering Frankia coriariae interactions with root exudates from three host plants.

Frankia coriariae BMG5.1 cells were incubated with root exudates derived from compatible (Coriaria myrtifolia), incompatible (Alnus glutinosa) and non-actinorhizal (Cucumis melo) host plants. Bacteria cells and their exoproteomes were analyzed by high-throughput proteomics using a Q-Exactive HF high resolution tandem mass spectrometer incorporating an ultra-high-field orbitrap analyzer. MS/MS spectra were assigned with two protein sequence databases derived from the closely-related genomes from strains BMG5.1 andDg1, the Frankia symbiont of Datisca glomerata. The tandem mass spectrometry data accompanying the manuscript describing the database searches and comparative analysis (Ktari et al., 2017, doi.org/10.3389/fmicb.2017.00720) [1] have been deposited to the ProteomeXchange with identifiers PXD005979 (whole cell proteomes) and PXD005980 (exoproteome data).

Host Plant Compatibility Shapes the Proteogenome of Frankia coriariae.

Molecular signaling networks in the actinorhizal rhizosphere select host-compatible Frankia strains, trigger the infection process and eventually the genesis of nitrogen-fixing nodules. The molecular triggers involved remain difficult to ascertain. Root exudates (RE) are highly dynamic substrates that play key roles in establishing the rhizosphere microbiome. RE are known to induce the secretion by rhizobia of Nod factors, polysaccharides, and other proteins in the case of legume symbiosis. Next-generation proteomic approach was here used to decipher the key bacterial signals matching the first-step recognition of host plant stimuli upon treatment of Frankia coriariae strain BMG5.1 with RE derived from compatible (Coriaria myrtifolia), incompatible (Alnus glutinosa), and non-actinorhizal (Cucumis melo) host plants. The Frankia proteome dynamics were mainly driven by host compatibility. Both metabolism and signal transduction were the dominant activities for BMG5.1 under the different RE conditions tested. A second set of proteins that were solely induced by C. myrtifolia RE and were mainly linked to cell wall remodeling, signal transduction and host signal processing activities. These proteins may footprint early steps in receptive recognition of host stimuli before subsequent events of symbiotic recruitment.

Permanent Draft Genome Sequences of Three Frankia sp. Strains That Are Atypical, Noninfective, Ineffective Isolates.

Here, we present draft genome sequences for three atypical Frankia strains (lineage 4) that were isolated from root nodules but are unable to reinfect actinorhizal plants. The genome sizes of Frankia sp. strains EUN1h, BMG5.36, and NRRL B16386 were 9.91, 11.20, and 9.43 Mbp, respectively.

Permanent Improved High-Quality Draft Genome Sequence of Nocardia casuarinae Strain BMG51109, an Endophyte of Actinorhizal Root Nodules of Casuarina glauca.

Here, we report the first genome sequence of a Nocardia plant endophyte, N. casuarinae strain BMG51109, isolated from Casuarina glauca root nodules. The improved high-quality draft genome sequence contains 8,787,999 bp with a 68.90% GC content and 7,307 predicted protein-coding genes.

Permanent Draft Genome Sequence of Nocardia sp. BMG111209, an Actinobacterium Isolated from Nodules of Casuarina glauca.

Nocardia sp. strain BMG111209 is a non-Frankia actinobacterium isolated from root nodules of Casuarina glauca in Tunisia. Here, we report the 9.1-Mbp draft genome sequence of Nocardia sp. strain BMG111209 with a G + C content of 69.19% and 8,122 candidate protein-encoding genes.

Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains.

The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch's postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation.

Contrasted reactivity to oxygen tensions in Frankia sp. strain CcI3 throughout nitrogen fixation and assimilation.

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixing Frankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions on Frankia sp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen-deficient conditions. The mRNA levels for the nitrogenase structural genes (nifHDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhC and hpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, the hup2 mRNA levels increased with hyperoxic environment, while hup1 mRNA levels remained relatively constant. Taken together, these results indicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection.

Draft Genome Sequence of Frankia sp. Strain BMG5.23, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina glauca Grown in Tunisia.

Nitrogen-fixing actinobacteria of the genus Frankia are symbionts of woody dicotyledonous plants termed actinorhizal plants. We report here a 5.27-Mbp draft genome sequence for Frankia sp. strain BMG5.23, a salt-tolerant nitrogen-fixing actinobacterium isolated from root nodules of Casuarina glauca collected in Tunisia.

Draft Genome Sequence of Frankia sp. Strain Thr, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina cunninghamiana Grown in Egypt.

Nitrogen-fixing actinobacteria of the genus Frankia are symbionts of woody dicotyledonous plants termed actinorhizal plants. We report here a 5.3-Mbp draft genome sequence for Frankia sp. stain Thr, a nitrogen-fixing actinobacterium isolated from root nodules of Casuarina cunninghamiana collected in Egypt.

Nocardia casuarinae sp. nov., an actinobacterial endophyte isolated from root nodules of Casuarina glauca.

An actinobacterium strain BMG51109a was isolated from surface sterilized root nodules of Casuarina glauca collected in Tunisia. The 16S rRNA gene sequence of strain BMG51109a showed most similarity (96.53-96.55 %) to the type strains of Nocardia transvalensis, N. aobensis and N. elegans. Chemotaxonomic analysis supported the assignment of the strain to Nocardia genus. The major menaquinone was MK-8(H4c) while the polar lipid profile contained diphosphatidylglycerol, phosphatidylmonomethylethanolamine, glycophospholipid, phosphatidylinositol, one uncharacterized phospholipid and three glycolipids. Whole-cell sugar analysis revealed the presence of meso-diaminopimelic acid, arabinose and galactose as diagnostic sugars, complemented by glucose, mannose and ribose. The major cellular fatty acids were tuberculostearic, oleic, palmitoleic and stearic acids. Physiological and biochemical tests showed that strain BMG51109a could be clearly distinguished from its closest phylogenetic neighbours. On the basis of these results, strain BMG51109a(T) (= DSM 45978(T) = CECT 8469(T)) is proposed as the type strain of the novel species Nocardia casuarinae sp. nov.