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Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

ABSTRACT: The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

SUBMITTER: Borremans B

PROVIDER: S-EPMC95457 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

Borremans B B Hobman J L JL Provoost A A Brown N L NL van Der Lelie D D

Journal of bacteriology 20011001 19

The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a ...[more]

PMID: 11544228

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Project description: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 possess 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 nickel-inducible 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.

Dataset Information

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

Publications

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

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