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Journal of Bacteriology, March 2000, p. 1399-1409, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

C. Tibazarwa,1,2 S. Wuertz,1,3 M. Mergeay,1 L. Wyns,2 and D. van der Lelie1,*

Environmental Technology Expertise Centre, Flemish Institute for Technological Research, B-2400 Mol,1 and Department of Ultrastructure, Free University of Brussels, Flemish Interuniversity Institute of Biotechnology, B-1640 St-Genesius-Rode,2 Belgium, and Institute of Water Quality Control and Waste Management, Technical University of Munich, D-85749 Garching, Germany3

Received 21 July 1999/Accepted 19 November 1999

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 Ni2+ and 2.0 mM Co2+ in a noncomplexing medium for metals. The two promoter regions PY (upstream cnrY) and PH (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.

* Corresponding author. Mailing address: Environmental Technology Expertise Centre, Flemish Institute for Technological Research (Vito), Boeretang 200, B-2400 Mol, Belgium. Phone: 0032 14 335166. Fax: 0032 14 580523. E-mail: vdlelied{at}vito.be.

Journal of Bacteriology, March 2000, p. 1399-1409, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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