Amir Ktari

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.

Genomic approaches toward understanding the actinorhizal symbiosis: an update on the status of the Frankia genomes

The actinorhizal symbiosis is a mutualistic relationship between an actinobacterium from the genus Frankia and a wide variety of dicotyledonous plants representing 8 different families of angiosperms. Molecular phylogenetic approaches have identified four major Frankia lineages that have distinctive plant host ranges. Since the first published three Frankia genomes, an effort was undertaken to provide full genomic databases covering all four well established Frankia lineages and to provide depth of the number of strains covered. Here, we report on the updated status of these sequencing efforts. At present, there are 25 complete or draft Frankia genomes that have been sequenced and annotated, and several others are now in the pipeline being sequenced. An overview of the Frankia genomes will be presented focusing on their general genomic properties including size of the pan- and core-gene pool, size relationship and genome plasticity. Furthermore, a description of biosynthetic potential and a discussion about genes (nitrogenase, hopanoid biosynthesis, truncated hemoglobin, hydrogenase uptake gene clusters) involved in the symbiosis will be discussed. The absence of common nod genes within these Frankia genomes provides clues about the host-microbe recognition process for the actinorhizal symbiosis

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

ABSTRACT 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.

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

ABSTRACT 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.

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

ABSTRACT 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.

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.

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

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

ABSTRACT 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.

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).