Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34

Abstract

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-freeA. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two-to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30%o similarity, respectively, with the corresponding proteins of czc.Alcaligenes eutrophus CH34 is a metal-resistant bacterium that carries two plasmids; pMOL28 (163 kb) determines resistance to nickel, cobalt, mercury, and chromate, and pMOL30 (238 kb) determines resistance to cadmium, zinc, cobalt, mercury, and copper (7,9,16,18). Metal-resistant bacteria isolated from low-grade ore deposits in Belgium and Zaire (7, 14) have several traits in common with strain CH34 and thus indicate the predominance of this type among metal-resistant bacteria. Nickel and cobalt resistance (cnr) encoded by pMOL28 has been studied in some detail. The pMOL28-encoded nickel and cobalt resistance is inducible (31) and is due to an energy-dependent specific efflux system (20,29,38). In rare mutants, nickel and cobalt resistance is expressed constitutively (32). Plasmid pMOL28 has been transferred to other wild-type strains of A. eutrophus, such as H16, N9A, and G29, and confers the ability to tolerate 3 mM NiCl2 and 5 mM CoCl2 to the transconjugants when they are grown on solid or in liquid media (16 degree of nickel resistance also was observed in Alcaligenes hydrogenophilus, Pseudomonas putida, and P. oleovorans. In all transconjugants the resistance to nickel and cobalt was constitutively expressed. Resistance was not expressed in Escherichia coli (30).The genes coding for chromate and cobalt-nickel resistance were cloned (18) from plasmid pMOL28, and the nucleotide sequence of the chromate resistance gene was determined (19). The genes of pMOL30, which determine resistance to cadmium, zinc, and cobalt (czc), have a...

Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.

“…It has been suggested that SigX may play a similar role in B. subtifis (17). (12,(32)(33)(34) (14), and preliminary sequence similarities suggest that the E. coli 0.E operon also encodes a negative regulator (M.A.L., data not shown). Although the precise mechanism of negative regulation in these systems has not been established, in the case of CarQ the stoichiometry ofthe a factor and its negative regulator is critically important.…”

Plants as extreme environments? Ni-resistant bacteria and Ni-hyperaccumulators of serpentine flora

“…Genetic determinants for Ni resistance have been first identified and characterized at the molecular level from Alcaligenes xylosoxidans 31A (known also as Ralstonia eutropha, then as Ralstonia metallidurans and now as Cupriavidus metallidurans 31A) and Alcaligenes eutrophus CH34 (now known as Cupriavidus metallidurans CH34) which were isolated from industrially contaminated sites (Mergeay et al 1978;Schmidt and Schlegel 1989). A. xylosoxidans 31A harbors two genetic determinants (ncc, nre) on plasmid pTOM9, while C. metallidurans CH34 contains one system only (cnr) (Liesegang et al 1993;Mergeay et al 2003;Nies 2000), but a large number of transporters and a regulatory apparatus are present also in its genome constituting an integrated (both plasmid-borne and chromosomal) system for resistance to metals (Mergeay et al 2003;von Rozycki and Nies 2008). In all these systems Ni resistance is due to an inducible efflux system, mediated by ATP binding cassette (ABC) transporters, that lower the intracellular Ni concentration by pumping out Ni cations from the cytoplasm (Mergeay et al 2003).…”

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

“…Resistance to Cd# + , Zn# + and Co# + has been shown to be located on a czc operon of the plasmid pMOL30 (240 kb), while Co# + combined with Ni# + resistance is located on the cnr operon of the plasmid pMOL28 (163 kb) (Liesegang et al, 1993 ;Mergeay et al, 1985 ;Nies et al, 1987 ;Siddiqui et al, 1989). Other plasmid-borne resistances to metals include resistance to copper (cop), lead ( pbr) and mercury (mer) on pMOL30 and resistance to chromate (chr) and mercury on pMOL28 (for a review see Mergeay, 2000).…”