A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel-and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

Summary Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen‐fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron‐based X‐ray fluorescence assays were carried out in the nodule‐specific M. truncatula ferroportin Medicago truncatula nodule‐specific gene Ferroportin2 (MtFPN2) is an iron‐efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early‐fixation zone of the nodules. Loss‐of‐function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen‐fixing bacteroids in M. truncatula nodules.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.