Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses▿

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus

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 N(2)-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

Molecular Methods for Research on Actinorhiza

Actinorhizal root nodules result from the interaction between a nitrogen-fixing actinomycete from the genus Frankia and roots of dicotyledonous trees and shrubs belonging to 25 genera within 8 plant families. Most actinorhizal plants can reach high rates of nitrogen fixation comparable to those found in root nodule symbiosis of the legumes. As a consequence, these trees are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. While the basic knowledge of these symbiotic associations is still poorly understood, actinorhizal symbioses emerged recently as original systems to explore developmental strategies to form nitrogen-fixing nodules. Many tools have been developed in recent years to explore the interaction between Frankia and actinorhizal plants including molecular biology, biochemistry, and genomics. However, technical difficulties are often encountered to explore these symbiotic interactions, mainly linked to the woody nature of the plant species and to the lack of genetic tools for their bacterial symbionts. In this chapter, we report an inventory of the main recent molecular tools and techniques developed for studying actinorhizae