Regulation of the cnr Cobalt and Nickel Resistance Determinant from Ralstonia sp. Strain CH34

Abstract

Ralstonia sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28. The cnr genes are organized in two clusters, cnrYXH and cnrCBA. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the cnrY and cnrC genes. These two promoters exhibit conserved sequences at the ؊10 (CCGTATA) and ؊35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni 2؉. Half-maximal activation from the cnrCBA operon was determined by using appropriate lacZ gene fusions and was shown to occur at an Ni 2؉ concentration of about 50 M.Ralstonia sp. strain CH34 (formerly Alcaligenes eutrophus strain CH34 [3]) contains at least seven determinants encoding resistances to toxic heavy metals, located either on the bacterial chromosome or on one of the two indigenous plasmids pMOL28 (180 kb [37]) and pMOL30 (238 kb [7,20]). The cnr determinant of plasmid pMOL28 mediates inducible resistance to Co 2ϩ and Ni 2ϩ in Ralstonia sp. strain CH34 (15). The cnr determinant is similar to ncc (nickel-cobalt-cadmium resistance) of Alcaligenes xylosoxidans 31A (34) and czc (cobaltzinc-cadmium resistance) on plasmid pMOL30 of Ralstonia sp. strain CH34 (28). All three resistances are based on cation efflux, which is best characterized for Czc (13,24,29). In analogy to Czc (9, 25, 28, 32), the products of the genes cnrA, cnrB, and cnrC are likely to form a membrane-bound protein complex catalyzing an energy-dependent efflux of Ni 2ϩ and Co 2ϩ , and the mechanism of action of the CnrCBA complex may be that of a proton/cation antiporter.Three regulators seem to control Cnr, an extracellular function (ECF) sigma factor (CnrH) and the products of two additional genes with unknown precise functions (CnrX and CnrY products, respectively) (15, 16). The genes cnrYXH are located upstream of cnrCBA and have the same direction of transcription. Transposon Tn5 insertion upstream of cnrH led to a constitutive expression of nickel resistance and to low zinc resistance as well (4, 15). However, this may be a polar effect and, additionally, a readthrough from a transposon promoter. As shown with Tn5-lacZ fusions (31), cnr is best induced by 128 M Ni 2ϩ . Other metals serve as less efficient inducers; however, this experiment was done with nickel-sensitive cnr-lacZ transposon-insertion mutants. In this study an improved cnrCBA-lacZ operon fusion was constructed, which mediates full nickel resistance and which was used to evaluate the physiology of cnr regulation.

NreB from Achromobacter xylosoxidans 31A Is a Nickel-Induced Transporter Conferring Nickel Resistance

“…Transposon mutagenesis was conducted as described previously (24). Total RNA of A. xylosoxidans was isolated as described previously (8,9). Northern blot analysis was performed as described by Große et al (9).…”

“…Well-studied examples include Ralstonia metallidurans CH34 and Achromobacter xylosoxidans 31A (8,24). The determinant responsible for nickel resistance in R. metallidurans CH34, cnr (cobalt-nickel resistance), encodes three regulatory genes (cnrY, cnrX, and cnrH) and three structural genes encoding the subunits of the Co-Ni efflux pump (cnrC, cnrB, and cnrA) (8,26). The cnr determinant is similar to the ncc determinant (nickel-cobalt-cadmium resistance) of A. xylosoxidans 31A.…”

Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34

“…The C. metallidurans type strain CH34 contains two megareplicons [one chromosome of 3?9 Mb, and one megaplasmid (MPL) of 2?6 Mb], and two large plasmids, pMOL28 (171 kb) and pMOL30 (234 kb), harbouring a variety of genes conferring resistance to Cd(II), Zn(II), Co(II), Ni(II), Cr(VI), Hg(II), Pb(II), Ag(I) and Cu(II) (Grass et al, 2000;Juhnke et al, 2002;Mergeay et al, 1985;Taghavi et al, 1997). The Joint Genome Institute (JGI) in California has recently sequenced the genome of this organism, and the draft sequence is now available at http://genome.jgi-psf.org/draft_microbes/ralme/ralme.home.…”

The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane Proteins

“…For instance, pumping out the surplus of cobalt and nickel cations essentially involves the resistance, nodulation, and cell division efflux pumps CnrCBA in C. metallidurans CH34 or NccCBA in C. metallidurans 31A (3,5). The transcription of the genes cnrCBA or nccCBA encoding the efflux pumps is under the control of the products of three additional genes, cnrYXH or nccYXH, also organized in operons (3,6,7). Although the Cnr and Ncc systems parallel each other in many respects, Cnr from C. metallidurans CH34 has emerged as the experimental model for their characterization (6 -10).…”

“…Metal sensing relies on CnrX, a dimeric type II transmembrane (TM) 3 protein that displays a 13-kDa-large C-terminal metal sensor domain in the periplasm 9 -12). Upon binding of nickel or cobalt to the sensor domain of CnrX, CnrY-mediated inhibition of the extracytoplasmic function factor CnrH (13) is relieved, and CnrH enables the RNA polymerase to transcribe cnrCBA resistance genes (6,7). To characterize the structural basis of metal sensing by CnrX, we have previously produced a soluble form of the periplasmic domain spanning residues 31-148.…”