The actinorhizal symbiosis of the earliest divergent Frankia cluster

In recent years, the need to reduce reliance on synthetic nitrogen fertilizer has led to extensive research on biological nitrogen fixation, especially on root nodule symbioses. My study focuses on actinorhizal symbioses, the symbiotic interactions between members of nitrogen-fixing soil actinobacteria from the genus Frankia and a diverse group of plants from eight families, collectively called actinorhizal plants. Frankia cluster II has been shown to be sister to all other clusters. Thus, one of my aims was to gain insight into this cluster to get more information about the evolution of actinorhizal symbioses. The first sequenced genome of a member from this cluster Candidatus Frankia datiscae Dg1 originated from Pakistan. This strain contains the canonical nod genes nodABC responsible for the synthesis of lipochitooligosaccharide Nod factors. In this thesis, we obtained three Frankia inocula from North America (USA), one from Europe (France), one from Asia (Japan) and one from Oceania (Papua New Guinea). Thirteen metagenomes were sequenced based on gDNA isolated from root nodules of Datisca glomerata (Datiscaceae), Ceanothus thyrsiflorus (Rhamnaceae), Coriaria myrtifolia and Coriaria arborea (Coriariaceae). This study shows that members of Frankia cluster II come in teams, helping to explain the ability of cluster II to nodulate a wide host range, four families from two orders. The inoculum from Papua New Guinea, the only sequenced strain from the Southern Hemisphere so far, contains a new Frankia species, which was proposed as Candidatus Frankia meridionalis. All cluster II strains in this study contain the canonical nod genes nodABC, with the exception of the strain from Papua New Guinea which contains only nodB’C. All North American metagenomes also contain the sulfotransferase gene nodH. This gene shows host plant-specific expression in that it was expressed in nodules of C. thyrsiflorus but not in D. glomerata. Phylogenetic analysis and transposase frequencies of the new genomes strongly support the hypothesis that the extension of the cluster II host range from Coriaria to Datisca occurred in Eurasia and that cluster II strains came to North America via the Bering Strait. To acquire more information of the influence of the host plant on the behavior of the microsymbionts, the bacterial metabolism in nodules of D. glomerata (Cucurbitales) and C. thyrsiflorus (Rosales) were compared at the level of transcription. The system to protect nitrogenase from oxygen in Ceanothus nodules seems to be more efficient than in Datisca nodules, whereas the bacterial nitrogen metabolism is likely to be similar in both host plants. The amino acid profile of D. glomerata nodules shows that the nitrogenous solutes are dominated by glutamate and arginine, supporting the suggestion that Frankia in D. glomerata nodules exports an assimilated form of nitrogen, most likely arginine. Thus, our data show that cluster II Frankia strains differ from all other Frankia clusters with regard to the presence of the canonical nod genes and their nitrogen metabolism in symbiosis. At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.