Regulation of the cnr Cobalt and Nickel Resistance Determinant of Ralstonia eutropha ( Alcaligenes eutrophus ) CH34

Abstract

The linked resistance to nickel and cobalt of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) is encoded by the cnr operon, which is localized on the megaplasmid pMOL28. The regulatory genes cnrYXH have been cloned, overexpressed, and purified in Escherichia coli. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in E. coli. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of E. coli core RNA polymerase enzyme, could retard at least two different promoter DNA targets, cnrYp and cnrHp, localized within the cnrYXH locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL M91650 description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of cnrYXH and the luxCDABE, luciferase reporter genes. Nickel and cobalt specifically induced the cnrYXH-luxCDABE fusion at optimal concentrations of 0.3 mM Ni 2؉ and 2.0 mM Co 2؉ in a noncomplexing medium for metals. The two promoter regions P Y (upstream cnrY) and P H (upstream cnrH) were probed and characterized using this vector and were found to control the nickelinducible regulatory response of the cnr operon. The cnrHp promoter was responsible for full transcription of the cnrCBA structural resistance genes, while the cnrYp promoter was necessary to obtain metal-inducible transcription from the cnrHp promoter. The zinc resistance phenotype (ZinB) of a spontaneous cnr mutant strain, AE963, was investigated and could be attributed to an insertion of IS1087, a member of the IS2 family of insertion elements, within the cnrY gene.The resistance of Ralstonia eutropha-like strain CH34 (Alcaligenes eutrophus CH34) against multiple heavy metals can be regarded as a phenomenon in its own right (15) and has found increasing applications within the field of environmental technology (29). The metal resistance determinants, which are localized predominantly on either of the two indigenous megaplasmids pMOL28 (15, 28) and pMOL30 (15), have been recently reviewed by Taghavi et al. (27). One important application has been the development of metal-specific biosensors based on in vivo and in vitro gene fusions of CH34 heavy metal resistance determinants. This was possible only through a detailed knowledge of the regulation of the metal resistances at the molecular level (2).The cnr operon, located on pMOL28 (13, 28), encodes a phenotype of inducible resistance to 5 mM Co 2ϩ and 3 mM Ni 2ϩ in minimal medium. R...

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

“…The sequence of the insertion in the cnrY1(Con) mutant gene contains a nearly complete duplication of the sequence CGCGACGCGTgGCG tGC (nonidentical base pairs are shown in lowercase) located directly downstream of the 14-bp insertion. Although the site of this putative target duplication is different from that of the insertion of IS1087 reported in the accompanying study (38), this duplication is a strong hint that an insertion sequence element was also responsible for the cnrY1(Con) mutation in strain DN195(pMOL28-4). The mutation leads to a frameshift resulting in the expression of a 134-amino-acid (aa) mutant protein which is identical in its amino-terminal 46 aa to CnrY but continues in another reading frame thereafter (15,38).…”

“…Although the site of this putative target duplication is different from that of the insertion of IS1087 reported in the accompanying study (38), this duplication is a strong hint that an insertion sequence element was also responsible for the cnrY1(Con) mutation in strain DN195(pMOL28-4). The mutation leads to a frameshift resulting in the expression of a 134-amino-acid (aa) mutant protein which is identical in its amino-terminal 46 aa to CnrY but continues in another reading frame thereafter (15,38). In contrast to CnrY, the mutant protein does not contain a possible transmembrane ␣-helix.…”

“…The PhoA data clearly demonstrate that the carboxy termini of CnrX and CnrY are in the periplasm. CnrH is indeed a sigma factor as shown by gel retardation assays (38) and runoff transcription (G. Grass, S. Kühnemund, and D. H. Nies, unpublished data). Thus, it is possible that CnrH is controlled by a CnrYX transmembrane anti-sigma factor complex which binds CnrH in the absence of Ni 2ϩ .…”

Honor thy symbionts

“…1). Previously characterized ECF-type factors control a variety of functions including expression of heat-shock genes in E. coli (67); biosynthesis of alginates (exopolysaccharides containing D-mannuronic and L-guluronic acids) in Pseudomonas aeruginosa (68); iron uptake in E. coli (69); nickel and cobalt efflux in Alcaligenes europhus (70); outer membrane protein synthesis in response to variations in osmolarity, barometric pressure, and temperature in Photobacterium spp. (71); and cellular responses to disulphide stress in Streptomyces coelicolor (72).…”

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

“…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.…”