Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster: They Come in Teams

Abstract

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type

“…The expression of murC1 and murC2 was analysed in the nodules of Datisca glomerata (C. Presl) Paill. induced by Candidatus Frankia californiensis Dg2 [21] and by Candidatus Frankia datiscae Cm1 [14], respectively. It was analysed in the nodules of Coriaria nepalensis Wall.…”

“…induced by Candiatus Frankia datiscae Dg1 [15], in the nodules of Coriaria arborea Linds. induced by Candidatus Frankia meriodionalis Cppng1 [14], and in the nodules of Coriaria myrtifolia induced by Frankia coriariae BMG5.1 [17]. The expression was also analysed in the nodules of Discaria trinervis (Gillies ex hook.)…”

“…Eight weeks after germination, the plants were inoculated with crushed nodules of D. glomerate, which were then used for the propagation of Dg1, Dg2, and Cm1, separately. Crushed nodules of C. arborea induced by Cppng1 [14] were used for inoculation with Cppng1. For inoculation with F. coriariae BMG5.1 [17] and F. discariae BCU110501 [22], in vitro cultures of Frankia were used, which had been grown in a BAP medium without nitrogen [24], adjusted to pH 9 for BMG5.1, at 28 • C for 35 and 28 d, respectively.…”

“…The genus can be split into four phylogenetically distinct clades, known as cluster-1 to cluster-4; the first three clusters contain the symbiotic strains [10]. The phylogenetic relationships between the clusters has been a point of discussion based on use of single marker genes [11,12], but recent studies using multiple phylogenetic markers or the core genome have shown that cluster-2 is the earliest divergent symbiotic clade [13][14][15][16]. Strains belonging to cluster-2 have a particular low saprotrophic potential.…”

“…Within cluster two, so far, only two strains could be grown in culture, namely: Frankia coriariae BMG5.1 [17] and the closely related strain BMG5.30 [18]. Recent studies on the Frankia genome sequences isolated from whole nodules, or from Frankia vesicles isolated from nodules, have led to the elucidation of the phylogeny of cluster-2 and the identification of several lineages within this cluster [13][14][15]. Analysis of these genomes revealed that murC, encoding the enzyme that adds L-alanine as the first amino acid to UDP-N-actetylmuramic acid in the course of cell wall PG biosynthesis, was present in two divergent copies in all of the genomes of the Frankia cluster-2 strains available.…”