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

Abstract

The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR. A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII. Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity. IMPORTANCEThe molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5=-ATATAGTATACCCCCCTATAGTATAT-3=). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens. Cobalt is required by coenzyme B 12 -dependent enzymes and several proteins (1, 2). However, cobalt overload can cause cellular toxicity by catalyzing the generation of reactive oxygen species (3, 4), which leads to iron and glutathione depletion, and thus disturbing iron homeostasis (4-6). Cobalt competes with iron in heme proteins (7) and inhibits the activity of iron-sulfur (Fe-S) proteins as shown in Escherichia coli (5) and Salmonella enterica (6). To avoid cobalt toxicity, levels of intracellular cobalt must be properly controlled. Cobalt trafficking systems in the cell, including uptake systems, efflux systems, and metallochaperones, help maintain cobalt at levels suitable for growth (8).To prevent intracellular cobalt overload-mediated toxicity, excessive amounts of cobalt are eliminated by efflux systems involving components such as the major facilitator superfamily (MFS), P 1B-4 -ATPase, resistance nodulation cell division (RND), cation/ proton antiporter, and cation diffusion facilitator (CDF). The E. coli RcnA (resistance to cobalt and nickel) efflux pump belongs to a unique family that is responsible for the detoxification of cobalt and nickel (9). The expression of rcnA is negatively regulated by RcnR (10). CoaT is a P 1B-...

Nickel and cobalt resistance properties of Sinorhizobium meliloti isolated from Medicago lupulina growing in gold mine tailing

“…In our previous work, we found that the expression of two putative genes SM0020-17742 and SM0020-17737 was induced by Cu 2+ through transcriptome sequencing. The 966-bp-long open reading frame of SM0020-17742 encodes a 321-amino-acid protein, and the deduced protein shows high identity with several previously characterized cobalt- and nickel-resistant proteins: DmeF of C. metallidurans (ABF07084, 37%) and DmeF of A. fabrum (AAK86697, 52%), so we designated the SM0020-17742 gene dme F. In C. metallidurans and A. tumefaciens , the DmeF protein has an important role in cobalt and nickel resistance ( Dokpikul et al, 2016 ; Munkelt, Grass & Nies, 2004 ). However, the phylogenetic tree based on the DmeF protein sequence showed that the DmeF proteins of S. meliloti CCNWSX0020, S. arboris and S. medicae are more closely related to each other than that of C. metallidurans and A. tumefaciens ( Fig.…”

“…Therefore, efflux systems, uptake systems, the synthesis of ligand compounds and metallochaperones for regulating cobalt and nickel homeostasis play a crucial role in most cobalt/nickel-resistant organisms. Among these cobalt/nickel-resistant systems, resistance nodulation cell division efflux pumps (RND) ( Stahler et al, 2006 ), cation diffusion facilitators (CDF) ( Dokpikul et al, 2016 ) and P 1b -type ATPases have been highlighted ( Rutherford, Cavet & Robinson, 1999 ).…”

Nickel Metalloregulators and Chaperones

“…DmeR is a Ni(II)-and Co(II)-responsive metalloregulator that has been identified in Rhizobium leguminosarum (R. leguminosarum), Agrobacterium tumefaciens (A. tumefaciens), Sinorhizobium meliloti [81][82][83]. The dmeRF operon encodes both DmeR and DmeF, a cation diffusion facilitator (CDF) [81].…”

“…DmeR has a RcnR-like finger print, HCHH ( Figure 6), and is therefore, expected to bind Ni(II) and Co(II) with six-coordinate octahedral geometry as seen in RcnR. It is interesting to note that cobalt stress induces the expression of iron-responsive genes in A. tumefaciens [82]. Additionally, RirA, an iron regulator, is essential for the bacteria to cope with nickel and cobalt toxicity [82].…”

“…It is interesting to note that cobalt stress induces the expression of iron-responsive genes in A. tumefaciens [82]. Additionally, RirA, an iron regulator, is essential for the bacteria to cope with nickel and cobalt toxicity [82].…”