Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N2-fixing root nodules on diverse and globally distributed angiosperms in the “actinorhizal” symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%–98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses

Early signaling in actinorhizal symbioses

Nitrogen-fixing root nodulation, confined to four plant orders, encompasses more than 14,000 Leguminosae species, and approximately 200 actinorhizal species forming symbioses with rhizobia (Rhizobium, Bradyrhizobium, etc.,) and Frankia bacterial species, respectively

Differential Frankia protein patterns induced by phenolic extracts from Myricaceae seeds

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the early symbiotic interactions between the bacterium Frankia and actinorhizal plants. Myricaceae, the most primitive actinorhizal family, was used as an experimental model to study specificity mechanisms because it includes species with either narrowor large specificity. Seed phenolic extracts from two Myricaceae species, Myrica gale, a narrow specificity host and Morella cerifera considered as a promiscuous host, were used to induce three Frankia strains ACN14a, M16467 and Ea112. The global protein pattern was altered by exposure to the plant extracts. The addition of 30 mg l21 of seed phenolic extracts provoked the inhibition of many protein biosynthesis whereas 1 and 10 mg l21 induced a global reprogramming of Frankia protein pattern. Changes in intensity of 115 spots in response to seed extracts were detected and analyzed by matrix-assisted laser desorption/ ionization time of flight mass spectrometry. Fifty proteins were efficiently identified with Frankia protein data banks deduced from the sequences of Frankia strains ACN14a and EaN1pec genomes. Differential proteins were involved in different metabolism pathways such as glycolysis and gluconeogenesis, transcription, fatty acids, carbohydrates, coenzymes and purines metabolisms. Chaperones biosynthesis and iron transport regulation, essential for nitrogen fixation, seem to be strain dependant. Several proteins possibly involved in the regulation of nodulation were also differentially expressed. The most obvious response was the upregulation of oxidative stress proteins such as FeSOD and Tellurium resistance proteins, suggesting a reorganization of Frankia metabolism to protect against host plant defense

The proteogenome of symbiotic Frankia alniFrankia\ alni in Alnus glutinosaAlnus\ glutinosa nodules

International audienceOmics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis

Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions

Frankia alni induces root nodules on Alnus, in which the bacterium differentiates into nitrogen (N)-fixing cells called vesicles. In culture, F. alni also undergoes major morphological changes as it alternates between N-replete and N-fixing conditions. Lack of biologically available N induces the synthesis of vesicles in which nitrogenase is protected from molecular oxygen by a thick lipid hopanoid envelope. Very little is known about the molecular basis of Frankia–host interaction as well as Frankia cell differentiation. The recent determination of the complete genome sequence of F. alni strain ACN14a has permitted us to characterize its proteome, particularly in the extracellular compartment, which could be involved in Frankia–host interaction, and in the switch from N-replete to N-fixing conditions. To that end, 126 bacterial proteins were analyzed by two-dimensional protein gel electrophoresis and identified by matrix-assisted laser desorption/ionization time of flight fingerprinting using a F. alni proteome database. Interestingly, the extracellular fraction contains some glycolytic enzymes lacking secretion signals, already reported to be extracellularly localized in some streptococci, as well as some abundant stress-resistance proteins. As expected, several proteins involved in N assimilation and oxidative defense system were upregulated in F. alni grown under N-fixing vs N-replete conditions. Furthermore, two Raf kinase inhibitor protein homologs that could play a role in cellular signaling, and a hemoglobin-like protein HbN that could be involved in detoxification of nitric oxide were also upregulated. More surprising, a succinate dehydrogenase was strongly downregulated, which could be linked to the need of pyruvate for the biosynthesis of hopanoids or to reduced oxygen diffusion in vesicles

Correction: Pujic et al. The Proteogenome of Symbiotic <i>Frankia alni</i> in <i>Alnus glutinosa</i> Nodules. <i>Microorganisms</i> 2022, <i>10</i>, 651

The authors wish to make the following corrections to this paper [...

Lectin genes in the Frankia alni genome

International audienceFrankia alni strain ACN14a's genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind speciWcally to sugar motifs. The genome of F. alni was found to contain 7 such lectincoding genes, Wve of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations

Differential Effects of Rare Specific Flavonoids on Compatible and Incompatible Strains in the Myrica gale-Frankia Actinorhizal Symbiosis▿ †

Plant secondary metabolites, and specifically phenolics, play important roles when plants interact with their environment and can act as weapons or positive signals during biotic interactions. One such interaction, the establishment of mutualistic nitrogen-fixing symbioses, typically involves phenolic-based recognition mechanisms between host plants and bacterial symbionts during the early stages of interaction. While these mechanisms are well studied in the rhizobia-legume symbiosis, little is known about the role of plant phenolics in the symbiosis between actinorhizal plants and Frankia genus strains. In this study, the responsiveness of Frankia strains to plant phenolics was correlated with their symbiotic compatibility. We used Myrica gale, a host species with narrow symbiont specificity, and a set of compatible and noncompatible Frankia strains. M. gale fruit exudate phenolics were extracted, and 8 dominant molecules were purified and identified as flavonoids by high-resolution spectroscopic techniques. Total fruit exudates, along with two purified dihydrochalcone molecules, induced modifications of bacterial growth and nitrogen fixation according to the symbiotic specificity of strains, enhancing compatible strains and inhibiting incompatible ones. Candidate genes involved in these effects were identified by a global transcriptomic approach using ACN14a strain whole-genome microarrays. Fruit exudates induced differential expression of 22 genes involved mostly in oxidative stress response and drug resistance, along with the overexpression of a whiB transcriptional regulator. This work provides evidence for the involvement of plant secondary metabolites in determining symbiotic specificity and expands our understanding of the mechanisms, leading to the establishment of actinorhizal symbioses

Omics of the early molecular dialogue between Frankia alni and Alnus glutinosa and the cellulase synton

The early Frankia-Alnus symbiotic molecular exchanges were analyzed in detail by protein and RNA omics. For this, Frankia cells were placed in the presence of Alnus roots but separated by a dialysis membrane for 64 h. The bacterial cells were then harvested and analyzed by high-throughput proteomics and transcriptomics (RNA-seq). The most upregulated gene clusters were found to be the potassium transporter operon kdp and an ABC transporter operon of uncharacterized function. The most upregulated proteins were found to be acyl dehydrogenases and the potassium transporter Kdp. These suggest a preadaptation to the impending stresses linked to the penetration into isotonic host tissues and a possible rearrangement of the membrane. Another cluster among the 60 most upregulated ones that comprised two cellulases and a cellulose synthase was conserved among the Frankia and other actinobacteria such as Streptomyces. Cellulase activity was detected on CMC all along the length of the root but not away from it. Frankia alni ACN14a was found to be unable to respire or grow on glucose as sole carbon source. The cellulose synthase was found active at the tip of hyphae in response to Alnus root exudates, resulting in a calcofluor stained tip