Functional and Expression Analysis of the Metal-Inducible dmeRF System from Rhizobium leguminosarum bv. viciae

Abstract

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.

Regulation of the Cobalt/Nickel Efflux OperondmeRFin Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance

“…DmeF is classified into the subgroup I Zn-CDFs (39). However, the DmeF proteins have been shown to exhibit broad metal specificity, with the ability to extrude Co(II) and Ni(II) (18,19). Like DmeF Cm and DmeF Rl , Agrobacterium tumefaciens DmeF (DmeF At ) exhibits six predicted transmembrane (TM) domains that contain two motifs, which are characteristic of the CDF family (the HX 3 H motif at TM2 and the HX 3 D motif at TM5) and are a unique characteristic of the subgroup I CDFs, in the histidine-rich stretch located between TM4 and TM5 (see Fig.…”

“…Thus far, only two DmeF proteins have been studied, from Cupriavidus metallidurans CH34 (DmeF Cm ) (18) and Rhizobium leguminosarum (DmeF Rl ) (19). DmeF is classified into the subgroup I Zn-CDFs (39).…”

“…The R. leguminosarum dmeRF mutant is hypersensitive to cobalt and nickel. The dmeRF genes have been shown to play an important role in R. leguminosarum during free-living and symbiotic interactions with legume plants under high-cobalt and high-nickel conditions (19). Cobalt stress and iron homeostasis are linked.…”

“…Cobalt transporters have not been characterized in A. tumefaciens. Rhizobium leguminosarum, a close relative of A. tumefaciens, belongs to the Alphaproteobacteria and contains the dmeRF operon that is responsible for exporting cobalt and nickel (19). R. leguminosarum DmeR represses dmeRF transcription.…”

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

“…In the original R. leguminosarum background a Ni-and Co-efflux system (DmeRF) is known to operate, and its role has been connected to the uptake of nickel. 42 Alternatively, another electrogenic efflux system could act as a charge-compensating mechanism by extruding positive charges out of the cell. Our uptake mixture is made of a potassium phosphate (KH 2 /K 2 HPO 4 ) buffer containing 10 mM MgCl 2 .…”