Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes

Abstract

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

The Nodule Microbiome: N2-Fixing Rhizobia Do Not Live Alone

“…Nodulating strains of Cupriavidus contain nodA (Chen et al 2005) and other nod genes. The Cupriavidus nod genes are similar to each other and form a distinct clade suggesting that they most likely had a single origin from a neotropical (Mimosoid) Burkholderia ancestor (Andam et al 2007;Bontemps et al 2010;De Meyer et al 2016).…”

“…Agrobacterium. Cummings et al (2009) identified a nodulating strain isolated from nodules of Sesbania cannabina as Rhizobium radiobacter Chen et al (2001Chen et al ( , 2003Chen et al ( , 2005, De Meyer et al (2016) (Agrobacterium tumefaciens) IRBG74, and Youseif et al (2014) isolated agrobacterial strains carrying both nod and nif genes enabling them to elicit soybean nodulation upon reinoculation. Methylobacterium.…”

“…Even though a-and b-rhizobia are evolutionary divergent, their symbiotic (nod and nif) genes are highly similar suggesting lateral transfer (Bontemps et al 2010;Chen et al 2003;De Meyer et al 2016;Moulin et al 2001). However, legume root nodules contain many other microbial residents.…”

The Structure and Evolution of Beta-Rhizobial Symbiotic Genes Deduced from Their Complete Genomes

“…Finally, we determined that three Alpha-rhizobia genera, Bradyrhizobium, Rhizobium and Azorhizobium shared nodU and nodS (except for R. leguminosarum and R. etli), and in this context it is interesting that Sesbania spp. nodulate with Rhizobium and Azorhizobium [58], but so far are not reported to do so with Bradyrhizobium. However, Bradyrhizobium housed the widest range of host-specific genes, sharing them with most other Rhizobia discovered in our study, so it is possible that strains of Bradyrhizobium that can nodulate Sesbania spp.…”

“…Although it is widely reported [58], some reports have indicated that horizontal gene transfer (HGT) has not been common even within Alpharhizobia, as revealed by the nodA and nodC phylogenies of some Ensifer and Rhizobium symbionts [8,31]. This lack of clarity as to the origin and evolution of symbiotic genes (nodulation and nitrogen fixation genes) in rhizobium sensu lato means that their origin/evolution in the Beta-rhizobia are still a subject of much debate.…”