Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Abstract

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.

Fig. 1.

Circular chromosomes of Frankia sp. HFPCcI3 (CCI), Frankia sp. ACN14a (ACN), and Frankia sp. EAN1pec (EAN), oriented to the dnaA gene. The inner ring indicates deviation of GC content from the genomic average. The outside outer ring shows the locations of secondary metabolic gene clusters that correlate with the cluster names provided in Table 1.

Fig. 2.

Putative Frankia chemical structures based on bioinformatic analysis of Frankia biosynthetic gene clusters. Compound numbers correlate with the “Predicted structure” data in Table 1.

Fig. 3.

Intact-cell MALDI-TOF analysis of Frankia sp. CcI3. Top, IC MALDI revealing prominent peptides ranging from 1,199 to 1,554 Da. Bottom, IC MALDI of Frankia sp. CcI3 grown on ¹⁵N-labeled medium depicting isotope shifts that roughly equate to the number of amino acid residues.